【作者】 胡向阳；

【导师】 张荣华；

【作者基本信息】 暨南大学 ， 中西医结合临床， 2008， 博士

【摘要】 目的:体外分离、培养大鼠骨髓间充质干细胞（Mesenchymal stem cells,MSCs）,研究MSCs生物学特性。观察丹参、藜芦水煎液诱导MSCs向神经元样细胞分化的作用,研究丹参、藜芦水煎液对分化后神经元样细胞SMNmRNA表达的影响;并采用血清药理学方法,研究丹参、藜芦含药血清诱导MSCs分化为神经元样细胞的作用。探索提高MSCs向神经元样细胞分化和上调分化后神经元样细胞SMNmRNA表达的干预因素,为进行神经元样细胞移植治疗脊髓。性肌萎缩症提供基础。方法:1.采用全骨髓贴壁法分离培养MSCs;通过形态学观察、绘制生长曲线、流式细胞仪进行MSCs表面抗原鉴定和检测MSCs生长周期来鉴定MSCs;建立体外培养的稳定MSCs细胞株系。2.观察丹参、藜芦水煎液诱导MSCs向神经元样细胞分化的作用,免疫组化法检测神经元样细胞Nestin、NSE、GFAP,摸索β-巯基乙醇、丹参水煎液、藜芦水煎液、丹参+藜芦水煎液的最佳诱导浓度,观察各组最佳浓度诱导MSCs向神经元样细胞分化的作用。3.观察丹参、藜芦水煎液对分化后神经元样细胞SMNmRNA表达的影响,FQ-PCR法检测空白组、β-巯基乙醇诱导组、丹参水煎液组、藜芦丹参水煎液组、丹参+藜芦水煎液组对分化后神经元样细胞SMNmRNA表达的影响。4.观察丹参、藜芦含药血清诱导MSCs向神经元样细胞分化的作用,免疫组化法检测神经元样细胞Nestin、NSE、GFAP,摸索丹参含药血清、藜芦含药血清、丹参+藜芦含药血清的最佳诱导浓度,观察各组最佳浓度诱导MSCs向神经元样细胞分化的作用。结果:1.全骨髓贴壁法分离培养的MSCs具有均质性好、增殖活力强、传代生长快的特点。本实验培养的MSCs经流式细胞仪检测,表达整合素家族成员的CD29和粘附分子CD44,不表达泛白细胞标志CD45;MSCs处于G₀-G₁期的细胞为87.63%,G₂-M期的细胞为3.57%,S期的细胞为8.80%:生长曲线均呈S形,接种1-3 d细胞的OD值变化不大,到第3 d细胞数目明显增加,第8 d到达顶点,之后减少;证实已成功建立稳定的MSCs株系。2.β-巯基乙醇、丹参水煎液、藜芦水煎液和丹参+藜芦水煎液最佳诱导MSCs向神经元样细胞分化浓度分别是β-巯基乙醇1mmol/L的无血清L-DMEM培养液、丹参水煎液120μl/ml10%胎牛血清L-DMEM培养液、藜芦水煎液140μl/ml 10%胎牛血清L-DMEM培养液和丹参+藜芦80μl/ml 10%胎牛血清L-DMEM培养液。空白组Nestin、NSE和GFAP表达均为阴性。丹参+藜芦水煎液组Ncstin阳性细胞率高于β-巯基乙醇组,P＜0.01;丹参+藜芦水煎液组NSE阳性细胞率高于β-巯基乙醇诱导组,P＜0.05;丹参+藜芦水煎液组Nestin、NSE阳性细胞率分别高于丹参水煎液和藜芦水煎液,P＜0.05;各实验组GFAP表达均为阴性。3.β-巯基乙醇组、丹参水煎液组、藜芦水煎液组、丹参+藜芦水煎液组分化后神经元样细胞SMNmRNA表达丰度值均高于空白组,P＜0.01;丹参+藜芦水煎液组SMNmRNA表达丰度值高于β-巯基乙醇组,P＜0.05:丹参+藜芦水煎液组SMNmRNA表达丰度值高于丹参水煎液组、藜芦水煎液组,P＜0.01;β-巯基乙醇组SMNmRNA表达丰度值高于丹参水煎液组、藜芦水煎液组,P＜0.05。4.丹参含药血清组、藜芦含药血清和丹参+藜芦含药血清最佳诱导MSCs向神经元样细胞分化浓度分别是丹参含药血清20μl/ml 10%胎牛血清L-DMEM培养液、藜芦含药血清25μl/ml 10%胎牛血清L-DMEM培养液和丹参+藜芦含药血清15μl/ml 10%胎牛血清L-DMEM培养液。空白组Ncstin、NSE和GFAP表达均为阴性。丹参+藜芦含药血清诱导组Nestin阳性细胞率高于β-巯基乙醇诱导组,P＜0.05;丹参+藜芦诱导组NSE阳性细胞率高于β-巯基乙醇诱导组,P＜0.01;各实验组GFAP表达均为阴性。结论:1.运用全骨髓贴壁法,可以建立体外稳定培养的MSCs株系。2.丹参水煎液、丹参+藜芦水煎液可以诱导体外培养的MSCs向神经元样细胞分化,藜芦水煎液诱导MSCs向神经元样细胞分化作用不明显,丹参+藜芦水煎液诱导MSCs向神经元.样细胞分化作用高于其它诱导组,MSCs没有诱导因素的作用不会向神经元样细胞分化。3.丹参、藜芦水煎液可以促进分化后神经元样细胞表达SMNmRNA,丹参+藜芦水煎液对神经元样细胞SMNmRNA表达作用强于丹参水煎液或藜芦水煎液,分化后的神经元样细胞在丹参+藜芦水煎液干预下有向运动神经元细胞进一步分化的趋向。4.丹参+藜芦含药血清组促MSCs向神经元样细胞分化作用弱于丹参+藜芦水煎液组,原因可能是丹参+藜芦组水煎液经过大鼠机体代谢,含药血清中促MSCs向神经元样细胞分化作用的有效组份（群）降低所致。更多还原

【Abstract】 Objective:Mesenchymal stem cells(MSCs) were isolated from the Spragu-Dawley rats and cμlutred in vitro,then to study the biological characteristics of MSCs.Compairing the effects of that MSCs were dieffrentiated into neuron-like cells,which inducing by the decoction and drug-containing serum of Salvia Mitiorrhiza,Veratrum nigrum.To investigate the expression of SMNmRNA in neuron-like cells,which inducing by the decoction Salvia Mitiorrhiza and Veratrum nigrum,and to analyse the mechanism of them.To explore the ideal factors which can elevate the differentiation percentage of MSCs dieffrentiated into neuron-like cells,and also to explore the ideal condition which can enhance the expression of SMNmRNA in neuron-like cells.It might offer reference for transplanting neuron-like cells to treat spinalm uscμlarat rophy(SMA).Methods:1.To investigate the effects of MSCs were isolated from the rats by means of the density-gradient centrifugation and the differential time adherent method.The stable and uniformal MSCs were instaurating in vitro.To observe the morphologic characteristics of MSCs,MTT method was used to investigate the proliferation activity of MSCs,the growth curve of MSCs was drawing according as proliferation activity of MSCs.The cell surface antigens and Cell cycle of MSCs were detected by flow cytometry(FCM).2.The effects of that MSCs were dieffrentiated into neuron-like cells induced by control group, classic group,Salvia Mitiorrhiza,Veratrum nigrum,Salvia Mitiorrhiza and Veratrum nigrum,was investigated,To explore the most effective concentration of the above groups on differentiation MSCs into neuron-like cells.Neuron-specific enolase(NSE),Nestin,gliall fibrillary acaidic protein(GFAP) of neuron-like cells were detected by immunocyto chemistry.The morphologic characteristics and the proliferation of neuron-like cells was observed.3.FQ-PCR was used to detect the expression of SMNmRNA in neuron-like cells,which inducing by control group,classic group,Salvia Mitiorrhiza decoction,Veratrum nigrum decoction, Salvia Mitiorrhiza and Veratrum nigrum decoction,To analyse expression of SMNmRNA in the above groups.4.To prepare different concentration serum and drug-containing serum(control group serum, Salvia Mitiorrhiza serum,Veratrum nigrum serum,Salvia Mitiorrhiza and Veratrum nigrum serum).To explore the the most effective concentration of the above groups on differentiation MSCs into neuron-like cells.NSE,GFAP,GFAP of neuron-like cells were detected by immunocyto chemistry.The morphologic characteristics and the proliferation of neuron-like cells was observed.Results:1.The MSCs isolated with differential time adherent method has characteristic of high homogenize,proliferating actively and differentiating quickly.The results of the surface antigens of MSCs detected by flow cytometer showed the positive expressions of CD29 and CD44 and the negative expressions of CD45,It is accord with the immunological characteristic of MSCs, It’s also substantiated that MSCs coμld be obtained and purified in vitro by our experimentation..2.The most effective differentiation concentration ofβ-mercaptoethanol(β-ME),Salvia Mitiorrhiza,Veratrum nigrum,Salvia Mitiorrhiza and Veratrum nigrum were L-DMEM cμlture medium containing 1mmol/1β-mercaptoethanol,L-DMEM cμlture medium containing 10% fetal calf seurm(FCS) and 120μl/ml Salvia Mitiorrhiza,L-DMEM cμlture medium containing 10%fetal calf seurm(FCS) and 140μl/ml Veratrum nigrum,L-DMEM cμlture medium containing 10%FCS and 80μl/ml Salvia Mitiorrhiza and Veratrum nigrum respectively.The induced expression rate of NSE and Nestin in the induced groups was higher than that in the control group,P＜0.01;The expression rate of Nestin in Salvia Mitiorrhiza and Veratrum nigrum was higher than that inβ-ME;The expression rate of NSE in Salvia Mitiorrhiza and Veratrum nigrum was higher than that inβ-ME,P＜0.05;but negatively with GFAP.3.The most effective differentiation concentration of Salvia Mitiorrhiza serum,Veratrurn nigrum serum,Salvia Mitiorrhiza and Veratrum nigrum serum were L-DMEM cμlture medium containing 10%fetal calf seurm(FCS) and 20μl/ml Salvia Mitiorrhiza serum,L-DMEM cμlture medium containing 10%fetal calf seurm(FCS) and 25μl/ml Veratrum nigrum serum,L-DMEM cμlture medium containing 10%FCS and 15μl/ml Salvia Mitiorrhiza and Veratrum nigrum serum respectively.The induced expression rate of NSE and Nestin in the induced groups was higher than that in the control group,P＜0.05;The expression rate of Nestin in Salvia Mitiorrhiza and Veratrum nigrum serum was higher than that inβ-ME;The expression rate of NSE in Salvia Mitiorrhiza and Veratrum nigrum serum was higher than that inβ-ME,P＜0.01;but negatively with GFAP.4.The relative expression abundance of SMNmRNA inβ-ME,Salvia Mitiorrhiza,Veratrum nigrum,Salvia Mitiorrhiza and Veratrum nigrum was higher than that in control group,P＜0.01.The relative expression abundance of SMNmRNA in Salvia Mitiorrhiza and Veratrum nigrum was higher than that inβ-ME,P＜0.05.Conclusion:1.The differential time adherent method was a ideal method to isolate MSCs from Spragu-Dawley rat.2.Salvia Mitiorrhiza decoction,Salvia Mitiorrhiza and Veratrum nigrum decoction coμld induce MSCs to differentiate into neuron-like cells in vitro.The effects of that MSCs were dieffrentiated into neuron-like cells induced byeratrum nigrum decoction was indistinctively. The differentiation effects of Salvia Mitiorrhiza and Veratrum nigrum decoction was higher than that in other groups,The reasons might be that there were some combination components and effectiveness in the Salvia Mitiorrhiza and Veratrum nigrum decoction,the contrary integration effects of Salvia Mitiorrhiza and Veratrum nigrum decoction need to be explore profoundly.3.The relative expression abundance of SMNmRNA in Salvia Mitiorrhiza and Veratrurn nigrum decoction was higher than that in other groups.The reasons might be that the SMN gene was in state of silencing,there was no transcription and expression of SMNmRNA.It’s might be the components and effectiveness in Salvia Mitiorrhiza and Veratrum nigrum decoction that activated the SMN gene of neuron-like cells,and enhanced the expression of SMNmRNA.4.The differentiation effects of Salvia Mitiorrhiza and Veratrum nigrum serum was lower than that in Salvia Mitiorrhiza and Veratrum nigrum decoction.The reasons might be that the induced components and effectiveness in Salvia Mitiorrhiza and Veratrum nigrum decoction were weaken by through the metabolism of rats.更多还原