骨髓间充质干细胞对酒精诱导海马神经元损伤的作用研究

The effect of bone marrow mesenchymal stem cell on alcohol-induced hippocampal neuron injury

目的通过体外观察骨髓间充质干细胞(BMMSC)对酒精诱导海马神经元损伤的作用,初步探讨BMMSC治疗酒精性痴呆(AAD)的可行性。方法取胎鼠海马进行原代神经元培养并进行神经元纯度鉴定。酒精组给予100mol/L酒精37℃培养24h,建立酒精诱导海马神经元损伤模型;BMMSC+酒精组在其培养液中同时加入BMMSC条件培养液(终浓度为50%)及100mol/L酒精,37℃培养24h;另设相同浓度的BMMSC条件培养液对照组(BMMSC组)及空白对照组(对照组)。采用四甲基偶氮唑蓝(MTT)比色法检测各组细胞存活率,以碘化丙啶(PI)/Hoechst33342染色观察细胞形态及凋亡情况。结果原代培养的海马神经元高表达神经元特异性烯醇化酶(NSE),低表达神经胶质纤维酸性蛋白(GFAP),提示成功培养了纯度较高的原代海马神经元。酒精组细胞存活率(0.80±0.09)低于对照组(1.00±0.11;t=4.128,P<0.01)及BMMSC组(1.04±0.07;t=-6.052,P<0.01);BMMSC+酒精组细胞存活率(0.92±0.11)高于酒精组(t=2.555,P<0.05)。BMMSC组及BMMSC+酒精组细胞存活率与对照组比较差异无统计学意义(均P>0.05)。酒精组可见较多的细胞核呈致密浓染及核碎裂的凋亡细胞,BMMSC+酒精组的凋亡细胞数量明显减少。结论 BMMSC可抑制酒精诱导的海马神经元凋亡发生,提示BMMSC移植治疗AAD可能具有一定可行性。

Objective To observe in vitro effects of bone marrow mesenchymal stem cell（BMMSC）on alcohol-induced hippocampal neuron injury and discuss the feasibility of alcohol-associated dementia（AAD）treatment with BMMSC transplantation.Methods Hippocampal neurons got from fetal rats were primarycultured for 6days and identified with immunofluorescence staining.Then they were divided into four groups.Some cells were treated with ethanol（100 mol/L）for 24 hin 37℃（the ethanol group）.BMMSC-conditioned medium（the concentration was 50%）was prepared and co-cultured with ethanol-treated cells for 24h（the BMMSC＋ethanol group）.The BMMSC group was treated with BMMSC-conditioned medium（the concentration was 50%）.For the control group,cells were treated only with DMEM medium.The cell viability was analyzed with MTT assay.The staining with propidium iodide（PI）/Hoechst33342 was used to observe cell apoptosis.Results Primary-cultured hippocampal neurons were NSE-positive and GFAP-negative,suggesting that primary hippocampal neurons with high purity were successfully cultivated.As compared with the control group（1.00±0.11）and the BMMSC group（1.04±0.07）,the cell viability of the ethanol group significantly decreased（0.80±0.09,P0.01）.As compared with the ethanol group,the cell viability of the BMMSC＋ethanol group significantly increased（0.92±0.11,P0.05）.Cell apoptosis was apparently induced by treatment of ethanol（100mol/L）for 24 h.However,co-cultured with BMMSC-conditioned medium（50%,24h）,these effects of ethanol on hippocampal neurons were prevented.Conclusions BMMSC plays a role to inhibit alcohol-induced hippocampal neuron apoptosis and it suggests that BMMSC transplantation is promising for AAD treatment.