| Part I Isolation and culture of adipose mesenchymal stem cells(ADSCs)Objectives:To isolate and culture adipose mesenchymal stem cells(ADSCs)from the inguinal adipose tissue of C57BL/6 mice and identify their biological characteristics.Methods:In aseptic environment,separate and extract the inguinal adipose tissues of mice,applicate 0.1%type IV collagenase to digest them,made them into single cell suspension,divide them into several culture flasks according to certain cell concentration,adding suitable amount containing 10%fetal bovine serum low glycemic DMEM culture medium,culture in the standard environment.Observe ADSCs morphology under an inverted microscope,and the osteogenesis and adipogenesis of ADSCs were induced to explore the potential ability of multi differentiation.The surface markers were analyzed and studied by flow cytometry.Results:Successfully cultured ADSCs from the mice have good cell morphology and strong growth ability,can be induced ADSCs differentiation into osteoblast and fat cells.ADSCs were positive express CD29、CD44 and Sca-1,negative express CD31.Part Ⅱ Study on the therapeutic effect and mechanism of VEGF-C-stimulated ADSCs on chronic experimental colitis in miceObjectives:To clarify the therapeutic effect of VEGF-C-stimulated ADSCs on 2,4,6-trinitrobenzene sulfonic acid(TNBS)induced chronic colitis and explore its mechanism.Methods:TNBS model of chronic colitis was constructed and randomly divided into four groups:blank control group(n=5)、TNBS group(n=5)、ADSCs group(n=5)、ADSCs+VEGF-C group(n=5).At the 1st and 21st days,ADSCs(106cells/mousend VEGF-C-stimulated ADSCs(106cells/mouse)were intraperitoneally injected.And body weight,colitis index and disease activity were monitored daily.On the 28th day,the mice were killed,and hematoxylin-eosin staining was made from colon tissue to evaluate colon pathology,PCNA immunohistochemical staining was used to evaluate cell proliferation,colon cell apoptosis was detected by TUNEL method,colon mucous cells were detected by Alcian Blue staining,colon collagen fiber cells were detected by Masson staining.And intestinal lymphatic vessel density was detected by LYVE-1 and podoplanin immunofluorescence staining.The expression of IL-6,IL-10,TNF-α,and INF-y in the colon tissues of each group was detected by qPCR.Western blot was used to verify the expression changes of VEGF-C and VEGFR-3 in the colon tissues of each group.Image J 1.6.0 was used for Image analysis.GraphPad Prism 7 and SPSS 25.0 were used for statistical analysis,and the data were presented in the form of means ± SD.One-way ANOVA or SNK-q tests were used to compare the differences between groups.P<0.05 indicates a statistically significant difference.Results:Compared with the TNBS group,the ADSCs group and the VEGF-C+ADSCs group significantly improved disease activity in mice with chronic colitis,including weight loss,diarrhea,inflammation,and prolonged survival(P<0.05)and significantly improved histological inflammation(including colitis score,histological score,P<0.05);In addition to weight change,the ADSCs+VEGF-C group was better than the ADSCs group(P<0.05);Evans blue drainage test showed that the colon lymphatic drainage function of ADSCs+VEGF-C group was significantly enhanced compared with that of ADSCs group(0.55±0.07μg vs 0.78±0.06μg,P=0.01).Compared with the model group,the ADSCs group and the ADSCs+VEGF-C group significantly inhibited the colon cell proliferation(51.2%±3.4%vs 35.3%±8.66%,P=0.0002;51.2%±3.4%vs 25.3%±4.3%,P<0.0001)and apoptosis(29.2%±4.9%vs 21.2%±3.5%,P=0.0019;29.2%±4.9%vs 15.8%±2.2%,P<0.0001)and collagen fiber hyperplasia(5.6%±0.9%vs 4.6%±0.3%,P=0.0031;5.6%±0.9%vs 3.3%±0.3%,P<0.0001).Meanwhile,the effect of ADSCs+VEGF-C group was significantly better than that of ADSCs group(P<0.05).The number of colonic mucous cells in the ADSCs group and the VEGF-C+ADSCs group increased significantly compared with the TNBS group(47±10 vs 64±4,P=0.0005;47±10 vs 71±2,P<0.0001).However,no significant difference was found between the two intervention groups.Immunofluorescence staining results the colon LVD of ADSCs+VEGF-C intervention group was significantly increased compared with that of ADSCs intervention group(206±15 vs 114±7,P<0.0001).qPCR results showed that mRNA expression levels of IL-6(64.30±24.06pg/ml vs 15.33±4.68pg/ml,P=0.0102)and TNF-a(45.28±6.00pg/ml vs 25.60±44.97pg/ml,P=0.0423)in the VEGF-C+ADSCs group were significantly lower than those in the ADSCs group.The mRNA expression of IL-10 was significantly higher than that of the ADSCs group(87.67±9.13pg/ml vs 56.08±7.93pg/ml,P<0.0001).The protein expression level of VEGF-C(1.39±0.09 vs 1.03±0.16,P=0.019)and VEGFR-3(1.23±0.08 vs 0.91±0.06,P=0.0005)in the colon tissues of mice in the VEGF-C+ADSCs group were significantly higher than that in the ADSCs group.Conclusions:For TNBS-induced chronic colitis,ADSCs stimulated by VEGF-C can significantly down-regulate the expression of proinflammatory cytokines and improve lymphatic drainage to significantly improve the effect of ADSCs on inhibiting intestinal inflammation. |
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