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目的分析生长分化因子11(GDF11)在体外对小鼠海马神经细胞系HT22增殖、分化的影响,探讨GDF11对神经细胞的作用机制。方法用完全培养基(含5%马血清、10%胎牛血清的高糖DMEM培养基)将小鼠海马神经细胞系HT22在37℃和5%CO_2条件下培养过夜后,更换为饥饿培养基(含1%胎牛血清和0.5%马血清的高糖DMEM)继续培养6 h后,分为实验组:弃去原有培养基,加入含有不同浓度GDF11(12.5、25、50、100 ng/m L)的饥饿培养基培养6、24或48 h;对照组:弃去原有培养基,加入与实验组相同体积的饥饿培养基培养6、24或48 h。采用增强型CCK8法检测细胞活力及增殖情况,通过实时定量PCR(qRT-PCR)检测细胞周期蛋白Cycling D2、神经干细胞标志蛋白Nestin、神经元标志蛋白βⅢ-Tubulin、星形胶质细胞标志蛋白GFAP及表皮生长因子受体EGFR、Notch1等信号通路蛋白的mRNA表达情况,通过Western blot方法分析GDF11对HT22细胞的Smad2/3、环磷腺苷效应元件结合蛋白Creb等蛋白的磷酸化的影响。结果处理24 h,实验组HT22细胞活力与对照组相近(P>0.05);处理48 h,25ng/m L r GDF11实验组与对照组细胞活力(%)为74.50!1.45 vs 69.17!0.73(P<0.05),其他实验组(r GDF11浓度分别为12.5、50和100 ng/m L)细胞增殖与对照组比较无明显差别(P>0.05)。qRT-PCR检测:实验组βⅢ-Tubulin基因表达上调明显(P<0.01),且有剂量依赖性;Nestin基因表达下调(P>0.05);GFAP基因表达明显上调(P<0.05);Cycling D2基因、EGFR基因和Notch1基因表达均明显下调(P<0.05)。Western blot分析:实验组Smad2/3蛋白和Creb蛋白的磷酸化与对照组相比明显上调(P<0.05)。结论 GDF11可能通过抑制Notch、EGFR信号通路,激活TGF-β、CREB信号通路等抑制小鼠海马神经细胞系HT22的增殖、促进其向神经元和星形胶质细胞的分化。
Objective To investigate the effects of GDF11 on the proliferation and differentiation of mouse hippocampal neuronal cell line HT22 in vitro and to explore the mechanism of GDF11 on neural cells. Methods The mouse hippocampal neuronal cell line HT22 was cultured in complete medium (5% horse serum and 10% fetal bovine serum high glucose DMEM) overnight at 37 ℃ and 5% CO 2, and then replaced with starvation medium (DMEM with 1% fetal bovine serum and 0.5% horse serum) were cultured for 6 h and then divided into experimental groups: the original medium was discarded and cells containing different concentrations of GDF11 (12.5, 25, 50, 100 ng / m L) for 6, 24 or 48 h. The control group: the original medium was discarded, and the same volume of starvation medium as the experimental group was added for 6, 24 or 48 h. Cell viability and proliferation were detected by enhanced CCK8 assay. Cyclin D2, Nestin, βⅢ-Tubulin, GFAP And epidermal growth factor receptor EGFR, Notch1 and other signaling pathways, and analyzed the effect of GDF11 on phosphorylation of Smad2 / 3 and cyclic adenosine monophosphate (CREB) binding protein Creb in HT22 cells by Western blot. Results After treatment for 24 h, the viability of HT22 cells in the experimental group was similar to that in the control group (P> 0.05). After treatment for 48 h, the cell viability (%) in the 25 ng / mL L GDF11 experimental group and the control group was 74.50! 1.45 vs 69.17! 0.73 <0.05). There was no significant difference in cell proliferation between the other experimental groups (rGFR11, 12.5, 50 and 100 ng / ml) compared with the control group (P> 0.05). qRT-PCR assay showed that the expression of βⅢ-Tubulin in experimental group was significantly up-regulated (P <0.01), and the expression of Nestin gene was down-regulated (P> 0.05); the expression of GFAP gene was up-regulated , EGFR gene and Notch1 gene were significantly down-regulated (P <0.05). Western blot analysis: Smad2 / 3 protein and Creb protein phosphorylation of the experimental group was significantly increased compared with the control group (P <0.05). Conclusion GDF11 may inhibit the proliferation of neuronal cell line HT22 in hippocampus and its differentiation into neurons and astrocytes by inhibiting Notch, EGFR signaling pathway, activating TGF-β and CREB signaling pathway.