摘要
目的探讨骨形成蛋白7(BMP-7)对单核细胞趋化蛋白1(MCP1)诱导体外培养人肾小管上皮细胞转分化的作用,并初步探讨该作用与转化生长因子β1(TGFβ1)、Smad3表达变化的关系。方法将体外培养HK2细胞分为以下各组:阴性对照组;阳性对照组:TGF-β1(5ng/ml)处理;MCP-1(0.1,1,10及50ng/ml)组,BMP7组(0.1,1,10,50ng/ml);MCP1(1ng/ml)加BMP-7(50ng/ml)组;MCP1(1ng/ml)加MCP1中和抗体(5μg/ml)组。应用半定量RTPCR方法检测HK-2细胞α平滑肌肌动蛋白(αSMA)mRNA表达,ELISA方法测定上清液中I型胶原分泌,Westernblot方法检测HK2细胞TGFβ1及Smad3表达。结果MCP1(0.1,1ng/ml)组HK2细胞αSMAmRNA表达较阴性对照组明显增强(P<0.01)。ELISA方法测定MCP1(0.1,1ng/ml)组上清液中I型胶原分泌明显高于阴性对照组(P<0.01)。MCP-1中和抗体与MCP-1(1ng/ml)共同作用24h后,HKC细胞αSMAmRNA表达几乎被完全抑制(P<0.001),而BMP7(50ng/ml)与MCP1(1ng/ml)共同作用24h后HK2细胞αSMAmRNA表达仅部分被抑制(P<0.01);MCP-1中和抗体或BMP7(50ng/ml)与MCP1(1ng/ml)共同作用24h后Ⅰ型胶原分泌较MCP1(1ng/ml)组明显降低(P<0.05)。Westernblot方法检测显示,MCP1(1ng/ml)组HK2细胞TGF-β1及Smad3表达水平均较阴性对照组明显上调(P<0.01)。MCP1中和抗体或BMP7(50ng/ml)与MCP-1(1ng/ml)共同作用24h后,HK2细胞TGF-β1表达均分别比MCP1(1ng/mL)单独作用明显下调,以MCP1中和抗体的作用更强(P<0.01,P<0.05);在MCP1中和抗体或BMP7作用24h后,Smad3表达也有类似下调(P<0.01,P<0.05)。结论MCP1能诱导体外培养的HK2细胞发生转分化,该作用可能与TGFβ1及Smad3表达上调有关。BMP7能部分抑制MCP1诱导HK2细胞转分化,该作用可能与TGFβ1及Smad3表达部分下调有关;同时提示MCP1诱导的转分化可能涉及非TGFβ依赖的细胞信号传导途径,需进一步研究。
Objective To examine the effect of Bone Morphogenetie protein-7 (BMP-7) on Monoeyte ehemoattraetant protein-1 ( MCP-1 ) induced epithelial-myofibroblast transition (EMT) in cultured renal proximal tubular cells (HK-2) and the relationship between TGF-β1-smad3 expressions and MCP-1 induced EMT. Methods The cultured HK-2 cells were divided into six groups: a,negative control, b, treated with TGF-β1 (5 ng/ml) as positive control, e, treated with MCP-1 (0. 1, 1, 10, 50 ng/ml), d, treated with BMP-7 (0. 1, 1, 10, 50 ng/ml) , e. co-treated with MCP-1 (1 ng/ml) and MCP-1 neutralized antibody (1 ng/ml) , f. co-treated with MCP-1 (1 ng/ml) and BMP-7 (50ng/ml). or-Smooth Muscle Aetin (α-SMA) mRNA expression of HK-2 cells was assessed with RT-PCR. Secretion of type Ⅰ collagen was assessed with RT-PCR and ELISA, respectively. TGF-β1 and Smad 3 expressions were assessed with Western blot. Results α-SMA mRNA expression significantly increased in HK-2 cells treated with MCP-1 (0. 1 ,1 ng/ml) compared with negative controls(5.97 ±0. 35,23.36 ± 1.37 vs. 0. 59 ±0. 38 ,P 〈0. 01 ). α-SMA mRNA expression of HK-2 ceils concomitantly treated with MCP-1 neutralized antibody or BMP-7 (50 ng/ml) and MCP-1 (1 ng/ml) significantly decreased than that in cells treated with MCP-1 (1 ng/ml) alone( 1.93 ±0. 34, 13.59 ±0. 38 vs. 36. 36 ± 1.37, P 〈 0. 01 ). Secretion of type Ⅰ collagen of the cells treated with MCP-1 (0. 1, 1 ng/ml) markedly increased compared with negative control (1751 ±34,1876 ± 45 vs. 1450 ± 62; P 〈 0.01 ). The secretion of type Ⅰ collagen of the supernatant were also significantly lower than that in cells treated with MCP-1 ( 1 ng/ml) alone ( 1462 ± 56, 1596 ± 34 vs. 1876 ± 45, P 〈 0. 05). The expression of TGF-β1 and Smad 3 of HK-2 cells treated with MCP-1 ( 1 ng/ml) were markedly higher than that of negative controls, respectively (36. 31 ± 1.37 vs. 0. 75 ± 0. 16, P 〈 0. 01 ; 56. 98 ± 2. 61 vs. 23.05 ± 1.82, P 〈0.01 ) The expressions of TGF-β1 and Smad 3 in HK-2 cells treated concomitantly with MCP-1 neutralized antibody or BMP-7 (50 ng/ml) and MCP-1 ( 1 ng/ml) were markedly decreased than that treated with MCP-1 alone, respectively. (4.61 ±0.74,23.74 ±2. 14 vs. 36.31 ± 1.37, P 〈 0.01 ;19. 63±1.65,37.06 ± 1.82 vs. 56. 98 ±2. 61 ,P 〈0. 01 ). The expressions of TGF-β1 and Smad 3 in HK-2 cells treated concomitantly with MCP-1 neutralized antibody or BMP-7 (50 ng/ml) and MCP-1 ( 1 ng/ ml) were markedly decreased than that treated with MCP-1 alone, respectively. (4.61±0.74,23.74 ± 2. 14 vs. 36. 31 ± 1.37, P〈0.01 ;19. 63 ± 1.65,37. 06 ± 1.82 vs. 56. 98 ±2.61 ,P〈0. 01 ). Conclusions The results documented that MCP-1 may induce EMT of HK-2 cells in vitro, and this effect is related to upregulated expression of TGF-β1 and Smad 3. BMP-7 may partially inhibite MCP-l-induced EMT and this effect is related to the downregulated expression of TGF-β1 and Smad 3 of the cells. The results also suggest that MCP-1 induced EMT may involve the TGF-β1-independant pathway of the cells.
出处
《中华医学杂志》
CAS
CSCD
北大核心
2005年第37期2607-2612,共6页
National Medical Journal of China
基金
北京市自然基金资助项目(7042043)