人脐血间充质干细胞成肌分化及对尿道括约肌功能影响的实验研究

发布时间：2018-01-12 14:21

本文关键词：人脐血间充质干细胞成肌分化及对尿道括约肌功能影响的实验研究　出处：《第三军医大学》2007年博士论文　论文类型：学位论文

【摘要】： 女性压力性尿失禁(Stress Urinary Incontinence,SUI)是一种常见疾病,其发生率随妇女年龄的增加而增加,影响妇女的生活质量和身心健康。SUI发生的主要原因是尿道高移动性和尿道括约肌功能障碍。女性SUI的治疗方法虽多,但均难从根本上纠正尿道括约肌功能障碍。本课题拟利用基因工程技术将生肌调节因子Myocardin基因转染人脐血间充质干细胞(UB-MSCs),并移植于SUI模型大鼠尿道括约肌周围,研究基因调控的UB-MSCs是否具有向平滑肌细胞分化的能力以及其在尿道括约肌周围存活、分布情况和对尿道括约肌功能的影响,为细胞移植治疗SUI提供理论依据。主要实验结果和结论如下: 1.采用密度梯度离心法从脐血中分离培养出间充质干细胞(UB-MSCs),并通过流式细胞仪对其表面抗原进行鉴定。UB-MSCs均稳定地表达相关的抗原标记CD29、CD44、CD105,但不表达造血细胞系的表面标记CD34、CD45,这与源于骨髓MSCs的表面抗原标记相一致。 2.构建了真核表达质粒载体pEGFP-Myocl。以pAdApt.Myocl为模板进行PCR和琼脂凝胶电泳,切胶回收纯化;反应产物经KpnⅠ和AgeⅠ双酶切后与线性化的载体用T4 DNA连接酶连接,形成pEGFP-Myocl,将之转入工程菌JM109,铺板,挑选阳性克隆,摇菌,提取质粒。目的片段测序分析,与Gene Bank报道序列完全一致。 3.应用脂质体转染的方法,将pEGFP-Myocl转入UB-MSCs中。转染后的细胞经G_(418)筛选,转染效率为15.3%。转染后的MSCs用RT-PCR可检测出Myocardin的表达;荧光显微镜下观察可见胞体内有报告基因产物的绿色荧光;免疫组化检测Myocardin以及其下游蛋白myosin表达为阳性,而对照组为阴性表达。这说明UB-MSCs在Myocardin的调控下可诱导为成平滑肌细胞。 4.通过模拟产伤和卵巢切除成功制作SUI大鼠动物模型。对模型大鼠进行尿动力学检测,最大膀胱容量和漏点压均明显低于正常;HE染色可见近端尿道括约肌萎缩,肌纤维有断裂。 5.将转染Myocardin基因的UB-MSCs扩增后局部植入模型鼠尿道括约肌周围,通过Brdu法观察发现其可在注射点附近短时生长,并表达肌相关蛋白Desmin;通过尿动力学指标的统计分析,细胞移植后SUI模型鼠的控尿功能可以得到改善,但此效应是移植细胞对损伤的修复还是单纯的填充效应,还需进一步研究。总之,本实验成功地构建了质粒载体pEGFP-Myocl,并将其成功地转染人UB-MSCs,使MSCs在Myocardin的调控下分化为成平滑肌细胞;并观察了向平滑肌分化的MSCs对SUI模型大鼠的尿道括约肌周围的分布、分化与生存情况以及对尿道括约肌功能的影响,初步探讨了细胞移植治疗SUI的可行性。本实验为SUI治疗的基础研究,旨在为SUI治疗提供新的思路。
[Abstract]:Female stress urinary incontinence (Stress Urinary, Incontinence, SUI) is a common disease, its incidence with age increasing, the main reason for women's health and life quality of.SUI is of high mobility and urethral sphincter dysfunction. Although there are many methods to treat the female SUI, but are difficult to fundamentally correct the urethral sphincter dysfunction. This project intends to use the technology of gene engineering myogenic regulatory factor Myocardin gene transfection of human umbilical cord blood mesenchymal stem cells (UB-MSCs), and transplanted into SUI rat model of urethral sphincter surrounding, the research of gene regulation whether UB-MSCs can differentiate into smooth muscle cells and its ability to survive in the urethral sphincter around the effect of the distribution, and urethral sphincter function, provide a theoretical basis for cell transplantation in the treatment of SUI.
The main experimental results and conclusions are as follows:
1. by density gradient centrifugation. Mesenchymal stem cells isolated from human umbilical cord blood (UB-MSCs), and were identified by flow cytometry for.UB-MSCs surface antigen for stable expression of antigen CD29, related CD44, CD105, surface marker CD34, but did not express hematopoietic cell line CD45, consistent surface this antigen from the bone marrow of MSCs.
2. to construct the eukaryotic expression plasmid vector pEGFP-Myocl. with pAdApt.Myocl as the template for PCR and agar gel electrophoresis, gel extraction purification; carrier reaction products by Kpn I and Age I digested and linearized by T4 DNA ligase, the formation of pEGFP-Myocl, will be transferred to the engineering bacteria JM109, plank, positive clones, shake bacteria, plasmid extraction. Analysis of DNA sequencing, and Gene Bank consistent with the sequence reported.
Methods 3. application of liposome transfection, pEGFP-Myocl was transformed into UB-MSCs. After transfection, the cells were treated with G_ (418) screening, the transfection efficiency was 15.3%. after transfection with MSCs RT-PCR can detect the expression of Myocardin; observation of green fluorescence reporter gene product in vivo were observed under fluorescence microscope; immunohistochemical detection of Myocardin and its the lower expression of myosin protein was positive, but negative in control group. This shows that UB-MSCs can be induced into smooth muscle cells under the control of Myocardin.
4., SUI rat model was successfully produced by simulating birth injury and ovariectomy. The maximal bladder volume and leak point pressure of model rats were significantly lower than those of normal rats. HE staining revealed proximal urethral sphincter atrophy and muscle fibers breaking.
Around 5. the transfected Myocardin gene was amplified by UB-MSCs after implantation of rat model of urethral sphincter, by the method of Brdu were observed in the growth in the short-term near injection points and the expression of muscle related protein Desmin; urodynamic indexes through statistical analysis, SUI model can be improved in the control of urinary function after transplantation, but this effect is repair of transplanted cells to injury or simply filling effect, further research is needed.
In conclusion, this study successfully constructed pEGFP-Myocl plasmid, and successfully transfected into UB-MSCs, MSCs under the regulation of Myocardin differentiation into smooth muscle cells; and to observe the differentiation of MSCs into smooth muscle around the urethral sphincter of SUI rat model of the distribution, differentiation and survival situation and effect on urethral sphincter the function, discussed the feasibility of cell transplantation for the treatment of SUI. Based on this experiment for the treatment of SUI, in order to provide new ideas for the treatment of SUI.

【学位授予单位】：第三军医大学
【学位级别】：博士
【学位授予年份】：2007
【分类号】：R329

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