肾小管上皮细胞原代培养及氧化应激凋亡模型的建立
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摘要
目的：①研究肾小管上皮细胞体外培养及鉴定方法②研究过氧化氢诱导的肾小管上皮细胞凋亡，为模拟肾缺血再灌注损伤时凋亡的研究建立良好的模型。
方法：①采用机械分离结合酶消化法获取肾小管，原代培养出肾小管上皮细胞。②利用动态观察、细胞化学染色法及肾小管上皮细胞的透射电镜观察进行肾小管上皮细胞的鉴定。③用含不同浓度过氧化氢（H2O2）的培养基对肾小管上皮细胞进行氧化应激培养。④利用凋亡细胞的形态变化及生物素原位末端标记法(TUNEL)观察氧化应激后肾小管上皮细胞的凋亡情况。
结果：1、肾小管段不同换液时间24h,48h,72h,96h,120h贴壁率分别为：0.1378±2.048E-02，0.3300±7.969E-02，0.5411±3.919E-02，0.4389±3.018E-02，经多组间两两比较，除72h与96h两组间两两比较差别无意义外（p>0.05）,其余各组间比较差别均有显著意义（p<0.05）。肾小管上皮细胞成功的原代培养：培养的动态观察，从2-3天开始有呈卵圆形的肾小管上皮细胞从贴壁的肾小管周围长出，并于第4-7天处于对数生长期。2、原代培养的细胞鉴定为肾小管上皮细胞：（1）、培养细胞经碱性磷酸酶染色均呈阳性。（2）、透射电镜观察见细胞面向液层的刷状缘有大量微绒毛形成。3、肾小管上皮细胞经不同浓度0mmol/L,0.6mmol/L,1.0mmol/L,1.4mmol/L H2O2应激后的细胞贴壁率分别为1.0000±0.000，0.9400±2.366, 0.9217±1.941, 0.8467±4.676。其中除0.6mmol/L与1.0mmol/L间差别无显著意义外(p>0.05),其余各组差别均有显著意义（p<0.05）。4、肾小管上皮细胞经H2O2氧化应激后出现不同程度的凋亡变化，生物素原位末端标记法（TUNEL）检测，过氧化氢应激浓度为0mmol/L,0.6mmol/L,1.0mmol/L,1.4mmol/L时，细胞的凋亡指数分别是3.000E-02±8.944E-03,0.1450±2.510E-02,0.2317±2.858E-02,0.2500±2.366E-02。其中除1.0mmol/L与1.4mmol/L两组间差别无显著意义（p>0.05）,其余各组间差别均有显著意义（p<0.05）。
结论：采用机械结合酶消化法可分离到较纯的肾小管，肾小管段培养72h首次换液细胞贴壁最佳,肾小管上皮细胞用常规方法可以成功培养及鉴定；肾小
管上皮细胞经H2O2氧化应激可以产生凋亡；在保持肾小管上皮细胞贴壁率较高的条件下,1.0mmol/L H2O2应激可作为体外研究肾缺血性损伤凋亡的良好模型。
Objective:①To investigate the method of primary culture and identify renal tubular epithelial cells②To study the apoptosis of renal tubular epithelial cells induced by Hydrogen peroxide (H2O2 ) and establish the satisfactory simulated model of apoptosis of injury of renal tubular epithelial cells in renal ischemic reperfusion.
Methods:①We harvested the renal tubular segments by machanical and chemical digestive method and primaryly cultured the renal tubular epithelial cells.②By dynamic observing ,cytochemistry staining ,and transmission electron microscope we identified the renal tubular epithelial cells.③Oxidizative stressed the renal tubular epithelial cells with medium containing different concentrations of H2O2.④We detected the apoptosis of renal tubular epithelial cells by the morphologic change and TUNEL(terminal-deoxynucleotidyl transferase mediated nick end labeling) after oxidative stress.
Results:①、The anchor rates of renal tubular respectively were 0.1378±2.048E-02，0.3300±7.969E-02，0.5411±3.919E-02，0.4389±3.018E-02 after different time of replacing medium and the difference (p<0.05) between each two groups had statistical significance except the two groups (72h) and (96h)(p>0.05).The primary culture of the renal tubular epithelial cells succeed: by dynamic observing ,the oval-like renal tubular epithelial cells start to grow around the anchoring renal tubular segment after cultured 2 to 3 days.The cells grow in logarithm growing phase from 4 to 7days.②The primary cultured cells were identified successfully as the renal tubular epithelial cells:(a)The cultured cells were all positive by alkali-phosphatase staining. (b)Under transmission electron microscopy the brush border of the renal tubular epithelial cells have a great many of microvilli .
③The renal tubular epithelial cells present different changes of apoptosic and the apoptosic cells confirmed by HE staining and DeadEndTM system detecting.The apoptosic indexes of the cells were 3.000E-02±8.944E-03,0.1450±2.510E-02,0.2317±2.858E-02,0.2500±2.366E-02 as the H2O2 stress concentrations were 0mmol/L, 0.6mmol/L, 1.0mmol/L, 1.4mmol/L respectively. The differences between each two groups had statistical significance (p<0.05) except the two groups 1.0mmol/L and 1.4mmol/L(p>0.05)。 ④The anchoring rates or renal tubular epithelial cells were 1.0000±0.000，0.9400±2.366, 0.9217±1.941, 0.8467±4.676 after stressed by H2O2 of different concentrations 0mmol/L, 0.6mmol/L, 1.0mmol/L,1.4mmol/L respectively. The differences between each two groups have statistical meaning (p<0.05) except the two groups (0.6mmol/L) and (1.0mmol/L)(p>0.05).
Conclusion:The machanical and chemical digestive method that we modified can obtain fairy pure renal tubular segments.The best growth time is the third day after the first replacing medium.The renal tubular epithelial cells can be cultured successfully with our culture method. The apoptosis of renal tubular epithelial cells can be induced by oxidative stress ( H2O2 ).On the condition of keeping the high anchoring rate of the cells , it is a good model to research apoptosis of the renal ischemic reperfusion injury with 1.0mmol/L H2O2 stress in vitro .
方法：①采用机械分离结合酶消化法获取肾小管，原代培养出肾小管上皮细胞。②利用动态观察、细胞化学染色法及肾小管上皮细胞的透射电镜观察进行肾小管上皮细胞的鉴定。③用含不同浓度过氧化氢（H2O2）的培养基对肾小管上皮细胞进行氧化应激培养。④利用凋亡细胞的形态变化及生物素原位末端标记法(TUNEL)观察氧化应激后肾小管上皮细胞的凋亡情况。
结果：1、肾小管段不同换液时间24h,48h,72h,96h,120h贴壁率分别为：0.1378±2.048E-02，0.3300±7.969E-02，0.5411±3.919E-02，0.4389±3.018E-02，经多组间两两比较，除72h与96h两组间两两比较差别无意义外（p>0.05）,其余各组间比较差别均有显著意义（p<0.05）。肾小管上皮细胞成功的原代培养：培养的动态观察，从2-3天开始有呈卵圆形的肾小管上皮细胞从贴壁的肾小管周围长出，并于第4-7天处于对数生长期。2、原代培养的细胞鉴定为肾小管上皮细胞：（1）、培养细胞经碱性磷酸酶染色均呈阳性。（2）、透射电镜观察见细胞面向液层的刷状缘有大量微绒毛形成。3、肾小管上皮细胞经不同浓度0mmol/L,0.6mmol/L,1.0mmol/L,1.4mmol/L H2O2应激后的细胞贴壁率分别为1.0000±0.000，0.9400±2.366, 0.9217±1.941, 0.8467±4.676。其中除0.6mmol/L与1.0mmol/L间差别无显著意义外(p>0.05),其余各组差别均有显著意义（p<0.05）。4、肾小管上皮细胞经H2O2氧化应激后出现不同程度的凋亡变化，生物素原位末端标记法（TUNEL）检测，过氧化氢应激浓度为0mmol/L,0.6mmol/L,1.0mmol/L,1.4mmol/L时，细胞的凋亡指数分别是3.000E-02±8.944E-03,0.1450±2.510E-02,0.2317±2.858E-02,0.2500±2.366E-02。其中除1.0mmol/L与1.4mmol/L两组间差别无显著意义（p>0.05）,其余各组间差别均有显著意义（p<0.05）。
结论：采用机械结合酶消化法可分离到较纯的肾小管，肾小管段培养72h首次换液细胞贴壁最佳,肾小管上皮细胞用常规方法可以成功培养及鉴定；肾小
管上皮细胞经H2O2氧化应激可以产生凋亡；在保持肾小管上皮细胞贴壁率较高的条件下,1.0mmol/L H2O2应激可作为体外研究肾缺血性损伤凋亡的良好模型。
Objective:①To investigate the method of primary culture and identify renal tubular epithelial cells②To study the apoptosis of renal tubular epithelial cells induced by Hydrogen peroxide (H2O2 ) and establish the satisfactory simulated model of apoptosis of injury of renal tubular epithelial cells in renal ischemic reperfusion.
Methods:①We harvested the renal tubular segments by machanical and chemical digestive method and primaryly cultured the renal tubular epithelial cells.②By dynamic observing ,cytochemistry staining ,and transmission electron microscope we identified the renal tubular epithelial cells.③Oxidizative stressed the renal tubular epithelial cells with medium containing different concentrations of H2O2.④We detected the apoptosis of renal tubular epithelial cells by the morphologic change and TUNEL(terminal-deoxynucleotidyl transferase mediated nick end labeling) after oxidative stress.
Results:①、The anchor rates of renal tubular respectively were 0.1378±2.048E-02，0.3300±7.969E-02，0.5411±3.919E-02，0.4389±3.018E-02 after different time of replacing medium and the difference (p<0.05) between each two groups had statistical significance except the two groups (72h) and (96h)(p>0.05).The primary culture of the renal tubular epithelial cells succeed: by dynamic observing ,the oval-like renal tubular epithelial cells start to grow around the anchoring renal tubular segment after cultured 2 to 3 days.The cells grow in logarithm growing phase from 4 to 7days.②The primary cultured cells were identified successfully as the renal tubular epithelial cells:(a)The cultured cells were all positive by alkali-phosphatase staining. (b)Under transmission electron microscopy the brush border of the renal tubular epithelial cells have a great many of microvilli .
③The renal tubular epithelial cells present different changes of apoptosic and the apoptosic cells confirmed by HE staining and DeadEndTM system detecting.The apoptosic indexes of the cells were 3.000E-02±8.944E-03,0.1450±2.510E-02,0.2317±2.858E-02,0.2500±2.366E-02 as the H2O2 stress concentrations were 0mmol/L, 0.6mmol/L, 1.0mmol/L, 1.4mmol/L respectively. The differences between each two groups had statistical significance (p<0.05) except the two groups 1.0mmol/L and 1.4mmol/L(p>0.05)。 ④The anchoring rates or renal tubular epithelial cells were 1.0000±0.000，0.9400±2.366, 0.9217±1.941, 0.8467±4.676 after stressed by H2O2 of different concentrations 0mmol/L, 0.6mmol/L, 1.0mmol/L,1.4mmol/L respectively. The differences between each two groups have statistical meaning (p<0.05) except the two groups (0.6mmol/L) and (1.0mmol/L)(p>0.05).
Conclusion:The machanical and chemical digestive method that we modified can obtain fairy pure renal tubular segments.The best growth time is the third day after the first replacing medium.The renal tubular epithelial cells can be cultured successfully with our culture method. The apoptosis of renal tubular epithelial cells can be induced by oxidative stress ( H2O2 ).On the condition of keeping the high anchoring rate of the cells , it is a good model to research apoptosis of the renal ischemic reperfusion injury with 1.0mmol/L H2O2 stress in vitro .
引文
1． BAYATI A,NYGREN K,et al:The longterm outcome of post-ishemic acute renal failure in the rat.A histopathological study of the untreated kidney . Acta Physiol Scand 1989，138:35-47
2． Weiberg JM.The cell biology of ischemic renal injury.Kidney Int 1991,39(3):476
3． Kerr,JFR,Wyllie AH, Currie :Apoptosis:A basic biological phenomenon with wide-ranging implications in tissue kinetics. Br J Cancer 1972,26:239-257
4． Schumer M,Colombel MC,et al.Morphologic,biochemical and molecular evidence of apoptosis during the reperfusion phase after brief periods of renal ischemia. Am J Pathol,1992，140:831
5． Searle J,Kerr JFR,Bishop CJ.Necrosis and apoptosis:Distinct modes of cell death with fundamentally different significance.Pathol Annu 1982,17(2):229-259
6． Arends MJ,Morris RG,Wyllic AH.Apoptosis ,the role of the endonuclease.Am J Pathol 1990,136:593-608
7． 赵明中,汪家瑞, 魏嘉平,等 复方丹参滴丸对大鼠心肌缺血再灌注时心肌细胞凋亡及调亡相关基因表达的影响. 中国临床药理学杂志 1999，15(4):288-291
8． 陈敏、郭仁寿，等. 肾缺血再灌注损伤中氧自由基与细胞凋亡. 实用儿科临床杂志 1999,5:283-284
9． 黄海长,王海燕,等 急性肾缺血再灌注后修复过程中的细胞凋亡, 中国病理生理杂志 1996.(4):354
10． 廖洪军,陈香美,等 缺血性肾损伤中细胞凋亡的初步观察, 中华医学杂志 1994,10:629-631
11． Detrisac CJ,et al.Tissue culture of human kidney epithelial cells of proximal tubular origin.Kidney Int 1984，25:383
12． Rudolfs K.Zalups,Lawrence H.Lash.METHODS IN RENAL TOXICOLOGY CRC Press ,Inc.1996，190
13． 司徒镇强，细胞培养， 西安：兴墨图书出版公司 1996，173
14． LAWRENCE H.LASH,In vitro methods of assessing renal damage.TOXICOLOGIC PATHOLOGY,1998，26(1)pp:33-42
LEILA CUTTLE，XIAO-JU ZHANG，ZOLTAN H. ENDRE,et al . Bcl-Xl
15． translocation in renal tubular epithelial cells in vitro protects distal cells from oxidative stress. Kidney Int 2001，59:1779-1788
16． 方福德等，分子生物学前沿技术，北京医科大学中国协和医科大学联合出版社，1998，7
17． 刘海峰、刘为纹等 幽门杆菌诱导胃粘膜上皮细胞凋亡和增殖与P53基因表达。世界华人消化杂志，2001，11
18． 田劲，陈香美，黎磊石。 冬虫夏草、大黄及肾大部分切除大鼠血清对肾小管上皮细胞生长的影响。中西医结合杂志。1991；9：547-549
19． 谢锦玉， 现代细胞化学技术及其在中西医药中的应用， 中医古籍出版社 1998，16
20． KAKO K,KATO M,MATSUOKA T,MUSTAPHA A:Depression of membrane-bound Na+-K+-ATPase activity induced by free radicals and by ischemia of kidney .Am J Physiol 1988，254:C330-C337
21． PALLER MS ,HEDLUND BE:The role of iron in postischemic renal failure in the rat. Kidney Int 1988，34:474-480
22． VAN WAARDE A,STROMSKI ME,THULIN G,GAUDIO KM, et al :Protection of the kidney against ischemic injury by inhibiton of 5'-nucleotidase. AM J Physiol 1989，256:F298-F305
23． CHAN L ,LEDINGHAM JGG,CLARKE J,ROSS BD:The importance of pH in acute renal failure,in Acute Renal Failure,edited by ELIAHOU HE ,London, Libbey,1982,pp58-61
24． YOUNG EW, HUMES HD:Calcium and acute renal failure.Miner Electrolyte Metab 1991,17:106-111
25． Sato N, Iwata S, et al. J Immunol,1995,154:3149-3203
26． Olsen S, Burdick JF,et al. Primary acute renal failure :acute tubular necrosis in the transplanted kidney morphology and pathogensis.Medicine Baltimore. 1989,68:173
27． Gobe GC,Axelsen RA, et al. Cellular events in experiment unilateral ischemic renal atrophy and in regeneration after contralateral nephrectomy. Lab Invest 1990,63:700
28． Allen J,Gobe GC, et al. Effects of hypoxia on morpholoical and biochemical characteritics of renal epithelial cell and tubular cultures. Renal Failure 1992,14:453
Foidart-Willems J,Dechenne C,Mahien P.Biosynthesis of basement
29． membrane collagen in culture of renal glomerular and tubular epithelial cells.Diabete Metab 1975 Dec 1(4):227-34
30． Rudolfs K.Zalups,Lawrence H.Lash.METHODS IN RENAL TOXICOLOGY CRC Press ,Inc.1996，190
31． 郑丰，黎磊石。大黄对体外肾小管细胞增殖的影响。中华医学杂志 1993，（73）6：343
32． 王东，吴雄飞，金锡御。大鼠肾小管上皮细胞的原代培养及传代。中华实验外科杂志 1999，16（2）：179
33． KADKHODAEE M，HANSON GR，et al. Detection of hydroxyl and carbon-centred radicals by EPR spectroscoopy after ischemia and reperfusion in the rat kidney. Free Radic Res 1996，25:31-42
34． KADKHODAEE M,GOBE G,et al. DNA frahmentation reduced by anti-oxidants following ischemia-reperfusion in the isolated perfused rat kidney.Nephrology 1998，4:163-175
35． 李建明，蔡黔，周红等。过氧化氢对肠上皮细胞线粒体的损伤作用。第三军医大学学报，2002，24（2）：142
36． Sandstrom PA, Tebbey PW, et al. Lipid hydroperoxides induce apoptosis in T cells displaying a HIV-associated glutathione peroxides deficiency. J Biol Chem,1994，269:798-801
37． 陆怡，许彩民，潘华珍。氧化诱导细胞凋亡初探。解剖学报，1995，26：398
38． 迟先煊，郭畹华等。过氧化氢诱发小脑皮质神经元凋亡。神经解剖学杂志，1998，14（1）：19-22
39． 陈凤华等 牛磺酸抑制过氧化氢诱导牛晶状体上皮细胞凋亡的实验研究。中华眼科杂志，2000，4:32
2． Weiberg JM.The cell biology of ischemic renal injury.Kidney Int 1991,39(3):476
3． Kerr,JFR,Wyllie AH, Currie :Apoptosis:A basic biological phenomenon with wide-ranging implications in tissue kinetics. Br J Cancer 1972,26:239-257
4． Schumer M,Colombel MC,et al.Morphologic,biochemical and molecular evidence of apoptosis during the reperfusion phase after brief periods of renal ischemia. Am J Pathol,1992，140:831
5． Searle J,Kerr JFR,Bishop CJ.Necrosis and apoptosis:Distinct modes of cell death with fundamentally different significance.Pathol Annu 1982,17(2):229-259
6． Arends MJ,Morris RG,Wyllic AH.Apoptosis ,the role of the endonuclease.Am J Pathol 1990,136:593-608
7． 赵明中,汪家瑞, 魏嘉平,等 复方丹参滴丸对大鼠心肌缺血再灌注时心肌细胞凋亡及调亡相关基因表达的影响. 中国临床药理学杂志 1999，15(4):288-291
8． 陈敏、郭仁寿，等. 肾缺血再灌注损伤中氧自由基与细胞凋亡. 实用儿科临床杂志 1999,5:283-284
9． 黄海长,王海燕,等 急性肾缺血再灌注后修复过程中的细胞凋亡, 中国病理生理杂志 1996.(4):354
10． 廖洪军,陈香美,等 缺血性肾损伤中细胞凋亡的初步观察, 中华医学杂志 1994,10:629-631
11． Detrisac CJ,et al.Tissue culture of human kidney epithelial cells of proximal tubular origin.Kidney Int 1984，25:383
12． Rudolfs K.Zalups,Lawrence H.Lash.METHODS IN RENAL TOXICOLOGY CRC Press ,Inc.1996，190
13． 司徒镇强，细胞培养， 西安：兴墨图书出版公司 1996，173
14． LAWRENCE H.LASH,In vitro methods of assessing renal damage.TOXICOLOGIC PATHOLOGY,1998，26(1)pp:33-42
LEILA CUTTLE，XIAO-JU ZHANG，ZOLTAN H. ENDRE,et al . Bcl-Xl
15． translocation in renal tubular epithelial cells in vitro protects distal cells from oxidative stress. Kidney Int 2001，59:1779-1788
16． 方福德等，分子生物学前沿技术，北京医科大学中国协和医科大学联合出版社，1998，7
17． 刘海峰、刘为纹等 幽门杆菌诱导胃粘膜上皮细胞凋亡和增殖与P53基因表达。世界华人消化杂志，2001，11
18． 田劲，陈香美，黎磊石。 冬虫夏草、大黄及肾大部分切除大鼠血清对肾小管上皮细胞生长的影响。中西医结合杂志。1991；9：547-549
19． 谢锦玉， 现代细胞化学技术及其在中西医药中的应用， 中医古籍出版社 1998，16
20． KAKO K,KATO M,MATSUOKA T,MUSTAPHA A:Depression of membrane-bound Na+-K+-ATPase activity induced by free radicals and by ischemia of kidney .Am J Physiol 1988，254:C330-C337
21． PALLER MS ,HEDLUND BE:The role of iron in postischemic renal failure in the rat. Kidney Int 1988，34:474-480
22． VAN WAARDE A,STROMSKI ME,THULIN G,GAUDIO KM, et al :Protection of the kidney against ischemic injury by inhibiton of 5'-nucleotidase. AM J Physiol 1989，256:F298-F305
23． CHAN L ,LEDINGHAM JGG,CLARKE J,ROSS BD:The importance of pH in acute renal failure,in Acute Renal Failure,edited by ELIAHOU HE ,London, Libbey,1982,pp58-61
24． YOUNG EW, HUMES HD:Calcium and acute renal failure.Miner Electrolyte Metab 1991,17:106-111
25． Sato N, Iwata S, et al. J Immunol,1995,154:3149-3203
26． Olsen S, Burdick JF,et al. Primary acute renal failure :acute tubular necrosis in the transplanted kidney morphology and pathogensis.Medicine Baltimore. 1989,68:173
27． Gobe GC,Axelsen RA, et al. Cellular events in experiment unilateral ischemic renal atrophy and in regeneration after contralateral nephrectomy. Lab Invest 1990,63:700
28． Allen J,Gobe GC, et al. Effects of hypoxia on morpholoical and biochemical characteritics of renal epithelial cell and tubular cultures. Renal Failure 1992,14:453
Foidart-Willems J,Dechenne C,Mahien P.Biosynthesis of basement
29． membrane collagen in culture of renal glomerular and tubular epithelial cells.Diabete Metab 1975 Dec 1(4):227-34
30． Rudolfs K.Zalups,Lawrence H.Lash.METHODS IN RENAL TOXICOLOGY CRC Press ,Inc.1996，190
31． 郑丰，黎磊石。大黄对体外肾小管细胞增殖的影响。中华医学杂志 1993，（73）6：343
32． 王东，吴雄飞，金锡御。大鼠肾小管上皮细胞的原代培养及传代。中华实验外科杂志 1999，16（2）：179
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