| [Objective]1.To provide experimental subjects for the therapeutic effect and mechanism of umbilical cord mesenchymal stem cells(UCMSC)in the treatment of IBD by constructing acute model and chronic model using DSS solution.2.To provide theoretical basis and method for the clinical treatment of IBD with UCMSC by evaluating the efficacy and mechanism of different sources of UCMSC and different injection routes for inflammatory bowel disease.[Methods]1.Preparations of UCMSC:1.UCMSC of KM mice:take 3 KM pregnant mice,and perform cervical dislocation,paunch,cut the umbilical cord,and remove the capsules and blood,etc.;cut into small pieces smaller than lmm3,inoculate in T25 culture flask.Then placed in a 5%C02 incubator.They were passaged till monolayer cells spread to 80%-90%of the bottomof the culture bottle.2.Human UCMSC:P2 cells provided by National-local engineering laboratory for stem cell and immunocyte biomedical technology.,were amplified 1:3.Human and mouse UCMSC were analyzed for the positive expression rate of UCMSC marker antigen by flow cytometry,and UCMSC were assayed for osteogenic,adipogenesis,and chondrogenic differentiation.P3 cells were diluted to 3 x 106/mL with a cryopreservation solution and stored in liquid nitrogen for later use.When used,UCMSC were diluted with physiological saline to a corresponding concentration of 5 x 106/ml and 1 x 107/ml.2.Establishment of animal model of IBD:? Acute model:healthy male C57BL/6 mice(age:7-8w;weight:20-22g)drinking 3%DSS aqueous solution freely for seven days,then sacrificed mice to obtain samples for relevant test and analysis.?Chronic model:KM mice were allowed to freely drink 3%DSS solution for 5 days.First let the mice freely drink 3%DSS solution for five days and then withdrawl for 10 days,following the "drinking DSS solution for 4 days,withdrawal(DSS)3 days" cycle for 5 times.Mice were sacrificed and relevant specimens were collected.During the modeling period,monitoring mouse body weight,fecal form,blood in the stool,and mortality and other indicators daily.After the end of modeling,the colon tissue was stained with HE,observed histological changes of the colon,while being performed histopathological scores.3.UCMSC transplantation for treatment of mouse IBD(1)Different injection methods for treatment of acute IBD model:According to the above method,36 mice were for acute IBD models were made and randomly divided into model control group,intraperitoneal injection group and intravenous injection group,12 in each group.At the same time,9 mice were set up as normal control group.Among them,model group received intraperitoneal injection of 100 ul normal saline on the first,third,and fifth days after the start of the experiment;intravenous group and the intraperitoneal group received injections of 5×106/ml UCMSC(100ul)respectively on the first,third,and fifth day.The mice were sacrificed and obtained samples on the 7th day after the start of the modeling.(2)Human and mouse UCMSC for chronic IBD model:according to the method above,45 acute IBD models were made and randomly divided into model control group,murine UCMSC group and human UCMSC group,15 in each group.normal control groups were also set up(n=9).Mice from murine UCMSC-treated group and the human UCMSC-treated group were injected with UCMSC and human UCMSC intraperitoneally,respectively(with a cell concentration of1×107/ml;100 ?l).Mice in the model group were injected with an equal volume of saline in the peritoneal cavity.(3)observation index:?structure of colon tissue of mice;?concentrations of IL-6 and TNF-a in serum;immunohistochemical staining of inflammatory cells;? staining of goblet cells in colon tissue:?staining of collagen fibers in colon tissue.? Mice's daily mental state and weight;?feces form;?blood in the stool.The mice were sacrificed and obtained samples on the 50th day after the start of the modeling.4.Analysis of the mechanism of umbilical cord mesenchymal stem cells on IBDUmbilical cord mesenchymal stem cells on the mechanism of IBDWestern blot was used to detect the expression of autophagy markers LC3A/B,tight junction protein Occludin,vascular endothelial growth factor VEGF-A and its receptor VEGFR-l;qPCR were used to analyze gene expressions of Claudin-1,Occludin,and inflammatory factor IL-6.UCMSC were localized by GFP-labeled UCMSCs and immunohistochemistry,gene relative expression of claudin-1,Occludin and i1-6 genes in colon tissues;expression levels of proteins of vegf-a,vegfr-1 and Occludin.[Results]1.Identification of UCMSCBoth human and KM mouse-derived UCMSC were fibrous and adherently growing.The KM mouse-derived UCMSC markers were CD 105 and CD90,the expressions of that were 99.9%and 100%,respectively,while the positive expression rate of CD34 was 0.239%.The expression rates of human UCMSC markers CD73,CD90,and CD 105 were 97.5%,98.2%,and 97.5%,respectively;the expression rates of CD34,HLA-DR and CD45+ were 0.861%,2.22%,and 0.695%,respectively.Both two kinds of UCMSC can be induced to differentiate into adipocytes,bone-like,and cartilage-like cells.2.Establishment of Model and EvaluationAcute model of IBD:After 4 days of drinking 3%DSS solution,mice developed blood in the stool.After 5 days,the weight of the mice began to decline,with heavily constipating,lethargy,clumps,and general anatomy showed that the mice had congestive edema and ulcers in the entire colon or part of the colon.Histopathology and staining showed destruction of intestinal epithelial structure,gland disorder,goblet cells,desposition of many collagen fibers throughout the intestinal wall.In the chronic IBD model,mice exhibited intermittent blood in the stool,weight was maintained at a low level(about 85%of the initial body weight),inflammatory cells infiltrated the layers of the intestine,glandular structures almost disappeared,and the intestinal lumen was narrowing.3.Efficacy of UCMSC(1)During the observation period,the weight loss percentages in intrapeitoneal injection UCMSC group,intravenous UCMSC group,and model control group were:11%,14%,and 17%,respectively.The mortality rates in intrapeitoneal injection UCMSC group,intravenous UCMSC group and model control group were:0%,8.3%,and 33%,respectively;was present.The serum concentrations of IL-6 in intrapeitoneal UCMSC group,intravenous UCMSC group,and model control group were:27.17±1.48(pg/ml),71.11±9.19(pg/ml),and 104.90±9.78(pg/ml),respectively(p<0.05).).The serum concentrations of TNFa in intrapeitoneal UCMSC group,intravenous UCMSC group,and model control group were:119.50± 12.33?153.67± 1.50?174.92 ±7.72(pg/ml)(p<0.05).qPCR results showed that the expressions of Claudinl and Occludin in intrapeitoneal UCMSC group were higher than that in the other three groups(p<0.05).The histopathologic scores of normal control group,intraperitoneal injection UCMSC group,intravenous UCMSC group and model group were 0.5±0.30;5.9±1.10;8.7±1.39;8.8±1.33(p<0.05).(2)The mortality rates of human and KM murine UCMSC group and model control group were 0%,13.3%,and 60%,respectively.The CD4+T cells,macrophages,and neutrophils in the colon tissue of human and KM mice UCMSC groups were less infiltrating,which was similar to that of the normal control group.The three types of white blood cells in model control group showed diffuse infiltration.The goblet cells in the human UCMSC-treated group and the KM-murinized UCMSC-treated group were more evenly distributed,with higher density,deeper staining,and less collagen fiber exudation.The situation was opposite in the model control group.The protein expression of LC3A/B in human and KM mouse UCMSC group was lower than that in other two groups;the expression of VEGF-A,VEGFR-1 and Occludin protein was increased in human UCMSC treatment group.[Conclusion]1.The KMMSC mouse UCMSC cells were successfully obtained by tissue culture method.UCMSC highly expressed CD90,CD105,and low expression of CD34.Human UCMSC express high levels of CD73,CD90,and CD105;low levels of CD34,CD45,and HLA-DR.Both KM mice and human UCMSC have the potential to differentiate into bone,cartilage,and lipids.2.The IBD acute and chronic models were successfully established using continuous or intermittent free drinking 3%DSS solution.3.Intraperitoneal injection and intravenous UCMSC treatment of IBD acute models have been shown to be effective,but the effect of intraperitoneal injection was better.4.Both human UCMSC and KM mouse-derived UCMSC can reduce the mortality of mice with IBD and reduce intestinal inflammation,suggesting that both sources of UCMSC have therapeutic effects.5.UCMSC promotes the expression of intestinal tight junction protein Occludin;downregulates the expression of autophagic marker protein LC3A/B in ileum;upregulates the expression of VEGF-A and VEGFR-1 at the injured site.6.UCMSC injected into the body,in the intestine,liver,spleen,lung,kidney organ tissue distribution,in which the number and density of the spleen is the largest. |
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