tPNS对hBMSCs诱导分化后神经元样细胞生长状态及凋亡的影响

发布时间：2018-08-04 14:43

【摘要】：目的：研究三七总皂苷(tPNS)与碱性成纤维细胞生长因子(bFGF)在人骨髓间充质干细胞(hBMSCs)分化为神经元样细胞过程中的诱导、抗凋亡作用及作用剂量。方法：hBMSCs取自健康成年志愿者，全骨髓贴壁法分离、培养、扩增、纯化hBMSCs，流式细胞仪检测hBMSCs表面的CD29、CD44、CD105、CD34分子，并测定细胞周期。取第3代hBMSCs，含20%胎牛血清的1mmol/Lβ-巯基乙醇(β-ME)预诱导24h，继而用20ng/mLbFGF（标准对照组）、1.0mg/mL tPNS（药物组）、20ng/mL bFGF+(0.5 mg/mL、1.0 mg/mL、2.0 mg/mL) tPNS（低、中、高剂量联合组）诱导液诱导，设立空白对照组。采用免疫细胞化学法鉴定神经细胞特异性抗原标记神经元特异性烯醇化酶(NSE)、微管相关蛋-2(MAP-2)和神经胶质纤维酸性蛋白(GFAP)的表达；MTT法检测各组诱导剂不同时间点对神经元样细胞活力的影响，TUNEL、Annexin V-FITC/PI凋亡试剂盒测定诱导后各组细胞凋亡率。结果: 1、成功分离培养出hBMSCs，流式细胞仪检测表面标记为：CD29(+)、CD44(+)、CD105(+)、CD34(-)，分离培养的细胞符合hBMSCs表型特征；测定细胞周期示：高比例的G0/G1期细胞提示hBMSCs具有高度分化潜能； 2、免疫细胞化学法测定诱导后细胞：联合组NSE、MAP-2阳性细胞比例高于空白对照组、标准对照组及药物组(P0.05)，，且bFGF与药物联合组各组之间随着药物剂量的增加神经元样细胞特异性标志物阳性率也增加(P0.05)，GFAP表达阴性； 3、MTT测定诱导后细胞活力示：联合组细胞的活性较对照组高(P0.05)，联合组间随着药物剂量的增加细胞活性也增高(P0.05)，且细胞活性高、存活时间延长。 4、分别用TUNEL、Annexin V-FITC/PI凋亡试剂盒检测诱导后各组细胞凋亡率，诱导后联合组细胞凋亡率明显下降(P0.05)，且联合组中随着药物剂量的增加凋亡率随之下降(P0.05)。结论：1、tPNS与bFGF均可诱导hBMSCs分化为神经元样细胞，联合组更能有效诱导使其表达神经细胞表面特异性抗原，且细胞活力较好，与药物剂量呈正相关； 2、tPNS与bFGF诱导hBMSCs分化为神经元样细胞后增殖能力强，联合组诱导后细胞增殖能力及活力更强，存活时间明显延长，与药物剂量呈正相关； 3、tPNS与bFGF诱导hBMSCs分化为神经元样细胞后凋亡率下降，tPNS与bFGF联合诱导可明显减少神经元样细胞凋亡率，与药物剂量呈负相关。
[Abstract]:Aim: to study the induction of panax notoginseng saponins (tPNS) and basic fibroblast growth factor (bFGF) in differentiation of human bone marrow mesenchymal stem cells (hBMSCs) into neuron-like cells. Methods: human hBMSCs were isolated from healthy adult volunteers, isolated, cultured, amplified and purified by whole bone marrow adherent method. Flow cytometry was used to detect CD29, CD44, CD105 and CD34 molecules on the surface of hBMSCs, and the cell cycle was measured. 1mmol/L 尾 -mercaptoethanol (尾 -ME) containing 20% fetal bovine serum was taken from the third generation hBMSCs for 24 hours, and then was induced by 1.0 mg / mL tPNS (drug group) with 20ng/mLbFGF (standard control group) and 20 mg / mL bFGF (0.5 mg / mL mLX 1.0 mg / mL 2.0 mg/mL) tPNS (). The expression of neuron-specific enolase (NSE), microtubule-associated egg -2 (MAP-2) and glial fibrillary acidic protein (GFAP) was detected by immunocytochemistry. The effect of inducer on neuron-like cell viability was detected by MTT assay. Apoptosis rate of each group was determined by Tunel Annexin V-FITC/PI assay. Results: 1. HBMSCs were isolated and cultured successfully, and the surface markers were detected by flow cytometry as: 1) CD29 () CD44 () CD44 () / CD105 () / CD34 (-). The results of cell cycle analysis showed that the high proportion of G0/G1 phase cells indicated that hBMSCs had high differentiation potential. 2.Immunocytochemistry method was used to determine the percentage of NSEP MAP-2 positive cells in the combined group than in the blank control group. The positive rate of neuron-like cell specific markers in the standard control group and drug group (P0.05) was also increased with the increase of drug dose (P0.05). 3MTT assay showed that the cell activity of the combined group was higher than that of the control group (P0.05), and the activity of the combined group was also increased with the increase of the drug dose (P0.05), and the cell activity of the combined group was higher than that of the control group (P0.05). The survival time was prolonged. 4. The apoptosis rate of each group was detected by Tunel Annexin V-FITC/PI apoptosis kit after induction. After induction, the apoptosis rate of combined group decreased significantly (P0.05), and the apoptosis rate decreased with the increase of drug dosage (P0.05). Conclusion both hBMSCs and bFGF can induce the differentiation of hBMSCs into neuron-like cells. The combined group can effectively induce the expression of neuronal surface specific antigen, and the cell viability is better, which is positively correlated with the dosage of the drug. 2the proliferation ability of hBMSCs differentiated into neuron-like cells induced by TPN and bFGF was stronger than that of the combined group, and the survival time was significantly prolonged, which was positively correlated with the dosage of drug. 3The apoptosis rate of hBMSCs differentiated into neuron-like cells induced by bFGF and tPNS was decreased. The apoptosis rate of neuron-like cells induced by tPNS and bFGF was significantly decreased, which was negatively correlated with drug dose.
【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R329

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