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。
Object: To explore the approaches to isolate, culture and identifythe bone marrow mesenchymal stem cells (BMSCs) of SD rat in vitro.
Methods: The BMSCs were enriched and expanded by whole bone marrowculture. The morphology of the BMSCs was examined and analyzed by lightmicroscopy and the purity and phemotype were analyzed by flow cytometry. Theability of osteogenic differentiation of the BMSCs under different inductionconditions was assessed．
Results: The BMSCs expanded by whole bone marrow culture were adherentcells with a similar spindle-shaped morphologh during different passages. Thephemotypes are CD44(96.49%), CD90(95.63%), CD45(0.64%), CD34(0.39%)respectively. The BMSCs could be induced to differentiate into osteoblasts．
Conclusion: Rat BMSCs can be isolated and expanded by whole bone marrowculture, which is an efficient and simple method with high purity and good stability.
Objective: Use glial cell line-derived neurotrophic factor (GDNF) andneurotrophin-3(NT-3) gene transfected rat bone marrow mesenchymal stem cells(BMSCs), to provide the experiment basis of treating neurological disease;
Methods: BMSCs were isolated by the whole bone marrow culture,Characterized by flowcytometry and osteogenic induction. BMSCs with GDNF and NT-3gene were transfected, and morphological changes were measured bymicroscope. The expression of neural specific markers were detected by RT-PCR andimmunofluorescence assay;
Results: BMSCs were cultured and purified in vitro. After the induction ofdifferentiation, BMSCs became round or cone-shaped with distinctive outgrowth ofprotrusions. These cells revealed neuron-like morphological changes, and most ofthese cells were intertwined into a network structure. RT-PCR showed positiveexpressions of GDNF, NT-3, NSE, nestin and MAP-2, and immunofluorescence assayshowed MAP-2and GFAP expression;
Conclusions: Genetically modified BMSCs of co-expressing GDNF and NT-3were able to differentiate into neuronal-like cells and express nerve markers. Thereprovides all experimental basis for gene therapy to treat nervous system-relateddisorders, such as Hirschsprung disease.
Object: The study which compare ion channel current betweenBMSCs and induced neuron-like cells by path clamp technique，and affirm theinduced-neuron electrophysiological function, provide experimental data for entericnervous system diseases therapy;
Methods: Whole-cell patch and perforated patch technique were performed toelucidate the electrical behavior of MSC and induced neuron-like cells by inductorGDNF and NT-3;
Results: Before differentiation, The IKDRcurrent peak is583.6536±74.75945pAat+60mV, current density is10.25393±1.313413pA/pF, the IKCacurrent peak is370.775±49.57507pA，current density is6.513967±0.87096pA/pF, the Itocurrent peak is467.03±68.44461pA，current density is8.205025±1.20247pA/pF; afterdifferentiation, The IKDRcurrent peak is583.6536±74.75945pA at+60mV, currentdensity is10.25393±1.313413pA/pF, the IKCacurrent peak is370.775±49.57507pA，current density is6.513967±0.87096pA/pF, the Itocurrent peak is467.03±68.44461pA，current density is8.205025±1.20247pA/pF; after differentiation, the IKDRcurrentpeak is850.32±53.5708pA, current density is18.72207±1.578505pA/pF, the IKCacurrent peak is452.6455±13.4805pA, current density is8.058586±0.943178pA/pF,theItocurrent peak is621.194±66.039pA, current density is15.00152±1.918339pA/pF.The changes of current peak and current density between before and afterdifferentiation are significant different (P<0.05);
Conclusion: the neuron-like cells differentiated from BMSCs called preliminaryelectrophysiology characteristic of neuron cells, it is in the process from immatureneuron to mature neuron gradually.
引文
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