GDNF和NT-3双基因诱导BMSCs分化为神经样细胞的实验研究

发布时间：2018-03-17 01:21

本文选题：大鼠　切入点：骨髓间充质干细胞　出处：《华中科技大学》2013年博士论文　论文类型：学位论文

【摘要】：目的：建立体外培养扩增、检测鉴定SD大鼠骨髓间充质干细胞(BMSCs)的方法，观察其生物学特性；方法：采取全骨髓培养法分离和纯化大鼠BMSCs，用显微镜观察细胞形体，用流式细胞仪检测细胞纯度，观察BMSCs在诱导条件下向成骨细胞分化的能力；结果：全骨髓培养法培养的BMSCs具有良好的贴壁性，流式细胞仪检测：CD44(96.49%)、CD90(95.63%)、CD45(0.64%)、CD34(0.39%)；第3代细胞经诱导剂诱导可向成骨细胞分化；结论：本实验用全贴壁筛选法培养出高纯度的BMSCs，增殖稳定，在条件诱导下具有多分化的潜能。目的：利用GDNF和NT-3双基因转染诱导大鼠骨髓间充质干细胞(BMSCs)分化为神经样细胞，为其治疗神经性疾病提供实验基础；方法：全骨髓法分离培养BMSCs，流式细胞术及成骨诱导预实验检测BMSCs纯度及特性。转染GDNF和NT-3双基因后，在显微镜下观察细胞形态变化；利用RT-PCR和免疫荧光检测神经细胞特异性标志物表达；结果：BMSCs能在体外成功分离培养，诱导分化后，BMSCs胞体变圆，伸出明显突起，并可见多数细胞相互交织成网状结构，呈神经细胞样形态。RT-PCR检测GDNF、NT-3、NSE、nestin、MAP-2基因表达，免疫荧光标记检测可见表达MAP-2和GFAP；结论：GDNF和NT-3双基因修饰诱导的BMSCs可分化为神经样细胞，并表达神经元的标志，为基因治疗神经系统疾病如先天性巨结肠提供实验基础。目的：本研究利用膜片钳技术，比较MSCs及诱导后神经样细胞之间通道电流的情况，进一步确认分化后神经样细胞的电生理功能，将为肠神经系统缺如疾病的治疗提供前期工作基础。方法：应用膜片钳技术，采用全细胞记录方式，对由GDNF和NT-3双基因诱导的分化前BMSCs和分化后的神经样细胞进行电生理功能检测；结果：分化前记录到延迟整流样钾电流(IKDR)在+60mV时电流大小为583.6536±74.75945pA，电流密度为10.25393±1.313413pA/pF；钙激活钾通道电流（IKCa）在+60mV时记录到电流峰值为370.775±49.57507pA，电流密度为6.513967±0.87096pA/pF；瞬时外向钾通道电流（Ito）在+60mV时电流峰值为467.03±68.44461pA，电流密度为8.205025±1.20247pA/pF；分化后IKDR在+60mV时电流大小为850.32±53.5708pA，电流密度为18.72207±1.578505pA/pF； IKCa在+60mV时电流峰值为452.6455±13.4805pA，电流密度为8.058586±0.943178pA/pF； Ito电流峰值为621.194±66.039pA，电流密度为15.00152±1.918339pA/pF；分化前BMSCs的IKDR和分化后神经样细胞的IKDR在+60mV时电流峰值相比较具有明显的统计学差异（t=2.721，P值=0.015）；分化前IKDRBMSCs电流密度和分化后神经样细胞IKDR电流密度相比较亦具有明显的统计学差异（t=2.441，P值=0.030）；结论：大鼠骨髓BMSCs诱导分化的神经元样细胞初步具有神经元的电生理特性，，是BMSCs由神经前体细胞向成熟神经元这一终点转化过程中由未成熟逐渐迈向成熟的过程。
[Abstract]:Objective: to establish a method for detection and identification of bone marrow mesenchymal stem cells (BMSCs) in SD rats in vitro, and to observe the biological characteristics of the bone marrow mesenchymal stem cells (BMSCs).
Methods: whole bone marrow culture was used to isolate and purify rat BMSCs. Cell morphology was observed by microscope. The purity of cells was detected by flow cytometry, and the ability of BMSCs to differentiate into osteoblasts under induction conditions was observed.
Results: BMSCs cultured in whole bone marrow culture showed good adherence. Flow cytometry showed that CD44 (96.49%), CD90 (95.63%), CD45 (0.64%), CD34 (0.39%), and third generation cells could differentiate into osteoblasts through inducers.
Conclusion: in this experiment, the highly purified BMSCs was cultured with full wall screening method, and the proliferation was stable, and it had the potential of multi differentiation under the condition of condition induced.
Objective: to induce rat bone marrow mesenchymal stem cells (BMSCs) to differentiate into neuron like cells by double gene transfection of GDNF and NT-3, and provide experimental basis for their treatment of neuropathic diseases.
Methods: BMSCs was isolated and cultured in whole bone marrow, and the purity and characteristics of BMSCs were detected by flow cytometry and osteogenic induction. After transfection of GDNF and NT-3 double genes, morphological changes of cells were observed under microscope. RT-PCR and immunofluorescence were used to detect the expression of specific markers of nerve cells.
Results: BMSCs can be successfully isolated and cultured in vitro after induced differentiation, BMSCs cells became round, protruding obvious protrusions, and most visible cells interwoven into the mesh structure, a neural cell like morphology of.RT-PCR NT-3, NSE, detection of GDNF, nestin, MAP-2 gene expression, immunofluorescence detection showed the expression of MAP-2 and GFAP;
Conclusion: GDNF and NT-3 double gene modified BMSCs can differentiate into neuron like cells and express neuronal markers, providing experimental basis for gene therapy of nervous system diseases such as Hirschsprung's disease.
Objective: in this study, patch clamp technique was used to compare the channel currents between MSCs and induced neuron like cells. Further confirmation of the electrophysiological function of neural cells after differentiation will provide a preliminary basis for the treatment of diseases of the intestinal nervous system.
Methods: patch clamp technique and whole cell recording were used to detect the electrophysiological function of pre differentiated BMSCs and differentiated neuron like cells induced by GDNF and NT-3 double genes.
Results: before differentiation recorded delayed rectifier-like potassium current (IKDR) in +60mV when the current size is 583.6536 + 74.75945pA, the current density was 10.25393 + 1.313413pA/pF; calcium activated potassium current (IKCa) in the +60mV record to the current peak was 370.775 + 49.57507pA, the current density was 6.513967 + 0.87096pA/pF; transient outward potassium current (Ito) at +60mV peak current was 467.03 + 68.44461pA, the current density was 8.205025 + 1.20247pA/pF; differentiation after size IKDR in +60mV current was 850.32 + 53.5708pA, the current density was 18.72207 + 1.578505pA/pF; IKCa current at +60mV peak current density was 452.6455 + 13.4805pA, 8.058586 + 0.943178pA/pF; the peak current of Ito was 621.194 + 66.039pA and the current density was 15.00152 + 1.918339pA/pF; BMSCs IKDR before differentiation and differentiation of neuron like cells of the IKDR current in the +60mV when compared with the peak Significant statistical difference (t=2.721, P value =0.015). The IKDRBMSCs current density before differentiation and the IKDR current density after differentiation were also significantly different (t=2.441, P value =0.030).
Conclusion: the neuron like cells derived from rat bone marrow BMSCs have the electrophysiological characteristics of neurons. It is the process from immature BMSCs to mature neurons in the process of transformation from neural precursor cells to mature neurons.

【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R725.7

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