| Background and aims:Crohn's disease(CD)is a chronic inflammatory granulomatous disease of unknown cause.It could invade the entire digestive tract from the mouth to the anus,and could occur in any part of the digestive tract,especially in the intestine,mainly at the end of the ileum and adjacent to the colon.The fistula formation is a characteristic clinical manifestation of CD,including internal fistula and external fistula.The internal fistula lead to other intestinal segments,mesentery,ureter,vagina,bladder,retroperitoneal,etc.While the external fistula is communicated with the outside through the abdominal wall or perianal skin.About 25%of CD patients will develop refractory fistula during the course of the disease,which affects their quality of life seriously.Mesenchymal stem cells(MSCs)are pluripotent stem cells with low immunogenicity and immunomodulatory ability.MSCs are a promising therapeutic strategy in inflammatory bowel disease(IBD).Therefore,we simulated the formation of enterocutaneous fistula in CD patients by establishing a mouse enterocutaneous fistula model,and conducted local application of MSCs for intervention.Observed its therapeutic effect,and to further explore its possible mechanism of action.Methods:First,we constructed a MSCs cell line with firefly luciferase(Fluc)-green fluorescent protein(GFP)double fusion reporter gene that can track cell movement and survival in real time by in vivo imaging.Then,the mouse enterocutaneous fistula model was established and randomly divided into phosphate buffer(PBS)group and MSCs treated group,with 8 mice in each group.After the transplantation of the same volume of PBS or MSCs(1×10~6)in the orifice of enterodermal fistula,the repair of the fistula tissue was detected at different time points by the percentage of weight loss,the macroscopic manifestation and the histopathology techniques.The survival of MSCs in vivo after transplantation,the content of reactive oxygen species(ROS),as well as angiogenesis at the orifice of enterodermal fistula were detected by bioluminescence imaging(BLI).fistula tissue was collected for real-time quantitative PCR to detect the expressions of inflammation-related factors and angiogenesis-related factors.At the same time,in order to evaluate the role of MSCs in angiogenesis under in vitro conditions,we treated human umbilical vein endothelial cells(HUVECs)with MSCs cultured supernatants to observe the proliferation,migration,and tubulization of HUVECs.Results:After the establishment of the enterocutaneous fistula model in mice,the formation of fistula was revealed by small animal X-ray barium contrast.After local injection of MSCs or PBS,living image of small animals at 1,3,5,7 days showed that MSCs could stably accumulate in the site of enterocutaneous fistula in mice,and survive in the site of fistula for about 1 week.At the same time,MSCs treatment can reduce the weight loss,promote weight recovery in mice,and accelerate the healing speed of fistula site in mice.Real-time quantitative PCR results showed that MSCs treatment could reduce the expression of pro-inflammatory factors in the fistula site,increase the expression of anti-inflammatory factors to inhibit fistula inflammation.And the expression of angiogenic factors in fistula site was enhanced.The results of bioluminescence imaging and chemiluminescence imaging showed that the ROS content in the fistula site of mice significantly decreased,and the angiogenesis ability markedly increased in the MSCs treatment group,which accelerated the healing of the fistula in mice.In addition,we demonstrated that MSCs supernatant can significantly promote the proliferation,migration and tubulization of HUVECs cells in vitro.Conclusions:In the model of enterocutaneous fistula in mice,local application of mesenchymal stem cells can reduce the percentage of body weight loss,reduce tissue inflammation,promote the angiogenesis of damaged tissue,and accelerate the healing of enterocutaneous fistula. |
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