| BackgroundWith the change of modern lifestyle and diet,obesity has become one of the most important chronic diseases that threaten human health.Studies have shown that obesity can induce inflammation and insulin resistance and is closely related to diabetes,cardiovascular disease,sleep respiratory dysfunction and some cancers.Obesity is a chronic metabolic disease which results from excessive accumulation of fat in the body due to the fact that the body's energy intake is greater than its energy consumption.There are two main forms of white adipose tissue(WAT)in the body:subcutaneous adipose tissue(subcutaneous adipose tissue,SAT)below the dermis,and visceral adipose tissue(VAT)around the viscera.For obese people,excessive accumulation of excess fat in the viscera is more harmful to the body.A certain amount of VAT is necessary for the body.VAT surrounds the human organs to support,stabilize and protect the organs.Compared with subcutaneous adipose tissue,visceral adipose tissue is more easily decomposed to cause the increase of plasma free fatty acid(free fatty acid,FFA).The high concentration of FFA in plasma inhibits the activation of insulin signaling pathway in the skeletal muscle and inhibits the expression of glucokinase and transporter in the liver,thus inducing insulin resistance.Therefore,visceral fat is also known as "the most dangerous fat".Since obesity caused by SAT accumulation causes a few complications,it is usually called "benign obesity".At present,the weight and fat content is no longer the only criterion for the diagnosis of obesity.The heterotopic distribution of lipids and inflammatory change have become one of the indicators to determine the severity and metabolic abnormalities of obesity.Adipose tissue is an important source of adult mesenchymal stem cells.Adipose derived mesenchymal stem cells(Adipose-derived mesenchymal stem cells,ADSCs),like other mesenchymal stem cells,have the potential for proliferation and pluripotent differentiation.In specific induction conditions,ADSCs can be differentiated into adipocytes,chondrocytes,osteoblasts,muscle cells and nerve cells.In recent years,ADSCs have been highly concerned in the field of tissue repair,and can be used for various tissue repair and regeneration treatment.As an important component of adipose tissue,ADSCs can participate in maintaining normal adipose tissue through self-renewal and differentiation into adipocytes.As mentioned above,although VAT and SAT are both white fat,their metabolic characteristics are markedly different.Excessive accumulation of VAT is more likely to cause disorder of glucose and lipid metabolism,leading to inflammatory and a series of metabolic related diseases.So what causes the difference between VAT and SAT?Is it related to the difference in self-renewal and adipogenic differentiation of ADSCs from these two fat depots?The aim of this study is to reveal the key factors that affect the biological behavior of VAT and SAT by studying the differences between the proliferation and adipogenic differentiation of S-ADSCs and V-ADSCs and to explore the causes of metabolic differences between VAT and SAT,and to reveal the causes and mechanisms of the metabolic syndrome.CD90(Cluster of Differentiation 90)is also called THY-1(Thymocyte differentiation antigen 1).Similar to the relative specific surface markers CD105,CD44,CD29 on mesenchymal stem cells,CD90 is expressed on the surface of ADSCs.As a highly conserved glycoprotein,CD90 is riveted on the surface of cell membrane through glycosyl phosphatidyl inositol,and is expressed on the surface of fibroblasts,hematopoietic cells,epithelial cells,tumor cells and mesenchymal stem cells.It is mainly involved in the interaction between cells and cells and cell and cell matrix.It plays an important role in cell proliferation,apoptosis,necrosis,adhesion,exudation and metastasis.As one of the markers of tumor stem cells,the role of CD90 in tumor cells is also very significant.CD90 positive and CD90 negative hepatoma carcinoma cells are subcutaneously injected into immune deficient mice,and the proliferation rate and tumorigenicity of CD90 positive hepatoma carcinoma cells are obviously higher than that of CD90 negative ovarian cancer cells.These phenomena suggest that CD90 plays an important role in regulating the proliferation of several tumor cells.The role of CD90 in ADSCs and whether it affects the proliferation and adipogenic differentiation of S-ADSCs and V-ADSCs,have not been studied.Therefore,in this study,we aim to explore the effects of CD90 on the proliferation and adipogenic differentiation of S-ADSCs and V-ADSCs.?.Isolation and culture of V-ADSCs and S-ADSCsC57BL/6 male mice were fed to 10-12 weeks and were killed after anesthesia.We carefully peeled off the adipose tissue around the epididymis(visceral fat)and subcutaneous fat(subcutaneous fat)of the groin in the super-clean bench.Two types of adipose tissues were weighed respectively and were cut with scissors,then were added into the corresponding volume of KRB(Krebs-Ringer buffer)solution containg collagenase.After digestion,the mixture was centrifugated,then the supernatant was dropped.The cell precipitation was resuspended with complete medium and the cells were cultured under the condition of 37 ? and 5%CO2-After 24h,the suspended cells were removed and the attached spindle cells were cultured as ADSCs.?.Study on the difference of proliferation and stemness between S-ADSCs and V-ADSCs1.Experiments related to proliferationS-ADSCs and V-ADSCs at the third generation were planted in 6-well plate for the detection of proliferation differences by EdU incorporation and colony formation test.2.Cell cycle detectionTo examine differences in cell cycle between S-ADSCs and V-ADSCs,the third generation cells were stained with propidium iodide(PI)solution and detected by flow cytometry.3.Detection of sternness gene expressionThe total RNA V-ADSCs and S-ADSCs at the third generation was extracted by fast200 RNA extraction kit.After reverse transcription,cDNA was used to detect the expression of sternness genes(Nanog,Oct4 and Sox2)by Real-time quatitive PCR.4.Detection of signal pathway related to proliferationS-ADSCs and V-ADSCs at the third generation were digested and plated in 6-well plate and incubated for 24 hours in the incubators.Protein lysate was used to extract protein from ADSCs,and western-blot was used to detect the protein expression level of AKT,p-AKT,STAT3,p-STAT3 and cyclinD1 in S-ADSCs and V-ADSCs.?.Study on the differences in adipogenic differentiation between S-ADSCs and V-ADSCs.1.Differences in adipogenic differentiationS-ADSCs and V-ADSCs were induced by adipogenic induction.The cells were collected for 0,4,8 and 12 days during the induction process,and RNA was extracted.The mRNA levels of white adipocyte markers PPAR-? C/EBP???P2 was detected by Real-time quatitive PCR.After induction,the cells were stained with Oil Red O,the adipogenic differentiation of S-ADSCs and V-ADSCs was observed and photographed under microscope.After taking pictures,the Oil Red O in adipocytes was dissolved with isopropanol and the OD values were measured at 500nm.2.Differences in mitotic clonal expansion induced by adipogenic inductionS-ADSCs and V-ADSCs at the third generation were digested and cultured to fusion state or over fusion state,and then were induced by adipogenic induction for 16 hours respectively,the mitotic clonal expansion of S-ADSCs and V-ADSCs was detected by EdU incorporation assay?.Detection of stem cell surface markers on V-ADSCs and S-ADSCs1?Detection of surface markers on stem cellsS-ADSCs and V-ADSCs at the fourth generation were collected,and the expression of CD90,CD 105 and CD44 on the surface of S-ADSCs and V-ADSCs cells were detected by flow cytometry.2.Detection of CD90 mRNA levelS-ADSCs and V-ADSCs at the third generation were digested and plated in 12-well plate and incubated for 24 hours in the incubators.The total RNA was extracted by fast200 kit,and the mRNA level of CD90 in V-ADSCs and S-ADSCs was detected by Real-time quatitive PCR after reverse transcription.3.Protein level detection of CD90S-ADSCs and V-ADSCs at the third generation were digested and planted 12-well plate.The proteins were extracted from ADSCs 24 hours later,and the protein level of CD90 in S-ADSCs and V-ADSCs was detected by western-blot.?.Effects of CD90 gene silence on the proliferation and stemness of S-ADSCs1.CD90 gene silencing experimentS-ADSCs were transfected with CD90 small interfering RNA(siCD90),and the transfection efficiency and interference efficiency were detected 24 hours later.2.Effects of CD90 gene silence on the proliferation of S-ADSCsEdU incorporation assayAfter transfected with siCD90 for 24h,S-ADSCs were digested and planted into 96-well plate and incubated overnight.Then the cells were incubated with EdU to test the effects of CD90 on the proliferation of S-ADSCs.CCK-8 experimentAfter transfected with siCD90 for 24h,S-ADSCs were digested and planted.After Oh,12h,24h,36h and 48h of culture,the cell viability was detected using Cell Counting Kit-8.3.Effects of CD90 gene silence on the cell cycle of S-ADSCs Cell cycle detectionAfter transfected with siCD90 for 24h,S-ADSCs were stained with PI solution,and the cell cycle were detected by flow cytometry.4.Effects of CD90 gene silence on the expression of stemness genes in S-ADSCsAfter transfected with siCD90 for 24h,S-ADSCs were collected and the total RNA was extracted by fast200 kit,and the expression of Nanog,Oct4 and Sox2 of S-ADSCs were detected by Real-time quatitive PCR.5.Effects of CD90 gene silence on proliferation-related signaling pathways.S-ADSCs were transfected with siCD90,24 hours later,the protein were extracted from the cells,and the protein levels of AKT,p-AKT and cyclinD1 were detected by western-blot.?.Effects of CD90 gene silence on the adipogenic differentiation of S-ADSCs1.CD90 gene silencing experimentS-ADSCs were transfected with shCD90 recombinant lentivirus,and the transfection efficiency and interference efficiency were detected 72 hours later.2.Effects of CD90 gene silence on the adipogenic cdifferentiation of S-ADSCsS-ADSCs at the third generation were transfected with shCD90 for 72h,then the cells were digested and planted.The mRNA levels of white adipocyte markers PPAR-?,C/EBP?,?P2 and adiponectin were detected by Real-time quatitive PCR.After induction,the cells were stained with Oil Red O,and photographed under microscope.3.Effects of CD90 gene silence on mitotic clonal expansion of S-ADSCs under adipogenic inductionS-ADSCs at the third generation were transfected with shCD90 recombinant lentivirus.After 72 hours,the cells were digested and planted.When the cells were cultured to cell fusion or over fusion state,the cells were stimulated with adipogenic induction for 16h,and then the mitotic clonal expansion of S-ADSCs was detected using EdU incorporation assay.?.The effects of CD90 on the activation of AKTFour groups of plasmids,pENTER-Thyl(pENTER-CD90)+pcDNA3-AKT-PH-GFP(AKT-PH can bind to PIP3 on cell membrane and activate AKT),pENTER-CD90+pcDNA3-AKT-PH[R25C]-GFP(The mutant of AKT-PH can not bind to PIP3),pENTER+pcDNA3-AKT-PH-GFP,pENTER+pcDNA3-AKT-PH[R25C]-GFP,were respectively transfected into HEK-293T cells.After 24 hours,immunofluorescence staining was performed,and the effects of CD90 on the AKT-PH transposition were observed using laser confocal microscope.?.Intervention experiment of shCD90 recombinant lentivirus in vivo1.CD90 gene silencing experimentS-ADSCs were transfected with shCD90 recombinant lentivirus,and the transfection efficiency and interference efficiency were detected 72 hours later.2.Injection of shCD90 recombinant lentivirus into miceC57BL/6 male mice at the age of 8 weeks were injected with 0.2%pentobarbital sodium.The inguinal subcutaneous fat pad was exposed and the lentivirus solution was injected into each fat pad.(control group:shControl lentivirus;experimental group:shCD90 recombinant lentivirus)3.Glucose tolerance test(GTT)GTT test was carried out on the third weeks after operation.Briefly,the mice were weighed and fasted at 17:00 p.m the day before GTT.GTT was carried out at 9:00 a.m.Each mouse was intraperitoneally injected with glucose according to the amount of 2g/kg body weight.Blood glucose levels were monitored with a glucose meter at 0,15,30,45,60,90,and 120min after injection,respectively.And the data were made for statistics.4.Weight monitoring in miceAfter shCD90 recombinant lentivirus was injected,the mice were weighed continuously for four weeks.5.Detection of adipocyte size and related gene expression in SATSAT were collected,fixed,embedded,sliced and stained with HE by Google biotech company.The morphology and size of adipocytes were observed under microscope.Then the size of adipocyte was measured and the final data were counted with histogram.The total RNA was extracted by Trizol,and mRNA was reversed to cDNA.The expression of leptin was detected by Real-time quatitive PCR.6.Detection of proliferation ability of S-ADSCs in miceAt the fourth week of operation,S-ADSCs of SAT were extracted and planted in 6-well plate.When the cells were purified to the second generation,S-ADSCs were collected and planted in 96-well plate.Then the cells were incubated with EdU to test proliferation of S-ADSCs.Results1.S-ADSCs proliferate more rapidly than V-ADSCsS-ADSCs from inguinal SAT and V-ADSCs from epididymal VAT of mice were used to detect proliferation by EdU incorporation assay and clone formation experiments.We found that the proliferation rate of S-ADSCs was significantly higher than that of V-ADSCs.The flow cytometry data also showed that the cell proportion of S-ADSCs in G1 phase was less than that of V-ADSCs,but the cell proportion of S phase was more than that of V-ADSCs.At the same time,the expression of sternness genes Nanog,Oct4 and Sox2 in S-ADSCs was significantly higher than that in V-ADSCs.Western-blot showed that the proliferation and sternness dominance of S-ADSCs were associated with significant activation of AKT pathway.2.S-ADSCs enhance the adipogenic differentiation by promoting mitotic clonal expansionOil Red O staining showed that the lipid formation of S-ADSCs was stronger than that of V-ADSCs after adipogenic induction.During the induction,the expression levels of PPAR-?,C/EBP?,?P2 and adiponectin were significantly upregulated in S-ADSCs compared with V-ADSCs.In response to adipogenic induction,the ability of mitotic clonal expansion of S-ADSCs was significantly stronger than that of V-ADSCs as determined by EdU incorporation assay.indicating that S-ADSCs enhanced its adipogenic differentiation by promoting mitotic clonal expansion.3.CD90 is highly expressed on S-ADSCs compared with V-ADSCsThe expression levels of stem cell-related markers CD90,CD 105 and CD44 on S-ADSCs and V-ADSCs were detected by flow cytometry.It was found that the expression of CD90 in S-ADSCs was significantly stronger than that in V-ADSCs.Then,we further confirmed the high expression of CD90 in S-ADSCs at mRNA and protein levels by Real-time quatitive PCR and western-Blot.4.Silence of CD90 gene decreases the proliferation and stemness ofS-ADSCs by inhibiting AKT activationSilence of CD90 gene in S-ADSCs using siCD90 significantly decreased their proliferation,inhibited the cell percentage in S phase of ADSCs,and downregulated expression levels of sternness markers.In addition,the activation of AKT and the expression of cyclinD1 were also obviously suppressed.5.Silence of CD90 gene reduces the adipogenic differentiation of S-ADSCs by inhibiting mitotic clonal expansionOil Red O staining showed that the lipid formation in S-ADSCs was obviously inhibited by CD90 gene silencing;correspondingly,the expression levels of white adipocyte-related genes PPAR-y,C/EBPa,aP2 and adiponectin were decreased.Moreover,EdU incorporation experiment showed that CD90 silencing could significantly inhibit the mitotic clonal expansion of S-ADSCs.These results indicated that CD90 regulated the adipogenic differentiation of S-ADSCs by regulating cell mitotic clonal expansion.6.CD90 promotes the transposition of AKT-PH to cell membraneWhen pENTER and pcDNA3-AKT-PH-GFP were transfected into HEK-293T cells,few AKT-PH was translocated from cytoplasm to the cell membrane;after overexpression of pENTER-CD90 and pcDNA3-AKT-PH-GFP,the expression of AKT-PH was obviously decreased in the cytoplasm,but increased on the cell membrane,with an apprent colocalization with CD90.7.Effects of CD90 gene silence on the adipose tissue and metabolic homeostasis in miceGTT was performed 3 weeks after the operation.The mice in shCD90 group showed a significant reduction in glucose tolerance compared with those in shControl group.Four weeks after the operation,SAT was prepared into paraffin section.The HE staining showed that the volume of adipocytes and the mRNA level of leptin were significantly increased in the shCD90 group compared with those in shControl group.S-ADSCs from shCD90 group showed decreasing of proliferation ability compared with those from shControl group.Conclusion1.S-ADSCs proliferate more rapidly than V-ADSCs due to the increase in AKT activation and G1/S phase transition.2.The adipogenic differentiation ability of S-ADSCs is stronger than that of V-ADSCs;S-ADSCs enhance their ability of adipogenic differentiation by promoting mitotic clonal expansion3.CD90 is highly expressed on S-ADSCs,which promotes the proliferation and adipogenic differentiation of S-ADSCs by promoting AKT-PH translocation to cell membrane and enhancing AKT activation4.Silence of CD90 gene in SAT inhibits the proliferation of S-ADSCs and induces the adipocyte hypertrophy,resulting in the reduction of glucose tolerance in mice... |
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