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Objective:To investigate the effects of ginsenoside Rg1 (GS-Rg1) on the secretion of exosomes (MSC-Exo) and expression of angiogenesis related microRNAs (miRNAs) in mesenchymal stem cells.Methods:Human umbilical cord blood mesenchymal stem cells (hUCBMSCs) were divided into experimental group and control group. The experimental group was treated with GS-RG1 at a final concentration of 40 mg/L, while the control group was treated with phosphate buffered saline (PBS) at the same volume. Both groups were cultured for 24 h. The morphology of MSC-Exo was observed by transmission electron microscopy; the characteristic surface markers were identified by Western blot; the concentration of MSC-Exo was detected by dicootanobutyric acid protein quantification method, and the expression of 8 miRNAs related to angiogenesis in MSC-Exo was detected by reverse transcription polymerase chain reaction (RT-PCR).Results:After 24 h of incubation, MSC-Exo with a circular membrane vesicle structure was visible. MSC-Exo was positive for the expression of the characteristic surface markers CD9, CD63 and TSG101. After 24 h of intervention, the concentration of MSC-Exo protein were (1.080±0.019)μg/μl and (0.881±0.032)μg/μl in the experimental group and control group, respectively, with statistically significant difference ( P<0.01). The expression of miR-126-3p, miR-21, miR-146a-5p and miR-125b-5p in the GS-Rg1 group were significantly higher than that in the control group, while the expression of miR-16-5p was significantly lower than that in the control group (all P<0.05). Conclusions:GS-Rg1 promotes the secretion of MSC-Exo and enhances the expression of angiogenesis-related miRNAs within Exo to promote angiogenesis.
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Objective:To investigate the effect of Astragaloside Ⅳ-mediated Endothelial progenitor cells derived exosomes (EPC-Exos) on the biological function of EPC-Exos damaged by high glicose.Methods:EPCs from human umbilical cord blood were isolated and cultured in vitro. the EPC-Exos secreted by EPCs were extracted by ultracentrifugation combined with ultrafiltration, and identified by specific markers CD9, CD63 and CD81, respectively. After the cells were cultured for 24 hours with AS-IV at 100 mg/L and PBS at the same volume, the morphological characteristics of EPC-Exos were observed by transmission electron microscope. Human endothelial cells were isolated, cultured and identified in vitro. The identified endothelial cells were pretreated with 30 mmol/L glucose for 120 h and randomly divided into experimental group and control group, at the same time set the normal group. The cells were cultured for 24 hours, the effects of EPC-Exos on proliferation, adhesion, migration and angiogenesis of endothelial cells damaged by high glucose were observed by using cell counting kit-8 (CCK-8) Cell Proliferation Assay Kit, cell scratch test, adhesion assay and in vitro angiogenesis assay by Matrigel. Results:Compared with the normal group, the proliferation, migration, adhesion and tubulogenesis of human endothelial cells in the control group were significantly lower ( t=24.35, 6.80, 10.65, 9.62, P<0.05). Compared with the control group, the proliferation, adhesion, migration and tubulogenesis of human endothelial cells in the experimental group were significantly enhanced ( t=30.68, 5.99, 5.40, 8.25, P<0.05). Conclusions:EPC-Exos mediated by AS-Ⅳ can significantly improve the biological function of human endothelial cells damaged by high glucose and has the potential to modulate endothelial neovascularization in diabetic rats.
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Objective:To investigate the impacts of astragaloside Ⅳ (AS-Ⅳ) on in- vitro proliferation and angioblastic differentiation of human umbilical cord blood-derived mesenchymal stem cells (hUCBMSCs), providing a basis for further research about the effects of AS-Ⅳ on mesenchymal stem cells (MSCs)-mediated angiogenesis. Methods:The hUCBMSCs were extracted from umbilical cord blood of normal full-term infants and subcultured. Osteoblasts, chondroblasts, and lipoblasts were induced, differentiated and identified. At the same time, the surface antigens CD44, CD73, and CD105 on hUCBMSCs were determined by flow cytometry. The successfully identified hUCBMSCs were cultured and treated with a series concentrations of AS-Ⅳ (0, 50, 100, 200, 300, and 400 mg/L). The optimum concentration of AS-Ⅳ for cell proliferation in hUCBMSCs was confirmed. In another experiment, hUCBMSCs were randomly divided into the experimental group and the control group. The cells in the experiment group were treated with the optimum concentration of AS-Ⅳ, and those in the control group were treated with equal volume of PBS. The impact of AS-Ⅳ on the proliferation of hUCBMSCs was detected using the cell counting kit (CCK-8). Besides, the impact of AS-Ⅳ on the angioblastic differentiation of hUCBMSCs was examined using the matrigel in- vitro tube formation assay. CD31 and von willebrand factor (vWF) expressions were determined using immunofluorescence after hUCBMSCs differentiated towards endothelial cells. Results:Under the light microscope, hUCBMSCs had clear edges and arranged orderly, showing a typical long fusiform structure. Flow cytometry confirmed that hUCBMSCs had surface markers of mesenchymal stem cells. The optimum concentration of AS-Ⅳ for the proliferation of MSCs was 300 mg/L. The OD values of the control and experimental groups were (0.51±0.01) and (0.98±0.05), respectively, with statistical significance ( t=15.96, P<0.05), indicating that the proliferation ability of the experimental group was enhanced. Compared with the control group, the tube density and the length of the tube network in vitro in the experimental group were higher, with statistically significant difference [(629.80±52.94)mm vs (110.36±13.19)mm, P<0.05]. Compared with the control group, the expression of CD31 and vWF increased in the experimental group after AS-Ⅳ induced hUCBMSCs differentiation ( t=13.64, 13.18, P<0.05). Conclusions:AS-Ⅳ has no toxicity to human umbilical cord blood mesenchymal stem cells, and can improve their proliferation function, and induce hUCBMSCs to differentiate into endothelial cells.
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Objective:To investigate the effects of mesenchymal stem cells-derived exosomes (MSC-Exos) secreted by mesenchymal stem cells (MSCs) induced by astragaloside IV (AS-IV) on the biological function and pyroptosis of human umbilical vein endothelial cells (HUVECs) injured by high glucose.Methods:After human umbilical cord blood mesenchymal stem cells (hUCBMSCs) were intervened with 400 mg/L of AS-IV, exosomes were extracted, and then the morphology and specific markers of exosomes were identified. Human umbilical vein endothelial cells (HUVECs) were cultured in a medium with a glucose concentration of 30 mmol/L to prepare a high glucose-impaired HUVECs model. High glucose-impaired HUVECs were randomly divided into experimental and model groups, with the experimental group intervened with 100 μg/ml of MSC-Exos and the model group intervened with an equal volume of PBS solution, while a blank control group was also set up. Cell counting Kit-8 (CCK-8) cell proliferation assay, adhesion assay, matrigel tube formation assay and scratch assay were used to detect the effects of AS-IV-mediated MSC-Exos on the proliferation, adhesion, tube formation and migrationability of HUVECs; Western blot and real time fluorescence quantitative polymerase chain reaction (qRT-PCR) were used to detect the protein and mRNA expression of scorch death-related molecules, such as Caspase-1, GSDMD (Gasdermin D) and NLRP3 in each group.Results:The proliferation, adhesion number, tube number and migration width of HUVECs cells were significantly lower than those in the blank group ( P<0.05); The expression of Caspase-1, GSDMD, NLRP3 protein and their mRNA increased significantly ( P<0.001); Under the intervention of MSC-Exos mediated by AS-IV, the cell proliferation, adhesion number, tube number and migration width of HUVECs were significantly higher than those in the model group ( P<0.05); The expression of Caspase-1, GSDMD, NLRP3 protein and their mRNA decreased, with statistically significant difference ( P<0.05). Conclusions:AS-IV mediated MSC-Exos can significantly improve the biological function of high glucose-impaired endothelial cells, and its mechanism may be related to anti-pyroptosis.
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Wound repair is a fundamental task that the whole field of the Burn and Plastic surgery pays urgent attention to and longs for a breakthrough. In this column, wound repair balance laws theory is expounded and we are expecting people in the field gradually began to value the use of balance law. Guided by the law of balance principle, people are required to conduct scientific research, improve clinical technique and develop new materials. The theory is designed to improve the level of scientific research and clinical diagnosis, and will set up a new milestone in the field of wound repair.
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Objective:To investigate the effect of astragaloside IV (AS-IV) on the secretion of stromal cell-derived factor-1α (SDF-1α) and CXC chemokine receptor 4 (CXCR4) by high glucose injured human umbilical vein endothelial cells (HUVECs), so as to lay a foundation for further study on AS-IV improving angiogenesis by regulating SDF-1 α/CXCR4 axis of endothelial cells.Methods:HUVECs were isolated and cultured from the umbilical vein of full-term healthy newborns and identified by von Willebrand factor (vWF) combined with 4-diamino-2-phenylindole (DAPI) nuclear staining. The obtained HUVECs was cultured in EGM-2 medium with 30 mmol/L glucose for 120 h to obtain high glucose damaged HUVECs. After intervention with different concentration gradients (25 mg/L, 50 mg/L, 100 mg/L, 200 mg/L, 400 mg/L) AS-IV for 72 hours, the contents of SDF-1α and CXCR4 were detected by enzyme linked immunosorbent assay (ELISA) method to determine the best concentration of AS-IV. The supernatant of damaged HUVECs were collected at 6, 12, 24, 48 and 72 hours after intervention with the best concentration of AS-IV, and the contents of SDF-1α and CXCR4 were detected by ELISA method to determine the best action time of AS-IV. The damaged HUVECs was randomly divided into experimental group and control group, and the blank group was set up at the same time. The experimental group was treated with the best concentration of AS-IV and the best time, the control group and the blank group were treated with the same volume of phosphate buffered saline (PBS) solution, and the contents of SDF-1α and CXCR4 in each group were detected by ELISA method.Results:The vWF factor on the cell membrane was green fluorescence, and the nucleus was blue after DAPI staining. When the fusion image showed green fluorescence, HUVECs were identified by blue fluorescence. The expression of SDF-1α in damaged HUVECs was the best when treated with AS-IV of 100 mg/L for 24 hours (1 642.87 pg/ml), and the expression of CXCR4 in damaged HUVECs was the best when treated with AS-IV of 50 mg/L for 48 hours (8.44 ng/ml). Compared with the control group, the contents of SDF-1α and CXCR4 in the experimental group were significantly increased, and the difference was statistically significant ( P<0.05). While the contents of SDF-1α and CXCR4 in the experiment group were slightly less than those in the blank group and there was no statistically significant difference ( P>0.05). Conclusions:AS-IV can promote the expression of SDF-1α and CXCR4 in HUVECs damaged by high glucose to return to normal physiological level, so as to play the role of vascular repair and neovascularization.
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In this paper,the author introduced the evolution of instruments related to gynecological singlesite laparoscopic surgery,from the perspective of historical development,focusing on the advantages and disadvantages of related instruments and the reasons for their improvement,as well as the impact on the development of gynecological single-site laparoscopic surgery.It is helpful for readers to better choose the appropriate instruments on the basis of understanding the relevant history.
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Objective The objective is to probe into the effects of astragaloside Ⅳ (AS-Ⅳ) on activity and proliferative function of endothelial progenitor cells (EPCs),which lays a foundation for further study on the effects of AS-Ⅳ on vascular neovascularization mediated by endothelial progenitor cells.Methods The mononuclear cells were isolated by the density gradient centrifugation in umbilical cord blood of full-term healthy infants,and EPCs were obtained by subculture and cell identification when the cells presented spindle shapes.The obtained EPCs were randomly divided into the experimental group and the control group.In the experimental group,EPCs were cultured by AS-Ⅳ with different concentration gradients (25 mg/L,50 mg/L,100 mg/L,200 mg/L and 400 mg/L),while in the control group,they were treated with the same amount of phosphate buffer saline (PBS) solutions.The effects of AS-Ⅳ on the proliferation of endothelial progenitor cells was studied by cell counting kit-8 (CCK-8) cell proliferation experiment,and the activity rate of EPCs cells was measured at the optimum concentration of EPCs proliferation.Results EPCs were successfully obtained after confirming nuclear staining test of CD31 antibody and 4',6-diamidi-no-2-phenylindole (DAPI).Further study showed that AS-Ⅳ can promote the proliferation of EPCs,and its optimal concentration of EPCs proliferation is 100 mg/L.Compared with the normal control group,the activity rate of endothelial progenitor cells after intervention of AS-Ⅳ was 98.7％,higher than 98.12％ in the control group,with significant difference (x2 =49.59,P ＜0.01).Conclusions AS-Ⅳ can enhance the activity of human EPCs and promote their proliferation in vitro.
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Objective By taking usage of bioinformatics screening methods,this medical research aimed at exploring how the miRNAs in endothelial progenitor cell exosomes relate to the regulation of angiogenesis.Methods miRNAs in endothelial progenitor cells exosomes and angiogenesis-related miRNA was intersected from the existing database to obtain the candidates of miRNA molecules related to angiogenesis in endothelial progenitor exosomes.Results 160 and 50 miRNAs in endothelial progenitor cell candidates were obtained through experimental data analysis and literature searching respectively.600 candidates of angiogenesis-related miRNAs were obtained through literature searching;the top 20 with the highest frequency were selected out.Finally,9 miRNA candidates (miR-126,miR-21,miR-221,miR-92a,miR-199a,miR-210,miR-214,miR-155,miR-146a) that may be highly expressed in endothelial progenitor exosomes and associated with angiogenesis were obtained for the following research.Conclusions Based on data analysis and literature searching,bioinformatics could screen out the target miRNAs for follow-up studies easily and reliably,it is worthy to be widely applied and popularized.
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Objective To prepare a new-type acellular dermal matrix (ADM) and research on its relevant performance,which would provide theoretical evidence for clinical application.Methods Skin of Bama suckling pig was taken as resource of skin,and technologies of physics,chemistry and biology were selected to prepare new-type ADM.To detect the external structure,physical and chemical property as well as biological property of the prepared new-type ADM,hematoxylin-eosin (HE) staining observation,scanning electron microscope observation,amino acid analysis,material porosity and hydrophilicity test,tensile strength and in vitro degradation experiment,cytotoxicity test,and animal experiment have been conducted.Results New-type ADM cells have been thoroughly removed and dermal matrix remains intact with collagen content of 95.55％,connective three-dimensional pore structure,(85.03 ± O.99) ％ of porosity,(24.56 ± 0.57) ° of contact angle implying new-type ADM was hydrophilic substance,(5.48 ± 0.44) Pa of tensile strength implying its moderate level of pulling force,in vitro degradation period reduced to (28.7 ± O.76) h,and ＞75％ relative growth rate (RGR).Cells grew and proliferated on new-type ADM and could be replaced by original tissue after degradation.Conclusions New-type ADM have overcome disadvantages of traditional preparation method in sabotaging dermal matrix structure and incompletely removing cells from matrix,which is qualified with higher level of collagen content and porosity.With improved biological property,greatly reduced inflammation immunoreactions,and accelerated degradation rate,new-type ADM is of higher level of clinical application value.
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MicroRNAs have been identified as a new class of regulatory molecules that affect many biological functions by interferring the target gene expressions. Latest studies demonstrate that microRNAs can influence many pivotal bio-processes and deeply involve in the metabolism of glucose, lipid and amino acid and biological oxidation. For glucose metabolism, microRNAs are related to insulin secretion, insulin sensitivity, glucose uptake, glycolysis, oxidation and mitochondrial function. For lipid matebolism, microRNAs can regulate the target genes related to lipid biosynthesis, catabolism and transportation. MicroRNAs can influence glutamine catabolism.
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Animals , Humans , Glucose , Metabolism , Glutamine , Metabolism , Insulin , Metabolism , Insulin Secretion , Lipid Metabolism , Physiology , Metabolism , Physiology , MicroRNAs , PhysiologyABSTRACT
Objective To investigate the mechanism of HSP70 that inhibits myocardial cell apoptosis in sepsis.Methods Myocardial cells in primary culture were randomly divided into control group,normal serum group,sepsis serum group,transported empty vector group and transported HSP70 group.The myocardial cells in transported HSP70 group have been transported by pcDNA3.1-HSP70 for 36 hours.The myocardial cells in every group have been cultured by respective serum for 2 hours and dyed by Hoechst 33258,and then calculate the rate of myocardial cells apoptosis.Using Western-blot to investigate the effect of overexpression of HSP70 on Caspase-3,8,9's activation and Bid's cracking.Results The rate of myocardial cells apoptosis after dealing in transported HSP70 group [ ( 12.48 ± 2.39 ) ％,( 23.96 ± 3.12 ) ％,( 25.40 ± 3.96) ％ ] is lower than in sepsis serum group [ ( 28.66 ± 2.24 ) ％,( 55.76 ± 5.69 ) ％,( 46.89±8.74)％,t =5.856,5.932,6.027,P ＜0.01,n =3] and lower than in transported empty vector group [(34.25±3.42)％,(50.71±6.38)％,(47.62+5.74)％,t =5.876,5.903,6.122,P ＜0.01,n =3],is higher than in control group,and in normal serum group(3.13％ ～ 6.75％ ,t =6.324,6.578,6.137,5.987,6.032,6.871,P ＜ 0.01,n =3 ).When Caspase-3,8,9 activating,the gray-scale of P11,P20 and P10 in transported HSP70 group( 12.5276 ± 2.1247,9.3481 ± 4.5423,16.1349 ± 6.0641 ) is lighter than that in sepsis serum group ( 27.1324 ± 2.1564,25.5643 ± 4.3018,36.5647 ± 6.7135,t =5.856,5.902,5.891,P ＜ 0.01,n =3 ) and lighter than in transported empty vector group (28.0314 ±2.0367,25.6413 ±4.1356,34.5648 ±5.9473,t =3.861,3.933,4.281,P ＜0.05,n =3),is deeper than in control group(8.0324 ± 1.5234,5.1246 ± 1.3274,2.0314 ±0.6423,t =3.286,3.867,4.031,P＜0.05,n =3) and in normal serum group(8.5649 ± 1.2136,6.0324 ± 1.0214,3.2146 ±0.1325,t =5.898,5.969,6.879,P ＜0.01,n =3).The gray-scale of tBid in transported HSP70 group( 12.0316 ±2.3641 ) is lighter than in sepsis serum group(27.0536 ± 5.3214),t =3.274 ( P ＜ 0.05,n =3 ) and lighter than in transported empty vector group(27.1034 ± 3.6741,t =3.301,P ＜ 0.05,n =3 ),is deeper than in control group ( 6.0347 ± 2.1304,t =5.924,P ＜ 0.01,n =3 ) and in normal serum group ( 7.3121± 1.3021,t =5.871,P ＜ 0.01,n =3 ).Conclusions HSP70 inhibit myocardial cells apoptosis in sepsis by intervened the death receptor pathway and mitochondrial pathway.
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<p><b>BACKGROUND</b>One of the major characteristics of the human immunodeficiency virus type 1 (HIV-1) is its unusually high degree of genetic variability, which involves in genetic diagnosis, subtyping, vaccine design, and epidemiology. HIV-1 CRF01_AE is a main prevalent HIV-1 recombinant strain in China. In this study, three full-length CRF01_AE genomes from Fujian Province, China were cloned, sequenced, and analyzed; and the further genetic diversity defining and epidemiologic analysis were carried out.</p><p><b>METHODS</b>Proviral DNA was extracted from non-cultured peripheral blood mononuclear cells, the near full-length HIV-1 genome was amplified and the PCR products were cloned into pCR-XL-TOPO vector and sequenced. 5'-long terminal repeat (LTR) and 3'-LTRs were amplified by additional independent PCR and cloned into pMD18T vector. Gene-based phylogenic tree was constructed and genetic distances were calculated by MEGA 3.1. Simplot was used for Bootscan analysis.</p><p><b>RESULTS</b>The phylogeny and genetic distance analysis of the three near full-length sequences confirmed that these three samples clustered with CRF01_AE isolates, more close to Thailand CRF01_AE strain CM240, and were distantly related to African CRF01_AE strain 90CF402. Analysis of their genomic organization revealed the presence of nine potential open reading frames. There were no major deletions, rearrangements, or insertions in the three sequences, but an in-frame stop codon was found in tat gene of Fj051. LTRs of the three sequences contained a few nucleotides mutation. We did not find new mosaic recombinant in the three sequences. The V3 motif was GPGQ in all the three sequences, and there were only few amino acids differences in all three V3 loop sequences.</p><p><b>CONCLUSION</b>This report reveals the background of the three full-length CRF01_AE genomes, the most dominantly circulating HIV-1 strain in Fujian Province, China. The work is essential for the design and development of an effective AIDS vaccine for the region.</p>