| PART Ⅰ Down-regulate Tnfaip6 Expression of Bone Marrow Mesenchymal Stem Cells by RNA Interference Rrecombinant AdenovirusObjective:To explore whether RNA interference(RNAi)recombinant adenovirus can down-regulate the expression of tumor necrosis factor induced protein 6(Tnfaip6)in bone marrow mesenchymal stem cells(MSCs).Methods:Added corresponding amounts of negative control adenovirus to infect mouse bone marrow MSCs with multiplicity of infection(MOI)of 50,100,200,and 400 respectively,and used complete medium or enhanced infection solution as the infection system to explore the optimal infection condition.According to this infection condition,added the corresponding amount of Tnfaip6 gene RNA interference recombinant adenovirus to infect MSCs.Observed the cell morphology and growth state after infection,and detected the expression of GFP in mouse bone marrow MSCs and the cell surface molecules(CD31,CD45,Sca-1)by flow cytometry 48 hours after infection to detect the infection rate and determine whether adenovirus affected the phenotype of MSCs.Then,detected the expression level of Tnfaip6 mRNA after transfection by reverse transcription quantitative polymerase chain reaction(RT-qPCR),and screened out the RNAi adenovirus with the highest interference efficiency at the mRNA level from three candidate targets.At last,mouse bone marrow MSCs were infected by the selected RNAi adenovirus and the expression levels of Tnfaip6 protein in mouse bone marrow MSCs were detected by Western blotting after RNAi.Results:Replacement of complete medium with enhanced infection solution and MOI 200 were the best infection condition for mouse bone marrow MSCs.The adenovirus infection rate was as high as about 90.5%.There were no obvious changes in cell morphology and growth status after infection.The positive rates of CD31,CD45,and Sca-1 on the surface of MSCs detected by flow cytometry were 0.4%,0.2%,and 99.1%,respectively.These results of cell surface molecules were in line with MSCs characteristics.The expression of Tnfaip6 mRNA and protein in mouse bone marrow MSCs were significantly down-regulated after RNAi(P<0.05).Conclusion:Tnfaip6 RNAi recombinant adenovirus can successfully infect mouse bone marrow MSCs,down-regulate their Tnfaip6 mRNA and protein expression levels,does not affect their growth and cell phenotype,and laid the foundation for follow-up experiments to explore the role of Tnfaip6 in the immune regulation of MSCs transplantation in inflammatory bowel disease(IBD).PART Ⅱ Verify the Regulation of Tnfaip6 Secreted by Bone Mesenchymal Stem Cells on Tfh and Tfr in IBD Model MiceObjective:MSCs transplantation has good prospects in IBD treatment,but the specific mechanism is unclear.This study plans to explore the regulation of bone marrow MSCs on follicular helper T cells(Tfh),follicular regulatory T cells(Tfr),and immune response in IBD,and verify whether this immune regulation depends on Tnfaip6 secreted by MSCs.Methods:Sixty female Balb/c mice were randomly divided into 6 groups of 10 mice each.TNBS presensitization and enema were administerd to induct a mouse model of acute experimental colitis,while the normal control group was given the same volume of PBS enema.The hair color,activities,weight changes,stool characteristics and fecal occult blood test results were recorded.On the next day of modeling(day 2),the normal control group and model group received a single intraperitoneal injection of 100μL PBS buffer.The MSCs group,Ad-MSCs group,and RNAi-MSCs group received a single intraperitoneal injection of 100μL PBS suspension containing 106MSCs,control adenovirus-infected MSCs,and Tnfaip6 RNAi adenovirus-infected MSCs respectively.Mice in the Tnfaip6 group were intraperitoneally injected with 100μL PBS containing 4 μg of recombinant mouse Tnfaip6 protein daily from day 2 to day 5.On day 8,mice were anesthetized,the eyeballs were removed,and blood was collected,then the spleen,mesenteric lymph nodes,and colon were taken out.Observed the colon tissue injury,and recorded the inflammation associated histological scores in colon tissue under microscope by HE staining.The proportion of CD4+CXCR5+PD-1+Tfh,CD4+CXCR5+Foxp3+Tfr in mesenteric lymph nodes and spleen were detected by flow cytometry.Relative expression levels of Tfh main transcription factor BCL-6,Tfr main transcription factor Foxp3 and related cytokines including IL-21,TNF-α,and IL-10 mRNA in colon tissue were detected by RT-qPCR.Serum IL-21,TNF-α,and IL-10 Tnfaip6 concentrations were detected by ELISA.Results:After injected with TNBS enema,mice in the model group,MSCs group,Ad-MSCs group,RNAi-MSCs group,and Tnfaip6 group all showed symptoms such as reduced intake and activity,hogback,rough hair,diarrhea,and hematochezia.Transplantation of MSCs or supplementation of exogenous Tnfaip6 protein improved above symptoms,while the symptoms of RNAi-MSCs group had no significant improvement.Compared with the normal group,the model group’s weight loss percentage,DAI,colon tissue injury and inflammation related histological scores,the relative mRNA expression levels of BCL-6,IL-21,and TNF-α in the colon tissue,serum IL-21,TNF-α and Tnfaip6 concentrations,the proportion of Tfh cells in mesenteric lymph nodes and spleen all increased significantly(P<0.05).The relative expression levels of Foxp3 and IL-10 mRNA in colon tissue,the concentration of serum IL-10,the proportion of Tfr cells in mesenteric lymph nodes and spleen were not statistically different from those in the normal group(P>0.05).The ratios of Tfh/Tfr in mesenteric lymph nodes and spleen were significantly higher than those in the normal group(P<0.05).Compared with the model group,mice in the MSCs group,Ad-MSCs group,and Tnfaip6 group all showed significant improvement in symptoms such as weight loss,reduced intake,rough hair,diarrhea,hematochezia,and gross colon injury.DAI,inflammation related scores of colon tissue injury,the relative expression levels of BCL-6,IL-21,and TNF-α mRNA in colon tissue,serum IL-21 and TNF-α concentrations,and the proportion of Tfh cells in mesenteric lymph nodes and spleen were all lower than the model group(P<0.05),the relative expression levels of Foxp3 and IL-10 mRNA in colon tissue,serum IL-10 and Tnfaip6 concentrations,and the proportion of Tfr cells in mesenteric lymph nodes and spleen were significantly higher than those in the model group.The ratios of Tfh/Tfr in mesenteric lymph nodes and spleen were lower than those in the model group(P<0.05).There were no statistically differences in the above indicators between the RNAi-MSCs group and the model group(P>0.05).Conclusion:Tfh increase during IBD active phase.Tfh decrease while Tfr increase during IBD remission phase.The imbalance of Tfh/Tfr ratio is closely related to the progression and outcome of IBD.Bone marrow MSCs transplantation may alleviate IBD symptoms and intestinal injury through inhibiting Tfh differentiation,promoting Tfr differentiation,improving the imbalance of Tfh/Tfr,then reducing the inflammatory response.This immunomodulatory effect of MSCs in IBD may depend on the secretion of Tnfaip6. |
