Ghrelin／GHS—R1a对鼠GH3细胞株增殖的影响及其作用机制

The proliferative effect of Ghrelin/GHS-R on rat GH3 cell line and its molecular mechanism

目的探讨Ghrelin／GHS-R1a对鼠GH3细胞株增殖作用的影响及其机制。方法采用噻唑蓝（MTT）比色法和免疫印迹（Western blot）法，检测1×10^-10～1×10^-6mol／L不同浓度的Ghrelin对GH3细胞增殖作用的影响，以及其作用GH3细胞株后总ERK和磷酸化ERK蛋白的表达，同时检测PKC抑制剂、酪氨酸激酶抑制剂和MEK抑制剂对Ghrelin／GHS—R1a作用GH3细胞后，其肿瘤细胞增殖抑制率和总ERK以及磷酸化ERK蛋白的表达。结果不同浓度（1×10^-10～1×10^-6）mol／L的Ghrelin作用GH3细胞测得的肿瘤细胞增殖率（％）分别为107±5、157±3、135±18、197±8和171±10，与对照组比较差异有统计学意义（P〈0．01）；而其磷酸化ERK和总ERK的灰度比值分别为0．412±0．064、0．612±0．045、0．573±0．038、0．675±0．034和0．631±0．053，与对照组比较差异有统计学意义（P〈0．05）。而PKC抑制剂GF109203X、酪氨酸磷酸激酶抑制剂Genistein和MAPK激酶抑制剂U0126对Ghrelin／GHS—R1a作用GH3细胞后，其肿瘤细胞增殖明显被抑制，差异有统计学意义（P〈0．01）。结论Ghrelin／GHS·R1a对GH3细胞株具有促增殖作用，且是通过激活MAPK途径中的ERK发挥作用的。

Objective To explore the proliferative effect of Ghrelin/GHS-Rla on rat GH3 cell line and its molecular mechanism. Methods The effect of ghrelin （ at 1 × 10^-10-1 × 10^-6 mol/L concentrations） on proliferation of GH3 ceils was studied by using MTF methods. The protein of total ERK and phosphorylated ERK was detected by Western blot. The activation of the MAPK pathway was studied by using Western blot and inhibitors of ERK, PKC and tyrosine phosphatase pathways. Results Ghrelin signifieandy increased proliferation at 1 × 10^-10 mol/L to 1 × 10^-6 mol/L concentrations （ 1 × 10^-10 mol/L for 107 ±5,1 × 10-9 mol/L for 157 ± 3,1 × 10^-8 mol/L for 135 ± 18,1 × 10^-7 mol/L for 197 ± 8, and 1 × 10^-6 mol/L for 171 ± 10, respectively） as compared with untreated controls （P 〈 0.01 ）. And ghrelin also caused a significant increase in phosphorylated ERK in Western-blot at 1 × 10^-10 mol/L to 1 × 10^-6 mol/ L concentrations （ 1 × 10^-10 mol/L for 0. 412 ± 0.064,1×10^-9 mol/L for 0. 612 ± 0. 045,1 × 10 ^-8 mol/L for 0. 573 ± 0. 038,1 × 10^-7 mol/L for 0.675 ± 0. 034, and 1 × 10^-6 mol/L for 0.631 ± 0.053 ;respectively） as compared with untreated controls （P 〈 0.05 ）. Ghrelin （ at 1 × 10^-7 mol/L concentration） caused a significant increase in phosphorylated ERK1/2. The positive effect of ghrelin on MTT was abolished by the MAPK kinase inhibitor U0126 ,the PKC inhibitor GF109203X and the tyrosine kinase inhibitor Genistein. This could also be clearly demonstrated by Western blot analysis, where a transient increase in ERK1/2 phosphorylation by ghrelin was attenuated by all three inhibitors. Conclusion This study has shown a novel role for ghrelin in stimulating the proliferation of rat GH3 cell line, suggesting that ERK activation is involved in mediating the effects of ghrelin on cell proliferation.