Ubiquitin-specific protease 42 regulates osteogenic differentiation of human adipose-derived stem cells / 北京大学学报(医学版)

Objective:To explore the effect of ubiquitin-specific protease 42(USP42)on osteogenic differentiation of human adipose-derived stem cells(hASCs)in vivo and in vitro.Methods:A combina-tion of experiments was carried out with genetic depletion of USP42 using a lentiviral strategy.Alkaline phosphatase(ALP)staining and quantification,alizarin red S(ARS)staining and quantification were used to determine the osteogenic differentiation ability of hASCs under osteogenic induction between the experimental group(knockdown group and overexpression group)and the control group.Quantitative re-verse transcription PCR(qRT-PCR)was used to detect the expression levels of osteogenesis related genes in the experimental group and control group,and Western blotting was used to detect the expression levels of osteogenesis related proteins in the experimental group and control group.Nude mice ectopic im-plantation experiment was used to evaluate the effect of USP42 on the osteogenic differentiation of hASCs in vivo.Results:The mRNA and protein expressions of USP42 in knockdown group were significantly lower than those in control group,and those in overexpression group were significantly higher than those in control group.After 7 days of osteogenic induction,the ALP activity in the knockdown group was sig-nificantly higher than that in the control group,and ALP activity in overexpression group was significantly lower than that in control group.After 14 days of osteogenic induction,ARS staining was significantly deeper in the knockdown group than in the control group,and significantly lighter in overexpression group than in the control group.The results of qRT-PCR showed that the mRNA expression levels of ALP,os-terix(OSX)and collagen type Ⅰ(COL Ⅰ)in the knockdown group were significantly higher than those in the control group after 14 days of osteogenic induction,and those in overexpression group were signifi-cantly lower than those in control group.The results of Western blotting showed that the expression levels of runt-related transcription factor 2(RUNX2),OSX and COL Ⅰ in the knockout group were significant-ly higher than those in the control group at 14 days after osteogenic induction,while the expression levels of RUNX2,OSX and COL Ⅰ in the overexpression group were significantly lower than those in the control group.Hematoxylin-eosin staining of subcutaneous grafts in nude mice showed that the percentage of osteoid area in the knockdown group was significantly higher than that in the control group.Conclusion:Knockdown of USP42 can significantly promote the osteogenic differentiation of hASCs in vitro and in vi-vo,and overexpression of USP42 significantly inhibits in vivo osteogenic differentiation of hASCs,and USP42 can provide a potential therapeutic target for bone tissue engineering.

Effect of adipose-derived stem cells with overexpression or silencing of lncRNA SNHG8 on vascular endothelial cell dysfunction / 中国病理生理杂志

AIM:To investigate the effects of adipose-derived stem cells(ADSCs)with overexpression or si-lencing of long noncoding RNA(lncRNA)SNHG8 on the viability,migration,angiogenesis,and the expression of vasoac-tive factors in human umbilical vein endothelial cells(HUVECs).METHODS:Identification of ADSCs derived from morbidly obese patients(O-ADSCs)was conducted using flow cytometry and induction of lipogenesis and osteogenesis.The expression of lncRNA SNHG8 in healthy human ADSCs(H-ADSCs)and O-ADSCs was detected by RT-qPCR.Tran-swell method was used to establish the indirect co-culture system of ADSCs and HUVECs for 48 h,and the cells were di-vided into O-ADSCs+HUVECs group,H-ADSCs+HUVECs group,and HUVECs alone group.The mRNA and protein ex-pression levels of angiotensin Ⅱ(Ang Ⅱ),endothelin-1(ET-1)and endothelial nitric oxide synthase(eNOS)in HUVECs were detected by RT-qPCR and Western blot.The lncRNA SNHG8 overexpression and silencing lentiviruses were con-structed and used to infect O-ADSCs.The indirect co-cultured ADSCs and HUVECs were divided into O-ADSCs-OE-SNHG8+ HUVECs group,O-ADSCs-OE-NC+HUVECs group,O-ADSCs-sh-SNHG8+HUVECs group,and O-ADSCs-sh-NC+HUVECs group.After co-culture for 48 h,the viability,migration and tubule formation of HUVECs were detected by CCK-8,scratch and angiogenesis assays,respectively.The mRNA and protein expression levels of Ang Ⅱ,ET-1 and eNOS in HU-VECs were detected by RT-qPCR and Western blot,respectively.The nitrate reductase method was used to detect the con-tent of NO in HUVECs.RESULTS:(1)The cultured cells were identified as ADSCs.(2)Compared with H-ADSCs,ln-cRNA SNHG8 expression was significantly up-regulated in O-ADSCs(P<0.01).(3)Compared with H-ADSCs+HUVECs group and HUVECs group,the mRNA and protein expression levels of Ang Ⅱ and ET-1 in HUVECs in O-ADSCs+HU-VECs group were up-regulated(P<0.01).(4)Overexpression of lncRNA SNHG8 in O-ADSCs enhanced the viability,mi-gration and tube formation ability of HUVECs,up-regulated the mRNA and protein expression levels of Ang Ⅱ and ET-1,down-regulated the mRNA and protein expression levels of eNOS,and decreased the content of NO in HUVECs(P<0.05).However,silencing of lncRNA SNHG8 in O-ADSCs exerted opposite results(P<0.05).CONCLUSION:(1)The O-ADSCs can promote endothelial cell viability,migration and tubule formation through paracrine effects.(2)The O-ADSCs with overexpression of lncRNA SNHG8 promote the imbalance of diastolic and contractile factors secreted by endo-thelial cells,and induce the dysfunction of vascular endothelial cells.

Research progress on dedifferentiated fat cells and their application in oral and maxillofacial bone tissue engineering / 口腔疾病防治

@#The identification of suitable seed cells represents a critical scientific problem to be solved in the field of oral and maxillofacial bone tissue regeneration. The application of adipose-derived stem cells (ASCs) in tissue and organ repair and regeneration has been studied extensively. In recent years, dedifferentiated fat (DFAT) cells have also shown broad application prospects in the field of bone tissue engineering. DFAT cells express stem cell-related markers and have the potential to differentiate into adipocytes, osteoblasts, chondrocytes, nerve cells, cardiomyocytes and endothelial cells. In addition, DFAT cells also have the advantages of minimally invasive acquisition, strong proliferation and high homogeneity. Currently, all studies involving the application of DFAT cells in scaffold-based and scaffold-free bone tissue engineering can confirm their effectiveness in promoting bone regeneration. However, cytological research still faces some challenges, including relatively low cell culture purity, unclear phenotypic characteristics and undefined dedifferentiation mechanisms. It is believed that with the continuous development and improvement of isolation, culture, identification and directional induction of osteogenic differentiation methods, DFAT cells are expected to become excellent seed cells in the field of oral and maxillofacial bone tissue engineering in the future.

The effect of gauze filtration on the content of adipose stem cells in fat gel / 中国医师杂志

Objective:To study the effect of gauze filtration on the content of adipose stem cells in fat gel.Methods:In March 2022, fat samples from 5 patients who underwent abdominal fat aspiration at the Hunan Provincial People′s Hospital were collected, prepared fat ged, the fat gel was filtered 1-5 times with 2 layers of gauze, and the amount of fat gel passing through a 1 ml threaded syringe with a 27 G needle was measured after each filtration. At the same time, the content of fat stem cells in the filtered fat gel was detected by flow cytometry.Results:As the number of filters increases, the amount of fat gel passing through gradually increased. The increasing trend was obvious after the first to fourth filters, and the increasing trend slowed down after the fifth filter. After statistical analysis, the differences between the first filter and the second filter, the second filter and the third filter, and the third filter and the fourth filter were statistically significant ( P<0.05). There was no statistically significant difference between the fourth filter and the fifth filter ( P>0.05). As the number of filters increased, the content of adipose stem cells in the fat gel gradually decreased. The decreasing trend was obvious after the first to third filters, and the decreasing trend slowed down after the fourth filter. After statistical analysis, the differences between the first filter and the second filter, as well as the second filter and the third filter, were statistically significant (all P<0.05). However, there was no statistically significant difference between the third filter and the fourth filter, and the fourth filter and the fifth filter (all P>0.05). Conclusions:The more times gauze is filtered, the better the permeability of fat gel. However, the content of adipose stem cells will significantly decrease in the first three filters. Therefore, the number of times gauze is filtered for fat gel should be minimized as much as possible.

The study of the whole bladder acellular matrix prepared by the perfusion decellularization system combined with the adipose-derived stem cells to construct the tissue engineering bladder / 中华泌尿外科杂志

Objective:To prepare the whole bladder acellular matrix (BAM) using the self-designed perfusion decellularization system, and evaluate the feasibility of constructing the tissue engineering bladder with the adipose-derived stem cells (ADSCs).Methods:This study was conducted from October 2020 to April 2021. The self-designed perfusion decellularization system was used, and four different decellularization protocols (group A, group B, group C and group D) were formulated, according to the flow direction of the perfusate and the action time of different decellularization solutions. Among them, the urethral orifice of the bladder tissue was used as the outflow tract of the perfusion fluid in groups A and B. The top of the bladder was cut off and used as the outflow tract of the perfusion fluid in groups C and D. In groups A and C, 1% Triton X-100 was treated for 6 h, and 1% sodium dodecyl sulfate (SDS) was treated for 2 h. In groups B and D, 1% Triton X-100 was treated for 7 h, and 1% sodium dodecyl sulfate (SDS) was treated for 1 h. In addition, the tissue in the normal bladder group was directly obtained from the natural bladder tissue, which did not require perfusion, cryopreservation and thawing. The fast and efficient decellularization protocol was screened out through HE, DAPI, Masson trichrome and Alcian Blue staining and quantitative analyses to prepare the whole bladder scaffold. The prepared BAM was used as the scaffold material, and the ADSCs were used as the seeding cells to construct the tissue engineering bladder. HE and DAPI staining were used to observe the distribution of ADSCs on the BAM.Results:HE and DAPI staining showed that there was no obvious nuclear residue in the group C. Masson trichrome and Alcian Blue staining showed that the collagen structure and glycosaminoglycan were well preserved in the group C. There was no significant difference in bladder wall thickness between the group C and the normal bladder group [(975.44±158.62)μm vs.(1 064.49±168.52)μm, P > 0.05]. The DNA content in the group C [(43.59 ±4.59) ng/mg] was lower than that in the normal bladder group, group A, group B and group D [(532.50±26.69), (135.17±6.99), (182.49±13.69) and(84.00±4.38)ng/mg], and the difference was statistically significant ( P<0.05). The collagen content [(10.98 ± 0.29)μg/mg] and glycosaminoglycan content [(2.30±0.18)μg/mg] in group C were not significantly different with those in the normal bladder group [(11.69±0.49) and (2.36±0.09)μg/mg, P>0.05]. Scanning electron microscopy showed that a large number of pore structures could be observed on the surface of the prepared BAM in groups A-D and were facilitated to cell adhesion. The isolated and cultured ADSCs were identified by flow cytometry to confirm the positive expression of CD90 and CD29, and the negative expression of CD45 and CD106. Live/dead staining and CCK-8 detection confirmed that the prepared BAM in the group C had no cytotoxicity. HE and DAPI staining showed that a large number of ADSCs were distributed on the surface and inside of the tissue engineering bladder. Conclusions:The whole bladder shape BAM prepared by the self-designed perfusion decellularization system could be used as the scaffold material for bladder tissue engineering, and the constructed tissue engineering bladder could be used for bladder repair and reconstruction.

Aucubin combined with ADSCs-exos protects TBHP-induced nucleus pulposus cells via TLR4/NF-κB pathway / 中国中药杂志

This paper aims to investigate the effects and mechanisms of adipose-derived stem cells-exosomes(ADSCs-exos) toge-ther with aucubin in protecting human-derived nucleus pulposus cells(NPCs) from inflammatory injury, senescence, and apoptosis. The tert-butyl hydroperoxide(TBHP)-induced NPCs were assigned into normal, model, aucubin, ADSCs-exos, and aucubin+ADSCs-exos groups. The cell viability was examined by cell counting kit-8(CCK-8), cell proliferation by EdU staining, cell senescence by senescence-associated-β-galactosidase(SA-β-Gal), and cell cycle and apoptosis by flow cytometry. Enzyme-linked immunosorbent assay was employed to examine the expression of interleukin-1β(IL-1β), IL-10, and tumor necrosis factor-α(TNF-α). Real-time fluorescence quantitative PCR and Western blot were employed to determine the mRNA and protein levels of aggregated proteoglycan(aggrecan), type Ⅱ collagen alpha 1(COL2A1), Toll-like receptor 4(TLR4), and nuclear factor-kappa B(NF-κB). The results showed that compared with the model group, the aucubin or ADSCs-exos group showed enhanced viability and proliferation of NPCs, decreased proportion of G_0/G_1 phase cells, increased proportion of S phase cells, reduced apoptosis and proportion of cells in senescence, lowered IL-1β and TNF-α levels, elevated IL-10 level, down-regulated mRNA and protein levels of TLR4 and NF-κB, and up-regulated mRNA and protein levels of aggrecan and COL2A1. Compared with the aucubin or ADSCs-exos group, the aucubin+ADSCs-exos combination further increased the viability and proliferation of NPCs, decreased the proportion of G_0/G_1 phase cells, increased the proportion of S phase cells, reduced the apoptosis and proportion of cells in senescence, lowered the IL-1β and TNF-α levels, elevated the IL-10 level, down-regulated the mRNA and protein levels of TLR4 and NF-κB, and up-regulated the mRNA and protein levels of aggrecan and COL2A1. In summary, both aucubin and ADSCs-exos could exert protective effects by inhibiting inflammatory responses, reducing apoptosis and senescence of NPCs, improving cell viability and proliferation as well as extracellular matrix synthesis, which may be associated with the inhibition of TLR4/NF-κB signaling pathway activation. The combination of both plays a synergistic role in the protective effects.

Effect of Kartogenin combined with adipose-derived stem cells on tendon-bone healing after anterior cruciate ligament reconstruction / 中国修复重建外科杂志

OBJECTIVE@#To investigate the effect of Kartogenin (KGN) combined with adipose-derived stem cells (ADSCs) on tendon-bone healing after anterior cruciate ligament (ACL) reconstruction in rabbits.@*METHODS@#After the primary ADSCs were cultured by passaging, the 3rd generation cells were cultured with 10 μmol/L KGN solution for 72 hours. The supernatant of KGN-ADSCs was harvested and mixed with fibrin glue at a ratio of 1∶1; the 3rd generation ADSCs were mixed with fibrin glue as a control. Eighty adult New Zealand white rabbits were taken and randomly divided into 4 groups: saline group (group A), ADSCs group (group B), KGN-ADSCs group (group C), and sham-operated group (group D). After the ACL reconstruction model was prepared in groups A-C, the saline, the mixture of ADSCs and fibrin glue, and the mixture of supernatant of KGN-ADSCs and fibrin glue were injected into the tendon-bone interface and tendon gap, respectively. ACL was only exposed without other treatment in group D. The general conditions of the animals were observed after operation. At 6 and 12 weeks, the tendon-bone interface tissues and ACL specimens were taken and the tendon-bone healing was observed by HE staining, c-Jun N-terminal kinase (JNK) immunohistochemical staining, and TUNEL apoptosis assay. The fibroblasts were counted, and the positive expression rate of JNK protein and apoptosis index (AI) were measured. At the same time point, the tensile strength test was performed to measure the maximum load and the maximum tensile distance to observe the biomechanical properties.@*RESULTS@#Twenty-eight rabbits were excluded from the study due to incision infection or death, and finally 12, 12, 12, and 16 rabbits in groups A-D were included in the study, respectively. After operation, the tendon-bone interface of groups A and B healed poorly, while group C healed well. At 6 and 12 weeks, the number of fibroblasts and positive expression rate of JNK protein in group C were significantly higher than those of groups A, B, and D (P<0.05). Compared with 6 weeks, the number of fibroblasts gradually decreased and the positive expression rate of JNK protein and AI decreased in group C at 12 weeks after operation, with significant differences (P<0.05). Biomechanical tests showed that the maximum loads at 6 and 12 weeks after operation in group C were higher than in groups A and B, but lower than those in group D, while the maximum tensile distance results were opposite, but the differences between groups were significant (P<0.05).@*CONCLUSION@#After ACL reconstruction, local injection of a mixture of KGN-ADSCs and fibrin glue can promote the tendon-bone healing and enhance the mechanical strength and tensile resistance of the tendon-bone interface.

Experimental study of subcutaneous adipose-derived stem cells inhibiting orthodontic root resorption / 中国修复重建外科杂志

OBJECTIVE@#To investigate the effect of human subcutaneous adipose-derived stem cells (hADSCs) local transplantation on orthodontically induced root resorption (OIRR) and provide theoretical and experimental basis for the clinical application of hADSCs to inhibit OIRR.@*METHODS@#Forty 8-week-old male Sprague Dawley rats were randomly divided into experimental group and control group, with 20 rats in each group, to establish the first molar mesial orthodontic tooth movement (OTM) model of rat right maxillary. The rats in the experimental group were injected with 25 μL of cell suspension containing 2.5×10 5 hADSCs on the 1st, 4th, 8th, and 12th day of modeling, while the rats in the control group were injected with 25 μL of PBS. The rat maxillary models were obtained before and after 7 and 14 days of force application, and 10 rats in each group were killed and sampled after 7 and 14 days of force application. The OTM distance was measured by stereomicroscope, the root morphology of the pressure side was observed by scanning electron microscope and the root resorption area ratio was measured. The root resorption and periodontal tissue remodeling of the pressure side were observed by HE staining and the root resorption index was calculated. The number of cementoclast and osteoclast in the periodontal tissue on the pressure side was counted by tartrate resistant acid phosphatase staining.@*RESULTS@#The TOM distance of both groups increased with the extension of the force application time, and there was no significant difference ( P<0.05). There was no significant difference in OTM distance between the experimental group and the control group after 7 and 14 days of force application ( P>0.05). Scanning electron microscope observation showed that small and shallow scattered resorption lacunae were observed on the root surface of the experimental group and the control group after 7 days of force application, and there was no significant difference in the root resorption area ratio between the two groups ( P>0.05); after 14 days of application, the root resorption lacunae deepened and became larger in both groups, and the root resorption area ratio in the experimental group was significantly lower than that in the control group ( P<0.05). The range and depth of root absorption in the experimental group were smaller and shallower than those in the control group, and the root absorption index in the experimental group was significantly lower than that in the control group after 14 days of force application ( P<0.05). The number of cementoclast in the experimental group was significantly lower than that in the control group after 7 and 14 days of force application ( P<0.05); the number of osteoclasts in the experimental group was significantly lower than that in the control group after 14 days of force application ( P<0.05).@*CONCLUSION@#Local transplantation of hADSCs may reduce the area and depth of root resorption by reducing the number of cementoclasts and osteoclasts during OTM in rats, thereby inhibiting orthodontic-derived root resorption.

The effect of ADSC on the activity of EPC under co-culture and its related mechanism / 安徽医科大学学报

Objective@#To investigate the effect of co-culture of adipose-derived stem cells(ADSC) and endothelial progenitor cells(EPC) on the activity of EPC and its related mechanism．@*Methods@#Rat ADSC and EPC were isolated，cultured，expanded and identified in vitro ．The experiment was divided into three groups : EPC group，EPC + ADSC co-culture group，and EPC + ADSC + PI3K-inhibitor group．After 48 hours of co-culture，the cells of the three groups were treated with Transwell．The effects of ADSC and EPC co-culture and PI3K / AKT pathway on EPC activity were evaluated by CCK-8 assay，scratch assay and angiogenesis assay，respectively．Western blot was used to detect vascular endothelial growth factor A (VEGFA) and endothelial nitric oxide synthase (eNOS) ，vascular endothelial-cadherin ( VE-cadherin) ，CD133 ，phospho-phosphatidylinositol 3-kinase ( phospho-phosphatidylinositol 3-kinase(p-PI3K) and phospho-protein Kinase B (p-AKT) expression levels in EPC to detect the effects of ADSC and EPC co-culture and PI3K / AKT pathway on the differentiation ability of EPC into mature endothelial cells. @*Results@#CCK-8 results showed that the absorbance at 450 nm of EPC in EPC + ADSC co-culture group was higher than that in EPC group and EPC + ADSC + PI3K-inhibitor group at different time points，and the difference was statistically significant (P＜0. 01) ．The scratch test showed that the relative scratch distance of EPC + ADSC co-culture group was smaller than that of EPC group and EPC + ADSC + PI3K-inhibitor group after 24 hours ，and the difference was statistically significant (P＜0. 01) ．Tube formation assay showed that the average number of tubelike structures formed in EPC + ADSC co-culture group was higher than that in EPC group and EPC + ADSC + PI3K-inhibitor group，and the difference was statistically significant (P＜0. 01) ．Western blot showed that the expression levels of VEGFA，eNOS，VE-cadherin，p-PI3K and p-AKT of EPC in EPC + ADSC co-culture group were higher than those in EPC group and EPC + ADSC + PI3K-inhibitor group．The expression level of CD133 in EPC group was lower than that in EPC + ADSC + PI3K-inhibitor group，and the difference was statistically significant (P ＜0. 01) ．@*Conclusion @#Co-culture of ADSC and EPC can improve the proliferation，migration，differentiation and vasogenic activity of EPC through the regulation of PI3K / AKT pathway.

Stem cell therapy for alopecia areata / 中华皮肤科杂志

It is currently considered that alopecia areata is caused by the impairment of immune privilege in hair follicles. Stem cells have immunoregulatory functions, and can secrete a variety of cytokines to promote immune privilege in hair follicles. Stem cell therapy, especially umbilical cord- and adipose-derived stem cell therapy, has been applied to a variety of preclinical and clinical studies on alopecia, providing a new approach to refractory alopecia areata.

Role of active ADM microparticle in skin wound healing of diabetic mice / 中华医学美学美容杂志

Objective:To evaluate the effects of adipose-derived stem cells (ADSCs) and ADM microparticle on diabetic wound healing.Methods:ADSCs was co-cultured with ADM microparticle in vitro. The models of diabetic nude mice were established by intraperitoneal injection of STZ and the full-thickness skin defects were designed on the back. All 24 diabetic mice were randomly divided into 4 group: experimental groups were transplanted with ADSCs and ADM microparticle and the other groups were transplanted with ADSCs, ADM microparticle and blank control group was set up. On the 7th and 14 th days, the wound healing rate of 3 mice randomly selected from each group was calculated, and the thickness between dermis and epidermis was measured by hematoxylin and eosin staining. The density of neovascularization was measured by immunohistochemical staining. The differences were compared between the groups.Results:Compared to the ADSCs groups, the mice of the experimental groups showed higher cell survival rate. The wound healing rate in the experimental groups was (86.0±2.7)% (7 days) and (98.5±1.1)% (14 days), thicker dermis-epidermis distance was (99.1±1.8) μm (7 days) and (124.3±4.3) μm (14 days) ( P<0.05), and higher density of neovascularization was noted. Conclusions:The transplantation with active ADM microparticle can significantly promote neovascularization and wound healing of diabetic wound.

Comparison of two different levels of adipose derived stem cells in fat transplantation in nude mice / 中华医学美学美容杂志

Objective:To explore the mechanism of fat formation induced by PLA-adipose derived stem cells (ADSC) and LAF-ADSC cells, and to provide a new direction and idea for cell-assisted granular fat transplantation.Methods:The liposuction operation of normal human body was performed in the plastic surgery operating room of Chengdu Badachu Medical Cosmetic Hospital. The liposuction obtained through the operation was placed still, and the upper granular adipose tissue was digested, isolated, cultured and identified by PLA-ADSC; the obtained lower liquid tissue was centrifuged, and the precipitate was digested, isolated, cultured and identified by LAF-ADSC; the differentiation characteristics and growth ability of PLA-ADSC and LAF-ADSC were compared. Then animal experiments were carried out: PLA-ADSC and LAF-ADSC were mixed with matrix glue Matrigel and transplanted into nude mice as experimental group 1 and experimental group 2. Matrix glue Matrigel was transplanted into nude mice as control group. The lipogenic ability of the three groups in nude mice was compared.Results:The cell experiment showed that the cells extracted from the static granular fat and the lower filtrate sediment could adhere to the wall and grew and subcultured smoothly. The cells from the above two different sources expressed CD44, CD73 and CD105, with positive rates of 99.5%, 99.99% and 99.7% respectively, while CD19, CD31 and CD45 were negative. The results of lipogenic induction and differentiation showed that there were lipid droplets in the cytoplasm, and the lipid droplets were orange red after cell oil red O staining. The results of animal experiment showed that three months after transplantation, the average graft volume of experimental group 1 was (0.070±0.009) cm 3, that of experimental group 2 was (0.067±0.007) cm 3, and that of the control group was (0.009±0.005) cm 3. The difference between the graft volume of experimental group 1 and experimental group 2 and the control group was statistically significant ( t=26.522 and t=37.183, all P<0.01), There was no significant difference in graft volume ( t=1.250, P>0.05). The wet weight of grafts in experimental group 1 was (0.200±0.021) g, that in experimental group 2 was (0.175±0.019) g, and that in the control group was (0.129±0.012) g, there was significant difference between experimental groups 1 and 2 and the control group ( t=11.601 and t=9.978, P<0.05), but there was no significant difference between experimental group 1 and experimental group 2 ( t=3.650, P>0.05). After oil red O staining, the grafts in experimental groups 1 and 2 were generally orange yellow, and the control group was scattered light yellow. The expression of CD31 in the experimental groups 1 and 2 was positive, and the expression of CD31 in control group was negative. Conclusions:Active ADSCs can be extracted from the granular fat layer and the lower filtrate of the static fat aspirate, and the ADSCs from both sources have good lipogenic ability in nude mice.

The study on the tissue engineered bladder patch constructed with the double-layer silk scaffold and adipose-derived stem cells for bladder repair and reconstruction / 中华泌尿外科杂志

Objective:To investigate the effect of tissue engineered bladder patch constructed by double-layer silk scaffold and adipose-derived stem cells (ADSCs) in the repair and reconstruction of bladder.Methods:This study was conducted from May 2020 to March 2021. The silk fibroin (SF) aqueous solution was obtained from silkworm cocoons, and a double-layer silk scaffold composed of silk fibroin film and silk fibroin sponge was further prepared. The rat ADSCs were isolated, cultured, and the ADSCs surface markers (CD29, CD90, CD45, CD106) were identified by flow cytometry. The ADSCs were planted on a double-layer silk scaffold to construct a tissue-engineered bladder patch. Thirty-six male SD rats were randomly divided into three groups: tissue engineered bladder patch group (SF-ADSCs group, n=15), double-layer silk scaffold group (SF group, n=15), control group ( n=6). The tissue engineered bladder patch (SF-ADSCs group) and double-layer silk scaffold (SF group) were wrapped on the omentum to promote vascularization. The vascularization was evaluated by HE and immunofluorescence staining. The wrapped tissue engineered bladder patch and double-layer silk scaffold were used to repair the defective bladder. In the control group (six rats), the incision was closed immediately after the bladder tissue fully exposed. At 4 weeks and 12 weeks after operation, the general morphology of bladder tissue and cystography were performed to evaluate the recovery of bladder morphology. After the graft was harvested, HE and Masson's trichrome staining and immunofluorescence staining were used to observe the regeneration of bladder wall tissue. Urodynamics was used to assess the recovery of bladder function at 12 weeks after operation. Results:The flow cytometry results confirmed that the isolated cells positively expressed CD29 and CD90, and there was no significant expression of CD45 and CD106. Gross observation and scanning electron microscope confirmed that the preparation of double-layer silk scaffold not only had a pore structure that was conducive to cell planting, but also had good toughness and was facilitated to surgical suture. The number (43.50±2.66) and area (0.73±0.03)% of vascular-like structures in the SF-ADSCs group after the omentum encapsulation was significantly higher than that in the SF group [(24.50±3.51), (0.55±0.05)%], and the difference was statistically significant ( P<0.05). At 4 weeks after bladder repair, the histological staining of the grafts in the SF-ADSCs and SF groups showed a large number of degraded fragments of double-layer silk scaffold. At 12 weeks, the morphology of the graft in the SF-ADSCs group showed uniform bladder morphology, which was similar to that of normal bladder tissue. Immunofluorescence staining showed that the continuous urothelial layer, abundant smooth muscle tissue, vascular structure and regenerated neurons could be observed in the SF-ADSCs group. Urodynamic test showed that the bladder maximum volume (0.74±0.03)ml and compliance (16.68±0.44)μl/cm H 2O in the SF-ADSCs group, which were better than that in the SF group [(0.47±0.05)ml, (14.89±0.37)μl/cm H 2O], but lower than that in the control group [(1.12±0.08)ml, (19.34±0.45)μl/cm H 2O], and the difference was statistically significant ( P<0.05). Conclusions:The tissue engineered bladder patch constructed with double-layer silk scaffolds and ADSCs could promote the morphological repair of bladder tissue, the regeneration of bladder wall structure and the recovery of bladder physiological function.

The role of LncRNA00602 in Ad36-induced differentiation of adipocytes / 中华内分泌代谢杂志

Objective:To investigate the possible role of long non-coding RNA (LncRNA) 00602 in promoting browning in adipocytes induced by adenovirus type 36 (Ad36).Methods:According to Ad36 infection, adipose tissue samples of obese patients were divided into Ad36-negative group and Ad36-infected group. Realtime fluorescent quantitative PCR (qRT-PCR) was used to detect the changes in the expression of LncRNA00602 mRNA in omental adipose tissue of the two groups, and analyze the differences between the two groups. The correlation between waist-to-hip ratio, systolic blood pressure, diastolic blood pressure, fasting blood glucose, triacylglyceride and other indicators of the patients in the group with LncRNA00602 mRNA expression were analyzed. HE staining was used to detect the size of adipocytes in the omental adipose tissue of the Ad36 negative group and the Ad36 infection group. qRT-PCR and Western blotting were used to detect the mRNA and protein expression levels of uncoupling protein 1 (UCP1) and PR domain containing 16 (PRDM16) in omental adipose tissue of two groups of patients. Human adipose-derived stem cells (hADSC) were isolated and cultured, using Ad36 to induce differentiation, and divided into control group and LncRNA00602 knockdown group. On 0, 2, and 4 days after LncRNA00602 knockdown, fluoroboron dipyrrole (BODIPY) and mitochondrial red fluorescence (Mito-Tracker Red) were used to stain intracellular lipid droplets and mitochondria. At the same time, qRT-PCR and Western blotting were used to detect changes in the expression of UCP1 and PRDM16.Results:The expression of LncRNA00602 gene in the Ad36 infection group was higher than that in the Ad36 negative group (all P<0.05). The expression of LncRNA00602 in the Ad36 negative group was not significantly different from the above clinical indicators, while the expression of LncRNA00602 was negatively correlated with serum fasting blood glucose and triacylglyceride ( r=-0.522, -0.486, P<0.05) in the Ad36 infection group; HE staining showed that the average adipocyte area of the Ad36 infection group was smaller than that of the Ad36 negative group. At the same time, UCP1 and PRDM16 gene expression were higher than the negative group (all P<0.05). At the cellular level, on the 2nd and 4th days after knockdown of LncRNA00602, the lipid droplet area of adipocytes in the LncRNA00602 knockdown group was larger than that of the control group, the number of mitochondria decreased compared with the control group, and difference was statistically significant ( P<0.05 or P<0.01); Compared with the control group, there was significantly lower expression of the browning marker genes UCP1, PRDM16, and protein in the adipocytes in the LncRNA00602 knockdown group (all P<0.05). Conclusion:In Ad36-induced adipocyte differentiation, LncRNA00602 may positively regulate the expression of UCP1, PRDM16 and lipid droplet metabolism, and promote the browning of adipocytes.

Effect of donor age on biological characteristics of adipose-derived stem cells / 中华医学美学美容杂志

Objective:To identify the influence of age factors on biological characteristics of human adipose-derived stem cells (ADSCs), and to compare the differences in morphology, aging characteristics, differentiation potential, surface marker expression, and proliferation ability of human ADSCs with different ages and the effect of human ADSCs of different ages on proliferation and migration of fibroblasts.Methods:Adipose tissues from 27 individuals of healthy beauty treatments, aged 0-59 years, were collected from Department of Burns and Plastic Surgery, the 940th Hospital of Joint Logistics Support Force from January 2020 to December 2020. According to the age of the donor, they were divided into 6 groups. 0-9 years old group (4 cases), 10-19 years old group (4 cases), 20-29 years old group (5 cases), 30- 39 years old group (5 cases), 40-49 years old group (5 cases), and 50-59 years old group (4 cases). We obtained and cultured the ADSCs of each group, subcultured to obtain the ADSCs, and observed the morphological characteristics of each group; the proliferation ability of each group of ADSCs was detected by CCK-8 method; the differentiation potential of each group of ADSCs was detected by in vitro adipogenesis and osteogenic induction. Flow cytometry was used to detect the surface marker expression levels of each group of ADSCs, and qPCR was used to detect the expression levels of HGF, PPAR-γ, type Ⅲ collagen and DbX2 in each group. We also collected the skin tissues of a healthy person from Department of Burns and Plastic Surgery of the 940th Hospital of Joint Logistics Support Force in 2020, obtained and cultured fibroblasts, subcultured to obtain the second generation of fibroblasts, and prepared each group of ADSCs conditioned medium (ADSCs-CM); the proliferation ability of fibroblasts cultured in each group of ADSCs-CM was detected by CCK-8 method, and the migration ability of fibroblasts cultured in each group of ADSCs-CM was detected by cell scratch method. SPSS 21.0 software was used for data analysis and P<0.05 was considered statistically significant. Results:The cells of all age groups were in a typical spindle shape. The cells of the younger age group showed small nuclei and the cell morphology were uniform and similar; the cells of the older age group had larger nuclei and uneven morphology. With aging, the proliferation ability of ADSCs gradually decreased. In vitro adipogenic differentiation induction results showed that with the increase in age, the ability of ADSCs to differentiate into adipocytes gradually decreased. In vitro osteogenic differentiation induction results also demonstrated that with the increase in age, the ability of ADSCs to differentiate into osteoblasts gradually decreased. The expression rates of positive surface markers of ADSCs in each group of ADSCs were all above 93%. With aging, the expression levels of PPAR-γ, HGF, and type Ⅲ collagen decreased, and the expression levels of DbX2 showed an upward trend with age. The number of β-galactosidase staining positive cells increased with age, while the difference between groups A, B and C was not statistically significant, and the difference between groups A and D, E, and F was statistically significant ( P<0.05). The ADSCs conditioned medium of each group was able to promote the proliferation of fibroblasts, but the difference between the groups was not statistically significant. With aging, the ability of ADSCs conditioned medium to promote the migration of fibroblasts gradually decreased. Conclusions:The proliferation ability and multidirectional differentiation ability of human ADSCs and the ability to secrete a variety of cytokines that promote the proliferation and migration of fibroblasts show a downward trend, but the adipogenic differentiation ability of ADSCs declines significantly after the age of 40 years. ADSCs in all age groups could promote the proliferation and migration of fibroblasts.

Research progress of hydrogel combined with mesenchymal stem cells in the treatment of spinal cord injury / 生物医学工程学杂志

Spinal cord injury (SCI) is a complex pathological process. Based on the encouraging results of preclinical experiments, some stem cell therapies have been translated into clinical practice. Mesenchymal stem cells (MSCs) have become one of the most important seed cells in the treatment of SCI due to their abundant sources, strong proliferation ability and low immunogenicity. However, the survival rate of MSCs transplanted to spinal cord injury is rather low, which hinders its further clinical application. In recent years, hydrogel materials have been widely used in tissue engineering because of their good biocompatibility and biodegradability. The treatment strategy of hydrogel combined with MSCs has made some progress in SCI repair. This review discusses the significance and the existing problems of MSCs in the repair of SCI. It also describes the research progress of hydrogel combined with MSCs in repairing SCI, and prospects its application in clinical research, aiming at providing reference and new ideas for future SCI treatment.

Extracellular vesicle activities regulating macrophage- and tissue-mediated injury and repair responses

Macrophages are typically identified as classically activated (M1) macrophages and alternatively activated (M2) macrophages, which respectively exhibit pro- and anti-inflammatory phenotypes, and the balance between these two subtypes plays a critical role in the regulation of tissue inflammation, injury, and repair processes. Recent studies indicate that tissue cells and macrophages interact

Addition Of Adipose Derived Stem Cell To Beta Tricalcium Phosphate and Human Cancellous Bone for Craniofacial Bone Tissue Engineering: An In Vitro Study

@#Introduction: Autologous bone graft remains the method of choice for correction of osseous defects despite its shortcomings related to its limited availability, donor side effects and post-surgical potential complications of the recipient. It is imperative to develop more innovative substitute that offers little to no adverse effects. We aimed to assess the impact of addition human adiposed derived stem cell to Beta tricalcium phosphate (βTCP) and human cancellous bone in vitro. Methods: Experimental study was carried out in vitro, where βTCP and human cancellous freeze-dried bone graft were seeded onto a 24-well microplate (each well containing 2x106 hADSCs). A colorimetric assay (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide/MTT) was carried out for three days using the second passage of hADSCs to calculate the cell viability using ELISA reader at optical density (OD) 590nm. Results: MTT Assay showed that the percentage of viable cells in both groups were more than 70%, of which the βTCP showed significantly higher percentage than cancellous bone groups. Conclusion: This study proved that the addition of human adipose derived stem cell to βTCP and human cancellous bone in vitro is harmless and significantly improve cell viability in vitro.

Roles of adipocytes and adipose-derived stem cells in the lipophilic metastasis of ovarian cancer / 中国肿瘤临床

Ovarian cancer is the most common cause of cancer-associated deaths among the gynecological malignancies. The 5-year survival rate of the patients is less than 30%. This high mortality rate is mainly due to the metastasis. Adipose-rich omentum is the main site of metastasis, suggesting that ovarian cancer may have the characteristic of "lipophilic" metastasis. Adipocytes and adipose-derived stem cells are the most important components of the omentum. These cell ssecrete cytokines and adipokines and modify glucose and lipid metabolism to induce migration of ovarian cancer cells to the omentum. Simultaneously, they create a suitable microenvironment for the metastasis and colonization of these cancer cells by interacting with vascular endothelial cells, tumor-associated macrophages, and cancer-associated fibroblasts. This article reviews the recent advances in the role of adipocytes and adipose-derived stem cells in the "lipophilic" metastasis of ovarian cancer, aiming at providing new ideas for preventing ovarian cancer metastases and improving the survival rate of patients with ovarian cancer.

Proteomics analysis of exosomes from adipose-derived stem cells in skin damage repair / 中国组织工程研究

BACKGROUND: There are lots of researches about using adipose-derived stem cells to promote damage repair, but the studies from the perspective of exosome from adipose-derived stem cells based on proteomics and bioinformatics technologies are rarely reported. OBJECTIVE: To analyze the differentially expressed proteins of exosome from adipose-derived stem cells between diabetic patients with leg ulcers and healthy adults through proteomics and bioinformatics technologies, so as to screen out the diagnostic and prognostic landmarks. METHODS: Adipose-derived stem cells were obtained from 25 diabetic patients (trial group) with leg ulcers who accepted debridement skin grafting at the Department of Plastic and Burn, Second Affiliated Hospital of Air Force Military Medical University from October 2018 to May 2019 and 10 healthy adults who concurrently accepted liposuction (control group). Exosomes from adipose-derived stem cells were extracted for identification. Three cases were randomly selected from each group for proteomics analysis. LFQ technology was used to conduct quantitative analysis. GO and Pathway analysis for the differentially expressed proteins were performed using clueGO (version 2.5.4) was used to do. The transactional analysis between proteins was conducted (String database, version 11.0, R software clusterProfi ler package, FDR < 0.25). RESULTS AND CONCLUSION: (1) Totally 231 quantitative differentially expressed proteins were screened out, 31 kinds of which had significant difference. There were 29 down-regulated proteins and 2 up-regulated proteins in the trial group. (2) Differential proteins had a high correlation with nutrition and metabolic diseases. Differentially expressed proteins were mostly expressed in extracellular vesicles and platelet alpha granules and were mainly involved in two biological processes-coagulation process and lipid transport. The main molecular functions of differential proteins were related to enzyme inhibitor activity and heparin binding capacity. (3) The pathway analysis of PPI and KEGG showed that the significantly down-regulated ITGA2B and ITGB3 proteins were closely related to the course of the disease in the trial group, both of which are expected to be the new targets in stem cell treatment for diabetes with leg ulcers.