小鼠神经干细胞移植治疗去神经节巨结肠实验研究

发布时间：2019-05-22 08:21

【摘要】： 第一部分：新生小鼠神经干细胞分离培养、鉴定及分化目的：探讨从新生小鼠大脑皮质分离培养出神经干细胞并在体外大量扩增的方法，为进一步研究神经干细胞移植治疗先天性巨结肠症提供可靠的细胞供体。方法：用机械吹打法从新生小鼠大脑皮质分离出神经干细胞，台酚蓝计数活细胞，应用添加B27、bFGF和EGF的无血清培养基进行原代及传代培养，MTT法测定神经干细胞增殖情况，取原代培养形成的神经球采用有限稀释法进行神经干细胞的单克隆培养，并将所获得的单克隆细胞传代培养。利用10％胎牛血清自然诱导神经干细胞分化，利用添加NGF的培养基研究神经干细胞向胆碱能神经元定向分化，倒置显微镜下观察其分化情况。运用SABC免疫细胞化学技术对原代、传2代及单克隆神经球行Nestin抗原检测，鉴定神经干细胞；对自然分化后的细胞行NF-200、GFAP和MBP检测，鉴定分化细胞的类型，并计算各型细胞的阳性率；对NGF定向诱导后的细胞行ChAT检测，并计算各组ChAT阳性细胞率。结果：原代培养成功得到悬浮生长的细胞球克隆，免疫组化检测显示该细胞克隆Nestin抗原表达强阳性，传代后可得到具有相同生物学特性的细胞群。通过有限稀释法可以培养出单克隆来源的细胞群，且该单克隆细胞同样Nestin抗原表达强阳性。原代、传代及单克隆来源的细胞群均具有持续增殖的能力，经胎牛血清诱导后均可分化为NF-200、GFAP和MBP表达阳性的神经元、星形胶质细胞和少突胶质细胞。NGF定向诱导可显著提高分化细胞中ChAT阳性细胞率(15．48％)，与FBS组(4．49％)相比差异有统计学意义。结论：利用无血清培养技术成功从新生小鼠大脑皮质分离培养出神经干细胞，并通过单克隆培养获得大量纯化可作为细胞移植供体的神经干细胞，NGF在体外培养环境中可显著提高神经干细胞向胆碱能神经元分化的比例。第二部分：JetPEI介导GDNF及EDNRB共转染神经干细胞实验研究目的：探讨神经干细胞转染的新方法，并观察转染后目的基因在神经干细胞内的表达情况，为联合基因导入神经干细胞移植治疗先天性巨结肠症奠定实验基础。方法：原代培养新生小鼠大脑皮质源性神经干细胞，运用JetPEI转染试剂将目的基因GDNF和EDNRB共转染至神经干细胞内，免疫荧光显微镜观察、流式细胞仪检测绿色荧光蛋白(GFP)表达情况，测定转染效率，RT-PCR检测目的基因mRNA表达情况。结果：成功培养扩增出可用于基因转染的神经干细胞，，转染后24小时即可在免疫荧光显微镜下观察到GFP的表达，流式细胞仪检测显示24、48、72小时转染效率分别为17．56％、26．38％，27．53％。RT-PCR显示目的基因在神经干细胞内成功表达，48和72小时mRNA表达量较高。结论：运用JetPEI成功将目的基因转染至神经干细胞内，且目的基因可以在神经干细胞内有效表达，为相关神经相关性疾病的基因治疗奠定了实验基础。第三部分：小鼠去神经节巨结肠模型的构建及鉴定目的：探索建立适于神经干细胞移植的巨结肠动物模型的方法，并观察研究该模型的的病理组织学特征。方法：90只雄性昆明小鼠随机分为正常对照组、生理盐水组(NS组)和苯扎氯铵组(BAC组)3组，BAC组以0．5％苯扎氯铵(BAC)处理降结肠浆膜层15min，NS组以生理盐水代替，正常对照组不做任何处理。术后通过大体解剖观察各组对象结肠变化，HE染色观察处理段结肠壁组织学改变，计数每mm肠管神经元数目。乙酰胆碱酯酶组织化学染色及NF-200免疫组织化学染色检测肌间神经丛消除情况。RT-PCR检测NF-200、GFAP、ChAT、nNOS mRNA表达水平。结果：大体解剖见正常对照组无异常；NS组腹腔内有轻微粘连，无肠腔狭窄；BAC组处理段结肠狭窄梗阻，近段结肠大量粪便堆积，呈不同程度的扩张。组织学检测见正常对照组及NS处理组结肠壁肠肌层排列有序，粘膜层及粘膜下层无损伤，肠神经节存在。BAC组粘膜及粘膜下层无明显病理性改变，平滑肌层增厚，肌间神经元数目明显减少，与正常对照组和NS组相比差异有统计学意义。乙酰胆碱酯酶组织化学染色见正常对照组及NS组肠肌间及部分粘膜下神经元及神经纤维染为棕黄色，BAC组AChE表达明显降低，肠肌间无阳性表达，粘膜下可见轻微着色。免疫组织化学染色显示BAC组肌间NF-200表达阴性，正常对照组及NS组NF-200表达阳性。半定量RT-PCR检测显示BAC组NF-200、GFAP、ChAT、nNOS mRNA表达量均明显下调，与其它两组相比差异有统计学意义。结论：运用0．5％苯扎氯铵成功选择性去除了小鼠结肠肌间神经丛，构建成与先天性巨结肠症具有相似病理特征的小鼠巨结肠模型，为下一步的神经干细胞移植治疗先天性巨结肠症奠定实验基础。第四部分：神经干细胞在巨结肠小鼠结肠壁内存活分化研究目的：研究神经干细胞在去神经节小鼠结肠壁内的存活分化情况，探讨神经干细胞移植治疗结肠无神经节细胞症的可行性。方法：0．5％苯扎氯铵(BAC)处理8周龄昆明小鼠结肠浆膜层选择性去除结肠壁神经节制作巨结肠模型，原代培养新生小鼠大脑皮质来源神经干细胞，Hoechst33342标记传代纯化后的神经干细胞。运用微量注射器将标记后的神经干细胞移植入模型鼠病变肠段，分别于术后第7、14、21、28天行大体观察，HE染色，免疫组织荧光检测，RT-PCR检测，观察小鼠生物学特性和神经干细胞存活分化情况。结果：原代培养神经干细胞Nestin表达阳性，体外培养可分化为神经元和神经胶质细胞。BAC处理后，HE染色及免疫组织化学染色显示小鼠结肠肌间神经从消失。神经干细胞移植后各观测时间点可见Hoechst33342标记阳性细胞，免疫组织荧光检测显示NSCs组术后第7天结肠壁存在Nestin表达阳性细胞，21天后可见NSE及GFAP表达阳性细胞，NS组有少量阳性细胞，神经元计数显示NSCs组神经元平均数目为137．50个／mm，明显高于NS组，差异有统计学意义。NSCs组ChAT、nNOS mRNA相对表达量明显高于NS组，差异有统计学意义。结论：移植后的神经干细胞可以在去神经节小鼠结肠壁内存活并分化为神经元及胶质细胞，部分恢复肠道神经的调节作用，为神经干细胞移植治疗先天性巨结肠症提供了实验依据。
[Abstract]:Part 1: Isolation, culture, identification and differentiation of neural stem cells from neonatal mice Objective: To study the isolation and culture of neural stem cells from the cerebral cortex of the newborn mice and to amplify the neural stem cells in vitro Methods: To provide reliable cells for further study of neural stem cell transplantation in the treatment of congenital megacolon Methods: The neural stem cells were isolated from the cerebral cortex of the newly-born mice by mechanical blowing, and the cells were counted. The serum-free medium supplemented with B27, bFGF and EGF was used for primary and secondary culture. In the case of cell proliferation, the neurospheres formed from the primary culture are subjected to the monoclonal culture of the neural stem cells using a limited dilution method, and the obtained monoclonal antibodies are fine The differentiation of neural stem cells induced by 10% fetal bovine serum was used to study the orientation and differentiation of neural stem cells to cholinergic neurons by the addition of NGF. The cell line NF-200, GFAP and MBP were detected and the types of the differentiated cells were identified by using the SABC immunocytochemistry technique. The types of differentiated cells were identified and the types of the cells were calculated. the positive rate of the cell, the cell line ChAT after the induction of NGF and the ChAT in each group, Results: The cell-ball clone of the cell clone was successfully obtained by primary culture, and the expression of the Nestin antigen in the cell was detected by immunohistochemistry. The cell population of the characteristics of the material. The cell population of the monoclonal antibody can be cultured by the limited dilution method, and the monoclonal cell is also Nesti. The expression of n-antigen is strongly positive. The primary, subcultured and monoclonal-derived cell groups have the ability to proliferate, and can be differentiated into NF-200, GFAP and MBP-positive neurons after the induction of fetal bovine serum, and the astrocytes are fine. The expression of NGF in the cells and oligodendrocytes significantly increased the ChAT-positive cell rate (15.48%) in the differentiated cells, compared with the FBS group (4.49%). Conclusion: The neural stem cells isolated from the cerebral cortex of the newborn mice were successfully isolated from the cerebral cortex of the newborn mice by the serum-free culture technique, and a large amount of purification can be obtained by the monoclonal culture. The neural stem cells of the donor are cultured in vitro, and the neural stem cells can be significantly increased in the in vitro culture environment. The proportion of cholinergic neuron differentiation. Part 2: JetPEI-mediated GDNF and ED The purpose of the NRB co-transfection of neural stem cells: a new method for the transfection of neural stem cells and the observation of transfection Expression of the post-target gene in neural stem cells and the introduction of neural stem cells for the combined gene The purpose of this study was to provide an experimental basis for the treatment of congenital megacolon. Methods: The primary cultured neonatal mouse cerebral cortex-derived neural stem cells, the target gene GDNF and EDNRB were co-transfected into neural stem cells by using the JethPEI transfection reagent, and the flow cytometry was used to detect the green stem cells. in that case of the expression of the fluorescent protein (GFP), the transfection efficiency was determined, The expression of the target gene was detected by RT-PCR. The results showed that the expression of GFP could be observed under the immunofluorescence microscope after 24 hours after the transfection of the neural stem cells which could be used for gene transfection. The results showed that 24,48,7 were detected by flow cytometry. The two-hour transfection efficiency was 17.56%, 26.38% and 27.53%, respectively. Conclusion: The target gene can be successfully transfected into neural stem cells by using JETPEI, and the target gene can be found in neural stem cells. The effect expression lays a foundation for the gene therapy of the related nerve-related diseases. Experimental basis. Part 3: Construction and identification of the model of the deganglionic megacolon in mice: to explore the establishment of a suitable neural stem Methods:90 male Kunming mice were randomly divided into three groups: the normal control group, the normal saline group (NS group) and the control group (BAC group). The serosal layer of the colon was reduced for 15 min, and the NS group was replaced by normal saline, and no treatment was done in the normal control group. Changes of the colon and HE staining to observe the histological changes of the colon wall in the treatment section, counting the number of neurons per mm of the intestinal canal. Histochemical staining of alkaline esterase and immunohistochemical staining of NF-200 to detect that elimination of intermuscular nerve plexus. -PCR was used to detect the level of NF-200, GFAP, ChAT, nNOS mRNA. Stenosis of the intestinal lumen; the obstruction of the colon in the treatment section of the BAC group, the accumulation of a large amount of stool in the proximal colon, and a different degree of expansion. The histological examination was found to be normal. In the control group and the NS treatment group, the intestinal muscularis of the colon wall was arranged in order, and the mucosa layer and the lower layer of the mucosa were not damaged, and the intestinal ganglion was present. There was no obvious pathological change in the mucosa and the submucosal layer of the BAC group. In the normal control group and the NS group, there was a significant difference in the thickness of the myometrium and the number of intermuscular neurons. The expression of AChE in the BAC group was significantly lower, and the expression of AChE in the BAC group was significantly lower. The expression of NF-200 in BAC group was negative, and the expression of NF-200 was positive in normal control group and NS group. Conclusion: The expression of ChAT and nNOS mRNA is down-regulated, and it is of statistical significance to the other two groups. The mouse giant colon model with the pathological characteristics and the next step of neural stem cell transplantation The experimental basis for the treatment of congenital megacolon. Part IV: The purpose of the study of the survival and differentiation of neural stem cells in the colon wall of the megacolon: the study of God The viability and differentiation of stem cells in the colon wall of the deganglionic mouse, and the feasibility of the transplantation of neural stem cells in the treatment of the non-ganglionic cell disease in the colon were discussed. Methods: The selective removal of the colon wall ganglion from the colon serosa layer of the 8-week-old Kunming mice was treated with 0.5% benzo-cloride (BAC). A colon model was used to culture the neural stem cells from the cerebral cortex of the newly-born mouse. Hoechst33342 was used for the passage and purification of the neural stem cells. The labeled neural stem cells were transplanted into the intestinal segments of the model mice by using a microsyringe. Gross observation, HE staining, fluorescence detection of immune tissue, RT-PCR detection, biological characteristics of mice and nerve trunk were observed. Results: The expression of Nestin in primary cultured neural stem cells was positive. In vitro, in vitro culture can be differentiated into neurons and glial cells. After BAC treatment, HE staining and immunohistochemical staining show the disappearance of the intermuscular nerve of the colon. The Hoechst33342 marked positive cells can be seen at each observation time point after the transplantation of the neural stem cells, and the fluorescence detection of the immune tissue shows the NSCs group. The positive cells of Nestin were expressed in the colon wall on the 7th day, and the positive cells of NSE and GFAP were observed in 21 days, and the NS group was less. The average number of neurons in NSCs was 137.50/ mm. The relative expression of ChAT and nNOS mRNA in NSCs was significantly higher than that in NS group.
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2009
【分类号】：R329

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