| Object i ve:1. To investigate the vivo methods of isolation, cultivation, purification and identification of human umbilical cord mesenchymal stem cells (hUC-MSCs).2. To study the vivo methods of intestinal mucosal epithelial damage model by Caco-2cells.3. To study the protective effects of Heme oxygenase-1(HO-1) transfected hUC-MSCs on injury of intestine and its related mechanism.Methods:1. hUC-MSCs were isolated, cultivated, and purified by culturing some small tissue blocks directly, and were identified by flow cytometry and the differentiation of the fat and bone tissue. Then, hUC-MSCs were transfected by recombinant adenovirus with the fluorescent marker gene of enhanced green fluorescent protein and the gene of HO-1, and form the stable expression of HO-1/hUC-MSCs. Further more, whether HO-1was transfected into the cells and expressed was testified by testing the content of HO-1mPNA by RT-PCR.2. Caco-2cells were used to simulate the intestinal mucosa epithelial in order to study the change of the structure and function of the intestinal mucosa epithelial. TNF alpha was used to damage Caco-2cells to establish the damage model of the intestinal mucosal epithelial. The optimum concentration and the best processing time of TNF alpha under this experimental condition was testified by immunofluorescence, RT-PCR and Western blot.3. Caco-2cells were used to simulate the intestinal mucosa epithelial, TNF alpha was used to damage Caco-2cells to establish the damage model of the intestinal mucosal epithelial, and indirect co-culture system of HO-1, Caco-2cells, hUC MSCs and HO-1/hUC-MSCs by Transwell Chambers. And, whether HO-1was transfected into the cells and expressed was testified by testing the content of HO-1mRNA by RT-PCR. The content change of the tight junction proteins Occludin and ZO1of Caco-2cells was tected by immunofluorescence, RT-PCR and Western blot and the content of the cytokines IL-10and IL-6was tected by ELISA.Results:1. hUC-MSCs were isolated, cultivated, and purified successfully, and most of the third generation of cell showed positive in CD29and CD44, but nearly negative in CD34, CD45.2. TNF alpha with the concentration of0μg/L,50μg/L, 100μg/L and200μg/L and with the time of0h,6h,12h and24h was used to damage Caco-2cells to establish successfully the damage model of the intestinal mucosal epithelial. The optimum concentration and the best processing time of TNF tested by immunofluorescence, RT-PCR and Western blot wre separately100μg/L and24h.3. The experimental was divided into6groups, the first group:only CaCo-2cells; the second group:TNF-a+Caco-2cells; the third group CaCo-2cells+TNF-α+Ad-HO-1; the fourth group:CaCo-2cells+TNF-a+hUC-MSCs; the fifth group:CaCo-2cells+TNF-α+hUC-MSCs+Ad-HO-1; the sixth group:CaCo-2cells+TNF-α+HO-1/hUC-MSCs. Immunofluorescence showed that the integrity of CaCo-2cell tight junction protein changed, manifestation of cell tight junction protein fracture, manifestation the decrease in the number of fluorescence; Compared with second group, immunofluorescence showed that the red and green fluorescence of the first, third, fourth, fifth, and sixth group increased, and the sixth group was more than the third, fourth and fifth group; RT-PCR showed that, Compared with control group.the expression amount of Occludin mRNA and ZO1mRNA of the first, third, fourth, fifth, and sixth group increased, and the sixth group was more than the third, fourth and fifth group, they were all statistically significant; ELISA showed that the level of IL-6of the third, fourth, fifth, and sixth group descended, with IL-10ascending, and the sixth group was the most obvious, they were all statistically significant,and the first group of expression is extremely low.Conclusions:1. By culturing some small tissue blocks directly, hUC-MSCs were isolated, cultivated, and purified successfully; The fluorescent marker gene of enhanced green fluorescent protein gene and HO-1were recombined by adenovirus, and adenovirus were packaged. Recombinant adenovirus were transfected into umbilical cord mesenchymal stem cells, which could form stable expression of HO-1/hUC-MSCs.2. TNF alpha was used to damage Caco-2cells to establish successfully the damage model of the intestinal mucosal epithelial.3. Transwell little chambers were adopted to establish the cell co-culture system of target cells with Caco-2indirectly; HO-1, hUC-MSCs, HO-1+hUC-MSCs could promote the expression of intestinal epithelial tight junction protein and mRNA. reduce the release of inflammatory cytokines and promote the release of anti-inflammatory factor, so as to protect the intestinal mucosa epithelial cells. What’s more, the above effect of HO-1/hUC-MSCs was more obvious than pure hUC-MSCs, pure HO-1and HO-1+hUC-MSCs. |
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