转染Nurrl基因的大鼠骨髓基质细胞的诱导分化

目的 通过克隆孤儿核受体(Nurrl)基因并转染大鼠的骨髓基质细胞(MSCs),探讨Nurrl基因对MSCs在三七总皂甙(tPNS)和全反式维甲酸(ATRA)的联合诱导下向神经元分化的影响.方法 构建pcDNA3.1-hygro-Nurrl表达质粒,应用脂质体Lipofectamine 2000转染MSCs.转染前MSCs以相同的密度铺6孔板,并随机设置4个实验组,分别是:Nurrl tPNS/ATRA组、tPNS/ATRA组、Nurrl组和对照组,利用免疫细胞化学方法检测Nurrl基因的表达.转染48 h对Nurrl tPNS/ATRA组和tPNS/ATRA组的细胞,依次给予β-疏基乙醇(BME)预诱导和tPNS/ATRA诱导,而Nurrl组和对照组的细胞除给予培养基代替tPNS/ATRA诱导剂外,其余步骤均相同;检测4组细胞的酪氨酸羟化酶(TH)、乙酰胆碱酯酶(AChE)和γ-氨基丁酸(GABA)抗体的免疫细胞化学结果并统计各组阳性细胞的比例.结果 免疫细胞化学显示,转染的骨髓基质细胞表达Nurrl蛋白;Nurrl tPNS/ATRA组TH阳性细胞比例明显高于tPNS/ATRA组,分别是(38.4±4.6)%和(5.9±3.4)%.结论 用tPNS/ATRA作诱导剂,转染Nurrl的MSCs诱导后TH阳性细胞的比例明显高于未转染组,差别有统计学意义;tPNS/ATRA诱导MSCs分化成神经元细胞的作用明确.

THE INDUCEMENT AND DIFFERENTIATION OF THE RATS' BONE MARROW STROMAL CELLS TRANSFECTED WITH NURRL GENE

Objective To study the effect of Nurrl gene on the differentiation of rats’ marrow stromal cells (MSCs) into neurous under the co inducement of total panax notoginserg saponins (tPNS) and all trans retineic acid (ATRA) by coloning the Nurrl gene and transfecting it into MSCs. Methods Expressing plasmids pcDNA31 hygro Nurrl were cloned, then transfected into MSCs with lipofectamine 2000. To begin with, MSCs were subcultured into 6 wells cultured plate at about 5×105 cells/well density and the wells were divided into four groups randomly which were Nurrl+tPNS/ATRA group, tPNS/ATRA group, Nurrl group and control group. Secondly, the plasmids were introduced to the MSCs in Nurrl+tPNS/ATRA group and Nurrl group,then protein expression of Nurrl was identified with immunocytochemistry. Thirdly, after the MSCs and plasmids had been co cultured for 48 hours, cells in Nurrl+tPNS/ATRA group and tPNS/ATRA group were induced with BME in advance then with tPNS/ATRA in due form. For cells in Nurrl and control group, the only difference was that tPNS/ATRA was replaced with the culture. Finally we compared the different percentage of positive cells in four groups with TH, AChE and GABA antibodies by immunocytochemistry method. Results The immunocytochemical test showed that the MSCs transfected with Nurrl gene expressed Nurrl protein. The percentage of positive cells of TH antibody in Nurrl+tPNS/ATRA group was (38.4±4.6)% distinctly higher than that of tPNS/ATRA group, which was (5.9±3.4)%. Conclusion With tPNS/ATRA induced and immunocytochemistry of TH, positive cells percentage in N