Effect Of Mesenchymal Stem Cells Modified With CXCR4 Combined With IL-1β SiRNA On Acute Liver Failure

Part 1:Over-expression of CXCR4 on mesenchymal stem cell improves migration to acute failing liver.Objective:To improve the engraftment efficiency of bone marrow mesenchymal stem cells modified with CXCR4 to the failing liver.Method:Lentiviral transduction was exploited to overexpress CXCR4(CXCR4-MSCs)or Null(Null-MSCs).The characteristics of CXCR4-MSCs and Null-MSCs were determined by RT-PCR,Western Bolt and flow cytometry.CXCR4-MSCs and Null-MSCs were infused intravenously 24 hours after administration of CCl4 in nude mice.The concentration of SDF-1α in damaged liver was detected by ELISA.Transwell migration assays were carried out to evaluate the migration ability of MSC toward SDF-1α.The distribution of the stem cells was analyzed by in vivo imaging.Results:Transfected MSCs over expressed CXCR4 in gene and protein level.The level of SDF-la in failing increased and maintained for over 3weeks.In vitro,CXCR4-MSCs showed better migration capability toward SDF-1α.In vivo imaging showed that CXCR4-MSCs migrated to the liver in greater numbers than Null-MSCs 5 days after acute liver failure(ALF).Conclusions:Genetically modified MSCs over-expressing CXCR4 showed a higher colonization in damaged liver,which provided a broad prospect for the treatment of MSC transplantation on acute liver failure.Part 2:In vivo the inhibition effect of IL-1β siRNA adenovirus vector on IL-1β in the liverObiective:To construct IL-1β siRNA adenovirus vector,explore its optimal dose and time point of interference effect on IL-1β in vivo.The immunogenicity of adenovirus vectors was determined.Method:Four pairs siRNA interference sequence for IL-1β was synthesized and cloned into pcDNA6.2-GW/EmGFP-miR vector.A series of chemically synthesized oligo was spliced to the desired sequence by PCR.The sequence was cloned into pMD-18T vector verified.The target gene was cloned into the vector pcDNA3.1+ to build the over expression vector.The interference vector and over expression vectors transfected simultaneously HEK293 model cells to evaluate the interference effect.The interfering vector with the best silence effect was selected and converted into a recombinant adenoviral vector.Adenovirus was packaged by 293A cells and collected to determine virus titers.Nude mice(n=20)were administrated with 2×107(20μl),5×107(50μl),1×108(100μl)or 2×108(200μl)ifu adenovirus vector by tail vein.The normal group(n=5)served as control group.36 hours later,relative expression of IL-1β in liver tissue was detected by qPCR to determine the best dose.Nude mice were treated with IL-1β siRNA adenovirus vector in best dose via tail vein and subsequently sacrificed at different time point to assay IL-1β gene expression by quantitative PCR.Healthy mice were treated with null adenovirus vector in best dose via tail vein,4h later serum INF-γ,TNF-αand IL-6 levels were detected by ELISA to assess the immunogenicity of adenovirus vectors.Results:Four pairs of IL-1β siRNA adenovirus vector were constructed,qPCR displayed the interference effects of vector 13MR0069-4 was the best and reached up to 69%.qPCR showed that interference efficiency of IL-1β siRNA adenovirus vector was about 90%when injected at a dose of 1×108 ifu,while increasing the number of vectors make no significant change on the interference efficiency.We found that interference efficiency of IL-1β siRNA reached peak at 36h after injected and then gradually attenuated,but at the 5th day,40%gene interference efficiency was observed.4h after null adenovirus vector were injected,serum INF-γ,TNF-α and IL-6 levels were detected and no significant change was observed.Conclusions:IL-1β siRNA adenoviral vector has a strong inhibitory effect on IL-1β.1×108 ifu was optimal interference dose and the interference efficiency reached peak at 36h.The vector does not induce non-specific immune response and can be used for further study.Part 3:Synergistic effect of CXCR4-MSC combined with IL-1βsiRNA adenoviral vector on acute liver failureObiective:To assess the synergistic effect of CXCR4-MSC and IL-1β siRNA adenoviral vector on acute liver failure.Method:20%v/v CCl4 soluted in olive oil was infused intraperitoneally with a dose of 8μl/g to induce acute liver failure model in nude mice.All mice were divided randomly into five groups:normal control group(n=10);ALF group(n=10);ALF + IL-1β siRNA group(n=10),1×108 ifu adenovirus were infused through tail vein 6 hours before administration of CCl4;ALF + MSC group(n=10),approximately 5×105 CXCR4-MSCs were injected intravenously 24 hours after administration of CC14;ALF + IL-1β siRNA + CXCR4-MSC group(n=10),1×108 ifu adenovirus were infused through tail vein 6 hours before administration of CCl4 and approximately 5×105 CXCR4-MSCs were injected intravenously 24 hours after administration of CCl4.10 mice from each group were picked up randomly for survival analysis.At different time points after intervention,mice in each group were sacrificed with an overdose of isoflurane.Liver function,serum levels of inflammatory factors,histopathology,apoptosis and proliferation of hepatocytes were detected.To assess the protective effect of IL-1β siRNA on MSC,GFP-positive cells in liver tissue were detected by immunohistochemistry and fluorescence microscopy.VEGF and HGF levels in liver were detected to explore the mechanism of combination therapy.Results:Compared with IL-1β siRNA alone or CXCR4-MSC alone,combination with IL-1β siRNA and CXCR4-MSC can promote liver function,significantly reduce transaminase levels and proinflammatory cytokines in vivo,promote hepatocyte proliferation and reduce apoptosis of hepatocyte.Immunohistochemistry and fluorescence microscopy revealed that IL-1βsiRNA+CXCR4-MSC significantly increased the number of survival MSCs than CXCR4-MSC.ELISA displayed IL-1β siRNA+CXCR4-MSC secreted more VEGF and HGF significantly than the other groups.Conclusions:IL-1β siRNA play a protective role for CXCR4-MSC transplantation.Combination with IL-1β siRNA and CXCR4-MSC has a synergistic effect.