体外诱导骨髓间充质干细胞分化为肾小管上皮样细胞的研究
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摘要
目的:探讨体外诱导骨髓间充质干细胞(mesenchyml stem cells, MSCs)向肾小管上皮样细胞分化的可能性。并观察不同条件下骨髓间充质干细胞体外诱导分化后的差异。
方法:抽取SD大鼠的骨髓,经密度梯度离心分离,联合贴壁筛选法获取纯化的骨髓间充质干细胞,以流式细胞仪鉴定间充质干细胞表面标志。取扩增3代的骨髓间充质干细胞,分别用含有胎牛血清(空白对照组);胎牛血清+缺血再灌注肾脏匀浆上清+全反式维甲酸ATRA(ATRA对照组);胎牛血清+缺血再灌注肾脏匀浆上清+表皮生长因子(EGF)(EGF组);胎牛血清+缺血再灌注肾脏匀浆上清+肝细胞生长因子(HGF)(HGF组);胎牛血清+缺血再灌注肾脏匀浆上清+骨形成蛋白(BMP-7)(BMP-7组);胎牛血清+缺血再灌注肾脏匀浆上清+促红细胞生成素(EPO)(EPO组);胎牛血清+缺血再灌注肾脏匀浆上清+上述四种因子(联合诱导组)的培养基进行培养。诱导7天后,倒置显微镜下观察细胞形态变化;化学染色检测细胞碱性磷酸酶表达,免疫细胞化学法检测cytokeratin-18蛋白的表达,免疫荧光细胞化学法检测E-cadherin蛋白的表达,RT-PCR检测E-cadherinmRNA的表达。
结果:
(1)流式细胞仪显示体外分离培养的第3代大鼠骨髓干细胞表面抗原表达: CD44阳性细胞表达率98.7%,CD90阳性细胞表达率98.1%, CD29阳性细胞表达率96.1%,而CD11b/c阳性细胞表达率14.7%, CD34阳性细胞表达率1.98%;(2)诱导7天后,与空白对照组相比,ATRA对照组和单因子诱导组(EGF组、HGF组、BMP-7组、EPO组)部分细胞变圆形、短梭形单层排列,联合诱导组的大部分细胞变圆形、短梭形,细胞密集处呈鹅卵石样排列;(3)碱性磷酸酶染色示:空白对照组细胞为阴性,ATRA对照组和单因子诱导组(EGF组、HGF组、BMP-7组、EPO组)有出现部分的阳性细胞,而在联合诱导组中阳性细胞数明显的增多;(4)免疫细胞化学示:空白对照组无表达,cytokeratin-18各组表达的阳性细胞率分别为:ATRA对照组27.40±2.70%;单因子EGF组29.60±4.51%;单因子HGF组26.20±3.70%;单因子BMP-7组26.80±5.00%;单因子EPO组27.80±3.03%;联合诱导组44.00±3.16%,与空白对照组相比均明显增加(P<0.01 vs空白对照组),其中联合诱导组增加更为明显;(5)免疫荧光细胞化学示:在高倍视野下,空白对照组无表达, E-cadherin各组表达的阳性细胞均数分别为:ATRA对照组16.40±2.69;单因子EGF组18.25±3.50;单因子HGF组19.24±3.41;单因子BMP-7组16.06±2.00;单因子EPO组19.33±3.57;联合诱导组30.26±5.16,与空白对照组相比均明显增加(P<0.01 vs空白对照组),其中联合诱导组增加更为明显;(6)RT-PCR方法检测到联合诱导组MSC的E-cadherin的mRNA表达明显高于ATRA对照组和单因子诱导组(P﹤0.01),而空白对照组不表达。
结论:以上四中因子在体外模拟的急性肾衰微环境中可诱导骨髓间充质干细胞分化为肾小管上皮样细胞。
AIM: To explore the possibility of mesenchymal stem cells(MSCs) differentiating into tubular epithelial-like cells in vitro, and to identified the contribution of MSCs to the renal repair of ischemia-reperfusion in vivo,and to investigate differentiation of rat bone marrow mesenchymal stem cells into tubular epithelial-like cells under different conditions.
METHODS: MSCs were obtained from rats. MSCs were isolated by gradient density centrifugation and plastic adherence and then purified, The surface markers were identified with flow cytometry after amplification in vitro. The purified MSCs of the third passage were respectively cultured in DMEM medium with fetal bovine serum (blank group), DMEM medium with fetal bovine serum and all-trans retinoic acid and ischemic reperfusion injured kidney tissue homogenate (ATRA control group), DMEM medium with fetal bovine serum and epidermal growth factor and ischemic reperfusion injured kidney tissue homogenate (EGF induction group), DMEM medium with fetal bovine serum and hepatocyte growth factor and ischemic reperfusion injured kidney tissue homogenate (HGF induction group), DMEM medium with fetal bovine serum and bone morphogenetic protein-7 and ischemic reperfusion injured kidney tissue homogenate (BMP-7 induction group), DMEM medium with fetal bovine serum and erythropoietin and ischemic reperfusion injured kidney tissue homogenate (EPO induction group),DMEM medium with fetal bovine serum and ischemic reperfusion injured kidney tissue homogenate and four factors above-mentioned (combined induction group). After 7 days, the cells were collected for alkaline phosphataestaining,cytokeratin-18 immunocytochemical analysis, E-cadherin immunofluorescence analysis, and the mRNA of E-cadherin was detected by RT-PCR.
RESULTS: (1)Cultured rat MSC at the passage 3 were positive for CD44、CD90 and CD29 (98.7%、98.1%、96.1%,respectively), but negative for CD11b/c and CD34 (14.7% and 1.98%, respectively). (2)Compared with blank group, the cells of ATRA control group and single factor induction group changed from spindle alike cells to round-like cells and elliptic cell. The cells of combined induction group became cobblestone-like cells after 7 days. (3)During differentiation of the tubular epithelial-like cell from the MSC, the counts of positive cells determined by AKP staining was increased. (4)Compared with blank group, the ratio of cytokeratin-18 positive cell in ATRA control group、EGF induction group、HGF induction group、BMP-7 induction group、EPO induction group and combined induction group were 27.40%±2.70%;29.60±4.51%;26.20±3.70%;26.80±5.00%;27.80±3.03%;44.00%±3.16%(P<0.01 vs blank group), the combined induction group increased obviously.(5)The mean of E-cadherin positive cell in ATRA control group、EGF induction group、HGF induction group、BMP-7 induction group、EPO induction group and combined induction group were 16.40±2.69 ; 18.25±3.50 ; 19.24±3.41 ;16.06±2.00;19.33±3.57;30.26±5.16 (P<0.01 vs blank group), the combined induction group increased obviously. (6)the mRNA expression of E-cadherin was increased obviously in combined induction group detected by RT-PCR.
CONCLUSION: MSC may differentiate into tubular epithelial-like cells under the induction condition of ischemic reperfusion injured kidney tissue homogenate and growth factors in vitro.
方法:抽取SD大鼠的骨髓,经密度梯度离心分离,联合贴壁筛选法获取纯化的骨髓间充质干细胞,以流式细胞仪鉴定间充质干细胞表面标志。取扩增3代的骨髓间充质干细胞,分别用含有胎牛血清(空白对照组);胎牛血清+缺血再灌注肾脏匀浆上清+全反式维甲酸ATRA(ATRA对照组);胎牛血清+缺血再灌注肾脏匀浆上清+表皮生长因子(EGF)(EGF组);胎牛血清+缺血再灌注肾脏匀浆上清+肝细胞生长因子(HGF)(HGF组);胎牛血清+缺血再灌注肾脏匀浆上清+骨形成蛋白(BMP-7)(BMP-7组);胎牛血清+缺血再灌注肾脏匀浆上清+促红细胞生成素(EPO)(EPO组);胎牛血清+缺血再灌注肾脏匀浆上清+上述四种因子(联合诱导组)的培养基进行培养。诱导7天后,倒置显微镜下观察细胞形态变化;化学染色检测细胞碱性磷酸酶表达,免疫细胞化学法检测cytokeratin-18蛋白的表达,免疫荧光细胞化学法检测E-cadherin蛋白的表达,RT-PCR检测E-cadherinmRNA的表达。
结果:
(1)流式细胞仪显示体外分离培养的第3代大鼠骨髓干细胞表面抗原表达: CD44阳性细胞表达率98.7%,CD90阳性细胞表达率98.1%, CD29阳性细胞表达率96.1%,而CD11b/c阳性细胞表达率14.7%, CD34阳性细胞表达率1.98%;(2)诱导7天后,与空白对照组相比,ATRA对照组和单因子诱导组(EGF组、HGF组、BMP-7组、EPO组)部分细胞变圆形、短梭形单层排列,联合诱导组的大部分细胞变圆形、短梭形,细胞密集处呈鹅卵石样排列;(3)碱性磷酸酶染色示:空白对照组细胞为阴性,ATRA对照组和单因子诱导组(EGF组、HGF组、BMP-7组、EPO组)有出现部分的阳性细胞,而在联合诱导组中阳性细胞数明显的增多;(4)免疫细胞化学示:空白对照组无表达,cytokeratin-18各组表达的阳性细胞率分别为:ATRA对照组27.40±2.70%;单因子EGF组29.60±4.51%;单因子HGF组26.20±3.70%;单因子BMP-7组26.80±5.00%;单因子EPO组27.80±3.03%;联合诱导组44.00±3.16%,与空白对照组相比均明显增加(P<0.01 vs空白对照组),其中联合诱导组增加更为明显;(5)免疫荧光细胞化学示:在高倍视野下,空白对照组无表达, E-cadherin各组表达的阳性细胞均数分别为:ATRA对照组16.40±2.69;单因子EGF组18.25±3.50;单因子HGF组19.24±3.41;单因子BMP-7组16.06±2.00;单因子EPO组19.33±3.57;联合诱导组30.26±5.16,与空白对照组相比均明显增加(P<0.01 vs空白对照组),其中联合诱导组增加更为明显;(6)RT-PCR方法检测到联合诱导组MSC的E-cadherin的mRNA表达明显高于ATRA对照组和单因子诱导组(P﹤0.01),而空白对照组不表达。
结论:以上四中因子在体外模拟的急性肾衰微环境中可诱导骨髓间充质干细胞分化为肾小管上皮样细胞。
AIM: To explore the possibility of mesenchymal stem cells(MSCs) differentiating into tubular epithelial-like cells in vitro, and to identified the contribution of MSCs to the renal repair of ischemia-reperfusion in vivo,and to investigate differentiation of rat bone marrow mesenchymal stem cells into tubular epithelial-like cells under different conditions.
METHODS: MSCs were obtained from rats. MSCs were isolated by gradient density centrifugation and plastic adherence and then purified, The surface markers were identified with flow cytometry after amplification in vitro. The purified MSCs of the third passage were respectively cultured in DMEM medium with fetal bovine serum (blank group), DMEM medium with fetal bovine serum and all-trans retinoic acid and ischemic reperfusion injured kidney tissue homogenate (ATRA control group), DMEM medium with fetal bovine serum and epidermal growth factor and ischemic reperfusion injured kidney tissue homogenate (EGF induction group), DMEM medium with fetal bovine serum and hepatocyte growth factor and ischemic reperfusion injured kidney tissue homogenate (HGF induction group), DMEM medium with fetal bovine serum and bone morphogenetic protein-7 and ischemic reperfusion injured kidney tissue homogenate (BMP-7 induction group), DMEM medium with fetal bovine serum and erythropoietin and ischemic reperfusion injured kidney tissue homogenate (EPO induction group),DMEM medium with fetal bovine serum and ischemic reperfusion injured kidney tissue homogenate and four factors above-mentioned (combined induction group). After 7 days, the cells were collected for alkaline phosphataestaining,cytokeratin-18 immunocytochemical analysis, E-cadherin immunofluorescence analysis, and the mRNA of E-cadherin was detected by RT-PCR.
RESULTS: (1)Cultured rat MSC at the passage 3 were positive for CD44、CD90 and CD29 (98.7%、98.1%、96.1%,respectively), but negative for CD11b/c and CD34 (14.7% and 1.98%, respectively). (2)Compared with blank group, the cells of ATRA control group and single factor induction group changed from spindle alike cells to round-like cells and elliptic cell. The cells of combined induction group became cobblestone-like cells after 7 days. (3)During differentiation of the tubular epithelial-like cell from the MSC, the counts of positive cells determined by AKP staining was increased. (4)Compared with blank group, the ratio of cytokeratin-18 positive cell in ATRA control group、EGF induction group、HGF induction group、BMP-7 induction group、EPO induction group and combined induction group were 27.40%±2.70%;29.60±4.51%;26.20±3.70%;26.80±5.00%;27.80±3.03%;44.00%±3.16%(P<0.01 vs blank group), the combined induction group increased obviously.(5)The mean of E-cadherin positive cell in ATRA control group、EGF induction group、HGF induction group、BMP-7 induction group、EPO induction group and combined induction group were 16.40±2.69 ; 18.25±3.50 ; 19.24±3.41 ;16.06±2.00;19.33±3.57;30.26±5.16 (P<0.01 vs blank group), the combined induction group increased obviously. (6)the mRNA expression of E-cadherin was increased obviously in combined induction group detected by RT-PCR.
CONCLUSION: MSC may differentiate into tubular epithelial-like cells under the induction condition of ischemic reperfusion injured kidney tissue homogenate and growth factors in vitro.
引文
[1]Sujata Kale, Anil Karihaloo, Paul R, et al. Bone marrow stem cells contribute to repair of the ischemically injured renal tubule [ J ]. J Clin Invest, 2003, 112: 42 - 49
[2] Poulsom R, Forties SJ, Dilke KH, et al. Bone marrow contributes to renal parenchymal turnover and regeneration. J pathol, 2001; 195: 229-235
[3] HerreraMB, BussolatiB, Bruno S, et al. Mesenchymal stem cells contribute to the renal repair of acute tubular ep ithelial injury. Int J MolMed, 2004, 14 (6) : 1035-1041.
[4]Pittenger MF, Mackay AM, Beck SC, et al. Multilineage potential of adult human mesenchymal stem cells. Science 1999, 284:143-147.
[5] Hanada K. Stimulatory efects of basic fibroblast growth factors and bone morphogenetic protein-2 on osteogenic differentiation of bone marrow derived mesenchymal stem cells [J]. J Bone Miner Res, 1997, 12: 1606-1641 [6]Star RA:Treament of acute renal failure .Kidney Int 54;1817-1831,1998.
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[8] Brzoska M,Geiger H,Gauer S,et al.Epithelial differentiation of human adipose tissue-derived adult stem cells.Biochem Biophys ResCommun.2005,330:142-150.
[9] Friedenstein AJ,Gorskaja JF,Kulagina NN.Fibroblast precursors in normal and irradiated mouse hematopoietic organs.Exp Hematol.1976:4(5):267-274.
[10]Pittenger MF,Mackay AM,Jaiswai SC,et al Multilineage potential of mesenchymal stem cells Science,1999,284(5411):43-147
[11]Deans RJ,Moseley AB.Mesenchymal stem cells:biology and potential clinical uses Exp Hematol,2000,28(8):875-884
[12] Fangming Lin, Kimberly C, Linheng Li, et al. Haematopoietic Stemcells Contrbuteto the Regeneration of Renal Tubules after Renal Ischemia-Reperfusion Injury Mice [J]. J Am Soc Nephrol, 2003, 14 (5): 1188.
[13] Kale S, Karihaloo A, Clark PR, et al. Bone marrow stem cells cotribute to repair of the ischemically injured renal tubule [J]. J Clin Invest, 2003, 112(1): 42-49.
[14]张婷,周云,张亚,等.体外诱导骨髓间充质干细胞向肾小管上皮细胞的分化.中国组织工程研究与临床康复[J].2007, 11(3): 478-481.
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[2] Poulsom R, Forties SJ, Dilke KH, et al. Bone marrow contributes to renal parenchymal turnover and regeneration. J pathol, 2001; 195: 229-235
[3] HerreraMB, BussolatiB, Bruno S, et al. Mesenchymal stem cells contribute to the renal repair of acute tubular ep ithelial injury. Int J MolMed, 2004, 14 (6) : 1035-1041.
[4]Pittenger MF, Mackay AM, Beck SC, et al. Multilineage potential of adult human mesenchymal stem cells. Science 1999, 284:143-147.
[5] Hanada K. Stimulatory efects of basic fibroblast growth factors and bone morphogenetic protein-2 on osteogenic differentiation of bone marrow derived mesenchymal stem cells [J]. J Bone Miner Res, 1997, 12: 1606-1641 [6]Star RA:Treament of acute renal failure .Kidney Int 54;1817-1831,1998.
[6] Douncis I S, Groomer E R. Chondrogenic phenotype of perichondrium–derivered chondropro- genitor cells is influenced bytransforming growth factor[J]. J Orthop Res, 1998, 15: 803
[7] Jang YY,Collector MI,Baylin SB,et al.Hematopoietic stem cells convert into liver cells within days without fusion.Nat Cell Biol.2004,6:532-539.
[8] Brzoska M,Geiger H,Gauer S,et al.Epithelial differentiation of human adipose tissue-derived adult stem cells.Biochem Biophys ResCommun.2005,330:142-150.
[9] Friedenstein AJ,Gorskaja JF,Kulagina NN.Fibroblast precursors in normal and irradiated mouse hematopoietic organs.Exp Hematol.1976:4(5):267-274.
[10]Pittenger MF,Mackay AM,Jaiswai SC,et al Multilineage potential of mesenchymal stem cells Science,1999,284(5411):43-147
[11]Deans RJ,Moseley AB.Mesenchymal stem cells:biology and potential clinical uses Exp Hematol,2000,28(8):875-884
[12] Fangming Lin, Kimberly C, Linheng Li, et al. Haematopoietic Stemcells Contrbuteto the Regeneration of Renal Tubules after Renal Ischemia-Reperfusion Injury Mice [J]. J Am Soc Nephrol, 2003, 14 (5): 1188.
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