【作者】 张飞；

【导师】 刘海涛；

【作者基本信息】 上海交通大学 ， 泌尿外科， 2015， 硕士

【摘要】 目的:糖尿病神经源性膀胱(Diabetic Neurogenic Bladder,DNB)是糖尿病引起的泌尿系统常见并发症,因临床治疗缺乏针对性,只能暂时缓解症状,不能中止DNB的进展和恢复膀胱功能。神经生长因子(Neuron Growth Factor,NGF)减少是DNB重要的致病因素。临床试验证实补充外源性NGF可有效改善DNB,但也存在NGF半衰期短、生物利用度低等问题。人脐带间充质干细胞(Human Umbilical Cord Mesenchymal Stem Cells,hUCMSCs)既具有多向分化潜能,又是一种良好的基因治疗的载体细胞。本课题拟在构建含人NGF的重组慢病毒载体并转染至人脐带MSCs的基础上,体外诱导分化含NGF的人脐带MSCs成为神经上皮祖细胞,使神经上皮祖细胞持续稳定表达NGF,从而使NGF和诱导分化后的人脐带MSCs分别发挥基因治疗和细胞治疗作用,为下一步的实验治疗奠定基础。方法:1.从新生儿脐带中提取hUCMSCs并鉴定其基本生物学特性,采用细胞计数仪计数制作生长曲线、流式细胞仪鉴定hUCMSCs细胞表面抗原,免疫荧光方法验证hUCMSCs的Nestin蛋白表达情况。2、诱导hUCMSCs向成骨方向及成脂方向分化。检测胶原表达、茜素红染色及ALP并评价成骨分化效果。油红O染色评价成脂分化效果。从而验证hUCMSCs的多向分化能力。3、过表达NGF的慢病毒载体构建,筛选最佳MOI后使用慢病毒转染hUCMSCs。荧光显微镜拍照,估算转染效率。4、利用添加bFGF、EGF的无血清D/F12培养基,经过两个阶段诱导过表达NGF的hUCMSCs向神经上皮祖细胞分化,采用形态学观察、免疫荧光、RT-PCR、Western-Blotting、ELISA等多种方法评价诱导效果。结果:1、原代hUCMSCs细胞形态为长梭形,呈漩涡状、鱼鳞样生长,倍增时间为32.1h,hUCMSCs表达间充质表面抗原CD105,CD29,CD13,CD90,不表达CD45,CD14,CD106,CD34,HLA-DR。HLA-DR不表达证明hUCMSCs免疫原性低。2、分别诱导分化hUCMSCs具有成骨和成脂分化潜能,证实hUCMSCs具有多向分化能力。3、构建过表达NGF的慢病毒载体并成功转染至hUCMSCs。4、经过两步诱导法,过表达NGF的hUCMSCs转变成大量神经样细胞团块。免疫荧光染色神经上皮祖细胞抗原标记Nestin和Musha-1高表达,RT-PCR显示Pax-6、Masha-1、Sox-1高表达。ELISA方法检测到培养液中NGF的表达。结论:1.证实提取的新生儿hUCMSCs具有较高的增殖能力及多向分化能力,体外培养多代的hUCMSCs仍然具备干细胞的基本生物学特性且免疫原性低。2.成功构建携带过表达NGF的慢病毒载体。3.利用基因转染技术成功构建过表达NGF的hUCMSCs,并在培养液中得到高效稳定的表达。4.成功诱导分化过表达NGF的hUCMSCs为神经上皮祖细胞,并在培养液中得到高效稳定的表达。5.本试验结果证明hUCMSCs是一种良好的基因治疗的载体细胞,为后续动物实验治疗奠定了基础。更多还原

【Abstract】 Aim: Diabetic neurogenic bladder(DNB) is a common complication of urinary system caused by diabetes. Due to a lack of pertinence in the clinical treatment, we can only temporarily relieve symptoms,but not suspend the progress of the DNB and restore bladder function. A lower expression level of NGF is a important pathogenic factor for DNB. Clinical trials confirm the added exogenous NGF can effectively improve DNB. But there is also a problem NGF has a short half-life and low bioavailability. Umbilical cord mesenchymal stem cells have a abilitry of Multi-directional differentiation potential, is a good vectors for gene therapy. After construction of lentiviral vector carrying NGF gene and differentiation of netural stem cells from h UCMSCs, using the technology of transfection, combined the function of gene therapy and cell therapy.At last, verify the mechanism on animal models.Method:1) Evaluation of the basic biological characteristics of h UCMSCs. The growth curve of h UCMSCs was counted by blood cell counting plate method. h UCMSCs cell surface was demonstrated by flow cytometry. Nestin protein expression was confirmed by immunofluorescence.2) Construction of lentiviral vector carrying NGF gene and its functional inspection.3) Induce h UCMSCs into osteogenic differentiation and adipogenic differentiation. The osteogenic differentiation ability was evaluated by collagen expression, ALP detection, Alizarin Red staining for three weeks and the adipogenic differentiation was detected by Oil Red Ostaining for three weeks.4) Through the inductive medium, h UCMSCs were induced into netural stem cells. The results were evaluated by morphological observation, RT-PCR, immunofluorescence.Results:1) Cell morphology of h UCMSCs was spindle long and swirling growth. Flow cytometry analysis showed that Men SCs are positive for CD105, CD29, CD13 and CD90 and negative for CD45, CD14, CD106, CD34 and HLA-DR. The expression of HLA-DR demonstrated that cells had low immunogenicity. Immunofluorescence showed that h UCMSCs expressed nestin.2) Recombinant lentiviral vector carrying NGF gene was constructed successfully.3) HUCMSCs had osteoblasts and adipogenic differentiation potential.4)Through the inductive medium, evaluated by morphological observation, RT-PCR, immunofluorescence, h UCMSCs were induced into netural stem cells.Conclusion:1) All the results showed that h UCMSCs could be obtained from a healthyhumam umbilical cord. HUCMSCs maintained the basic biological properties of stem cells and high amplification capacity. In addition, h UCMSCs showed low immunogenicity and pluripotent ability. The experiments further demonstrated that HUCMSCs had the potential to differentiate into netural stem cells, Our findings provided some theoretical basis for the clinical application for the treatment of diabetes and also demonstrated that HUCMSCs were ideal seed cells for cell therapy.更多还原