NRG1FP对小鼠精原细胞系GC-1细胞增殖的影响及作用机制研究

Effects of NRG1FP on the proliferation of mouse spermatogonia GC-1 cells and its mechanism

为了研究神经调节蛋白1功能肽段(NRG1FP)对小鼠精原细胞系GC-1细胞增殖的影响及其作用机制,试验采用Western-blot验证精原细胞系GC-1细胞中是否存在NRG1受体——ErbB2和ErbB4。利用大肠杆菌原核表达体系制备NRG1FP蛋白,用NRG1FP和/或细胞外钙离子(Ca^(2+))作用于精原细胞系GC-1细胞后,采用CCK-8试剂盒评价细胞增殖情况,采用Fura-2AM荧光染色观察和测定细胞质Ca^(2+)浓度变化,Western-blot测定基质相互作用分子1(STIM1)、钙通道蛋白Orai1、钙调蛋白(CaM)、CaM依赖性蛋白激酶Ⅳ(CaMKⅣ)、环磷酸腺苷反应元件结合蛋白(CREB)的表达量以及CaMKⅣ、CREB的磷酸化水平。结果表明:精原细胞系GC-1细胞中存在NRG1受体——ErbB2和ErbB4蛋白。大肠杆菌原核表达系统制备的NRG1FP蛋白可显著促进精原细胞系GC-1细胞增殖(P<0.05),且在细胞外存在Ca^(2+)条件下增殖活性最高,显著高于对照组(P<0.05)。NRG1FP蛋白可触发钙池操纵性钙内流(SOCE)。STIM1、Orai1、CaM相对表达量及p-CaMKⅣ、p-CREB蛋白的相对表达量显著上调(P<0.05)。说明NRG1FP可调控SOCE并激活CaM/CaMKⅣ/CREB信号通路,进而促进小鼠精原细胞系GC-1细胞的增殖。

In order to investigate the effects of neuregulin 1 functional peptide(NRG1 FP) on the proliferation of mouse GC-1 spermatogonia and its mechanism, Western-blot was used to verify the presence of NRG1 receptors ErbB2 and ErbB4 in spermatic GC-1 cells. Mice recombinant NRG1 FP was prepared by Escherichia coli expression system and treated with NRG1 FP and/or extracellular Ca^(2+). The cell proliferation was evaluated by Cell Counting Kit-8, and the cytoplasmic Ca^(2+) concentration was observed and measured by Fura-2 AM fluorescent staining. The expression levels of stromal interaction molecule 1(STIM1), calcium channel protein Orai1, calmodulin(CaM), CaM-dependent protein kinase IV(CaMKIV), cyclic adenosine monophosphate response element-binding protein(CREB) and phosphorylation levels of CaMKⅣ, CREB were determined by Western-blot analysis. The results showed that NRG1 receptors, ErbB2 and ErbB4 protein existed in spermatogonial GC-1 cells. NRG1 FP prepared by prokaryotic expression system could significantly promote the proliferation of GC-1 cells(P<0.05), and the proliferation activity of GC-T cells was the highest in the presence of extracellular Ca^(2+), which was significantly higher than that in the aontrot group(P<0.05). NRG1 FP could trigger store-operated calcium entry(SOCE). The relative expression of STIM1, Orai 1, CaM and protein relative expression of p-CaMKⅣ, p-CREB were significantly up-regulated(P<0.05). It indicated that NRG1 FP could regulate SOCE and activate the CaM/CaMKⅣ/CREB signaling pathway, thus promoting the proliferation of mouse spermatogon GC-1 cells.