The Repair Effects Of Transplanting Bone Marrow Derived Endothelial Progenitor Cells Transfected With TERT Gene In5/6Nephrectomy Rats

Objective:To construct the recombinant plasmid pZsGreen1-C1-TERT that carried telomerase reverse transcriptase (TERT) gene. Transfect the marrow derived endothelial progenitor cells with pZsGreen1-C1-TERT and explore the influence of TERT gene transfer to the cellular biological features. To investigate the repair effects and possible mechanism of transplanting EPCs and EPCs transfected with TERT gene in5/6nephrectomy rats.Methods:1. Construction the recombinant plasmid of pZsGreen1-C1-TERT: Amplified and extracted TERT gene fragment from the total RNA of the immature rat liver by RT-PCR assay, and then was conjugated with pZsGreen1-C1plasmid. Digested recombinant plasmid by restriction enzyme Bgl I and EcoR I for sequence analysis via the gel electrophoresiss.2. Isolation and cultivation of the EPCs:The bone marrow derived mononuclear cells were separated by density gradient centrifugation assay and cultured with EGM-2culture medium.(2)The endothelial progenitor cells were identified by cellular morphology, immune-fluorescence assay for the surface antigen detection, DIL-ac-LDL uptake and FITC-UEA-I binding capacity.3. Transfected EPCs with pZsGreen1-C1-TERT plasmid:The plasmid pZsGreenl-C1-TERT and empty pZsGreen1-C1were transfected via liposome Lipofectamine TM2000system. MTT assay was employed to detect the proliferation rate of endothelial progenitor cells; the flow cytometry, for early apoptosis detection; Western blot and Real-Time PCR, for the TERT protein and mRNA expression.4. The repair effects of transplanting EPCs and EPCs transfected with TERT gene in5/6nephrectomy rats:152healthy female SD rats aged6weeks were divided into five groups:sham group, model group, EPCs group (EPCs-N). pZsGreen1-C1-EFCs group (pZ-EPCs-N) and pZsGreen1-C1-TERT-EPCs group (pZ-TERT-EPCs-N). The sham group underwent anesthesia and isolated the kidney, but not nephrectomy, the rest groups underwent5/6nephrectomy. Rats of every group, respectively, were injected PBS, PBS, EPCs, pZsGreen1-C1-EPCs, pZsGreen1-C1-TERT-EPCs. At different times, the kidney, liver, spleen, heart and perpheral blood of pZsGreen1-C1-EPCs group and pZ-TERT-EPCs-N group were collected to observe Green1using the inverted fluorescence microscope. At the time of4weeks,8weeks,12weeks after injection, the urine and blood of rats were collected to examine urine protein in24h, blood urea nitrogen, serum cretinine and endogenous creatinine clearance rate. The kidney of all rats were collected to have pathological analysis, the peritubular capillaries counts and observe the expression of TERT, TGF-β1, cytokeratin, vimentin、 α-SMA、VEGF through techniques of Real-Time PCR and Western blotting.Results:1. The TERT cDNA amplified from RNA of the immature rat liver was about3399bp. The sequence of the recombinant plasmid of pZsGreen1-C1-TERT were proved identical to the reported cDNA sequence by DNA sequence analysis.2. Bone marrow derived mononuclear cells showed endothelial progenitor cells morphology, expressed CD133, vWF, and VEGFR-2, and could uptake DIL-ac-LDL and bind FITC-UEA-I simultaneously.3. The EPCs transfected with plasmids pZsGreen1-C1-TERT and pZsGreen1-C1display green fluorescence under fluorescence microscopy, with transfection rate70%. The endothelial progenitor cells transfected with pZs Green1-C1-TERT-EPCs hold an increased proliferation rate and decreased apoptosis rate than EPCs and EPCs transfected by pZsGreen1-C1on the28th day (P<0.05). TERT gene expression increased in pZsGreen1-C1-TERT transfected EPCs than that without transfection (P<0.05).4.(1) Green1labeled pZsGreen1-C1-EPCs and pZsGreen1-C1-TERT-EPCs were found in the remnant kidneys.(2) Compared with those for sham rats, model rats displayed the less body weight increase, lower creatinine clearance rate, and higher urinary protein, serum creatinine and serum urea lever(P<0.05). Compared with model rats, rats in EPCs-N,pZ-EPCs-N and pZ-TERT-EPCs-N groups displayed lower serum creatinine, lower serum urea and higher creatinine clearance rate (P<0.05). The pZ-TERT-EPCs-N group had the more remarkable effects(P<0.05).(3) Compared with those in the sham rats, model rats displayed the higher renal interstitial pathologic lesion scores and less peritubular capillaries counts(P<0.05). While the renal interstitial pathologic lesion scores decreased and peritubular capillaries density increased for the rats received cells transplantations, remarkablely for pZ-TERT-EPCs-N rats (P<0.05).(4) There was no expression of TERT mRNA or TERT protein in model or sham group rats. There were low lever expressions of TERT mRNA and TERT protein in the kidney of EPCs-N and pZ-EPCs-N rats at the time of4weeks after transplantation. While there were obvious expressions of TERT mRNA and TERT protein at4w,8w and12w(P<0.05).(5) The kidney expressions of TGF-β1 mRNA and protein were higher in model group than in sham group (P<0.05), with the tendency of elevation with the time prolongs. The expressions were decreased in EPCs-N, pZ-EPCs-N and pZ-TERT-EPCs-N groups than that in model group, especially for pZ-TERT-EPCs-N groups (P<0.05).(6) Compared with sham rats, the model rats displayed the progressive higher expressions of vimentin and a-SMA, and lower expression of cytokeratin. While in the EPCs-N, and pZ-EPCs-N group, these changes were decreased than those in model group, and the pZ-TERT-EPCs-N rats obtained more obvious effects (P<0.05).(7) The expressions of VEGF mRNA and protein in model group were obvious lower than that in sham rats, while they were higher in EPCs-N, pZ-EPCs-N and pZ-TERT-EPCs-N groups compared with model group.(8) The positive correlations were found between the peritubular capillaries counts and the creatinine clearance, the expressions of cytokeratin and VEGF. The negative correlations were found between the peritubular capillaries counts and the urinary protein, serum creatinine lever, serum urea lever, and the expression of TGF-β1, vimentin and α-SMA.Conclusion:1. The recombinant plasmid pZsGreen1-C1-TERT was successfully completed. 2. Isolated and cultured endothelial progenitor cells successfully in vitro. Transfection of pZsGreenl-Cl-TERT could upregulate the expression of TERT and enhance proliferation ability of EPCs.3. Transplantation of bone marrow EPCs could repair the renal pathological damage and improve renal function of5/6nephrectomy rats through up-regulating VEGF expression, protecting renal interstitial microvascular injury, down-regulating TGF-β1and repressing EMT. Transplantation of EPCs transfected by TERT gene could repair the renal pathological damage and improve renal function of5/6nephrectomy rats more effectively.