PKC和MAPK的激活在TGF-β_1诱导人近端肾小管细胞发生细胞肥大中的作用  

Involvement of PKC and MAPK in TGF-β_1 stimulated cell hypertrophy in human proximal tubular cell

作　　者：罗冬冬[1] 刘必成[1] 文玉杰[1] 刘宏[1] 马坤岭[1] 刘殿阁[1] 

机构地区：[1]东南大学附属中大医院肾脏病研究所,江苏南京210009

出　　处：《东南大学学报（医学版）》2005年第2期71-75,共5页Journal of Southeast University(Medical Science Edition)

基　　金：江苏省自然科学基金资助项目 (BK2 0 0 2 0 52 ) ;"1 35"工程医学重点人才基金资助项目 (RC2 0 0 2 0 64)。

摘　　要：目的 :研究细胞内蛋白激酶C(PKC)和有丝分裂原活化蛋白激酶 (MAPK)途径的激活在转化生长因子 - β1 (TGF - β1 )诱导人近端肾小管上皮细胞 (HK2 细胞 )发生细胞肥大中的作用。方法 :HK2 细胞培养液中预先加入MAPK特异性抑制剂(MEK1 )PD 980 5 9或PKC特异性抑制剂STS ,然后给予TGF - β1 刺激 ,8h后用MTT法检测细胞增殖、[3 H ] 亮氨酸掺入试验检测细胞内蛋白水平、流式细胞仪检测细胞周期分布。结果 :TGF - β1 抑制细胞的增殖、促进细胞内蛋白的合成及促使细胞生长停滞在G0 G1 期 [(TGF - β1 :0 .719± 0 .0 3 2vs .对照组 :1.199± 0 .0 2 2 ,P <0 .0 1) ;(TGF - β1 :2 86.0 0± 10 .15vs .对照组 :2 0 6.2 5± 19.14 ,P <0 .0 1) ;(TGF - β1 :81.63 3 %± 3 .477%vs .对照组 :60 .497%± 4.813 %,P <0 .0 1) ]。PD 980 5 9和STS均能显著抑制TGF - β1 诱导的细胞 [3 H] 亮氨酸掺入增加 (TGF -β1 +PD 980 5 9:2 16.0 0± 13 .13 ,TGF - β1 +STS :2 3 1.5 0± 10 .5 0vs .TGF - β1 :2 86.0 0± 10 .15 ,P分别小于 0 .0 5和 0 .0 1)。TGF - β1 的以上作用均能被STS和PD 980 5 9逆转 (TGF - β1 +STS :67.610 %± 3 .73 2 %,TGF -β1 +PD 980 5 9:65 .62 0 %± 5 .62 5 %vs .TGF- β1Objective To study the role of PKC and MAPK in TGF-β_1 stimulated cell hypertrophy in human proximal tubular cell. Methods All experiments were performed with human transformed proximal tubular epithelial cell line HK_2 under serum-free condition. Growth-arrested HK_2 cells were pre-treated with MEK_1 inhibitor, PD98059 or PKC inhibitor Staurosporine aglycone (STS) before adding TGF-β_1. After 8 hours, incorporation of [ 3H]-leucine was used to assess the level of protein synthesis and MTT was used to determine cell proliferation. Fluorescence-activated cell sorter (FACS) analysis was used to analyze cell cycle distribution.Results TGF-β_1 inhibited cell proliferation (TGF-β_1 group: 0.719±0.032 vs. Control: 1.199±0.022, P<0.01), enhanced incorporation of [ 3H]-leucine [TGF-β_1 group: 286.00±10.15 vs. Control: 206.25±19.14 cpm·(105 cell) -1, P<0.01] and arrested cells in the G_1 phase of the cell cycle (TGF-β_1 group: 81.633%±3.477% vs. Control: 60.497%±4.813% P<0.01). STS could potently attenuate TGF-β_1 induced incorporation of [ 3H]-leucine [TGF-β_1+PD 98059: 216.00±13.13; TGF-β_1+STS: 231.50±10.50 vs. TGF-β_1:286.00±10.15 cpm·(105 cell) -1,P<0.05, 0.01 respectively]. Both STS and PD98059 could reverse the inhibition of TGF-β_1 on cell proliferation in HK_2 cell (TGF-β_1+STS: 1.009±0.022; TGF-β_1+PD 98059: 0.947±0.022 vs. TGF-β_1: 0.719±0.032, P<0.01). FACS demonstrated TGF-β_1 arrested cell cycle at G_0/G_1 phrase, which was reversed when cells were exposed to serum-free medium containing STS+TGF-β_1 or PD98059+TGF-β_1 (Percentage of cells present in G_0/G_1 phrase: TGF-β_1+STS: 67.610%±3.732%; TGF-β_1+ PD 98059: 65.620%±5.625% vs. TGF-β_1: 81.633%±3.477% P<0.01). Conclusion Our results suggest that TGF-β_1induced cell hypertrophy in HK_2 cell may require activation of both PKC and MAPK.

关 键 词：TGF-β1 PD98059 MAPK PKC 诱导 对照组 细胞肥大 有丝分裂原活化蛋白激酶 细胞培养液 细胞内 

分 类 号：R735[医药卫生—肿瘤] R692[医药卫生—临床医学]