Effect Of CDX2 Overexpression On MUC2 Protein In Colonic Tissue Of Rats With Ulcerative Colitis

Objective To construct carrying CDX2 gene of rat bone mesenchymal stem cells,exploring a means of gene therapy for rat ulcerative colitis models.To investigate the effect of CDX2 gene overexpression on MUC2 protein in rat model of ulcerative colitis;Methods 1.Construction and identification of recombinant lentiviral vector of CDX2 gene Primers of CDX2 were designed according to the gene information of Gen Bank.Gene fragments of CDX2 were amplified by polymerase chain reaction(PCR);The gene fragment and linear plasmid vector were connected by In-Fusion technology and transformed into competent cells.The positive clones of lentiviral expression vector were obtained after screening and followed by sequencing.The recombinant vector was used to transfect 293 T cells and package virus,and then the virus titers were determined.2.Identification of rat BMSCs were infected with the recombinant lentiviral vector of CDX2 gene BMSCs were isolated and cultured in vitro.The immuneophenotype of BMSCs was detected by flow cytometry.The lentivirus vector containing CDX2 gene was transfected into rat BMSCs.The CDX2 m RNA and protein expression level was detected by RT-PCR and Western Blot.3.Induction of rat UC model and experimental grouping The female rat ulcerative colitis disease model induced by 5% 2,4,6-three trinitrobenzene sulfonic acid(2,4,6-TNBS)/ ethanol;the lentivirus transfected cell phenotype by FACS detection;then respectively 1ml normal saline,bone marrow mesenchymal stem cells,bone marrow mesenchymal stem cells transfected with empty lentivirus and bone marrow mesenchymal stem cells transfected with lentivirus carrying CDX2 were transplanted into UC model rats by tail vein injection respectively,additional untreated rats as normal control group.4.Quantitative score of rats in different groups after intervention Score of acute disease activity index(DAI)1 of rats in different groups;After 7 days by stem cell transplanted,the gross morphology changes of colonic tissue were observed by naked eye and light microscope(HE staining),and the colon tissue morphological damage index(CMDI)and the tissue damage index(TDI)were quantified.5.In addition,the expression of EGFP protein in frozen tissue sections was observed under fluorescence microscope.6.Detection of SRY、CDX2、MUC2 expression in rat colonic tissue PCR technique was used to detect the expression of SRY gene in male rats,and the expression of CDX2 m RNA and protein were detected by RT-PCR and Western Blot in different groups.The expression of MUC2 protein in different groups was detected by Western Blot technique.Results 1.The results of PCR,DNA sequencing and WB confirmed that the gene recombinant lentiviral vector of Ubi-CDX2-MCS-3FLAG-CMV-EGFP GV365 had been successfully constructed,and the titer reached 2×108TU/ml.2.BMSCs were isolated and cultured successfully.The FACS showed that CD29,CD73 and CD90 were positive,CD34 and CD45 were negative,fitting the phenotype of BMSCs.RT-PCR and Western blot showed that CDX2 gene was transfected into rat BMSCs and stably expressed.All of which showed the CDX2-BMSCs were constructed successfully.3.After 5%TNBS/ ethanol enema treatment,the rats appeared anorexia,weight loss,diarrhea and other symptoms of colitis.On the third day,DAI score of each group was significantly higher than the normal group,but no significant difference between experimental groups(F=0.39,P>0.05);FSCS showed that lentivirus transfected cells still showed the immunophenotype of bone marrow mesenchymal stem cells.4.In the seventh days after transplantation treatment by different bone marrow mesenchymal stem cells,the DAI,CMDI,TDI score of group E were significantly lower than B,C,D groups(P<0.05);C,D groups were lower than that of B group(P<0.05);instead of,the group C and D,no significant difference between the two groups(P>0.05).5.The frozen sections of each group were observed under fluorescence microscope.The results showed that only D and E two groups showed a large number of fluorescence,and the other groups had no fluorescence.6.PCR detection of SRY gene found visible bands at 213 bp in C,D and E group,which showed that the male rat bone marrow mesenchymal stem cells transplanted into rats and homing to colon tissue.Quantitative detection of CDX2 m RNA,CDX2 protein and MUC2 protein in rat colonic tissue by QRT-PCR and WB,the expression in group E was higher than that in B,C and D group(P<0.05),the expression in group B was the lowest,which was significantly lower than that of other groups(P<0.05),and there was no difference between group C and D(P>0.05).Conclusion 1.BMSCs that carries and stably expresses CDX2 gene has been created and transplanted into female rats;2.The rat model of ulcerative colitis was successfully induced by 5%TNBS/ ethanol,which laid the foundation for further experiment;3.For the first time,the overexpression of the target gene CDX2 was transfected into the rats by lentiviral vector with the bone marrow mesenchymal stem cells(MSCs)which as the carrier cells,to study the effect of CDX2 gene on the MUC2 protein in rat UC model;4.The therapeutic effect of bone marrow mesenchymal stem cells on ulcerative colitis and the relationship between CDX2 gene and ulcerative colitis were also confirmed.