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Objective:To investigate the clinical and genetic features in a family with type 2 congenital generalized lipodystrophy, and to improve the understanging of this disease.Methods:The clinical symptoms, results of the laboratory, and radiography examinations of the patient and his family members were analyzed. The whole exome sequencing and Sanger validation were used to determine the genetic cause of the disease.Results:Generalized lipodystrophy, impaired liver function, severe hypertriglyceridemia, and acanthosis nigricans were found in the proband. His serum leptin level was much lower than normal value. The proband and three members of this family were confirmed to have insertion mutation at exon 5 of BSCL2 gene. The site was mutated from TTC to TCGGTC, resulting in the replacement of glutamate by aspartate and arginine. The mutation in proband was homozygote, and his father, mother, and brother were heterozygous.Conclusions:The mutation in exon 5 c. 545_546insCCG of BSCL2 gene leads to the occurrence of type 2 congenital generalized lipodystrophy.
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This study investigated the mechanism by which icaritin (ICT) inhibits exosomes-induced lung metastasis of B16BL6 mouse melanoma cells. The culture supernatant of B16BL6 cells was collected for extraction of exosomes by ultracentrifugation and their characterization by transmission electron microscopy and Western blotting. Exosomal protein was quantified by BCA. A wound-healing assay was used to determine the effect of ICT on the migratory ability of B16BL6 cells induced by exosomes. After establishing an experimental melanoma lung metastasis model in C57BL/6 mice, we used H&E staining to study the ability of ICT to inhibit exosomes-induced melanoma metastasis. Animal experiments were approved by the Ethics Committee of Nanjing University of Chinese Medicine. ELISA and immunofluorescence were used to detect pro-inflammatory factors interleukin 6 (IL-6), S100 calcium-binding protein A8/A9 complex (S100A8/A9), serum amyloid A (SAA) and fibronectin in metastatic tumors. The expression of metastatic tissue-related proteins stimulator of interferon gene (STING), phospho-STING (p-STING), TANK-binding kinase 1 (TBK1) and phospho-TBK1 (p-TBK1) was detected by immunohistochemistry or Western blotting. The results showed that the particle size of exosomes was 149.33 ± 2.68 nm, the polydispersity index (PDI) was 0.192 ± 0.02, the zeta potential was -32.22 ± 0.50 mV, and the particles had classic tea tray-like membrane structure under TEM. The protein concentration of exosomes was measured to be 838.66 ± 62.14 μg·mL-1. The results of the cell scratch test showed that ICT can inhibit exosomes-induced migration of B16BL6 cells at a concentration of 5, 10, and 20 μmol·L-1. In vivo experimental results also showed that ICT can inhibit exosomes-induced metastasis of melanoma to the lungs and can significantly inhibit the expression of pro-inflammatory factors S100A8/A9, SAA and IL-6 in lung tissue, and inhibit the expression of p-STING and p-TBK1 in metastatic lung tissue. Taken together, these results indicated that ICT can significantly inhibit exosomes-induced tumor metastasis, and the inhibition is related to the inactivation of STING in metastatic foci.
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Background and Objectives@#Mesenchymal stromal cells (MSCs)-based treatment for degeneration of intervertebral disc (IVD) has been proposed recently. We here addressed whether MSC secreted factors can rejuvenate nucleus pulposus- derived stem/progenitor cells from degenerated disc (D-NPSCs) in vitro. @*Methods@#and Results: We analyzed the expression of MSCs and NP cell specific surface markers, pluripotency related genes, multilineage potential and cell proliferative capacity of D-NPSCs upon incubation with the conditioned medium which was collected from the umbilical cord derived MSCs (UCMSCs). Our results indicated that the conditioned medium restore the stemness of D-NPSCs by up-regulating the expression level of CD29 and CD105, pluripotency related genes OCT4 and Nanog, and NP progenitor marker Tie2. The increased stemness was accompanied by promoted cell proliferative capacity and improved osteogenic and chondrogenic differentiation potential. @*Conclusions@#Our findings suggested that the UCMSCs derived conditioned medium might be used to rejuvenate the degenerated NP stem/progenitor cells.
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Cyclic GMP-AMP synthase (cGAS)-stimulator of interferon gene (STING) signaling pathway as an essential immune response pathway in cytoplasm, can find cytoplasmic DNA to regulate the innate immune and adaptive immune response. Studies have shown that the signaling pathway can be activated by both tumor self-DNA and genomic instability, thus to promote or inhibit the development and metastasis of tumors. Therefore, the role of cGAS-STING in tumor genesis, development and metastasis will be systematically expounded from the structures, physiological functions, inhibitors and agonists of cGAS and STING as well as its pathway transduction regulation in this paper. The paper aims to offer theoretical basis and reference for targeting cGAS-STING anti-tumor drugs in clinical practice and cancer clinical and cancer research workers.
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This study aimed to prepare an anti-metastatic diallyl trisulfide-exosome (DATS-Exo) drug delivery system. Exosomes in the supernatants of lung metastasis mouse melanoma B16BL6 cell line culture were extracted by ultracentrifugation. The quantity of exosomes was determined by transmission electron microscopy (TEM), Malvern particle size meter, and BCA assay. Expression levels of exosome-specific biomarkers CD9, TSG101, Flotillin 1 and lung organotropic biomarker α6 were detected by Western blot. The sonication method was used to load DATS into exosomes. The uptake of exosomes by B16BL6 cells and lung tissue was observed by laser confocal microscopy. Wound healing assay was used to evaluate the anti-migration effect of DATS-Exo in vitro. Experimental lung metastasis model was established to evaluate the anti-metastasis effect of DATS-Exo in vivo. Animal experiments have been approved by the Ethics Committee of Nanjing University of Chinese Medicine. The results showed that the particle size of DATS-Exo was 112.3 ± 1.98 nm, polydispersity index (PDI) was 0.24 ± 0.08, zeta potential was -24.33 ± 4.11 mV, and the particles have classic tea tray-like membrane structure under TEM. The protein concentration of DATS-Exo was measured to be 1 312.33 ± 6.27 μg·mL-1. The drug loading rate was about 0.33% ± 0.02%. The exosomes could be taken up by B16BL6 cells and the lung tissue. Compared with the free DATS group, DATS-Exo had a better inhibitory effect on tumor metastasis in vitro and in vivo. Taken together, these results indicate that exosomes derived from the lung metastasis tumor cells have lung organotropic characteristic and drug-loading properties. Using these kind of exosomes as carrier for anti-tumor drug delivery can be a novel and effective strategy of anti-metastatic therapy.
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AIM:To explore a novel method to isolate human nucleus pulposus mesenchymal stem cells (hNP-MSCs) in vitro and to identify their biological characteristics.METHODS:The explant culture method was employed to isolate hNP-MSCs from nucleus pulposus tissue obtained by percutaneous endoscopic lumbar discectomy (PELD).The isolated cells were passaged for purification and cultured in vitro followed by morphological observation.The cell proliferation ability was detected by CCK-8 assay.Growth curves of the cells were drawn and surface antigens were detected by flow cytometry.The cells at the 3rd~6th passages were induced for adipogenic, osteogenic and chondrogenic differentiation, and examined by oil red O staining, alizarin red staining and Alcian blue staining.RESULTS:The cells with self-renewal were obtained from nucleus pulposus tissue obtained by PELD.The results of flow cytometry analysis revealed that the cells were positive for CD29, CD44, CD90, CD73 and CD105, but negative for CD34 and CD45.The proliferative capacity was consistent with the growth characteristics of MSCs and multilineage differentiation potential was identified.CONCLUSION:A novel method to efficiently isolate and culture hNP-MSCs,PELD combined with explant culture method,was established, which would promote the study of regenerative medicine based on hNP-MSCs.
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[Summary] According to urinary albumin excretion rates ( UAER) , 256 patients with type 2 diabete mellitus (T2DM) were divided into normal albuminuria (NA), microalbuminuria (MA), and clinical nephropathy (CN) groups while 108 healthy subjects as control group. The analysis of variance of single factor was applied to examine lipoprotein(a), triglyceride, total cholesterol(TC), low-density lipoprotein cholesterol(LDL-C), cystatin C, and homocysteine. The correlations of lipoprotein ( a ) with urinary albumin excretion rate ( UAER ) and glomerular filtration rate ( eGFR ) were analyzed by Pearson correlation analysis. The sensitivities of lipoprotein ( a ) were evaluated in diagnosis of diabetic nephropathy ( DN) by receiver operating characteristic curve. The results showed that lipoprotein ( a) levels in NA, MA, and CN groups were gradually increased, with a significant increase in CN group(P<0. 05). Pearson correlation analysis revealed that lipoprotein (a) was positively correlated with systolic pressure, TC, cystatin C, and UAER(all P<0. 05) and negatively correlated with fasting blood glucose and eGFR (P<0. 05). The area under the ROC curve of lipoprotein(a) was 0. 639, with the sensitivity 66. 4% and specificity 55. 9% in the optimal cutoff value of 8. 41 mg/dl. These results suggest that lipoprotein(a) may serve as an index for monitoring DN based on its better correlation with UAER and eGFR.
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[Summary] The genomic DNA was extracted from peripheral blood leukocyte of the patient with thyroid hormone resistance syndrome and 14 members of his family.The exons 1-10 of thyroid hormone receptor β (TRβ) gene were amplified by PCR.The products of PCR were sequenced directly to detect the gene mutation.The results showed that 3 members of this family were confirmed to have the C→T transition mutation at nucleotide 1 303 site within exon 10 of TRβ gene,and the missense mutation results in the substitution of histidine to tyrosine (H435Y).The heterozygous mutation may lead to the occurrence of thyroid hormone resistance syndrome.
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[ ABSTRACT] AIM:To develop the cell model of polymer/liquid crystal and to study the effect of their elasticity on the adhesion of rat bone marrow mesenchymal stem cells (rBM-MSCs).METHODS: Using the method of solvent e-vaporation induced phase separation, the cell model of polymer/liquid crystal was constructed.The surface morphology and phase separation structure were determined by polarized optical microscopy ( POM) , scanning electron microscopy ( SEM) and small angle X-ray scattering ( SAXS ) .rBM-MSCs were separated and expanded by adherent culture.The surface markers of rBM-MSCs were detected by flow cytometry.The cells were induced to osteogenic differentiation and adipogenic differentiation for 2 weeks.After 3 passages, the cells were divided into 4 groups, including total PU control group, 10%membrane group, 30%membrane group and 50%membrane group.The cells were then incubated with rhodamine phalloi-din for cytoskeleton staining and were observed under the confocal laser scanning microscope after cultured for 24 h.RE-SULTS:The cell model of polymer/liquid crystal was constructed successfully using the method of solvent evaporation in-duced phase separation.Flow cytometry results showed that the rBM-MSCs positively expressed CD29, CD44 and CD90, and negatively expressed CD34 and CD45.After stained with alizarin red S and oil red O, the calcium nodule and lipid droplets in rBM-MSCs were observed obviously.The cytoskeleton staining result indicated that the area in total PU control group, 10%membrane group and 30%membrane group were greater, and the actin microfilaments were also clearer than that in 50%membrane group.CONCLUSION:The cell model with suitable content of liquid crystal made a contribution to the rBM-MSCs’ adhesion, but too much liquid crystal inhibits cell adhesion.
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[ABSTRACT]AIM:Toexploretheeffectoftheelasticmodulusandsizesofliquidcrystal(LC)phasesonosteo-genic differentiation based on OPC/PU composite substrate by mimicking the microenvironment in rat bone mesenchymal stem cells (rBMSCs).METHODS: A series of composite substrates with different elastic modulus were constructed via modulation of LC content in the composites .The surface phase structure was observed by polarized microscopy , and the mechanical property was measured by a universal material testing machine .Furthermore, the laser confocal microscope was employed to observe the spreading , polarization and the cytoskeleton arrangement of the rBMSCs .The proliferation of rBM-SCs was evaluated by CCK-8 assay.The specific mRNA expression of osteogenic differentiation such as collagen Ⅰ, and osteopontin on the composite membranes was detected by real-time PCR.RESULTS:The size and number of LC phase in-creased and the elastic modulus of the composite substrates decreased with the increase of the LC content .The rBMSCs ex-hibited better characteristics of initial adhesion , spreading and proliferation on the OPC 10-PU and OPC30-PU in the early and medium culturing .The rBMSCs displayed higher expression of collagen Ⅰ and osteopontin on the OPC10-PU in the early and medium osteogenic induction , while the high expression of these osteogenic genes occured on the OPC 30-PU and OPC50-PU in later osteogenic induction .The emphasis of genetic expression was switched from collagen Ⅰin the early and medium osteogenic induction to osteopontin in the later stage .CONCLUSION:When the content of LC remained low in the composite substrates , rBMSCs mainly responded to the mechanical stimuli induced by substrate stiffness and exhibited distinguished cellular behaviors;with the increase in the LC content , rBMSCs had strong interactions with LC by sensing the viscoelasticity of LC , probably resulted from the contribution of both substrate stiffness and the viscoelasticity of LC phase .
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<p><b>OBJECTIVE</b>To investigate the change of expression level of metastasis suppressor gene Kiss-1 in the colorectal cancer cell line SW480 after radiation, and to determine its association with the proliferation and apoptosis of SW480 cells.</p><p><b>METHODS</b>SW480 cells were divided into control group (0 Gy) and study groups (2, 4, 6, 8 Gy). Cells in the study groups were irradiated by 6-MV X-ray radiation for 48 hours. Immunohistochemistry and real-time PCR methods were used to investigate the influence of radiation on Kiss-1 gene expression of SW480. Colony formation assay was used to detect the proliferation of SW480. Flow cytometry-Annexin- V/PI assay was used to observe the change of the apoptosis rate.</p><p><b>RESULTS</b>Compared with the control group, Kiss-1 protein expression increased after radiation of 6, 8 Gy (P<0.05), but no significant changes were observed after radiation of 2, 4 Gy(P>0.05). Kiss-1 gene mRNA level increased after radiation of 2, 4, 6 Gy, while no obvious change was observed for 8 Gy radiation. The apoptosis rates increased for 4, 6, 8 Gy radiation(P<0.05), however, there was no significant difference for 2 Gy radiation (P<0.05).</p><p><b>CONCLUSION</b>Radiation may increase Kiss-1 gene expression in SW480 cells, which results in decreases proliferation and increases apoptosis in residual surviving cells.</p>
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Humans , Apoptosis , Cell Line, Tumor , Cell Proliferation , Colorectal Neoplasms , Metabolism , Pathology , Kisspeptins , Genetics , Metabolism , Radiation Effects , RNA, Messenger , Genetics , X-RaysABSTRACT
BACKGROUND: The development of cartilage tissue engineering provides novel ideas for treatment of articular cartilage defects and implements construction of tissue-engineered cartilage in vivo.OBJECTIVE: To investigate the feasibility of constructing tissue-engineered osteochondral composite through bone marrow stem cells(BMSCs) cultured on the poly(lactide-co-glycolic acid) (PLGA), which was modified with collagen and cellular growth factors.METHODS: PLGA was made by phase separation technique, composited with collagen Ⅱ, basic fibroblast growth factor, and transforming growth factor-β1. The BMSCs of passage 3 were cultured on the above scaffolds. Thirty-six SD rats were randomly divided into experimental, control, and blank groups. These three groups received implantation of BMSCs composited with growth factors and collagen-PLGA, implantation of BMSCs composited with collagen-PLGA, and implantation of collagen-PLGA into the muscle, respectively. At 4, 8, and 12 weeks after surgery, cell directional differentiation and growth were examined by gross observation, hematoxylin-eosin staining, toluidine blue staining, collagen Ⅱ staining, and scanning electron microscope.RESULTS AND CONCLUSION: Gross observation showed that there were many chondroid tissues in the experimental group and fibrous tissues in the control and black groups. Stainings and electron microscope revealed that many chondroblasts and a few osteoclasts appeared in the composite of the experimental group. Toluidine blue and collagen Ⅱ stainings were positive in the experimental group and negative in the control and blank groups. These findings demonstrate that PLGA modified with collagen had a good cellular compatibility. BMSCs cultured on PLGA, which was modified with collagen and cellular growth factors, can construct the tissue-angineered osteochondral composite in rats.
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BACKGROUND: The structure of nanometer chitosan-sodium/collagen (nano-CS/COL) is similar to that of the extracellular matrix (ECM) in the nanometer level. Whether this can promote the adhesion and growth of bone marrow mesenchymal stem cells (MSCs) and the calcification?OBJECTIVE: To investigate the in vitro histocompatibility of nano-CS/COL. DESIGN: Single sample observation.SETTING: Department of Orthopaedics, First Hospital, Jinan University. MATERIALS: This study was performed at the Experimental Center, First Hospital Affiliated to Jinan University between March 2007 and July 2007. Ten 4-week-old female SD rats, of SPF grade, weighing 200 g, were provided by the Guangdong Provincial Laboratory Center [Permission No. SCXK (yue) 2003-0002]. The protocol was carried out in accordance with animal ethics guidelines for the use and care of animals. Nano-CS/COL METHODS: Bone marrow MSCs were isolated from SD rats and cultured. Cell surface antigen was detected by loss cellanalyticalmethod.Nano-CS/COLscaffold waspreparedbypolyelectrolyte confocallaser-scanning microscopy. The well-grown cells of the third passage were co-cultured in vitro on the nano-CS/COL scaffold. Taking simple nano-CS/COL scaffold material as control, the histocompatibility of scaffold material and cells were comprehensively evaluated by cell adherence rate, growth curve, cell activity and cycle, and scanning electron microscope observation.MAIN OUTCOME MEASURES: ① Identification of cell surface antigen marker after isolation and culture of bone marrow MSCs. ②The histocompatibility of nano-CS/COL material and bone marrow MSCs 2, 4 and 8 days after nano-CS/COL material compounded with cells. ③Determination of adherence rate of cells to nano-CS/COL material. ? Cell circle and activity detected 5 days after nano-CS/COL material compounding with cells. RESULTS: ① Detection results of cell surface antigen marker: The expression of CD29, CD106, CD44, CD34 and CD45 was 90.86%, 73.38%, 82.61%, 0.76% and 0.60%, respectively. ②Histocompatibility of bone marrow MSCs and nano-CS/COL material: It was shown under the scanning electron microscope that nano-CS/COL scaffold presented porous three-dimensional structure, and different sizes of macropoles and interconnected small pores. The interval porosity determined by quality assay was 85%-90%, and aperture averaged 150 μm (range 50 - 300u m). Two days after bone marrow MSCs compounded to nano-CS/COL scaffold, bone marrow MSCs presented globular shape and were scattered; Four days later, bone marrow MSCs presented shuttle shape, extended and anchored on the surface of nano-CS/COL by pseudopods; Eight days later, bone marrow MSCs proliferated and fused each other, and they secreted a lot of extracellular matrix, then which covered most material particles. ③ The adherence rate of bone marrow MSCs to nano-CS/COL: Bone marrow MSCs and nano-CS/COL were co-cultured 2 and 6 hours separately. The adherence rate of bone marrow MSCs was higher to nano-CS/COL scaffold than to simple chitosan scaffold. ④ Comparison of cells and cell cycle between on nano-CS/COL scaffold and on the chitosan scaffold: On the nano-CS/COL scaffold, cell activity was 96.67%, cell cycle at G0-G1 was 90.81%, at G2-M was 0.52% and at S was 8.66%. G2/G1 was 1.81. On the simple chitosan scaffold, cell activity was 95.27%, cell cycle at G0-G1 was 87.14%, at G2-M was 9.69%, and at S was 4.16%. G2/G1 was 1.80.CONCLUSION: Nano-CS/COL scaffold can be used as tissue engineering biomaterials because bone marrow MSCs can well grow on it.
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BACKGROUND:There is a great difference of grade size of macrobead in various joint diseases; therefore, it can be used to determine state of joint diseases initially.OBJECTTVE : To explore the physical properties of synovial fluid nano-particles and their correlations with the occurrence of knee osteoarthritis (KOA).DESIGN: Controlled experimental study based on synovial fluid samples.PARTICIPANTS: A total of 99 synovial fluid samples were collected from normal subjects and KOA patients with various KOA severities. Among them, 41 were normal synovial fluids, 58 were KOA.METHODS: Synovial fluid samples from individuals with and without KOA were obtained. Using the technology of quasi-elastic laser scattering, nano-particle size and its distribution were estimated, and the dynamic/static light scattering spectrometric analyzer allowed the measurement of particles Zeta potentials. A correlation analysis between the particle size, Zeta potentials and the onset of KOA was attempted.MAIN OUTCOME MEASURES:① Grade size and distribution of microsome in synovial fluid;② Zeta potentials and distribution of microsome in synovial fluid; ③ grade size and clinical correlation of microsome in synovial fluid.RESULTS: ① The mean nano-particle diameter in the synovial fluid of KOA patients were significantly greater than those of normal joints [(297±84), (63±23) nm, P ＜ 0.001]. The distribution curve of KOA synovial fluid nano-particle size was normal knee and (-15.84 ±3.34) mV of KOA patients, and there was a significant difference (P ＜ 0.001). This suggestedthat the Zeta potentials in the synovial fluid of KOA patients were significantly greater than those of normal joints. ③ The average particle size and Zeta potential of synovial fluid strongly correlated with the integrity of the joint of KOA (rp =0.797 2,0.631 9, P＜ 0.01).CONCLUSION: The nano-particle size and Zeta potential of synovial fluid are significantly correlated with the development of KOA, and this can reflect the severity of KOA.
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Objective To investigate expression of Human papillomavirus (HPV) 11 type E7 protein antigen in prokaryotic cells and its potential use for the serodiagnosis of condyloma acuminatum (CA).Methods The full-length gene encoding for HPV11 E7 protein was amplified by PCR,and cloned into vector pET32a(+) to form recombinant pET32a(+)/HPVll E7 plasmid.The fusion His-E7 protein was expressed and analyzed by using SDS-PAGE and Western blotting.Using ELISA assay,HPV11 E7 fusion protein were also used to screen human serum IgG antibody from 93 patients with CA,43 patients with cervix cancer and 58 healthy control subjects.Results Highly expressed fusion His-E7 protein was obtained,and purified protein served as a special diagnostic antigen to screen human serum antibody for CA serodiagnosis.It showed that CA group,cervix cancer group and healthy control human serum IgG antibody average value were 1.545?0.131,0.586?0.155 and 0.674?0.150 respectively,positive rate were 76.3% (71/93),11.6% (5/43) and 5.2% (3/58).There was significantly difference between the CA group to compare cervix cancer group and healthy control (P