中国全科医学
中國全科醫學
중국전과의학
CHINESE GENERAL PRACTICE
2015年
20期
2433-2438
,共6页
陈艳霞%杨丽萍%吴险峰%秦晓华%黄翀%房向东%涂卫平
陳豔霞%楊麗萍%吳險峰%秦曉華%黃翀%房嚮東%塗衛平
진염하%양려평%오험봉%진효화%황충%방향동%도위평
糖尿病肾病%红细胞生成素%细胞转分化%白细胞介素6%肿瘤坏死因子α%RhoA/ROCK信号通路
糖尿病腎病%紅細胞生成素%細胞轉分化%白細胞介素6%腫瘤壞死因子α%RhoA/ROCK信號通路
당뇨병신병%홍세포생성소%세포전분화%백세포개소6%종류배사인자α%RhoA/ROCK신호통로
Diabetic nephropathies%Erythropoietin%Cell transdifferentiation%Interleukin - 6%Tumor necrosis factor-alpha%RhoA/ROCK signaling pathway
目的：探讨重组人红细胞生成素（ rhEPO）对高糖诱导的正常人肾小管上皮细胞（ HK-2细胞）转分化过程中炎性因子的变化及其可能机制。方法体外培养HK-2细胞，采用随机数字表法分为空白对照组（未加任何刺激物）、高糖诱导组（高糖终浓度为30 mmol/L）、甘露醇对照组（甘露醇为24.5 mmol/L）、rhEPO对照组（ rhEPO终浓度为20 U/ml ）、不同浓度 rhEPO 干预组（ rhEPO 终浓度分别为5、10、20 U/ml +高糖）及 Rho 激酶抑制剂（Y27632）组（Y27632终浓度为30μmol/L，加入Y2763230 min后加高糖，高糖终浓度为30 mmol/L），以上各组均培养24 h。采用RT-PCR检测各组细胞RhoA mRNA、ROCK1 mRNA表达水平；采用细胞免疫荧光法检测E-钙黏蛋白（E-cadherin）、α平滑肌肌动蛋白（α-SMA）表达水平；采用酶联免疫吸附实验（ELISA）检测人纤维连接蛋白（FN）、白介素6（IL-6）、肿瘤坏死因子α（TNF-α）的蛋白表达水平。结果各组RhoA mRNA、ROCK1 mRNA表达比较，差异均有统计学意义（ P<0.05）；其中高糖诱导组与5 U/ml rhEPO组RhoA mRNA、ROCK1 mRNA表达均高于空白对照组，10 U/ml rhEPO组、20 U/ml rhEPO组及Rho激酶抑制剂组RhoA、ROCK1 mRNA表达均低于空白对照组（P<0.05）；不同浓度rhEPO组RhoA mRNA、ROCK1 mRNA表达均低于高糖诱导组（P<0.05），Rho激酶抑制剂组ROCK1 mRNA表达低于高糖诱导组（ P<0.05）；10 U/ml rhEPO组与20 U/ml rhEPO组RhoA mRNA、ROCK1 mRNA表达均低于5 U/ml rhEPO组（P<0.05）；20 U/ml rhEPO组RhoA mRNA、ROCK1 mRNA表达均低于10 U/ml rhEPO组（P<0.05）。各组α-SMA、E-cadherin、FN、IL-6及TNF-α蛋白表达比较，差异均有统计学意义（P<0.05）；其中高糖诱导组、不同浓度rhEPO组、Rho激酶抑制剂组α-SMA、FN、IL-6及TNF-α蛋白表达均高于空白对照组， E-cadherin均低于空白对照组（ P<0.05）；不同浓度rhEPO组、Rho激酶抑制剂组α-SMA、FN、IL-6及TNF-α蛋白表达均低于高糖诱导组，E-cadherin均高于高糖诱导组（P<0.05）；10 U/ml rhEPO组、20 U/ml rhEPO组α-SMA、FN、IL-6及TNF-α蛋白表达均低于5 U/ml rhEPO组，E-cadherin均高于5 U/ml rhEPO组（P<0.05）；Rho激酶抑制剂组α-SMA、FN蛋白表达均低于5 U/ml rhEPO组，E-cadherin均高于5 U/ml rhEPO组（P<0.05）；20 U/ml rhEPO组α-SMA、FN、IL-6及TNF-α蛋白表达均低于10 U/ml rhEPO组，E-cadherin均高于10 U/ml rhEPO组（P<0.05）；Rho激酶抑制剂组α-SMA、E-cadherin、FN蛋白表达均低于10 U/ml rhEPO组（P<0.05）。Pearson直线相关结果分析，高糖诱导组、不同浓度rhEPO干预组RhoA mRNA与ROCK1 mRNA表达水平呈正相关（ r=0.885、0.901、0.886、0.868，P<0.05）。结论 rhEPO可抑制高糖诱导的HK-2细胞转分化，rhEPO还可以通过减少炎性因子的产生，减轻炎性反应，从而延缓糖尿病肾病（ DN）的进展，延缓肾间质纤维化，其机制可能与RhoA/ROCK信号通路有关。
目的：探討重組人紅細胞生成素（ rhEPO）對高糖誘導的正常人腎小管上皮細胞（ HK-2細胞）轉分化過程中炎性因子的變化及其可能機製。方法體外培養HK-2細胞，採用隨機數字錶法分為空白對照組（未加任何刺激物）、高糖誘導組（高糖終濃度為30 mmol/L）、甘露醇對照組（甘露醇為24.5 mmol/L）、rhEPO對照組（ rhEPO終濃度為20 U/ml ）、不同濃度 rhEPO 榦預組（ rhEPO 終濃度分彆為5、10、20 U/ml +高糖）及 Rho 激酶抑製劑（Y27632）組（Y27632終濃度為30μmol/L，加入Y2763230 min後加高糖，高糖終濃度為30 mmol/L），以上各組均培養24 h。採用RT-PCR檢測各組細胞RhoA mRNA、ROCK1 mRNA錶達水平；採用細胞免疫熒光法檢測E-鈣黏蛋白（E-cadherin）、α平滑肌肌動蛋白（α-SMA）錶達水平；採用酶聯免疫吸附實驗（ELISA）檢測人纖維連接蛋白（FN）、白介素6（IL-6）、腫瘤壞死因子α（TNF-α）的蛋白錶達水平。結果各組RhoA mRNA、ROCK1 mRNA錶達比較，差異均有統計學意義（ P<0.05）；其中高糖誘導組與5 U/ml rhEPO組RhoA mRNA、ROCK1 mRNA錶達均高于空白對照組，10 U/ml rhEPO組、20 U/ml rhEPO組及Rho激酶抑製劑組RhoA、ROCK1 mRNA錶達均低于空白對照組（P<0.05）；不同濃度rhEPO組RhoA mRNA、ROCK1 mRNA錶達均低于高糖誘導組（P<0.05），Rho激酶抑製劑組ROCK1 mRNA錶達低于高糖誘導組（ P<0.05）；10 U/ml rhEPO組與20 U/ml rhEPO組RhoA mRNA、ROCK1 mRNA錶達均低于5 U/ml rhEPO組（P<0.05）；20 U/ml rhEPO組RhoA mRNA、ROCK1 mRNA錶達均低于10 U/ml rhEPO組（P<0.05）。各組α-SMA、E-cadherin、FN、IL-6及TNF-α蛋白錶達比較，差異均有統計學意義（P<0.05）；其中高糖誘導組、不同濃度rhEPO組、Rho激酶抑製劑組α-SMA、FN、IL-6及TNF-α蛋白錶達均高于空白對照組， E-cadherin均低于空白對照組（ P<0.05）；不同濃度rhEPO組、Rho激酶抑製劑組α-SMA、FN、IL-6及TNF-α蛋白錶達均低于高糖誘導組，E-cadherin均高于高糖誘導組（P<0.05）；10 U/ml rhEPO組、20 U/ml rhEPO組α-SMA、FN、IL-6及TNF-α蛋白錶達均低于5 U/ml rhEPO組，E-cadherin均高于5 U/ml rhEPO組（P<0.05）；Rho激酶抑製劑組α-SMA、FN蛋白錶達均低于5 U/ml rhEPO組，E-cadherin均高于5 U/ml rhEPO組（P<0.05）；20 U/ml rhEPO組α-SMA、FN、IL-6及TNF-α蛋白錶達均低于10 U/ml rhEPO組，E-cadherin均高于10 U/ml rhEPO組（P<0.05）；Rho激酶抑製劑組α-SMA、E-cadherin、FN蛋白錶達均低于10 U/ml rhEPO組（P<0.05）。Pearson直線相關結果分析，高糖誘導組、不同濃度rhEPO榦預組RhoA mRNA與ROCK1 mRNA錶達水平呈正相關（ r=0.885、0.901、0.886、0.868，P<0.05）。結論 rhEPO可抑製高糖誘導的HK-2細胞轉分化，rhEPO還可以通過減少炎性因子的產生，減輕炎性反應，從而延緩糖尿病腎病（ DN）的進展，延緩腎間質纖維化，其機製可能與RhoA/ROCK信號通路有關。
목적：탐토중조인홍세포생성소（ rhEPO）대고당유도적정상인신소관상피세포（ HK-2세포）전분화과정중염성인자적변화급기가능궤제。방법체외배양HK-2세포，채용수궤수자표법분위공백대조조（미가임하자격물）、고당유도조（고당종농도위30 mmol/L）、감로순대조조（감로순위24.5 mmol/L）、rhEPO대조조（ rhEPO종농도위20 U/ml ）、불동농도 rhEPO 간예조（ rhEPO 종농도분별위5、10、20 U/ml +고당）급 Rho 격매억제제（Y27632）조（Y27632종농도위30μmol/L，가입Y2763230 min후가고당，고당종농도위30 mmol/L），이상각조균배양24 h。채용RT-PCR검측각조세포RhoA mRNA、ROCK1 mRNA표체수평；채용세포면역형광법검측E-개점단백（E-cadherin）、α평활기기동단백（α-SMA）표체수평；채용매련면역흡부실험（ELISA）검측인섬유련접단백（FN）、백개소6（IL-6）、종류배사인자α（TNF-α）적단백표체수평。결과각조RhoA mRNA、ROCK1 mRNA표체비교，차이균유통계학의의（ P<0.05）；기중고당유도조여5 U/ml rhEPO조RhoA mRNA、ROCK1 mRNA표체균고우공백대조조，10 U/ml rhEPO조、20 U/ml rhEPO조급Rho격매억제제조RhoA、ROCK1 mRNA표체균저우공백대조조（P<0.05）；불동농도rhEPO조RhoA mRNA、ROCK1 mRNA표체균저우고당유도조（P<0.05），Rho격매억제제조ROCK1 mRNA표체저우고당유도조（ P<0.05）；10 U/ml rhEPO조여20 U/ml rhEPO조RhoA mRNA、ROCK1 mRNA표체균저우5 U/ml rhEPO조（P<0.05）；20 U/ml rhEPO조RhoA mRNA、ROCK1 mRNA표체균저우10 U/ml rhEPO조（P<0.05）。각조α-SMA、E-cadherin、FN、IL-6급TNF-α단백표체비교，차이균유통계학의의（P<0.05）；기중고당유도조、불동농도rhEPO조、Rho격매억제제조α-SMA、FN、IL-6급TNF-α단백표체균고우공백대조조， E-cadherin균저우공백대조조（ P<0.05）；불동농도rhEPO조、Rho격매억제제조α-SMA、FN、IL-6급TNF-α단백표체균저우고당유도조，E-cadherin균고우고당유도조（P<0.05）；10 U/ml rhEPO조、20 U/ml rhEPO조α-SMA、FN、IL-6급TNF-α단백표체균저우5 U/ml rhEPO조，E-cadherin균고우5 U/ml rhEPO조（P<0.05）；Rho격매억제제조α-SMA、FN단백표체균저우5 U/ml rhEPO조，E-cadherin균고우5 U/ml rhEPO조（P<0.05）；20 U/ml rhEPO조α-SMA、FN、IL-6급TNF-α단백표체균저우10 U/ml rhEPO조，E-cadherin균고우10 U/ml rhEPO조（P<0.05）；Rho격매억제제조α-SMA、E-cadherin、FN단백표체균저우10 U/ml rhEPO조（P<0.05）。Pearson직선상관결과분석，고당유도조、불동농도rhEPO간예조RhoA mRNA여ROCK1 mRNA표체수평정정상관（ r=0.885、0.901、0.886、0.868，P<0.05）。결론 rhEPO가억제고당유도적HK-2세포전분화，rhEPO환가이통과감소염성인자적산생，감경염성반응，종이연완당뇨병신병（ DN）적진전，연완신간질섬유화，기궤제가능여RhoA/ROCK신호통로유관。
Objective To investigate the effect and possible mechanism of rhEPO in the transdifferentiation of human kidney proximal tubular epithelial cells( HK -2 cells) induced by high glucose and the changes of inflammatory cytokines. Methods By using random number table,HK-2 cells cultured in vitro were divided into several groups:blank control group ( with no irritants),high glucose induced group( with a high glucose final concentration of 30 mmol/L),mannitol control group ( with a mannitol concentration of 24. 5 mmol/L),rhEPO control group( with a rhEPO final concentration of 20 U/ml),three rhEPO intervention groups(with a rhEPO final concentration of 5,10 and 20 U/ml respectively and high glucose added),Rho kinase inhibitor(Y27632)group(in which Y27632 with a final concentration of 30 μmol/L was added and high glucose was added 30 minutes later with a final concentration of 30 mmol/L). After 24 h in virto culture,reverse transcription polymerase chain reaction(RT-PCR)was used to evaluate the mRNA levels of RhoA and ROCK. The levels of E-cadherin and α-smooth muscle actin(α-SMA) proteins were assessed by immunofluorescent test. ELISA was used to measure the levels of Fibronectin(FN),IL-6 and TNF-αproteins. Results The groups were all significantly different(P<0. 05)in the mRNA levels of RhoA and ROCK;the high glucose induced group and the 5 U/ml rhEPO intervention group were higher ( P<0. 05 ) than the blank control group in the mRNA levels of RhoA and ROCK;the 10 U/ml rhEPO intervention group and the 20 U/ml rhEPO intervention group and the Y27632 group were lower ( P <0. 05 ) than the blank control group in the mRNA levels of RhoA and ROCK;the three rhEPO intervention groups were lower ( P <0. 05 ) than the high glucose induced group in the mRNA levels of RhoA and ROCK;the Y27632 group was lower(P<0. 05)than the high glucose induced group in the mRNA levels of ROCK;the 10 U/ml rhEPO intervention group and the 20 U/ml rhEPO intervention group were lower(P<0. 05)than the 5 U/ml rhEPO intervention group in the mRNA levels of RhoA and ROCK;the 20 U/ml rhEPO intervention group was lower (P<0. 05)than the 10 U/ml rhEPO intervention group in the mRNA levels of RhoA and ROCK. The groups were significantly different(P<0. 05)in the protein levels of α -SMA,E -cadherin,FN,IL -6 and TNF -α;the high glucose induced group,the three rhEPO intervention groups and the Y27632 group were higher(P<0. 05)in the protein levels of α-SMA, FN,IL-6 and TNF-αand were lower(P<0. 05)in E-cadherin than the blank control group;the three rhEPO intervention groups and the Y27632 group were lower(P <0. 05)in the protein levels of α -SMA,FN,IL -6 and TNF -α and were higher(P<0. 05)in E-cadherin than the high glucose induced group;the 10 U/ml rhEPO intervention group and the 20 U/ml rhEPO intervention group were lower(P<0. 05)in the protein levels ofα-SMA,FN,IL-6 and TNF-αand higher(P<0. 05)in E-cadherin than the 5 U/ml rhEPO intervention group;the Y27632 group was lower(P<0. 05)in the protein levels ofα-SMA and FN and were higher(P<0. 05)in E-cadherin than the 5 U/ml rhEPO intervention group;the 20 U/ml rhEPO intervention group was lower(P<0. 05)in the protein levels of α-SMA,FN,IL-6 and TNF-αand was higher(P<0. 05) in E - cadherin than the 10 U/ml rhEPO;the Y27632 group was lower ( P <0. 05 ) than the 10 U/ml rhEPO intervention group in the protein levels ofα-SMA,E-cadherin and FN. The pearson linear correlation analysis showed that the mRNA level of RhoA was positively correlated with the mRNA level of ROCK1 in the high glucose group and the three rhEPO intervention groups(r=0. 885,0. 901,0. 886,0. 868;P<0. 05). Conclusion rhEPO could inhibit the transdifferentiation of HK-2 cells induced by high glucose to delay the fibrosis of renal. rhEPO can also reduce the production of inflammatory cytokines,the generation of inflammatory cytokines and inflammatory response, thus delaying the progression of diabetic nephropathy and the fibrosis of renal interstitium. The mechanism may be related to RhoA/ROCK signaling pathway.
