| Objective:Construction of rat bone marrow mesenchymal stem cells(BM-MSCs)overexpressing interleukin(IL)10 and study its therapeutic effect on dextran sulfate sodium salt(DSS)-induced inflammatory bowel disease(IBD)in mice.Methods:1、The whole bone marrow adherence method was used to extract and culture rat BMMSCs;2、Osteogenic adipogenic induction identifies the differentiation capacity of BM MSCs;The expression of cell surface markers(CD11b,CD29,CD45,and CD90)was assessed by flow cytometry;3、Lentivirus(LV)transfection was used to construct BM-MSCs overexpressing IL-10.BM-MSCs were divided into three groups: MSCs group(untransfected BM-MSCs),LVMSCs group(transfected with lentivirus containing EGFP gene),IL-10-LV-MSCs group(transfected with lentivirus containing IL-10&EGFP gene),osteogenic adipogenic induction and flow cytometry to identify the differentiation ability and the expression levels of surface markers(CD11b,CD29,CD45 and CD90)and EFGP of LV-MSCs and IL-10-LV-MSCs,CCK8 was used to measure the changes in the number of cells in the three groups,and q PCR and ELISA were used to measure the expression of IL-10;4、Animal experiments were divided into five groups: Control group,DSS+PBS group(4% DSS-induced IBD treated with PBS),DSS+MSCs group(4% DSS-induced IBD treated with MSCs),DSS+LV-MSCs group(4% DSS-induced IBD treated with LV-MSCs)and DSS+IL-10-MSCs group(4% DSS-induced IBD treated with IL-10-LV-MSCs),after inducing IBD in mice with 4% DSS solution,the mice were treated by three tail vein injections,and the survival time,disease activity index(DAI)and body weight change of the five groups of mice were monitored,and the lesion site was observed by HE staining and colon macroscopically.The expression levels of tumor necrosis factor(TNF-α),IL-1β and IL-6 m RNA in colon tissue were detected by q PCR;5、LV-MSCs and IL-10-LV-MSCs were co-cultured with IBD mouse spleen lymphocytes to detect lymphocyte subsets T helper cells(Th)1,Th17 and regulatory T cells(Treg),LV-MSCs and IL-10-LV-MSCs were co-cultured with RAW264.7 to detect the secretion level of TNF-α in the culture supernatant.Results:1、The extracted BM-MSCs have the typical long spindle shape of stem cells,lipid droplets and calcium nodules are formed after induction and differentiation,and the cells highly express CD29 and CD90,low expression of CD11 b and CD45;2、After LV-MSCs and IL-10-LV-MSCs were induced to differentiate,lipid droplets and calcium nodules were formed.The cells expressed high levels of CD29 and CD90,and low levels of CD11 b and CD45.CCK8 showed that LV transfection had no effect on the number of BM-MSCs cells(P>0.05).Flow cytometry showed that the expression rate of EGFP in LV-MSCs and IL-10-LV-MSCs cells was over 75% after puromycin selection..The results of q PCR and ELISA showed that the expression level of IL-10 m RNA and the content of IL-10 in the cell culture supernatant in the LV-MSCs group had no significant changes(P>0.05,P>0.05),while the IL-10-LV-MSCs group had no significant changes(P>0.05,P>0.05).The expression level of IL-10 m RNA in cells and the content of IL-10 in cell culture supernatant were significantly increased(P<0.01,P<0.001);3、Survival curve,DAI and body weight change showed that: the weight of mice in DSS+PBS group continued to decrease,DAI continued to increase,and the survival time did not exceed 8 days.The weight of the mice in the DSS+MSCs and DSS+LV-MSCs groups tended to decrease gradually and occasionally rebounded,and there was no significant difference between the two groups(P>0.05).Days no longer rise,and the survival time is extended to 10 days.The body weight of DSS+IL-10-LV-MSCs mice no longer decreased on the 7th day,and the body weight increased significantly on the 11 th day.It was lower than the other three groups from the 4th day on DAI,and it began to decrease significantly on the6 th day.There was a significant difference among the three groups(P<0.001),and the score dropped below 3 on the 9th day(P<0.0001),and the survival time exceeded 10 days.Colon macroscopic observation and length statistics showed that the colorectum of the mice in the DSS+PBS group was atrophied and shortened,and blood was seen in the intestine.The length of the colorectum of the mice in the DSS+MSCs and DSS+LV-MSCs groups was slightly longer than that in the DSS+PBS group,and there was blood in the intestines.(P<0.01)There was no significant difference between the two groups(P>0.05).The colon length of the mice in the DSS+IL-10-LV-MSCs group was significantly longer than that in the DSS+PBS group(P<0.001).Formed feces can be seen in the intestine;4、The results of q PCR showed that the m RNA expressions of IL-1β and IL-6 in the DSS+MSCs and DSS+LV-MSCs groups were significantly lower than those in the DSS+PBS group(P<0.001),and there was no significant difference between the two groups(P>0.05),the expression of TNF-α m RNA in the DSS+MSCs and DSS+LV-MSCs groups was significantly higher than that in the DSS+PBS group(P<0.001),and there was no significant difference between the two groups(P>0.05).Compared with the DSS+MSCs and DSS+LV-MSCs groups,the DSS+IL-10-LV-MSCs group significantly decreased the TNF-α,IL-1β and IL-6(P<0.001,P<0.001,P<0.001);5、The results of in vitro co-culture showed that compared with the control group,the LV-MSCs group decreased the percentage of Th1 cells in the spleen lymphocytes of IBD mice but had no significant difference(P>0.05),and significantly increased the percentage of Th17 and Treg cells(P<0.01),significantly increased the secretion of TNF-α in RAW264.7 cells(P<0.01),and the IL-10-LV-MSCs group significantly decreased the percentage of Th1 cells compared with the control group(P<0.01),significantly increased the percentage of Treg cells(P<0.01),but had no significant effect on the number of Th17cells(P>0.05),and significantly decreased the secretion of TNF-α in RAW264.7 cells(P<0.0001).Conclusion:1、The cells extracted and cultured by the whole bone marrow adherence method have the biological characteristics of BM-MSCs;2、The way of LV transfection will not affect the biological characteristics and cell proliferation ability of BM-MSCs,and can stably express EGFP and IL-10 genes;3、BM-MSCs overexpressing IL-10 has a significant therapeutic effect on IBD mice;4、BM-MSCs overexpressing IL-10 may function by reducing the expression of colonic pro-inflammatory factors TNF-α,IL-1β and IL-6 m RNA,inhibiting Th1 cell activation,promoting Treg cell differentiation and inhibiting macrophage secretion of TNF-α. |
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