TERT基因转染骨髓内皮祖细胞移植对5/6肾切除大鼠肾脏损伤的修复研究
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摘要
目的：构建携带端粒酶逆转录酶(telomerase reverse transcriptase, TERT)基因的真核表达载体pZsGreen1-C1-TERT重组质粒,观察转染TERT基因后对SD大鼠骨髓内皮祖细胞(EPCs)生物学效应的影响,研究EPCs和转染TERT基因的EPCs移植对5/6肾切除模型大鼠的肾脏修复作用及其可能机制。
方法：
1.构建携带TERT基因的真核表达载体pZsGreen1-C1-TERT质粒：采用RT-PCR方法从幼鼠肝脏总RNA中扩增出TERT片段,通过双酶切方法将其与pZsGreen1-C1载体连接,得至pZsGreen1-C1-TERT重组质粒。重组质粒经限制性内切酶BglI和EcoR Ⅰ酶切,进行凝胶电泳鉴定和DNA序列分析。
2.分离、培养EPCs:采用密度梯度离心法分离SD大鼠骨髓单个核细胞,EGM-2特异性培基诱导培养EPCs。通过细胞形态观察、细胞免疫荧光细胞表面标志鉴定、摄取DIL-ac-LDL和结合FITC-UEA-Ⅰ能力试验鉴定EPCs。
3.转染pZsGreen1-C1-TERT质粒至EPCs:采用脂质体Lipofectam-ineTM2000将重组质粒pZsGreen1-C1-TERT转染至EPCs(pZsGreen1-C1-TERT-EPCs),并pZsGreen1-C1转染至EPCs(pZsGreen1-C1-EPCs)设为空质粒对照组。MTT法检测细胞增殖活性,流式细胞术检测早期凋亡情况,Western blot和Real-Time PCR检测细胞内TERT mRNA和蛋白表达。
4.检测EPCs和转染TERT基因的EPCs移植对5/6肾切除模型大鼠的肾脏保护作用及其机制：雌性6周龄SD大鼠随机分为假手术组(Sham,24只)、5/6肾切除模型组(Model,24只)、EPCs移植组(EPCs-N,24只)、pZsGreen1-C1-EPCs移植组(pZ-EPCs-N,40只)、pZsGreen1-C1-TERT-EPCs移植组(pZ-TERT-EPCs-N,40只)。各组于5/6肾切除术后一周分别尾静脉注射PBS、PBS、EPCs、pZsGreen1-C1-EPCs和pZsGreen1-C1-TERT-EPCs细胞悬液1ml。于注射后3d、1w、2W、3W、4W、6W、8W、12w各处死2只pZ-EPCs-N组和pZ-TERT-EPCs-N组大鼠,取新鲜肾脏、肝脏、脾脏、心脏及血涂片,在倒置荧光显微镜下观察各组织绿色荧光蛋白Green1的表达。同时于注射后4W、8w、12w收集尿液和血液用于检测24h尿蛋白定量、血肌酐、尿素氮和内生肌酐清除率,收集肾组织进行肾脏组织病理分析,计算CD34阳性管周毛细血管密度,用Western blotting、Real-Time PCR方法检测TERT、TGF-β1、角蛋白、波形蛋白、α-SMA、VEGF蛋白和基因表达。
结果：
1.用RT-PCR方法从幼鼠肝脏总RNA中扩增出的基因为3399bp,与所需大小TERT cDNA一致。对重组质粒pZsGreen1-C1-TERT进行酶切、凝胶电泳和DNA序列分析,结果显示基因大小和基因序列与目的基因TERT一致。
2.骨髓单个核细胞经过EGM-2诱导后呈现内皮祖细胞形态,细胞表面同时表达CD133、vWF和VEGFR-2,能够同时摄取DIL-ac-LDL和结合FITC-UEA-Ⅰ。
3.重组质粒pZsGreen1-C1-TERT和空质粒pZsGreen1-C1转染内皮祖细胞在荧光显微镜下显示绿色荧光,转染率为70%。EPCs组和pZsGreen1-C1-EPCs组细胞在14d、21d和28d均有较低水平的TERT mRNA和蛋白表达,且28d时表达量较14d表达量均明显降低(P均<0.05)。培养第28d时,pZsGreen1-C1-TERT-EPCs组细胞增殖活性明显高于EPCs组和pZsGreen1-C1-EPCs组细胞(P均<0.05)、凋亡比例显著低于EPCs组和pZsGreen1-C1-EPCs组细胞(P均<0.05)。
4.(1)尾静脉注射后,表达Green1的移植细胞pZsGreen1-C1-EPCs和pZsGreen1-C1-TERT-EPCs逐渐定位于残肾中。(2)在各时间点,Model组大鼠体重增长和肌酐清除率均明显低于Sham组(P<0.05),24h尿蛋白定量、血清尿素氮、肌酐水平均高于Sham组(P<0.05);EPCs-N组、pZ-EPCs-N组和pZ-TERT-EPCs-N组在第8w和第12w血清尿素氮、肌酐水平较Model组降低(P<0.05),肌酐清除率较Model组升高(P<0.05),尤其以12w时pZ-TERT-EPCs-N组明显(P<0.05)。(3)与Sham组相比,Model组肾间质病理评分明显增高(P<0.05),肾间质管周毛细血管密度降低(P<0.05),且随时间延长病变逐步加重；细胞移植后三组大鼠肾脏病理损伤较Model组均降低(P<0.05),管周毛细血管密度有不同程度增高；12w时pZ-TERT-EPCs-N较EPCs-N组、pZ-EPCs-N组治疗效果更明显。(4)TERT mRNA和蛋白在Model组和Sham组肾组织中无表达,在EPCs-N组、pZ-EPCs-N组仅第4w有少量表达,在pZ-TERT-EPCs-N组4W、8w和12w均有表达,且均高于EPCs-N组、pZ-EPCs-N组第4w时表达量(P<0.05)。(5)TGF-β1mRNA和蛋白在Sham组仅低量表达,Model组4W、8W、12w表达逐渐增高(P<0.05)；各移植组表达量较Model组均有降低(P<0.05),pZ-TERT-EPCs-N组降低最明显(P<0.05)。(6)与Sham组相比,Model组大鼠开始进行性高表达波形蛋白和α-SMA,低表达角蛋白(P<0.05)；与Model组相比,EPCs-N组和pZ-EPCs-N组波形蛋白、α-SMA表达上调和角蛋白表达下调水平均有减弱(P<0.05),pZ-TERT-EPCs-N组在第12w时效果更显著。(7)VEGF表达在Model组表达量明显低于Sham组,EPCs-N组、pZ-EPCs-N和pZ-TERT-EPCs-N组VEGF基因和蛋白水平表达较Model组均增高(P<0.05)。(8)相关性分析显示,管周毛细血管密度与24h尿蛋白定量、血清尿素氮、肌酐均呈显著负相关,与肌酐清除率呈正相关,与肾间质病理评分显著负相关,与TGF-β1、波形蛋白和α-SMA的表达均呈显著负相关,与角蛋白和VEGF表达呈显著正相关。
结论：
1.成功构建了重组质粒pZsGreen1-C1-TERTo
2.成功将骨髓单个核细胞诱导分化为骨髓EPCs,并导入TERT基因重组质粒,构建了具有高表达TERT基因、高增殖活性的pZsGreen1-C1-TERT-EPCs。
3.尾静脉注射移植骨髓EPCs能够显著修复5/6肾切除大鼠模型的肾脏病理损伤,改善肾功能,其机制可能与上调VEGF表达和保护肾间质微血管损伤、以及下调TGF-β1表达和阻遏肾小管上皮-间充质细胞转化(EMT)的发生有关。TERT基因重组的EPCs能够显著提升EPCs的肾脏损伤修复作用。
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.
方法：
1.构建携带TERT基因的真核表达载体pZsGreen1-C1-TERT质粒：采用RT-PCR方法从幼鼠肝脏总RNA中扩增出TERT片段,通过双酶切方法将其与pZsGreen1-C1载体连接,得至pZsGreen1-C1-TERT重组质粒。重组质粒经限制性内切酶BglI和EcoR Ⅰ酶切,进行凝胶电泳鉴定和DNA序列分析。
2.分离、培养EPCs:采用密度梯度离心法分离SD大鼠骨髓单个核细胞,EGM-2特异性培基诱导培养EPCs。通过细胞形态观察、细胞免疫荧光细胞表面标志鉴定、摄取DIL-ac-LDL和结合FITC-UEA-Ⅰ能力试验鉴定EPCs。
3.转染pZsGreen1-C1-TERT质粒至EPCs:采用脂质体Lipofectam-ineTM2000将重组质粒pZsGreen1-C1-TERT转染至EPCs(pZsGreen1-C1-TERT-EPCs),并pZsGreen1-C1转染至EPCs(pZsGreen1-C1-EPCs)设为空质粒对照组。MTT法检测细胞增殖活性,流式细胞术检测早期凋亡情况,Western blot和Real-Time PCR检测细胞内TERT mRNA和蛋白表达。
4.检测EPCs和转染TERT基因的EPCs移植对5/6肾切除模型大鼠的肾脏保护作用及其机制：雌性6周龄SD大鼠随机分为假手术组(Sham,24只)、5/6肾切除模型组(Model,24只)、EPCs移植组(EPCs-N,24只)、pZsGreen1-C1-EPCs移植组(pZ-EPCs-N,40只)、pZsGreen1-C1-TERT-EPCs移植组(pZ-TERT-EPCs-N,40只)。各组于5/6肾切除术后一周分别尾静脉注射PBS、PBS、EPCs、pZsGreen1-C1-EPCs和pZsGreen1-C1-TERT-EPCs细胞悬液1ml。于注射后3d、1w、2W、3W、4W、6W、8W、12w各处死2只pZ-EPCs-N组和pZ-TERT-EPCs-N组大鼠,取新鲜肾脏、肝脏、脾脏、心脏及血涂片,在倒置荧光显微镜下观察各组织绿色荧光蛋白Green1的表达。同时于注射后4W、8w、12w收集尿液和血液用于检测24h尿蛋白定量、血肌酐、尿素氮和内生肌酐清除率,收集肾组织进行肾脏组织病理分析,计算CD34阳性管周毛细血管密度,用Western blotting、Real-Time PCR方法检测TERT、TGF-β1、角蛋白、波形蛋白、α-SMA、VEGF蛋白和基因表达。
结果：
1.用RT-PCR方法从幼鼠肝脏总RNA中扩增出的基因为3399bp,与所需大小TERT cDNA一致。对重组质粒pZsGreen1-C1-TERT进行酶切、凝胶电泳和DNA序列分析,结果显示基因大小和基因序列与目的基因TERT一致。
2.骨髓单个核细胞经过EGM-2诱导后呈现内皮祖细胞形态,细胞表面同时表达CD133、vWF和VEGFR-2,能够同时摄取DIL-ac-LDL和结合FITC-UEA-Ⅰ。
3.重组质粒pZsGreen1-C1-TERT和空质粒pZsGreen1-C1转染内皮祖细胞在荧光显微镜下显示绿色荧光,转染率为70%。EPCs组和pZsGreen1-C1-EPCs组细胞在14d、21d和28d均有较低水平的TERT mRNA和蛋白表达,且28d时表达量较14d表达量均明显降低(P均<0.05)。培养第28d时,pZsGreen1-C1-TERT-EPCs组细胞增殖活性明显高于EPCs组和pZsGreen1-C1-EPCs组细胞(P均<0.05)、凋亡比例显著低于EPCs组和pZsGreen1-C1-EPCs组细胞(P均<0.05)。
4.(1)尾静脉注射后,表达Green1的移植细胞pZsGreen1-C1-EPCs和pZsGreen1-C1-TERT-EPCs逐渐定位于残肾中。(2)在各时间点,Model组大鼠体重增长和肌酐清除率均明显低于Sham组(P<0.05),24h尿蛋白定量、血清尿素氮、肌酐水平均高于Sham组(P<0.05);EPCs-N组、pZ-EPCs-N组和pZ-TERT-EPCs-N组在第8w和第12w血清尿素氮、肌酐水平较Model组降低(P<0.05),肌酐清除率较Model组升高(P<0.05),尤其以12w时pZ-TERT-EPCs-N组明显(P<0.05)。(3)与Sham组相比,Model组肾间质病理评分明显增高(P<0.05),肾间质管周毛细血管密度降低(P<0.05),且随时间延长病变逐步加重；细胞移植后三组大鼠肾脏病理损伤较Model组均降低(P<0.05),管周毛细血管密度有不同程度增高；12w时pZ-TERT-EPCs-N较EPCs-N组、pZ-EPCs-N组治疗效果更明显。(4)TERT mRNA和蛋白在Model组和Sham组肾组织中无表达,在EPCs-N组、pZ-EPCs-N组仅第4w有少量表达,在pZ-TERT-EPCs-N组4W、8w和12w均有表达,且均高于EPCs-N组、pZ-EPCs-N组第4w时表达量(P<0.05)。(5)TGF-β1mRNA和蛋白在Sham组仅低量表达,Model组4W、8W、12w表达逐渐增高(P<0.05)；各移植组表达量较Model组均有降低(P<0.05),pZ-TERT-EPCs-N组降低最明显(P<0.05)。(6)与Sham组相比,Model组大鼠开始进行性高表达波形蛋白和α-SMA,低表达角蛋白(P<0.05)；与Model组相比,EPCs-N组和pZ-EPCs-N组波形蛋白、α-SMA表达上调和角蛋白表达下调水平均有减弱(P<0.05),pZ-TERT-EPCs-N组在第12w时效果更显著。(7)VEGF表达在Model组表达量明显低于Sham组,EPCs-N组、pZ-EPCs-N和pZ-TERT-EPCs-N组VEGF基因和蛋白水平表达较Model组均增高(P<0.05)。(8)相关性分析显示,管周毛细血管密度与24h尿蛋白定量、血清尿素氮、肌酐均呈显著负相关,与肌酐清除率呈正相关,与肾间质病理评分显著负相关,与TGF-β1、波形蛋白和α-SMA的表达均呈显著负相关,与角蛋白和VEGF表达呈显著正相关。
结论：
1.成功构建了重组质粒pZsGreen1-C1-TERTo
2.成功将骨髓单个核细胞诱导分化为骨髓EPCs,并导入TERT基因重组质粒,构建了具有高表达TERT基因、高增殖活性的pZsGreen1-C1-TERT-EPCs。
3.尾静脉注射移植骨髓EPCs能够显著修复5/6肾切除大鼠模型的肾脏病理损伤,改善肾功能,其机制可能与上调VEGF表达和保护肾间质微血管损伤、以及下调TGF-β1表达和阻遏肾小管上皮-间充质细胞转化(EMT)的发生有关。TERT基因重组的EPCs能够显著提升EPCs的肾脏损伤修复作用。
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.
引文
[1]Nangaku M. Chronic hypoxia and tubulointerstitial injury:a final common pathway to end-stage renal failure[J]. J Am Soc Nephrol,2006,17(1):17-25.
[2]Ishii Y, Sawada T, Kubota K, et al. Loss of peritubular capillaries in the development of chronic allograft nephropathy[J]. Transplant Proc,2005,37(2):981-983.
[3]Muller G A, Strutz F M. Renal fibroblast heterogeneity[J]. Kidney Int Suppl,1995,50:S33-S36.
[4]Mucsi I, Rosivall L. Epithelial-mesenchymal transition in renal tubular cells in the pathogenesis of progressive tubulo-interstitial fibrosis[J]. Acta Physiol Hung,2007,94(1-2):117-131.
[5]Sun D, Feng J, Dai C, et al. Role of peritubular capillary loss and hypoxia in progressive tubulointerstitial fibrosis in a rat model of aristolochic acid nephropathy[J]. Am J Nephrol,2006,26(4):363-371.
[6]Kang D H, Hughes J, Mazzali M, et al. Impaired angiogenesis in the remnant kidney model:Ⅱ. Vascular endothelial growth factor administration reduces renal fibrosis and stabilizes renal function[J]. J Am Soc Nephrol,2001,12(7):1448-1457.
[7]Kang D H, Johnson R J. Vascular endothelial growth factor:a new player in the pathogenesis of renal fibrosis[J]. Curr Opin Nephrol Hypertens,2003,12(1):43-49.
[8]Kang D H, Kanellis J, Hugo C, et al. Role of the microvascular endothelium in progressive renal disease[J]. J Am Soc Nephrol,2002,13(3):806-816.
[9]Gluhovschi C, Gluhovschi G, Potencz E, et al. The endothelial cell markers von Willebrand Factor (vWF), CD31 and CD34 are lost in glomerulonephritis and no longer correlate with the morphological indices of glomerular sclerosis, interstitial fibrosis, activity and chronicity[J]. Folia Histochem Cytobiol,2010,48(2):230-236.
[10]Rautou P E, Vion A C, Amabile N, et al. Microparticles, vascular function, and atherothrombosis[J]. Circ Res,2011,109(5):593-606.
[11]Fliser D. Perspectives in renal disease progression:the endothelium as a treatment target in chronic kidney disease[J]. J Nephrol,2010,23(4):369-376.
[12]Schatteman G C, Dunnwald M, Jiao C. Biology of bone marrow-derived endothelial cell precursors[J]. Am J Physiol Heart Circ Physiol,2007,292(1):H1-H18.
[13]Asahara T, Murohara T, Sullivan A, et al. Isolation of putative progenitor endothelial cells for angiogenesis[J]. Science,1997,275(5302):964-967.
[14]Ziegelhoeffer T, Fernandez B, Kostin S, et al. Bone marrow-derived cells do not incorporate into the adult growing vasculature[J]. Circ Res,2004,94(2):230-238.
[15]Rafii S, Lyden D. Therapeutic stem and progenitor cell transplantation for organ vascularization and regeneration[J]. Nat Med,2003,9(6):702-712.
[16]Hur J, Yoon C H, Kim H S, et al. Characterization of two types of endothelial progenitor cells and their different contributions to neovasculogenesis[J]. Arterioscler Thromb Vasc Biol,2004,24(2):288-293.
[17]Kamihata H, Matsubara H, Nishiue T, et al. Implantation of bone marrow mononuclear cells into ischemic myocardium enhances collateral perfusion and regional function via side supply of angioblasts, angiogenic ligands, and cytokines[J]. Circulation,2001,104(9):1046-1052.
[18]Kinnaird T, Stabile E, Burnett M S, et al. Marrow-derived stromal cells express genes encoding a broad spectrum of arteriogenic cytokines and promote in vitro and in vivo arteriogenesis through paracrine mechanisms[J]. Circ Res,2004,94(5):678-685.
[19]Rehman J, Li J, Orschell C M, et al. Peripheral blood "endothelial progenitor cells" are derived from monocyte/macrophages and secrete angiogenic growth factors[J]. Circulation,2003,107(8):1164-1169.
[20]Chade A R, Zhu X, Lavi R, et al. Endothelial progenitor cells restore renal function in chronic experimental renovascular disease[J]. Circulation,2009,119(4):547-557.
[21]Reyes M, Dudek A, Jahagirdar B, et al. Origin of endothelial progenitors in human postnatal bone marrow[J]. J Clin Invest,2002,109(3):337-346.
[22]Takahashi T, Kalka C, Masuda H, et al. Ischemia- and cytokine-induced mobilization of bone marrow-derived endothelial progenitor cells for neovascularization[J]. Nat Med,1999,5(4):434-438.
[23]Murohara T, Ikeda H, Duan J, et al. Transplanted cord blood-derived endothelial precursor cells augment postnatal neovascularization[J]. J Clin Invest,2000,105(11):1527-1536.
[24]Yang C, Zhang Z H, Li Z J, et al. Enhancement of neovascularization with cord blood CD133+ cell-derived endothelial progenitor cell transplantation[J]. Thromb Haemost,2004,91(6):1202-1212.
[25]Peichev M, Naiyer A J, Pereira D, et al. Expression of VEGFR-2 and AC133 by circulating human CD34(+) cells identifies a population of functional endothelial precursors[J]. Blood,2000,95(3):952-958.
[26]Hu Y, Zhang Z, Torsney E, et al. Abundant progenitor cells in the adventitia contribute to atherosclerosis of vein grafts in ApoE-deficient mice[J]. J Clin Invest,2004,113(9):1258-1265.
[27]Sainz J, A1 H Z A, Caligiuri G, et al. Isolation of "side population" progenitor cells from healthy arteries of adult mice[J]. Arterioscler Thromb Vasc Biol,2006,26(2):281-286.
[28]Planat-Benard V, Silvestre J S, Cousin B, et al. Plasticity of human adipose lineage cells toward endothelial cells:physiological and therapeutic perspectives[J]. Circulation,2004,109(5):656-663.
[29]Shi Q, Rafii S, Wu M H, et al. Evidence for circulating bone marrow-derived endothelial cells [J]. Blood,1998,92(2):362-367.
[30]Martinez-Estrada O M, Munoz-Santos Y, Julve J, et al. Human adipose tissue as a source of Flk-1+cells:new method of differentiation and expansion[J]. Cardiovasc Res,2005,65(2):328-333.
[31]Sharpe E R, Teleron A A, Li B, et al. The origin and in vivo significance of murine and human culture-expanded endothelial progenitor cells[J]. Am J Pathol,2006,168(5):1710-1721.
[32]Flamme I, Risau W. Induction of vasculogenesis and hematopoiesis in vitro[J]. Development,1992,116(2):435-439.
[33]Weiss M J, Orkin S H. In vitro differentiation of murine embryonic stem cells. New approaches to old problems[J]. J Clin Invest,1996,97(3):591-595.
[34]Isner J M, Kalka C, Kawamoto A, et al. Bone marrow as a source of endothelial cells for natural and iatrogenic vascular repair[J]. Ann N Y Acad Sci,2001,953:75-84.
[35]Wright W E, Shay J W. Telomere biology in aging and cancer[J]. J Am Geriatr Soc,2005,53(9 Suppl):S292-S294.
[36]Hayflick L, Moorhead P S. The serial cultivation of human diploid cell strains[J]. Exp Cell Res,1961,25:585-621.
[37]Choi S W, Yang J E, Kang S J, et al. Herpetic infection on the vulva associated with eccrine squamous syringometaplasia in malignant lymphoma[J]. Acta Derm Venereol,1999,79(6):500-501.
[38]Bodnar A G, Ouellette M, Frolkis M, et al. Extension of life-span by introduction of telomerase into normal human cells[J]. Science,1998,279(5349):349-352.
[39]Hayflick L, Moorhead P S. The serial cultivation of human diploid cell strains[J]. Exp Cell Res,1961,25:585-621.
[40]Hornsby P J. Senescence and life span[J]. Pflugers Arch,2010,459(2):291-299.
[41]Chiu C P, Dragowska W, Kim N W, et al. Differential expression of telomerase activity in hematopoietic progenitors from adult human bone marrow[J]. Stem Cells,1996,14(2):239-248.
[42]Gancarcikova M, Zemanova Z, Brezinova J, et al. The role of telomeres and telomerase complex in haematological neoplasia:the length of telomeres as a marker of carcinogenesis and prognosis of disease[J]. Prague Med Rep,2010,111(2):91-105.
[43]Blasco M A. Telomere length, stem cells and aging[J]. Nat Chem Biol,2007,3(10):640-649.
[44]Yi X, Tesmer V M, Savre-Train I, et al. Both transcriptional and posttranscriptional mechanisms regulate human telomerase template RNA levels[J]. Mol Cell Biol,1999,19(6):3989-3997.
[45]Haber D A. Telomeres, cancer, and immortality[J]. N Engl J Med,1995,332(14):955-956.
[46]Anderson W F, Blaese R M, Culver K. The ADA human gene therapy clinical protocol:Points to Consider response with clinical protocol, July 6,1990[J]. Hum Gene Ther,1990,1(3):331-362.
[47]El-Aneed A. An overview of current delivery systems in cancer gene therapy[J]. J Control Release,2004,94(1):1-14.
[48]Cerone M A, Ward R J, Londono-Vallejo J A, et al. Telomerase RNA mutated in autosomal dyskeratosis congenita reconstitutes a weakly active telomerase enzyme defective in telomere elongation[J]. Cell Cycle,2005,4(4):585-589.
[49]Blackburn E H. Structure and function of telomeres[J]. Nature,1991,350(6319):569-573.
[50]Shore D. Telomerase and telomere-binding proteins:controlling the endgame[J]. Trends Biochem Sci,1997,22(7):233-235.
[51]Harley C B, Futcher A B, Greider C W. Telomeres shorten during ageing of human fibroblasts[J]. Nature,1990,345(6274):458-460.
[52]Greider C W, Blackburn E H. The telomere terminal transferase of Tetrahymena is a ribonucleoprotein enzyme with two kinds of primer specificity[J]. Cell,1987,51(6):887-898.
[53]Morin G B. The human telomere terminal transferase enzyme is a ribonucleoprotein that synthesizes TTAGGG repeats[J]. Cell,1989,59(3):521-529.
[54]Nakamura T, Bratton D L, Murphy R C. Analysis of epoxyeicosatrienoic and monohydroxyeicosatetraenoic acids esterified to phospholipids in human red blood cells by electrospray tandem mass spectrometry[J]. J Mass Spectrom,1997,32(8):888-896.
[55]Pearce V P, Sherrell J, Lou Z, et al. Immortalization of epithelial progenitor cells mediated by resveratrol[J]. Oncogene,2008,27(17):2365-2374.
[56]Boker W, Yin Z, Drosse I, et al. Introducing a single-cell-derived human mesenchymal stem cell line expressing hTERT after lentiviral gene transfer[J]. J Cell Mol Med,2008,12(4):1347-1359.
[57]Nakamura T M, Morin G B, Chapman K B, et al. Telomerase catalytic subunit homologs from fission yeast and human[J]. Science,1997,277(5328):955-959.
[58]Vandendriessche T, Vanslembrouck V, Goovaerts I, et al. Long-term expression of human coagulation factor Ⅷ and correction of hemophilia A after in vivo retroviral gene transfer in factor Ⅷ-deficient mice[J]. Proc Natl Acad Sci U S A,1999,96(18):10379-10384.
[59]Matz M V, Fradkov A F, Labas Y A, et al. Fluorescent proteins from nonbioluminescent Anthozoa species[J]. Nat Biotechnol,1999,17(10):969-973.
[60]Haas J A, Knox F G. Effect of meclofenamate or ketoconazole on the natriuretic response to increased pressure[J]. J Lab Clin Med,1996,128(2):202-207
[1]Schatteman G C, Dunnwald M, Jiao C. Biology of bone marrow-derived endothelial cell precursors[J]. Am J Physiol Heart Circ Physiol,2007,292(1):H1-H18.
[2]Asahara T, Murohara T, Sullivan A, et al. Isolation of putative progenitor endothelial cells for angiogenesis[J]. Science,1997,275(5302):964-967.
[3]Ziegelhoeffer T, Fernandez B, Kostin S, et al. Bone marrow-derived cells do not incorporate into the adult growing vasculature[J]. Circ Res,2004,94(2):230-238.
[4]Rafii S, Lyden D. Therapeutic stem and progenitor cell transplantation for organ vascularization and regeneration[J]. Nat Med,2003,9(6):702-712.
[5]Hur J, Yoon C H, Kim H S, et al. Characterization of two types of endothelial progenitor cells and their different contributions to neovasculogenesis[J]. Arterioscler Thromb Vasc Biol,2004,24(2):288-293.
[6]Kamihata H, Matsubara H, Nishiue T, et al. Implantation of bone marrow mononuclear cells into ischemic myocardium enhances collateral perfusion and regional function via side supply of angioblasts, angiogenic ligands, and cytokines[J]. Circulation,2001,104(9):1046-1052.
[7]Kinnaird T, Stabile E, Burnett M S, et al. Marrow-derived stromal cells express genes encoding a broad spectrum of arteriogenic cytokines and promote in vitro and in vivo arteriogenesis through paracrine mechanisms[J]. Circ Res,2004,94(5):678-685.
[8]Rehman J, Li J, Orschell C M, et al. Peripheral blood "endothelial progenitor cells" are derived from monocyte/macrophages and secrete angiogenic growth factors[J]. Circulation,2003,107(8):1164-1169.
[9]Murasawa S, Llevadot J, Silver M, et al. Constitutive human telomerase reverse transcriptase expression enhances regenerative properties of endothelial progenitor cells[J]. Circulation,2002,106(9):1133-1139.
[10]Takano H, Murasawa S, Asahara T. Functional and gene expression analysis of hTERT overexpressed endothelial cells[J]. Biologics,2008,2(3):547-554.
[11]Livak K J, Schmittgen T D. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method[J]. Methods,2001,25(4):402-408.
[12]Risau W, Flamme I. Vasculogenesis[J]. Annu Rev Cell Dev Biol,1995,11:73-91.
[13]Asahara T, Murohara T, Sullivan A, et al. Isolation of putative progenitor endothelial cells for angiogenesis[J]. Science,1997,275(5302):964-967.
[14]Reyes M, Dudek A, Jahagirdar B, et al. Origin of endothelial progenitors in human postnatal bone marrow[J]. J Clin Invest,2002,109(3):337-346.
[15]Takahashi T, Kalka C, Masuda H, et al. Ischemia-and cytokine-induced mobilization of bone marrow-derived endothelial progenitor cells for neovascularization[J]. Nat Med,1999,5(4):434-438.
[16]Murohara T, Ikeda H, Duan J, et al. Transplanted cord blood-derived endothelial precursor cells augment postnatal neovascularization[J]. J Clin Invest,2000,105(11):1527-1536.
[17]Yang C, Zhang Z H, Li Z J, et al. Enhancement of neovascularization with cord blood CD 133+ cell-derived endothelial progenitor cell transplantation[J]. Thromb Haemost,2004,91(6):1202-1212.
[18]Peichev M, Naiyer A J, Pereira D, et al. Expression of VEGFR-2 and AC 133 by circulating human CD34(+) cells identifies a population of functional endothelial precursors [J]. Blood,2000,95(3):952-958.
[19]Hu Y, Zhang Z, Torsney E, et al. Abundant progenitor cells in the adventitia contribute to atherosclerosis of vein grafts in ApoE-deficient mice[J]. J Clin Invest,2004,113(9):1258-1265.
[20]Sainz J, A1 H Z A, Caligiuri G, et al. Isolation of "side population" progenitor cells from healthy arteries of adult mice[J]. Arterioscler Thromb Vasc Biol,2006,26(2):281-286.
[21]Planat-Benard V, Silvestre J S, Cousin B, et al. Plasticity of human adipose lineage cells toward endothelial cells:physiological and therapeutic perspectives [J]. Circulation,2004,109(5):656-663.
[22]Shi Q, Rafii S, Wu M H, et al. Evidence for circulating bone marrow-derived endothelial cells[J]. Blood,1998,92(2):362-367.
[23]Martinez-Estrada O M, Munoz-Santos Y, Julve J, et al. Human adipose tissue as a source of Flk-1+cells:new method of differentiation and expansion[J]. Cardiovasc Res,2005,65(2):328-333.
[24]Mancinelli F, Tamburini A, Spagnoli A, et al. Optimizing umbilical cord blood collection:impact of obstetric factors versus quality of cord blood units[J]. Transplant Proc,2006,38(4):1174-1176.
[25]Murohara T, Ikeda H, Duan J, et al. Transplanted cord blood-derived endothelial precursor cells augment postnatal neovascularization[J]. J Clin Invest,2000,105(11):1527-1536.
[26]Peichev M, Naiyer A J, Pereira D, et al. Expression of VEGFR-2 and AC 133 by circulating human CD34(+) cells identifies a population of functional endothelial precursors[J]. Blood,2000,95(3):952-958.
[27]Jeon O, Hwang K C, Yoo K J, et al. Combined sustained delivery of basic fibroblast growth factor and administration of granulocyte colony-stimulating factor:synergistic effect on angiogenesis in mouse ischemic limbs[J]. J Endovasc Ther,2006,13(2):175-181.
[28]Sharpe E R, Teleron A A, Li B, et al. The origin and in vivo significance of murine and human culture-expanded endothelial progenitor cells[J]. Am J Pathol,2006,168(5):1710-1721.
[29]Flamme I, Risau W. Induction of vasculogenesis and hematopoiesis in vitro[J]. Development,1992,116(2):435-439.
[30]Weiss M J, Orkin S H. In vitro differentiation of murine embryonic stem cells. New approaches to old problems[J]. J Clin Invest,1996,97(3):591-595.
[31]Isner J M, Kalka C, Kawamoto A, et al. Bone marrow as a source of endothelial cells for natural and iatrogenic vascular repair[J]. Ann N Y Acad Sci,2001,953:75-84.
[32]Reyes M, Dudek A, Jahagirdar B, et al. Origin of endothelial progenitors in human postnatal bone marrow[J]. J Clin Invest,2002,109(3):337-346.
[33]Asahara T, Murohara T, Sullivan A, et al. Isolation of putative progenitor endothelial cells for angiogenesis[J]. Science,1997,275(5302):964-967.
[34]Asahara T. [Endothelial progenitor cells for vascular medicine][J]. Yakugaku Zasshi,2007,127(5):841-845.
[35]何小解,许自川,郑洪,等.大鼠骨髓内皮祖细胞分离与体外培养条件研究[J].世界科技研 究与发展,2008(2)：198-201.
[36]Teleron A A, Carlson B, Young P P. Blood donor white blood cell reduction filters as a source of human peripheral blood-derived endothelial progenitor cells[J]. Transfusion,2005,45(1):21-25.
[37]肖刚峰,张怀勤,季亢挺,等.单个核细胞的接种密度对贴壁筛选法培养内皮祖细胞增殖的影响[J].细胞与分子免疫学杂志,2006(2)：233-234.
[38]Shintani S, Murohara T, Ikeda H, et al. Augmentation of postnatal neovascularization with autologous bone marrow transplantation[J].Circulation,2001,103(6):897-903.
[39]Urbich C, Dimmeler S. Endothelial progenitor cells:characterization and role in vascular biology[J]. Circ Res,2004,95(4):343-353.
[40]Vasa M, Fichtlscherer S, Aicher A, et al. Number and migratory activity of circulating endothelial progenitor cells inversely correlate with risk factors for coronary artery disease[J]. Circ Res,2001,89(1):E1-E7.
[41]Kalka C, Masuda H, Takahashi T, et al. Vascular endothelial growth factor(165) gene transfer augments circulating endothelial progenitor cells in human subjects[J]. Circ Res,2000,86(12):1198-1202.
[42]Wagner D D, Olmsted J B, Marder V J. Immunolocalization of von Willebrand protein in Weibel-Palade bodies of human endothelial cells[J]. J Cell Biol,1982,95(1):355-360.
[43]Ewenstein B M, Warhol M J, Handin R I, et al. Composition of the von Willebrand factor storage organelle (Weibel-Palade body) isolated from cultured human umbilical vein endothelial cells[J]. J Cell Biol,1987,104(5):1423-1433.
[44]Wright W E, Shay J W. Telomere biology in aging and cancer[J]. J Am Geriatr Soc,2005,53(9 Suppl):S292-S294.
[45]Hayflick L, Moorhead P S. The serial cultivation of human diploid cell strains[J]. Exp Cell Res,1961,25:585-621.
[46]Brenner A J, Stampfer M R, Aldaz C M. Increased p16 expression with first senescence arrest in human mammary epithelial cells and extended growth capacity with p16 inactivation[J]. Oncogene,1998,17(2):199-205.
[47]Shay J W, Wright W E. Use of telomerase to create bioengineered tissues[J]. Ann N Y Acad Sci,2005,1057:479-491.
[48]Chang M W, Grillari J, Mayrhofer C, et al. Comparison of early passage, senescent and hTERT immortalized endothelial cells[J]. Exp Cell Res,2005,309(1):121-136.
[49]Bodnar A G, Ouellette M, Frolkis M, et al. Extension of life-span by introduction of telomerase into normal human cells[J]. Science,1998,279(5349):349-352.
[50]Durand G, Poeggeler B, Boker J, et al. Fine-tuning the amphiphilicity:a crucial parameter in the design of potent alpha-phenyl-N-tert-butylnitrone analogues[J]. J Med Chem,2007,50(17):3976-3979.
[51]Kiyono T, Foster S A, Koop J I, et al. Both Rb/p16INK4a inactivation and telomerase activity are required to immortalize human epithelial cells[J]. Nature,1998,396(6706):84-88.
[52]Nisato R E, Harrison J A, Buser R, et al. Generation and characterization of telomerase-transfected human lymphatic endothelial cells with an extended life span[J]. Am J Pathol,2004,165(1):11-24.
[53]Freedman D A, Folkman J. Maintenance of G1 checkpoint controls in telomerase-immortalized endothelial cells[J]. Cell Cycle,2004,3(6):811-816
[1]Nangaku M. Chronic hypoxia and tubulointerstitial injury:a final common pathway to end-stage renal failure[J]. J Am Soc Nephrol,2006,17(1):17-25.
[2]Becker G J, Hewitson T D. The role of tubulointerstitial injury in chronic renal failure[J]. Curr Opin Nephrol Hypertens,2000,9(2):133-138.
[3]Ishii Y, Sawada T, Kubota K, et al. Loss of peritubular capillaries in the development of chronic allograft nephropathy[J]. Transplant Proc,2005,37(2):981-983.
[4]Sun D, Feng J, Dai C, et al. Role of peritubular capillary loss and hypoxia in progressive tubulointerstitial fibrosis in a rat model of aristolochic acid nephropathy[J]. Am J Nephrol,2006,26(4):363-371.
[5]Kang D H, Hughes J, Mazzali M, et al. Impaired angiogenesis in the remnant kidney model:Ⅱ. Vascular endothelial growth factor administration reduces renal fibrosis and stabilizes renal function[J]. J Am Soc Nephrol,2001,12(7):1448-1457.
[6]Asahara T, Murohara T, Sullivan A, et al. Isolation of putative progenitor endothelial cells for angiogenesis[J]. Science,1997,275(5302):964-967.
[7]Huang W J, Xiao F Y, Zhang H Q, et al. [Effects of endothelial progenitor cells and endothelial outgrowth cells in repair of injured carotid vessel:a comparative study with rabbits][J]. Zhonghua Yi Xue Za Zhi,2007,87(44):3143-3147.
[8]Kawamoto A, Tkebuchava T, Yamaguchi J, et al. Intramyocardial transplantation of autologous endothelial progenitor cells for therapeutic neovascularization of myocardial ischemia[J]. Circulation,2003,107(3):461-468.
[9]Magri D, Fancher T T, Fitzgerald T N, et al. Endothelial progenitor cells:a primer for vascular surgeons[J].Vascular,2007,15(6):384-394.
[10]Kalka C, Masuda H, Takahashi T, et al. Vascular endothelial growth factor(165) gene transfer augments circulating endothelial progenitor cells in human subjects[J]. Circ Res,2000,86(12):1198-1202.
[11]Maurer M H, Thomas C, Burgers H F, et al. Transplantation of adult neural progenitor cells transfected with vascular endothelial growth factor rescues grafted cells in the rat brain[J]. Int J Biol Sci,2008,4(1):1-7.
[12]Wright W E, Shay J W. Telomere biology in aging and cancer[J]. J Am Geriatr Soc,2005,53(9 Suppl):S292-S294.
[13]Durand G, Poeggeler B, Boker J, et al. Fine-tuning the amphiphilicity:a crucial parameter in the design of potent alpha-phenyl-N-tert-butylnitrone analogues[J]. J Med Chem,2007,50(17):3976-3979.
[14]Murasawa S, Llevadot J, Silver M, et al. Constitutive human telomerase reverse transcriptase expression enhances regenerative properties of endothelial progenitor cells[J]. Circulation,2002,106(9):1133-1139.
[15]Eddy A A. Molecular basis of renal fibrosis[J]. Pediatr Nephrol,2000,15(3-4):290-301.
[16]Muller G A, Strutz F M. Renal fibroblast heterogeneity [J]. Kidney Int Supp1,1995,50:S33-S36.
[17]Rhyu D Y, Yang Y, Ha H, et al. Role of reactive oxygen species in TGF-betal-induced mitogen-activated protein kinase activation and epithelial-mesenchymal transition in renal tubular epithelial cells[J]. J Am Soc Nephrol,2005,16(3):667-675.
[18]Copeland J W, Beaumont B W, Merrilees M J, et al. Epithelial-to-mesenchymal transition of human proximal tubular epithelial cells:effects of rapamycin, mycophenolate, cyclosporin, azathioprine, and methylprednisolone[J]. Transplantation,2007,83(6):809-814.
[19]Galichon P, Hertig A. Epithelial to mesenchymal transition as a biomarker in renal fibrosis:are we ready for the bedside?[J]. Fibrogenesis Tissue Repair,2011,4:11.
[20]杨念生,武庆庆,姜宗培,等.维甲酸抑制大鼠单侧输尿管梗阻模型肾间质纤维化[J].中华肾脏病杂志,2004(3).
[21]杨念生,武庆庆,黄越芳,等.血管紧张素Ⅱ对大鼠残肾模型微血管的影响[J].中华肾脏病杂志,2003(6).
[22]张宁,龚侃,果宏峰,等.散发性肾透明细胞癌组织中VHL基因突变、血管内皮生长因子表达及其与微血管形成的关系[J].中华医学杂志,2004(19)：40-44.
[23]Tanaka T, Matsumoto M, Inagi R, et al. Induction of protective genes by cobalt ameliorates tubulointerstitial injury in the progressive Thyl nephritis[J]. Kidney Int,2005,68(6):2714-2725.
[24]Ohashi R, Shimizu A, Masuda Y, et al. Peritubular capillary regression during the progression of experimental obstructive nephropathy[J]. J Am Soc Nephrol,2002,13(7):1795-1805.
[25]Kliem V, Johnson R J, Alpers C E, et al. Mechanisms involved in the pathogenesis of tubulointerstitial fibrosis in 5/6-nephrectomized rats[J]. Kidney Int,1996,49(3):666-678.
[26]Bohle A, Mackensen-Haen S, Wehrmann M. Significance of postglomerular capillaries in the pathogenesis of chronic renal failure[J]. Kidney Blood Press Res,1996,19(3-4):191-195.
[27]Namikoshi T, Satoh M, Horike H, et al. Implication of peritubular capillary loss and altered expression of vascular endothelial growth factor in IgA nephropathy[J]. Nephron Physiol,20065 102(1):p9-p16.
[28]Mironidou-Tzouveleki M, Tsartsalis S, Tomos C. Vascular endothelial growth factor (VEGF) in the pathogenesis of diabetic nephropathy of type 1 diabetes mellitus[J]. Curr Drug Targets,2011,12(1):107-114.
[29]Zeng X Q, Xu Z C, Dang X Q, et al. [Expression of vascular endothelial growth factor is related to microvessel injury of renal interstitium in children with Henoch Schonlein purpura nephritis][J]. Zhongguo Dang Dai Er Ke Za Zhi,2009,11(9):717-721.
[30]Malmstrom N K, Kallio E A, Rintala J M, et al. Vascular endothelial growth factor in chronic rat allograft nephropathy[J]. Transpl Immunol,2008,19(2):136-144.
[31]Modelli D A L, Viero R M, Carvalho M F. Role of peritubular capillaries and vascular endothelial growth factor in chronic allograft nephropathy[J]. Transplant Proc,2009,41(9):3720-3725.
[32]Kang D H, Johnson R J. Vascular endothelial growth factor:a new player in the pathogenesis of renal fibrosis[J]. Curr Opin Nephrol Hypertens,2003,12(1):43-49.
[33]Kang D H, Kanellis J, Hugo C, et al. Role of the microvascular endothelium in progressive renal disease[J]. J Am Soc Nephrol,2002,13(3):806-816.
[34]Ferrara N, Henzel W J. Pituitary follicular cells secrete a novel heparin-binding growth factor specific for vascular endothelial cells[J]. Biochem Biophys Res Commun,1989,161(2):851-858.
[35]Schrijvers B F, Flyvbjerg A, De Vriese A S. The role of vascular endothelial growth factor (VEGF) in renal pathophysiology[J]. Kidney Int,2004,65(6):2003-2017.
[36]Yang J, Liu Y. Dissection of key events in tubular epithelial to myofibroblast transition and its implications in renal interstitial fibrosis[J]. Am J Pathol,2001,159(4):1465-1475.
[37]Forino M, Torregrossa R, Ceol M, et al. TGFbetal induces epithelial-mesenchymal transition, but not myofibroblast transdifferentiation of human kidney tubular epithelial cells in primary culture[J]. Int J Exp Pathol,2006,87(3):197-208.
[38]Koesters R, Kaissling B, Lehir M, et al. Tubular overexpression of transforming growth factor-betal induces autophagy and fibrosis but not mesenchymal transition of renal epithelial cells[J]. Am J Pathol,2010,177(2):632-643.
[39]Wang J Y, Bao H Y, Huang S M, et al. [Mechanisms of decorin inhibiting epithelial-to-mesenchymal transition induced by transforming growth factor betal in renal tubular epithelial cells][J]. Zhonghua Er Ke Za Zhi,2010,48(1):50-54.
[40]Minardi D, Lucarini G, Filosa A, et al. Prognostic role of tumor necrosis, microvessel density, vascular endothelial growth factor and hypoxia inducible factor-1alpha in patients with clear cell renal carcinoma after radical nephrectomy in a long term follow-up[J]. Int J Immunopathol Pharmacol,2008,21(2):447-455.
[41]Metsuyanim S, Levy R, Davidovits M, et al. Molecular evaluation of circulating endothelial progenitor cells in children undergoing hemodialysis and after kidney transplantation[J]. Pediatr Res,2009,65(2):221-225.
[42]Eizawa T, Murakami Y, Matsui K, et al. Circulating endothelial progenitor cells are reduced in hemodialysis patients[J]. Curr Med Res Opin,2003,19(7):627-633.
[43]Choi J H, Kim K L, Huh W, et al. Decreased number and impaired angiogenic function of endothelial progenitor cells in patients with chronic renal failure[J]. Arterioscler Thromb Vasc Biol,2004,24(7):1246-1252.
[44]de Groot K, Bahlmann F H, Bahlmann E, et al. Kidney graft function determines endothelial progenitor cell number in renal transplant recipients[J]. Transplantation,2005,79(8):941-945.
[45]Metsuyanim S, Levy R, Davidovits M, et al. Molecular evaluation of circulating endothelial progenitor cells in children undergoing hemodialysis and after kidney transplantation[J]. Pediatr Res,2009,65(2):221-225.
[46]Zeng L, Xiao Q, Margariti A, et al. HDAC3 is crucial in shear- and VEGF-induced stem cell differentiation toward endothelial cells[J]. J Cell Biol,2006,174(7):1059-1069.
[47]Hess D C, Hill W D, Martin-Studdard A, et al. Bone marrow as a source of endothelial cells and NeuN-expressing cells After stroke[J]. Stroke,2002,33(5):1362-1368.
[48]Wu X, Rabkin-Aikawa E, Guleserian K J, et al. Tissue-engineered microvessels on three-dimensional biodegradable scaffolds using human endothelial progenitor cells[J]. Am J Physiol Heart Circ Physiol,2004,287(2):H480-H487.
[49]Chade A R, Zhu X, Lavi R, et al. Endothelial progenitor cells restore renal function in chronic experimental renovascular disease[J]. Circulation,2009,119(4):547-557.
[50]Brenner A J, Stampfer M R, Aldaz C M. Increased p16 expression with first senescence arrest in human mammary epithelial cells and extended growth capacity with p16 inactivation[J]. Oncogene,1998,17(2):199-205.
[51]Chang M W, Grillari J, Mayrhofer C, et al. Comparison of early passage, senescent and hTERT immortalized endothelial cells[J]. Exp Cell Res,2005,309(1):121-136.
[52]Bodnar A G, Ouellette M, Frolkis M, et al. Extension of life-span by introduction of telomerase into normal human cells[J]. Science,1998,279(5349):349-352.
[1]Weissman I L. Stem cells:units of development, units of regeneration, and units in evolution[J]. Cell,2000,100(1):157-168.
[2]Metcalf D. Hematopoietic stem cells:old and new[J]. Biomed Pharmacother,2001,55(2):75-78.
[3]Mcculloch E A, Till J E. Perspectives on the properties of stem cells[J]. Nat Med,2005,11 (10):1026-1028.
[4]Steenhard B M, Isom K S, Cazcarro P, et al. Integration of embryonic stem cells in metanephric kidney organ culture[J]. J Am Soc Nephrol,2005,16(6):1623-1631.
[5]Yamamoto M, Cui L, Johkura K, et al. Branching ducts similar to mesonephric ducts or ureteric buds in teratomas originating from mouse embryonic stem cells[J]. Am J Physiol Renal Physiol,2006,290(1):F52-F60.
[6]Kim D, Dressler G R. Nephrogenic factors promote differentiation of mouse embryonic stem cells into renal epithelia[J]. J Am Soc Nephrol,2005,16(12):3527-3534.
[7]Sagrinati C, Ronconi E, Lazzeri E, et al. Stem-cell approaches for kidney repair:choosing the right cells[J]. Trends Mol Med,2008,14(7):277-285.
[8]Perin L, Sedrakyan S, Da S S, et al. Characterization of human amniotic fluid stem cells and their pluripotential capability [J]. Methods Cell Biol,2008,86:85-99.
[9]Perin L, Giuliani S, Jin D, et al. Renal differentiation of amniotic fluid stem cells[J]. Cell Prolif,2007,40(6):936-948.
[10]Hauser P V, De Fazio R, Bruno S, et al. Stem cells derived from human amniotic fluid contribute to acute kidney injury recovery[J]. Am J Pathol,2010,177(4):2011-2021.
[11]Herrera M B, Bussolati B, Bruno S, et al. Mesenchymal stem cells contribute to the renal repair of acute tubular epithelial injury[J]. Int J Mol Med,2004,14(6):1035-1041.
[12]Steenhard B M, Isom K S, Cazcarro P, et al. Integration of embryonic stem cells in metanephric kidney organ culture[J]. J Am Soc Nephrol,2005,16(6):1623-1631.
[13]Dekel B, Burakova T, Ben-Hur H, et al. Engraftment of human kidney tissue in rat radiation chimera:Ⅱ. Human fetal kidneys display reduced immunogenicity to adoptively transferred human peripheral blood mononuclear cells and exhibit rapid growth and development[J]. Transplantation,1997,64(11):1550-1558.
[14]Starter M B, Fahrner K J, Barksdale E J, et al. Correlation of fetal kidney and testis congenic graft survival with reduced major histocompatibility complex burden[J]. Transplantation,1989,47(4):651-660.
[15]Rogers S A, Lowell J A, Hammerman N A, et al. Transplantation of developing metanephroi into adult rats[J]. Kidney Int,1998,54(1):27-37.
[16]Woolf A S, Hornbruch A, Fine L G. Integration of new embryonic nephrons into the kidney[J]. Am J Kidney Dis,1991,17(6):611-614.
[17]Rogers S A, Liapis H, Hammerman M R. Transplantation of metanephroi across the major histocompatibility complex in rats[J]. Am J Physiol Regul Integr Comp Physiol,2001,280(1):R132-R136.
[18]Rogers S A, Lowell J A, Hammerman N A, et al. Transplantation of developing metanephroi into adult rats[J]. Kidney Int,1998,54(l):27-37.
[19]Dekel B, Burakova T, Ben-Hur H, et al. Engraftment of human kidney tissue in rat radiation chimera:Ⅱ. Human fetal kidneys display reduced immunogenicity to adoptively transferred human peripheral blood mononuclear cells and exhibit rapid growth and development[J]. Transplantation,1997,64(11):1550-1558.
[20]Dekel B, Amariglio N, Kaminski N, et al. Engraftment and differentiation of human metanephroi into functional mature nephrons after transplantation into mice is accompanied by a profile of gene expression similar to normal human kidney development[J]. J Am Soc Nephrol,2002,13(4):977-990.
[21]Minguell J J, Erices A, Conget P. Mesenchymal stem cells[J]. Exp Biol Med (Maywood),2001,226(6):507-520.
[22]Ito T, Suzuki A, Imai E, et al. Bone marrow is a reservoir of repopulating mesangial cells during glomerular remodeling[J]. J Am Soc Nephrol,2001,12(12):2625-2635.
[23]Imasawa T, Utsunomiya Y, Kawamura T, et al. The potential of bone marrow-derived cells to differentiate to glomerular mesangial cells[J]. J Am Soc Nephrol,2001,12(7):1401-1409.
[24]Rasmusson I, Ringden O, Sundberg B, et al. Mesenchymal stem cells inhibit lymphocyte proliferation by mitogens and alloantigens by different mechanisms[J]. Exp Cell Res,2005,305(1):33-41.
[25]Le Blanc K. Immunomodulatory effects of fetal and adult mesenchymal stem cells[J]. Cytotherapy,2003,5(6):485-489.
[26]Djouad F, Plence P, Bony C, et al. Immunosuppressive effect of mesenchymal stem cells favors tumor growth in allogeneic animals[J]. Blood,2003,102(10):3837-3844.
[27]Mackay A M, Beck S C, Murphy J M, et al. Chondrogenic differentiation of cultured human mesenchymal stem cells from marrow[J]. Tissue Eng,1998,4(4):415-428.
[28]Poulsom R, Forbes S J, Hodivala-Dilke K, et al. Bone marrow contributes to renal parenchymal turnover and regeneration [J]. J Pathol,2001,195(2):229-235.
[29]Gupta S, Verfaillie C, Chmielewski D, et al. A role for extrarenal cells in the regeneration following acute renal failure[J]. Kidney Int,2002,62(4):1285-1290.
[30]Lin F, Cordes K, Li L, et al. Hematopoietic stem cells contribute to the regeneration of renal tubules after renal ischemia-reperfusion injury in mice[J]. J Am Soc Nephrol,2003,14(5):1188-1199.
[31]Duffield J S, Park K M, Hsiao L L, et al. Restoration of tubular epithelial cells during repair of the postischemic kidney occurs independently of bone marrow-derived stem cells[J]. J Clin Invest,2005,115(7):1743-1755.
[32]Lin F, Moran A, Igarashi P. Intrarenal cells, not bone marrow-derived cells, are the major source for regeneration in postischemic kidney[J]. J Clin Invest.2005,115(7):1756-1764.
[33]Szczypka M S, Westover A J, Clouthier S G, et al. Rare incorporation of bone marrow-derived cells into kidney after folic acid-induced injury[J]. Stem Cells,2005,23(1):44-54.
[34]Nishida M, Fujimoto S, Toiyama K, et al. Effect of hematopoietic cytokines on renal function in cisplatin-induced ARF in mice[J]. Biochem Biophys Res Commun,2004,324(1):341-347.
[35]Humphreys B D, Valerius M T, Kobayashi A, et al. Intrinsic epithelial cells repair the kidney after injury[J]. Cell Stem Cell,2008,2(3):284-291.
[36]Imasawa T, Utsunomiya Y, Kawamura T, et al. Evidence suggesting the involvement of hematopoietic stem cells in the pathogenesis of IgA nephropathy[J]. Biochem Biophys Res Commun,1998,249(3):605-611.
[37]Nishimura M, Toki J, Sugiura K, et al. Focal segmental glomerular sclerosis, a type of intractable chronic glomerulonephritis, is a stem cell disorder[J]. J Exp Med,1994,179(3):1053-1058.
[38]Ikehara S. Bone marrow transplantation for autoimmune diseases[J]. Acta Haematol,1998,99(3):116-132.
[39]陆春,李娟,熊文杰,等.异基因造血干细胞移植治疗BXSB狼疮鼠[J].中华皮肤科杂志,2003(1)：30-32.
[40]Ende N, Chen R, Reddi A S. Transplantation of human umbilical cord blood cells improves glycemia and glomerular hypertrophy in type 2 diabetic mice[J]. Biochem Biophys Res Commun,2004,321(1):168-171.
[41]Lee R H, Seo M J, Reger R L, et al. Multipotent stromal cells from human marrow home to and promote repair of pancreatic islets and renal glomeruli in diabetic NOD/scid mice[J]. Proc Natl Acad Sci U S A,2006,103(46):17438-17443.
[42]Ezquer F E, Ezquer M E, Parrau D B, et al. Systemic administration of multipotent mesenchymal stromal cells reverts hyperglycemia and prevents nephropathy in type 1 diabetic mice[J]. Biol Blood Marrow Transplant,2008,14(6):631-640.
[43]Kita K, Gauglitz G G, Phan T T, et al. Isolation and characterization of mesenchymal stem cells from the sub-amniotic human umbilical cord lining membrane[J]. Stem Cells Dev,2010,19(4):491-502.
[44]Zhang X, Hirai M, Cantero S, et al. Isolation and characterization of mesenchymal stem cells from human umbilical cord blood:reevaluation of critical factors for successful isolation and high ability to proliferate and differentiate to chondrocytes as compared to mesenchymal stem cells from bone marrow and adipose tissue[J]. J Cell Biochem,2011,112(4):1206-1218.
[45]殷莉波,赵文秀,尹震宇,等.人脂肪间充质干细胞的分离培养及其鉴定[J].中国组织工程研究与临床康复,2010(32)：5997-6000.
[46]Carvalho K A, Oliveira L, Malvezzi M, et al. Immunophenotypic expression by flow cytometric analysis of cocultured skeletal muscle and bone marrow mesenchymal stem cells for therapy into myocardium[J]. Transplant Proc,2008,40(3):842-844.
[47]毕薇薇,黄若洋,刘洋.人胎儿肝脏间充质干细胞的体外培养及生物学特性研究[J].中国医药生物技术,2010(4)：277-280.
[48]Morigi M, Rota C, Montemurro T, et al. Life-sparing effect of human cord blood-mesenchymal stem cells in experimental acute kidney injury[J]. Stem Cells,2010,28(3):513-522.
[49]Li W, Vogel C F, Fujiyoshi P, et al. Development of a human adipocyte model derived from human mesenchymal stem cells (hMSC) as a tool for toxicological studies on the action of TCDD[J]. Biol Chem,2008,389(2):169-177.
[50]Baer P C, Bereiter-Hahn J, Missler C, et al. Conditioned medium from renal tubular epithelial cells initiates differentiation of human mesenchymal stem cells[J]. Cell Prolif,2009,42(1):29-37.
[51]Rumpold H, Wolf D, Koeck R, et al. Endothelial progenitor cells:a source for therapeutic vasculogenesis?[J]. J Cell Mol Med,2004,8(4):509-518.
[52]Becherucci F, Mazzinghi B, Ronconi E, et al. The role of endothelial progenitor cells in acute kidney injury[J]. Blood Purif,2009,27(3):261-270.
[53]Mohandas R, Segal M S. Endothelial progenitor cells and endothelial vesicles-what is the significance for patients with chronic kidney disease?[J]. Blood Purif,2010,29(2):158-162.
[54]Bussolati B, Bruno S, Grange C, et al. Isolation of renal progenitor cells from adult human kidney[J]. Am J Pathol,2005,166(2):545-555.
[55]Bai H W, Shi B Y, Qian Y Y, et al. Endothelial cell chimerism by fluorescence in situ hybridization in gender mismatched renal allograft biopsies[J]. Chin Med J (Engl),2007,120(10):859-862.
[56]Chade A R, Zhu X, Lavi R, et al. Endothelial progenitor cells restore renal function in chronic experimental renovascular disease[J]. Circulation,2009,119(4):547-557.
[57]Togel F, Weiss K, Yang Y, et al. Vasculotropic, paracrine actions of infused mesenchymal stem cells are important to the recovery from acute kidney injury[J]. Am J Physiol Renal Physiol,2007,292(5):F 1626-F1635.
[58]Stroo I, Stokman G, Teske G J, et al. Chemokine expression in renal ischemia/reperfusion injury is most profound during the reparative phase[J]. Int Immunol,2010,22(6):433-442.
[59]Patschan S A, Patschan D, Temme J, et al. Endothelial progenitor cells (EPC) in sepsis with acute renal dysfunction (ARD)[J]. Crit Care,2011,15(2):R94.
[60]Ma Y Y, Sun D, Li J, et al. Transplantation of endothelial progenitor cells alleviates renal interstitial fibrosis in a mouse model of unilateral ureteral obstruction[J]. Life Sci,2010,86(21-22):798-807.
[61]Morrison S J, White P M, Zock C, et al. Prospective identification, isolation by flow cytometry, and in vivo self-renewal of multipotent mammalian neural crest stem cells[J]. Cell,1999,96(5):737-749.
[62]Joseph N M, Mukouyama Y S, Mosher J T, et al. Neural crest stem cells undergo multilineage differentiation in developing peripheral nerves to generate endoneurial fibroblasts in addition to Schwann cells[J]. Development,2004,131(22):5599-5612.
[63]Oliver J A, Maarouf O, Cheema F H, et al. The renal papilla is a niche for adult kidney stem cells[J]. J Clin Invest,2004,114(6):795-804.
[64]Yamashita S, Maeshima A, Nojima Y. Involvement of renal progenitor tubular cells in epithelial-to-mesenchymal transition in fibrotic rat kidneys[J]. J Am Soc Nephrol,2005,16(7):2044-2051.
[65]Lazzeri E, Crescioli C, Ronconi E, et al. Regenerative potential of embryonic renal multipotent progenitors in acute renal failure[J]. J Am Soc Nephrol,2007,18(12):3128-3138.
[66]Mazzinghi B, Ronconi E, Lazzeri E, et al. Essential but differential role for CXCR4 and CXCR7 in the therapeutic homing of human renal progenitor cells[J]. J Exp Med,2008,205(2):479-490.
[2]Ishii Y, Sawada T, Kubota K, et al. Loss of peritubular capillaries in the development of chronic allograft nephropathy[J]. Transplant Proc,2005,37(2):981-983.
[3]Muller G A, Strutz F M. Renal fibroblast heterogeneity[J]. Kidney Int Suppl,1995,50:S33-S36.
[4]Mucsi I, Rosivall L. Epithelial-mesenchymal transition in renal tubular cells in the pathogenesis of progressive tubulo-interstitial fibrosis[J]. Acta Physiol Hung,2007,94(1-2):117-131.
[5]Sun D, Feng J, Dai C, et al. Role of peritubular capillary loss and hypoxia in progressive tubulointerstitial fibrosis in a rat model of aristolochic acid nephropathy[J]. Am J Nephrol,2006,26(4):363-371.
[6]Kang D H, Hughes J, Mazzali M, et al. Impaired angiogenesis in the remnant kidney model:Ⅱ. Vascular endothelial growth factor administration reduces renal fibrosis and stabilizes renal function[J]. J Am Soc Nephrol,2001,12(7):1448-1457.
[7]Kang D H, Johnson R J. Vascular endothelial growth factor:a new player in the pathogenesis of renal fibrosis[J]. Curr Opin Nephrol Hypertens,2003,12(1):43-49.
[8]Kang D H, Kanellis J, Hugo C, et al. Role of the microvascular endothelium in progressive renal disease[J]. J Am Soc Nephrol,2002,13(3):806-816.
[9]Gluhovschi C, Gluhovschi G, Potencz E, et al. The endothelial cell markers von Willebrand Factor (vWF), CD31 and CD34 are lost in glomerulonephritis and no longer correlate with the morphological indices of glomerular sclerosis, interstitial fibrosis, activity and chronicity[J]. Folia Histochem Cytobiol,2010,48(2):230-236.
[10]Rautou P E, Vion A C, Amabile N, et al. Microparticles, vascular function, and atherothrombosis[J]. Circ Res,2011,109(5):593-606.
[11]Fliser D. Perspectives in renal disease progression:the endothelium as a treatment target in chronic kidney disease[J]. J Nephrol,2010,23(4):369-376.
[12]Schatteman G C, Dunnwald M, Jiao C. Biology of bone marrow-derived endothelial cell precursors[J]. Am J Physiol Heart Circ Physiol,2007,292(1):H1-H18.
[13]Asahara T, Murohara T, Sullivan A, et al. Isolation of putative progenitor endothelial cells for angiogenesis[J]. Science,1997,275(5302):964-967.
[14]Ziegelhoeffer T, Fernandez B, Kostin S, et al. Bone marrow-derived cells do not incorporate into the adult growing vasculature[J]. Circ Res,2004,94(2):230-238.
[15]Rafii S, Lyden D. Therapeutic stem and progenitor cell transplantation for organ vascularization and regeneration[J]. Nat Med,2003,9(6):702-712.
[16]Hur J, Yoon C H, Kim H S, et al. Characterization of two types of endothelial progenitor cells and their different contributions to neovasculogenesis[J]. Arterioscler Thromb Vasc Biol,2004,24(2):288-293.
[17]Kamihata H, Matsubara H, Nishiue T, et al. Implantation of bone marrow mononuclear cells into ischemic myocardium enhances collateral perfusion and regional function via side supply of angioblasts, angiogenic ligands, and cytokines[J]. Circulation,2001,104(9):1046-1052.
[18]Kinnaird T, Stabile E, Burnett M S, et al. Marrow-derived stromal cells express genes encoding a broad spectrum of arteriogenic cytokines and promote in vitro and in vivo arteriogenesis through paracrine mechanisms[J]. Circ Res,2004,94(5):678-685.
[19]Rehman J, Li J, Orschell C M, et al. Peripheral blood "endothelial progenitor cells" are derived from monocyte/macrophages and secrete angiogenic growth factors[J]. Circulation,2003,107(8):1164-1169.
[20]Chade A R, Zhu X, Lavi R, et al. Endothelial progenitor cells restore renal function in chronic experimental renovascular disease[J]. Circulation,2009,119(4):547-557.
[21]Reyes M, Dudek A, Jahagirdar B, et al. Origin of endothelial progenitors in human postnatal bone marrow[J]. J Clin Invest,2002,109(3):337-346.
[22]Takahashi T, Kalka C, Masuda H, et al. Ischemia- and cytokine-induced mobilization of bone marrow-derived endothelial progenitor cells for neovascularization[J]. Nat Med,1999,5(4):434-438.
[23]Murohara T, Ikeda H, Duan J, et al. Transplanted cord blood-derived endothelial precursor cells augment postnatal neovascularization[J]. J Clin Invest,2000,105(11):1527-1536.
[24]Yang C, Zhang Z H, Li Z J, et al. Enhancement of neovascularization with cord blood CD133+ cell-derived endothelial progenitor cell transplantation[J]. Thromb Haemost,2004,91(6):1202-1212.
[25]Peichev M, Naiyer A J, Pereira D, et al. Expression of VEGFR-2 and AC133 by circulating human CD34(+) cells identifies a population of functional endothelial precursors[J]. Blood,2000,95(3):952-958.
[26]Hu Y, Zhang Z, Torsney E, et al. Abundant progenitor cells in the adventitia contribute to atherosclerosis of vein grafts in ApoE-deficient mice[J]. J Clin Invest,2004,113(9):1258-1265.
[27]Sainz J, A1 H Z A, Caligiuri G, et al. Isolation of "side population" progenitor cells from healthy arteries of adult mice[J]. Arterioscler Thromb Vasc Biol,2006,26(2):281-286.
[28]Planat-Benard V, Silvestre J S, Cousin B, et al. Plasticity of human adipose lineage cells toward endothelial cells:physiological and therapeutic perspectives[J]. Circulation,2004,109(5):656-663.
[29]Shi Q, Rafii S, Wu M H, et al. Evidence for circulating bone marrow-derived endothelial cells [J]. Blood,1998,92(2):362-367.
[30]Martinez-Estrada O M, Munoz-Santos Y, Julve J, et al. Human adipose tissue as a source of Flk-1+cells:new method of differentiation and expansion[J]. Cardiovasc Res,2005,65(2):328-333.
[31]Sharpe E R, Teleron A A, Li B, et al. The origin and in vivo significance of murine and human culture-expanded endothelial progenitor cells[J]. Am J Pathol,2006,168(5):1710-1721.
[32]Flamme I, Risau W. Induction of vasculogenesis and hematopoiesis in vitro[J]. Development,1992,116(2):435-439.
[33]Weiss M J, Orkin S H. In vitro differentiation of murine embryonic stem cells. New approaches to old problems[J]. J Clin Invest,1996,97(3):591-595.
[34]Isner J M, Kalka C, Kawamoto A, et al. Bone marrow as a source of endothelial cells for natural and iatrogenic vascular repair[J]. Ann N Y Acad Sci,2001,953:75-84.
[35]Wright W E, Shay J W. Telomere biology in aging and cancer[J]. J Am Geriatr Soc,2005,53(9 Suppl):S292-S294.
[36]Hayflick L, Moorhead P S. The serial cultivation of human diploid cell strains[J]. Exp Cell Res,1961,25:585-621.
[37]Choi S W, Yang J E, Kang S J, et al. Herpetic infection on the vulva associated with eccrine squamous syringometaplasia in malignant lymphoma[J]. Acta Derm Venereol,1999,79(6):500-501.
[38]Bodnar A G, Ouellette M, Frolkis M, et al. Extension of life-span by introduction of telomerase into normal human cells[J]. Science,1998,279(5349):349-352.
[39]Hayflick L, Moorhead P S. The serial cultivation of human diploid cell strains[J]. Exp Cell Res,1961,25:585-621.
[40]Hornsby P J. Senescence and life span[J]. Pflugers Arch,2010,459(2):291-299.
[41]Chiu C P, Dragowska W, Kim N W, et al. Differential expression of telomerase activity in hematopoietic progenitors from adult human bone marrow[J]. Stem Cells,1996,14(2):239-248.
[42]Gancarcikova M, Zemanova Z, Brezinova J, et al. The role of telomeres and telomerase complex in haematological neoplasia:the length of telomeres as a marker of carcinogenesis and prognosis of disease[J]. Prague Med Rep,2010,111(2):91-105.
[43]Blasco M A. Telomere length, stem cells and aging[J]. Nat Chem Biol,2007,3(10):640-649.
[44]Yi X, Tesmer V M, Savre-Train I, et al. Both transcriptional and posttranscriptional mechanisms regulate human telomerase template RNA levels[J]. Mol Cell Biol,1999,19(6):3989-3997.
[45]Haber D A. Telomeres, cancer, and immortality[J]. N Engl J Med,1995,332(14):955-956.
[46]Anderson W F, Blaese R M, Culver K. The ADA human gene therapy clinical protocol:Points to Consider response with clinical protocol, July 6,1990[J]. Hum Gene Ther,1990,1(3):331-362.
[47]El-Aneed A. An overview of current delivery systems in cancer gene therapy[J]. J Control Release,2004,94(1):1-14.
[48]Cerone M A, Ward R J, Londono-Vallejo J A, et al. Telomerase RNA mutated in autosomal dyskeratosis congenita reconstitutes a weakly active telomerase enzyme defective in telomere elongation[J]. Cell Cycle,2005,4(4):585-589.
[49]Blackburn E H. Structure and function of telomeres[J]. Nature,1991,350(6319):569-573.
[50]Shore D. Telomerase and telomere-binding proteins:controlling the endgame[J]. Trends Biochem Sci,1997,22(7):233-235.
[51]Harley C B, Futcher A B, Greider C W. Telomeres shorten during ageing of human fibroblasts[J]. Nature,1990,345(6274):458-460.
[52]Greider C W, Blackburn E H. The telomere terminal transferase of Tetrahymena is a ribonucleoprotein enzyme with two kinds of primer specificity[J]. Cell,1987,51(6):887-898.
[53]Morin G B. The human telomere terminal transferase enzyme is a ribonucleoprotein that synthesizes TTAGGG repeats[J]. Cell,1989,59(3):521-529.
[54]Nakamura T, Bratton D L, Murphy R C. Analysis of epoxyeicosatrienoic and monohydroxyeicosatetraenoic acids esterified to phospholipids in human red blood cells by electrospray tandem mass spectrometry[J]. J Mass Spectrom,1997,32(8):888-896.
[55]Pearce V P, Sherrell J, Lou Z, et al. Immortalization of epithelial progenitor cells mediated by resveratrol[J]. Oncogene,2008,27(17):2365-2374.
[56]Boker W, Yin Z, Drosse I, et al. Introducing a single-cell-derived human mesenchymal stem cell line expressing hTERT after lentiviral gene transfer[J]. J Cell Mol Med,2008,12(4):1347-1359.
[57]Nakamura T M, Morin G B, Chapman K B, et al. Telomerase catalytic subunit homologs from fission yeast and human[J]. Science,1997,277(5328):955-959.
[58]Vandendriessche T, Vanslembrouck V, Goovaerts I, et al. Long-term expression of human coagulation factor Ⅷ and correction of hemophilia A after in vivo retroviral gene transfer in factor Ⅷ-deficient mice[J]. Proc Natl Acad Sci U S A,1999,96(18):10379-10384.
[59]Matz M V, Fradkov A F, Labas Y A, et al. Fluorescent proteins from nonbioluminescent Anthozoa species[J]. Nat Biotechnol,1999,17(10):969-973.
[60]Haas J A, Knox F G. Effect of meclofenamate or ketoconazole on the natriuretic response to increased pressure[J]. J Lab Clin Med,1996,128(2):202-207
[1]Schatteman G C, Dunnwald M, Jiao C. Biology of bone marrow-derived endothelial cell precursors[J]. Am J Physiol Heart Circ Physiol,2007,292(1):H1-H18.
[2]Asahara T, Murohara T, Sullivan A, et al. Isolation of putative progenitor endothelial cells for angiogenesis[J]. Science,1997,275(5302):964-967.
[3]Ziegelhoeffer T, Fernandez B, Kostin S, et al. Bone marrow-derived cells do not incorporate into the adult growing vasculature[J]. Circ Res,2004,94(2):230-238.
[4]Rafii S, Lyden D. Therapeutic stem and progenitor cell transplantation for organ vascularization and regeneration[J]. Nat Med,2003,9(6):702-712.
[5]Hur J, Yoon C H, Kim H S, et al. Characterization of two types of endothelial progenitor cells and their different contributions to neovasculogenesis[J]. Arterioscler Thromb Vasc Biol,2004,24(2):288-293.
[6]Kamihata H, Matsubara H, Nishiue T, et al. Implantation of bone marrow mononuclear cells into ischemic myocardium enhances collateral perfusion and regional function via side supply of angioblasts, angiogenic ligands, and cytokines[J]. Circulation,2001,104(9):1046-1052.
[7]Kinnaird T, Stabile E, Burnett M S, et al. Marrow-derived stromal cells express genes encoding a broad spectrum of arteriogenic cytokines and promote in vitro and in vivo arteriogenesis through paracrine mechanisms[J]. Circ Res,2004,94(5):678-685.
[8]Rehman J, Li J, Orschell C M, et al. Peripheral blood "endothelial progenitor cells" are derived from monocyte/macrophages and secrete angiogenic growth factors[J]. Circulation,2003,107(8):1164-1169.
[9]Murasawa S, Llevadot J, Silver M, et al. Constitutive human telomerase reverse transcriptase expression enhances regenerative properties of endothelial progenitor cells[J]. Circulation,2002,106(9):1133-1139.
[10]Takano H, Murasawa S, Asahara T. Functional and gene expression analysis of hTERT overexpressed endothelial cells[J]. Biologics,2008,2(3):547-554.
[11]Livak K J, Schmittgen T D. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method[J]. Methods,2001,25(4):402-408.
[12]Risau W, Flamme I. Vasculogenesis[J]. Annu Rev Cell Dev Biol,1995,11:73-91.
[13]Asahara T, Murohara T, Sullivan A, et al. Isolation of putative progenitor endothelial cells for angiogenesis[J]. Science,1997,275(5302):964-967.
[14]Reyes M, Dudek A, Jahagirdar B, et al. Origin of endothelial progenitors in human postnatal bone marrow[J]. J Clin Invest,2002,109(3):337-346.
[15]Takahashi T, Kalka C, Masuda H, et al. Ischemia-and cytokine-induced mobilization of bone marrow-derived endothelial progenitor cells for neovascularization[J]. Nat Med,1999,5(4):434-438.
[16]Murohara T, Ikeda H, Duan J, et al. Transplanted cord blood-derived endothelial precursor cells augment postnatal neovascularization[J]. J Clin Invest,2000,105(11):1527-1536.
[17]Yang C, Zhang Z H, Li Z J, et al. Enhancement of neovascularization with cord blood CD 133+ cell-derived endothelial progenitor cell transplantation[J]. Thromb Haemost,2004,91(6):1202-1212.
[18]Peichev M, Naiyer A J, Pereira D, et al. Expression of VEGFR-2 and AC 133 by circulating human CD34(+) cells identifies a population of functional endothelial precursors [J]. Blood,2000,95(3):952-958.
[19]Hu Y, Zhang Z, Torsney E, et al. Abundant progenitor cells in the adventitia contribute to atherosclerosis of vein grafts in ApoE-deficient mice[J]. J Clin Invest,2004,113(9):1258-1265.
[20]Sainz J, A1 H Z A, Caligiuri G, et al. Isolation of "side population" progenitor cells from healthy arteries of adult mice[J]. Arterioscler Thromb Vasc Biol,2006,26(2):281-286.
[21]Planat-Benard V, Silvestre J S, Cousin B, et al. Plasticity of human adipose lineage cells toward endothelial cells:physiological and therapeutic perspectives [J]. Circulation,2004,109(5):656-663.
[22]Shi Q, Rafii S, Wu M H, et al. Evidence for circulating bone marrow-derived endothelial cells[J]. Blood,1998,92(2):362-367.
[23]Martinez-Estrada O M, Munoz-Santos Y, Julve J, et al. Human adipose tissue as a source of Flk-1+cells:new method of differentiation and expansion[J]. Cardiovasc Res,2005,65(2):328-333.
[24]Mancinelli F, Tamburini A, Spagnoli A, et al. Optimizing umbilical cord blood collection:impact of obstetric factors versus quality of cord blood units[J]. Transplant Proc,2006,38(4):1174-1176.
[25]Murohara T, Ikeda H, Duan J, et al. Transplanted cord blood-derived endothelial precursor cells augment postnatal neovascularization[J]. J Clin Invest,2000,105(11):1527-1536.
[26]Peichev M, Naiyer A J, Pereira D, et al. Expression of VEGFR-2 and AC 133 by circulating human CD34(+) cells identifies a population of functional endothelial precursors[J]. Blood,2000,95(3):952-958.
[27]Jeon O, Hwang K C, Yoo K J, et al. Combined sustained delivery of basic fibroblast growth factor and administration of granulocyte colony-stimulating factor:synergistic effect on angiogenesis in mouse ischemic limbs[J]. J Endovasc Ther,2006,13(2):175-181.
[28]Sharpe E R, Teleron A A, Li B, et al. The origin and in vivo significance of murine and human culture-expanded endothelial progenitor cells[J]. Am J Pathol,2006,168(5):1710-1721.
[29]Flamme I, Risau W. Induction of vasculogenesis and hematopoiesis in vitro[J]. Development,1992,116(2):435-439.
[30]Weiss M J, Orkin S H. In vitro differentiation of murine embryonic stem cells. New approaches to old problems[J]. J Clin Invest,1996,97(3):591-595.
[31]Isner J M, Kalka C, Kawamoto A, et al. Bone marrow as a source of endothelial cells for natural and iatrogenic vascular repair[J]. Ann N Y Acad Sci,2001,953:75-84.
[32]Reyes M, Dudek A, Jahagirdar B, et al. Origin of endothelial progenitors in human postnatal bone marrow[J]. J Clin Invest,2002,109(3):337-346.
[33]Asahara T, Murohara T, Sullivan A, et al. Isolation of putative progenitor endothelial cells for angiogenesis[J]. Science,1997,275(5302):964-967.
[34]Asahara T. [Endothelial progenitor cells for vascular medicine][J]. Yakugaku Zasshi,2007,127(5):841-845.
[35]何小解,许自川,郑洪,等.大鼠骨髓内皮祖细胞分离与体外培养条件研究[J].世界科技研 究与发展,2008(2)：198-201.
[36]Teleron A A, Carlson B, Young P P. Blood donor white blood cell reduction filters as a source of human peripheral blood-derived endothelial progenitor cells[J]. Transfusion,2005,45(1):21-25.
[37]肖刚峰,张怀勤,季亢挺,等.单个核细胞的接种密度对贴壁筛选法培养内皮祖细胞增殖的影响[J].细胞与分子免疫学杂志,2006(2)：233-234.
[38]Shintani S, Murohara T, Ikeda H, et al. Augmentation of postnatal neovascularization with autologous bone marrow transplantation[J].Circulation,2001,103(6):897-903.
[39]Urbich C, Dimmeler S. Endothelial progenitor cells:characterization and role in vascular biology[J]. Circ Res,2004,95(4):343-353.
[40]Vasa M, Fichtlscherer S, Aicher A, et al. Number and migratory activity of circulating endothelial progenitor cells inversely correlate with risk factors for coronary artery disease[J]. Circ Res,2001,89(1):E1-E7.
[41]Kalka C, Masuda H, Takahashi T, et al. Vascular endothelial growth factor(165) gene transfer augments circulating endothelial progenitor cells in human subjects[J]. Circ Res,2000,86(12):1198-1202.
[42]Wagner D D, Olmsted J B, Marder V J. Immunolocalization of von Willebrand protein in Weibel-Palade bodies of human endothelial cells[J]. J Cell Biol,1982,95(1):355-360.
[43]Ewenstein B M, Warhol M J, Handin R I, et al. Composition of the von Willebrand factor storage organelle (Weibel-Palade body) isolated from cultured human umbilical vein endothelial cells[J]. J Cell Biol,1987,104(5):1423-1433.
[44]Wright W E, Shay J W. Telomere biology in aging and cancer[J]. J Am Geriatr Soc,2005,53(9 Suppl):S292-S294.
[45]Hayflick L, Moorhead P S. The serial cultivation of human diploid cell strains[J]. Exp Cell Res,1961,25:585-621.
[46]Brenner A J, Stampfer M R, Aldaz C M. Increased p16 expression with first senescence arrest in human mammary epithelial cells and extended growth capacity with p16 inactivation[J]. Oncogene,1998,17(2):199-205.
[47]Shay J W, Wright W E. Use of telomerase to create bioengineered tissues[J]. Ann N Y Acad Sci,2005,1057:479-491.
[48]Chang M W, Grillari J, Mayrhofer C, et al. Comparison of early passage, senescent and hTERT immortalized endothelial cells[J]. Exp Cell Res,2005,309(1):121-136.
[49]Bodnar A G, Ouellette M, Frolkis M, et al. Extension of life-span by introduction of telomerase into normal human cells[J]. Science,1998,279(5349):349-352.
[50]Durand G, Poeggeler B, Boker J, et al. Fine-tuning the amphiphilicity:a crucial parameter in the design of potent alpha-phenyl-N-tert-butylnitrone analogues[J]. J Med Chem,2007,50(17):3976-3979.
[51]Kiyono T, Foster S A, Koop J I, et al. Both Rb/p16INK4a inactivation and telomerase activity are required to immortalize human epithelial cells[J]. Nature,1998,396(6706):84-88.
[52]Nisato R E, Harrison J A, Buser R, et al. Generation and characterization of telomerase-transfected human lymphatic endothelial cells with an extended life span[J]. Am J Pathol,2004,165(1):11-24.
[53]Freedman D A, Folkman J. Maintenance of G1 checkpoint controls in telomerase-immortalized endothelial cells[J]. Cell Cycle,2004,3(6):811-816
[1]Nangaku M. Chronic hypoxia and tubulointerstitial injury:a final common pathway to end-stage renal failure[J]. J Am Soc Nephrol,2006,17(1):17-25.
[2]Becker G J, Hewitson T D. The role of tubulointerstitial injury in chronic renal failure[J]. Curr Opin Nephrol Hypertens,2000,9(2):133-138.
[3]Ishii Y, Sawada T, Kubota K, et al. Loss of peritubular capillaries in the development of chronic allograft nephropathy[J]. Transplant Proc,2005,37(2):981-983.
[4]Sun D, Feng J, Dai C, et al. Role of peritubular capillary loss and hypoxia in progressive tubulointerstitial fibrosis in a rat model of aristolochic acid nephropathy[J]. Am J Nephrol,2006,26(4):363-371.
[5]Kang D H, Hughes J, Mazzali M, et al. Impaired angiogenesis in the remnant kidney model:Ⅱ. Vascular endothelial growth factor administration reduces renal fibrosis and stabilizes renal function[J]. J Am Soc Nephrol,2001,12(7):1448-1457.
[6]Asahara T, Murohara T, Sullivan A, et al. Isolation of putative progenitor endothelial cells for angiogenesis[J]. Science,1997,275(5302):964-967.
[7]Huang W J, Xiao F Y, Zhang H Q, et al. [Effects of endothelial progenitor cells and endothelial outgrowth cells in repair of injured carotid vessel:a comparative study with rabbits][J]. Zhonghua Yi Xue Za Zhi,2007,87(44):3143-3147.
[8]Kawamoto A, Tkebuchava T, Yamaguchi J, et al. Intramyocardial transplantation of autologous endothelial progenitor cells for therapeutic neovascularization of myocardial ischemia[J]. Circulation,2003,107(3):461-468.
[9]Magri D, Fancher T T, Fitzgerald T N, et al. Endothelial progenitor cells:a primer for vascular surgeons[J].Vascular,2007,15(6):384-394.
[10]Kalka C, Masuda H, Takahashi T, et al. Vascular endothelial growth factor(165) gene transfer augments circulating endothelial progenitor cells in human subjects[J]. Circ Res,2000,86(12):1198-1202.
[11]Maurer M H, Thomas C, Burgers H F, et al. Transplantation of adult neural progenitor cells transfected with vascular endothelial growth factor rescues grafted cells in the rat brain[J]. Int J Biol Sci,2008,4(1):1-7.
[12]Wright W E, Shay J W. Telomere biology in aging and cancer[J]. J Am Geriatr Soc,2005,53(9 Suppl):S292-S294.
[13]Durand G, Poeggeler B, Boker J, et al. Fine-tuning the amphiphilicity:a crucial parameter in the design of potent alpha-phenyl-N-tert-butylnitrone analogues[J]. J Med Chem,2007,50(17):3976-3979.
[14]Murasawa S, Llevadot J, Silver M, et al. Constitutive human telomerase reverse transcriptase expression enhances regenerative properties of endothelial progenitor cells[J]. Circulation,2002,106(9):1133-1139.
[15]Eddy A A. Molecular basis of renal fibrosis[J]. Pediatr Nephrol,2000,15(3-4):290-301.
[16]Muller G A, Strutz F M. Renal fibroblast heterogeneity [J]. Kidney Int Supp1,1995,50:S33-S36.
[17]Rhyu D Y, Yang Y, Ha H, et al. Role of reactive oxygen species in TGF-betal-induced mitogen-activated protein kinase activation and epithelial-mesenchymal transition in renal tubular epithelial cells[J]. J Am Soc Nephrol,2005,16(3):667-675.
[18]Copeland J W, Beaumont B W, Merrilees M J, et al. Epithelial-to-mesenchymal transition of human proximal tubular epithelial cells:effects of rapamycin, mycophenolate, cyclosporin, azathioprine, and methylprednisolone[J]. Transplantation,2007,83(6):809-814.
[19]Galichon P, Hertig A. Epithelial to mesenchymal transition as a biomarker in renal fibrosis:are we ready for the bedside?[J]. Fibrogenesis Tissue Repair,2011,4:11.
[20]杨念生,武庆庆,姜宗培,等.维甲酸抑制大鼠单侧输尿管梗阻模型肾间质纤维化[J].中华肾脏病杂志,2004(3).
[21]杨念生,武庆庆,黄越芳,等.血管紧张素Ⅱ对大鼠残肾模型微血管的影响[J].中华肾脏病杂志,2003(6).
[22]张宁,龚侃,果宏峰,等.散发性肾透明细胞癌组织中VHL基因突变、血管内皮生长因子表达及其与微血管形成的关系[J].中华医学杂志,2004(19)：40-44.
[23]Tanaka T, Matsumoto M, Inagi R, et al. Induction of protective genes by cobalt ameliorates tubulointerstitial injury in the progressive Thyl nephritis[J]. Kidney Int,2005,68(6):2714-2725.
[24]Ohashi R, Shimizu A, Masuda Y, et al. Peritubular capillary regression during the progression of experimental obstructive nephropathy[J]. J Am Soc Nephrol,2002,13(7):1795-1805.
[25]Kliem V, Johnson R J, Alpers C E, et al. Mechanisms involved in the pathogenesis of tubulointerstitial fibrosis in 5/6-nephrectomized rats[J]. Kidney Int,1996,49(3):666-678.
[26]Bohle A, Mackensen-Haen S, Wehrmann M. Significance of postglomerular capillaries in the pathogenesis of chronic renal failure[J]. Kidney Blood Press Res,1996,19(3-4):191-195.
[27]Namikoshi T, Satoh M, Horike H, et al. Implication of peritubular capillary loss and altered expression of vascular endothelial growth factor in IgA nephropathy[J]. Nephron Physiol,20065 102(1):p9-p16.
[28]Mironidou-Tzouveleki M, Tsartsalis S, Tomos C. Vascular endothelial growth factor (VEGF) in the pathogenesis of diabetic nephropathy of type 1 diabetes mellitus[J]. Curr Drug Targets,2011,12(1):107-114.
[29]Zeng X Q, Xu Z C, Dang X Q, et al. [Expression of vascular endothelial growth factor is related to microvessel injury of renal interstitium in children with Henoch Schonlein purpura nephritis][J]. Zhongguo Dang Dai Er Ke Za Zhi,2009,11(9):717-721.
[30]Malmstrom N K, Kallio E A, Rintala J M, et al. Vascular endothelial growth factor in chronic rat allograft nephropathy[J]. Transpl Immunol,2008,19(2):136-144.
[31]Modelli D A L, Viero R M, Carvalho M F. Role of peritubular capillaries and vascular endothelial growth factor in chronic allograft nephropathy[J]. Transplant Proc,2009,41(9):3720-3725.
[32]Kang D H, Johnson R J. Vascular endothelial growth factor:a new player in the pathogenesis of renal fibrosis[J]. Curr Opin Nephrol Hypertens,2003,12(1):43-49.
[33]Kang D H, Kanellis J, Hugo C, et al. Role of the microvascular endothelium in progressive renal disease[J]. J Am Soc Nephrol,2002,13(3):806-816.
[34]Ferrara N, Henzel W J. Pituitary follicular cells secrete a novel heparin-binding growth factor specific for vascular endothelial cells[J]. Biochem Biophys Res Commun,1989,161(2):851-858.
[35]Schrijvers B F, Flyvbjerg A, De Vriese A S. The role of vascular endothelial growth factor (VEGF) in renal pathophysiology[J]. Kidney Int,2004,65(6):2003-2017.
[36]Yang J, Liu Y. Dissection of key events in tubular epithelial to myofibroblast transition and its implications in renal interstitial fibrosis[J]. Am J Pathol,2001,159(4):1465-1475.
[37]Forino M, Torregrossa R, Ceol M, et al. TGFbetal induces epithelial-mesenchymal transition, but not myofibroblast transdifferentiation of human kidney tubular epithelial cells in primary culture[J]. Int J Exp Pathol,2006,87(3):197-208.
[38]Koesters R, Kaissling B, Lehir M, et al. Tubular overexpression of transforming growth factor-betal induces autophagy and fibrosis but not mesenchymal transition of renal epithelial cells[J]. Am J Pathol,2010,177(2):632-643.
[39]Wang J Y, Bao H Y, Huang S M, et al. [Mechanisms of decorin inhibiting epithelial-to-mesenchymal transition induced by transforming growth factor betal in renal tubular epithelial cells][J]. Zhonghua Er Ke Za Zhi,2010,48(1):50-54.
[40]Minardi D, Lucarini G, Filosa A, et al. Prognostic role of tumor necrosis, microvessel density, vascular endothelial growth factor and hypoxia inducible factor-1alpha in patients with clear cell renal carcinoma after radical nephrectomy in a long term follow-up[J]. Int J Immunopathol Pharmacol,2008,21(2):447-455.
[41]Metsuyanim S, Levy R, Davidovits M, et al. Molecular evaluation of circulating endothelial progenitor cells in children undergoing hemodialysis and after kidney transplantation[J]. Pediatr Res,2009,65(2):221-225.
[42]Eizawa T, Murakami Y, Matsui K, et al. Circulating endothelial progenitor cells are reduced in hemodialysis patients[J]. Curr Med Res Opin,2003,19(7):627-633.
[43]Choi J H, Kim K L, Huh W, et al. Decreased number and impaired angiogenic function of endothelial progenitor cells in patients with chronic renal failure[J]. Arterioscler Thromb Vasc Biol,2004,24(7):1246-1252.
[44]de Groot K, Bahlmann F H, Bahlmann E, et al. Kidney graft function determines endothelial progenitor cell number in renal transplant recipients[J]. Transplantation,2005,79(8):941-945.
[45]Metsuyanim S, Levy R, Davidovits M, et al. Molecular evaluation of circulating endothelial progenitor cells in children undergoing hemodialysis and after kidney transplantation[J]. Pediatr Res,2009,65(2):221-225.
[46]Zeng L, Xiao Q, Margariti A, et al. HDAC3 is crucial in shear- and VEGF-induced stem cell differentiation toward endothelial cells[J]. J Cell Biol,2006,174(7):1059-1069.
[47]Hess D C, Hill W D, Martin-Studdard A, et al. Bone marrow as a source of endothelial cells and NeuN-expressing cells After stroke[J]. Stroke,2002,33(5):1362-1368.
[48]Wu X, Rabkin-Aikawa E, Guleserian K J, et al. Tissue-engineered microvessels on three-dimensional biodegradable scaffolds using human endothelial progenitor cells[J]. Am J Physiol Heart Circ Physiol,2004,287(2):H480-H487.
[49]Chade A R, Zhu X, Lavi R, et al. Endothelial progenitor cells restore renal function in chronic experimental renovascular disease[J]. Circulation,2009,119(4):547-557.
[50]Brenner A J, Stampfer M R, Aldaz C M. Increased p16 expression with first senescence arrest in human mammary epithelial cells and extended growth capacity with p16 inactivation[J]. Oncogene,1998,17(2):199-205.
[51]Chang M W, Grillari J, Mayrhofer C, et al. Comparison of early passage, senescent and hTERT immortalized endothelial cells[J]. Exp Cell Res,2005,309(1):121-136.
[52]Bodnar A G, Ouellette M, Frolkis M, et al. Extension of life-span by introduction of telomerase into normal human cells[J]. Science,1998,279(5349):349-352.
[1]Weissman I L. Stem cells:units of development, units of regeneration, and units in evolution[J]. Cell,2000,100(1):157-168.
[2]Metcalf D. Hematopoietic stem cells:old and new[J]. Biomed Pharmacother,2001,55(2):75-78.
[3]Mcculloch E A, Till J E. Perspectives on the properties of stem cells[J]. Nat Med,2005,11 (10):1026-1028.
[4]Steenhard B M, Isom K S, Cazcarro P, et al. Integration of embryonic stem cells in metanephric kidney organ culture[J]. J Am Soc Nephrol,2005,16(6):1623-1631.
[5]Yamamoto M, Cui L, Johkura K, et al. Branching ducts similar to mesonephric ducts or ureteric buds in teratomas originating from mouse embryonic stem cells[J]. Am J Physiol Renal Physiol,2006,290(1):F52-F60.
[6]Kim D, Dressler G R. Nephrogenic factors promote differentiation of mouse embryonic stem cells into renal epithelia[J]. J Am Soc Nephrol,2005,16(12):3527-3534.
[7]Sagrinati C, Ronconi E, Lazzeri E, et al. Stem-cell approaches for kidney repair:choosing the right cells[J]. Trends Mol Med,2008,14(7):277-285.
[8]Perin L, Sedrakyan S, Da S S, et al. Characterization of human amniotic fluid stem cells and their pluripotential capability [J]. Methods Cell Biol,2008,86:85-99.
[9]Perin L, Giuliani S, Jin D, et al. Renal differentiation of amniotic fluid stem cells[J]. Cell Prolif,2007,40(6):936-948.
[10]Hauser P V, De Fazio R, Bruno S, et al. Stem cells derived from human amniotic fluid contribute to acute kidney injury recovery[J]. Am J Pathol,2010,177(4):2011-2021.
[11]Herrera M B, Bussolati B, Bruno S, et al. Mesenchymal stem cells contribute to the renal repair of acute tubular epithelial injury[J]. Int J Mol Med,2004,14(6):1035-1041.
[12]Steenhard B M, Isom K S, Cazcarro P, et al. Integration of embryonic stem cells in metanephric kidney organ culture[J]. J Am Soc Nephrol,2005,16(6):1623-1631.
[13]Dekel B, Burakova T, Ben-Hur H, et al. Engraftment of human kidney tissue in rat radiation chimera:Ⅱ. Human fetal kidneys display reduced immunogenicity to adoptively transferred human peripheral blood mononuclear cells and exhibit rapid growth and development[J]. Transplantation,1997,64(11):1550-1558.
[14]Starter M B, Fahrner K J, Barksdale E J, et al. Correlation of fetal kidney and testis congenic graft survival with reduced major histocompatibility complex burden[J]. Transplantation,1989,47(4):651-660.
[15]Rogers S A, Lowell J A, Hammerman N A, et al. Transplantation of developing metanephroi into adult rats[J]. Kidney Int,1998,54(1):27-37.
[16]Woolf A S, Hornbruch A, Fine L G. Integration of new embryonic nephrons into the kidney[J]. Am J Kidney Dis,1991,17(6):611-614.
[17]Rogers S A, Liapis H, Hammerman M R. Transplantation of metanephroi across the major histocompatibility complex in rats[J]. Am J Physiol Regul Integr Comp Physiol,2001,280(1):R132-R136.
[18]Rogers S A, Lowell J A, Hammerman N A, et al. Transplantation of developing metanephroi into adult rats[J]. Kidney Int,1998,54(l):27-37.
[19]Dekel B, Burakova T, Ben-Hur H, et al. Engraftment of human kidney tissue in rat radiation chimera:Ⅱ. Human fetal kidneys display reduced immunogenicity to adoptively transferred human peripheral blood mononuclear cells and exhibit rapid growth and development[J]. Transplantation,1997,64(11):1550-1558.
[20]Dekel B, Amariglio N, Kaminski N, et al. Engraftment and differentiation of human metanephroi into functional mature nephrons after transplantation into mice is accompanied by a profile of gene expression similar to normal human kidney development[J]. J Am Soc Nephrol,2002,13(4):977-990.
[21]Minguell J J, Erices A, Conget P. Mesenchymal stem cells[J]. Exp Biol Med (Maywood),2001,226(6):507-520.
[22]Ito T, Suzuki A, Imai E, et al. Bone marrow is a reservoir of repopulating mesangial cells during glomerular remodeling[J]. J Am Soc Nephrol,2001,12(12):2625-2635.
[23]Imasawa T, Utsunomiya Y, Kawamura T, et al. The potential of bone marrow-derived cells to differentiate to glomerular mesangial cells[J]. J Am Soc Nephrol,2001,12(7):1401-1409.
[24]Rasmusson I, Ringden O, Sundberg B, et al. Mesenchymal stem cells inhibit lymphocyte proliferation by mitogens and alloantigens by different mechanisms[J]. Exp Cell Res,2005,305(1):33-41.
[25]Le Blanc K. Immunomodulatory effects of fetal and adult mesenchymal stem cells[J]. Cytotherapy,2003,5(6):485-489.
[26]Djouad F, Plence P, Bony C, et al. Immunosuppressive effect of mesenchymal stem cells favors tumor growth in allogeneic animals[J]. Blood,2003,102(10):3837-3844.
[27]Mackay A M, Beck S C, Murphy J M, et al. Chondrogenic differentiation of cultured human mesenchymal stem cells from marrow[J]. Tissue Eng,1998,4(4):415-428.
[28]Poulsom R, Forbes S J, Hodivala-Dilke K, et al. Bone marrow contributes to renal parenchymal turnover and regeneration [J]. J Pathol,2001,195(2):229-235.
[29]Gupta S, Verfaillie C, Chmielewski D, et al. A role for extrarenal cells in the regeneration following acute renal failure[J]. Kidney Int,2002,62(4):1285-1290.
[30]Lin F, Cordes K, Li L, et al. Hematopoietic stem cells contribute to the regeneration of renal tubules after renal ischemia-reperfusion injury in mice[J]. J Am Soc Nephrol,2003,14(5):1188-1199.
[31]Duffield J S, Park K M, Hsiao L L, et al. Restoration of tubular epithelial cells during repair of the postischemic kidney occurs independently of bone marrow-derived stem cells[J]. J Clin Invest,2005,115(7):1743-1755.
[32]Lin F, Moran A, Igarashi P. Intrarenal cells, not bone marrow-derived cells, are the major source for regeneration in postischemic kidney[J]. J Clin Invest.2005,115(7):1756-1764.
[33]Szczypka M S, Westover A J, Clouthier S G, et al. Rare incorporation of bone marrow-derived cells into kidney after folic acid-induced injury[J]. Stem Cells,2005,23(1):44-54.
[34]Nishida M, Fujimoto S, Toiyama K, et al. Effect of hematopoietic cytokines on renal function in cisplatin-induced ARF in mice[J]. Biochem Biophys Res Commun,2004,324(1):341-347.
[35]Humphreys B D, Valerius M T, Kobayashi A, et al. Intrinsic epithelial cells repair the kidney after injury[J]. Cell Stem Cell,2008,2(3):284-291.
[36]Imasawa T, Utsunomiya Y, Kawamura T, et al. Evidence suggesting the involvement of hematopoietic stem cells in the pathogenesis of IgA nephropathy[J]. Biochem Biophys Res Commun,1998,249(3):605-611.
[37]Nishimura M, Toki J, Sugiura K, et al. Focal segmental glomerular sclerosis, a type of intractable chronic glomerulonephritis, is a stem cell disorder[J]. J Exp Med,1994,179(3):1053-1058.
[38]Ikehara S. Bone marrow transplantation for autoimmune diseases[J]. Acta Haematol,1998,99(3):116-132.
[39]陆春,李娟,熊文杰,等.异基因造血干细胞移植治疗BXSB狼疮鼠[J].中华皮肤科杂志,2003(1)：30-32.
[40]Ende N, Chen R, Reddi A S. Transplantation of human umbilical cord blood cells improves glycemia and glomerular hypertrophy in type 2 diabetic mice[J]. Biochem Biophys Res Commun,2004,321(1):168-171.
[41]Lee R H, Seo M J, Reger R L, et al. Multipotent stromal cells from human marrow home to and promote repair of pancreatic islets and renal glomeruli in diabetic NOD/scid mice[J]. Proc Natl Acad Sci U S A,2006,103(46):17438-17443.
[42]Ezquer F E, Ezquer M E, Parrau D B, et al. Systemic administration of multipotent mesenchymal stromal cells reverts hyperglycemia and prevents nephropathy in type 1 diabetic mice[J]. Biol Blood Marrow Transplant,2008,14(6):631-640.
[43]Kita K, Gauglitz G G, Phan T T, et al. Isolation and characterization of mesenchymal stem cells from the sub-amniotic human umbilical cord lining membrane[J]. Stem Cells Dev,2010,19(4):491-502.
[44]Zhang X, Hirai M, Cantero S, et al. Isolation and characterization of mesenchymal stem cells from human umbilical cord blood:reevaluation of critical factors for successful isolation and high ability to proliferate and differentiate to chondrocytes as compared to mesenchymal stem cells from bone marrow and adipose tissue[J]. J Cell Biochem,2011,112(4):1206-1218.
[45]殷莉波,赵文秀,尹震宇,等.人脂肪间充质干细胞的分离培养及其鉴定[J].中国组织工程研究与临床康复,2010(32)：5997-6000.
[46]Carvalho K A, Oliveira L, Malvezzi M, et al. Immunophenotypic expression by flow cytometric analysis of cocultured skeletal muscle and bone marrow mesenchymal stem cells for therapy into myocardium[J]. Transplant Proc,2008,40(3):842-844.
[47]毕薇薇,黄若洋,刘洋.人胎儿肝脏间充质干细胞的体外培养及生物学特性研究[J].中国医药生物技术,2010(4)：277-280.
[48]Morigi M, Rota C, Montemurro T, et al. Life-sparing effect of human cord blood-mesenchymal stem cells in experimental acute kidney injury[J]. Stem Cells,2010,28(3):513-522.
[49]Li W, Vogel C F, Fujiyoshi P, et al. Development of a human adipocyte model derived from human mesenchymal stem cells (hMSC) as a tool for toxicological studies on the action of TCDD[J]. Biol Chem,2008,389(2):169-177.
[50]Baer P C, Bereiter-Hahn J, Missler C, et al. Conditioned medium from renal tubular epithelial cells initiates differentiation of human mesenchymal stem cells[J]. Cell Prolif,2009,42(1):29-37.
[51]Rumpold H, Wolf D, Koeck R, et al. Endothelial progenitor cells:a source for therapeutic vasculogenesis?[J]. J Cell Mol Med,2004,8(4):509-518.
[52]Becherucci F, Mazzinghi B, Ronconi E, et al. The role of endothelial progenitor cells in acute kidney injury[J]. Blood Purif,2009,27(3):261-270.
[53]Mohandas R, Segal M S. Endothelial progenitor cells and endothelial vesicles-what is the significance for patients with chronic kidney disease?[J]. Blood Purif,2010,29(2):158-162.
[54]Bussolati B, Bruno S, Grange C, et al. Isolation of renal progenitor cells from adult human kidney[J]. Am J Pathol,2005,166(2):545-555.
[55]Bai H W, Shi B Y, Qian Y Y, et al. Endothelial cell chimerism by fluorescence in situ hybridization in gender mismatched renal allograft biopsies[J]. Chin Med J (Engl),2007,120(10):859-862.
[56]Chade A R, Zhu X, Lavi R, et al. Endothelial progenitor cells restore renal function in chronic experimental renovascular disease[J]. Circulation,2009,119(4):547-557.
[57]Togel F, Weiss K, Yang Y, et al. Vasculotropic, paracrine actions of infused mesenchymal stem cells are important to the recovery from acute kidney injury[J]. Am J Physiol Renal Physiol,2007,292(5):F 1626-F1635.
[58]Stroo I, Stokman G, Teske G J, et al. Chemokine expression in renal ischemia/reperfusion injury is most profound during the reparative phase[J]. Int Immunol,2010,22(6):433-442.
[59]Patschan S A, Patschan D, Temme J, et al. Endothelial progenitor cells (EPC) in sepsis with acute renal dysfunction (ARD)[J]. Crit Care,2011,15(2):R94.
[60]Ma Y Y, Sun D, Li J, et al. Transplantation of endothelial progenitor cells alleviates renal interstitial fibrosis in a mouse model of unilateral ureteral obstruction[J]. Life Sci,2010,86(21-22):798-807.
[61]Morrison S J, White P M, Zock C, et al. Prospective identification, isolation by flow cytometry, and in vivo self-renewal of multipotent mammalian neural crest stem cells[J]. Cell,1999,96(5):737-749.
[62]Joseph N M, Mukouyama Y S, Mosher J T, et al. Neural crest stem cells undergo multilineage differentiation in developing peripheral nerves to generate endoneurial fibroblasts in addition to Schwann cells[J]. Development,2004,131(22):5599-5612.
[63]Oliver J A, Maarouf O, Cheema F H, et al. The renal papilla is a niche for adult kidney stem cells[J]. J Clin Invest,2004,114(6):795-804.
[64]Yamashita S, Maeshima A, Nojima Y. Involvement of renal progenitor tubular cells in epithelial-to-mesenchymal transition in fibrotic rat kidneys[J]. J Am Soc Nephrol,2005,16(7):2044-2051.
[65]Lazzeri E, Crescioli C, Ronconi E, et al. Regenerative potential of embryonic renal multipotent progenitors in acute renal failure[J]. J Am Soc Nephrol,2007,18(12):3128-3138.
[66]Mazzinghi B, Ronconi E, Lazzeri E, et al. Essential but differential role for CXCR4 and CXCR7 in the therapeutic homing of human renal progenitor cells[J]. J Exp Med,2008,205(2):479-490.