【作者】 陈景波；

【导师】 王国斌；

【作者基本信息】 华中科技大学 ， 外科学， 2009， 博士

【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。更多还原

【Abstract】 PartⅠ: Culture and identification of neural stem cells derived from new born mouseObjective: To isolate and culture the purified monoclonal neural stem cells from the cerebral cortex of new born mice. To offer an ideal cell donor for the treatment of Hirschsprung disease with neural stem cells graft.Method: The cerebral cortex of new born mice was isolated and dissociated to single-cell suspension by mechanical trituration. The dissociated single cells were cultured in serum-free medium with B27, bFGF and EGF after trypan-blue dye exclusion test. The growth curve of neural stem cells was draw with MTT assay. After the formation of neurospheres, single-cell clone culture was performed by limiting dilution and the proliferated single-cell clones were harvested for subculture. To evaluate the potential differentiation ability of neural stem cells, the cells were cultured in medium containing 10%fetal bovine serum (FBS). NGF was supplemented into the culture medium to induce the differentiation of the neural stem cells into cholinergic neurone. Immunocytochemistry was used to detect the specific marker of neural stem cells—Nestin of the primary, passage culture and monoclonal neurospheres. The specific antigens NF-200, GFAP and MBP were detected to identify the neurons, astrocyte and oligodendrocyte. The ChAT was detected to identify the positive rate of cholinergic neurone.Results: The dissociated cells formed floating neuraspheres in suspension cultures. The primary and passaged neurospheres expressed Nestin antigen positively. By limiting dilution, we cultured the cell lines from single-cell clone and the monoclonal neurospheres expressed Nestin too. All the neurospheres had capabilities of self-renew, proliferation and the potentiality of differentiation into NF-200, GFAP and MBP positive cells. NGF can induce 15.48% of neural stem cells into cholinergic neurone. The difference between FBS and NGF group had statistical significance.Conclusion: Monoclonal neural stem cells which have the ability of proliferation and multi-directional differentiation can be isolated and cultured from the cerebral cortex of new born mice by limiting dilution. The positive rate of cholinergic neurone can significantly increased by adding NGF into the culture medium. PartⅡ: Study on cotransfection of GDNF and EDNRB into neural stem cells mediated by JetPEIObjective: To investigate the optimizing method for neural stem cells (NSCs) transfection and the expression of extrinsic genes in neural stem cells.Method: neural stem cells derived from new born mouse was cultured in Vitro.Extrinsic genes GDNF and EDNRB were cotransfected into primary cultured neural stem cells by using JetPEI. The expression of green fluorescent protein (GFP) was measured with fluorescence microscope and flow cytometer. The relative expression of GDNF and EDNRB mRNA was detected by RT-PCR.Results: Bright green fluorescence of the transfected cells could be observed underfluorescence microscope after 24h of transfection. Flow cytometer analysis showed that the efficiency of cotransfection was 17.56%、26.38%, 27.53 % in 24h, 48h, 72h respectively. Semi-quantitative RT-PCR confirmed that GDNF and EDNRB mRNA expressed successfully in the neural stem cells.Conclusions: The tartet genes were successfully cotransfected into neural stem cellsby using JetPEI. This provides a feasible technological platform for the polygene therapy of neural degenerative diseases. PartⅢ: Establishment and identification of aganglionosis mouse modelObjective: To establish an aganglionosis mouse model suitable for neural stem cells transplantation study and explore its histopathologic characteristics.Method: 90 male Kunming mice were randomly divided into normal control group, Normal saline (NS) group and benzalkonium chloride (BAC) group. In BAC group, 0.5% benzalkonium chloride was applied onto the serous layer of colon descendens for 15 minutes and normal saline was used instead of BAC in NS group. No treatment was applied to the normal control group. The change of colon in each group was observed by gross anatomy. The observation of histologic characteristics and neuron count was performed by HE staining. Acetylcholinesterase histochemical stain and Immunohistos-taining for NF-200 was used to evaluate the denervation of myenteric nerve plexus. The mRNA expression of NF-200、GFAP、ChAT and nNOS was analyze by semiquantitative RT-PCR.Results: Gross anatomy observation showed that there was no abnormality in normal control group, little conglutination and no stenosis of colon in NS group. In BAC group, the treated segment was presented as stenosis and obstruction. The Proximal part was presented as compensatory enlargement with the retention of excrement. HE staining showed no visible change in normal control group and NS group. No pathological change was observed in the mucous membrane and submucous layer of BAC group. The number of myenteric neurons decreased obviously compared with the other groups and the differences had statistical significance. Immunohistochemical staining showed that there were no NF-200 positive cells in the myenteron. Semiquantitative RT-PCR showed the down regulation of mRNA expression of NF-200、GFAP、ChAT and nNOS in BAC group compared with the other groups and the differences had statistical significance. Conclusion: The aganglionosis mouse model was established by selective chemical ablation of the myenteric nerve plexus of colon descendens. The chemically-induced colonic aganglionosis in this model provides the basis for future studies of neural stem cell transplantation therapy for HD. PartⅣ: Study on the survival and differentiation of neural stem cells in the colonic myenteron of aganglionic miceObjective: To study the survival and differentiation of neural stem cells in the colonic myenteron of aganglionic mice. To elucidate the possibility and the biological significance of intracolonic grafting of neural stem cells (NSCs) as a therapeutic strategy for aganglionosis.Methods: The descending colon serous layer of Kunming mice, 8 weeks old, was treated by 0.5% benzalkonium chloride (BAC) to selectively ablate the myenteric nerve plexus. Neural stem cells derived from the cerebral cortex of neonatal mice was cultured and labeled by Hoechst33342. The labeled NSCs were transplanted into the denervated colon by using the microinjector. The biocharacteristics of mice and the survival and differentiation of grafted cells was observed by gross anatomy, HE staining, immunohistofluorescence and RT-PCR.Results: The primary cultured NSCs were characterized as Nestin positive and can differentiate into neurons and glial cells. HE and immunohistochemistry staining showed that the myenteric plexuses of colon disappeared after treated by 0.5% BAC. The grafted cells were visualized in colon sections under fluorescence microscope. Immunohistofluorescence assay showed that there were Nestin positive cells 7 days after transplantation and NSE, GFAP positive cells 21 days after transplantation. Neurons count showed that there were 137.50 neurons per mm in NSCs group and only 54.00 neurons per mm in NS group. The differences had statistical significance. Semiquantitative RT-PCR showed the up-regulation of mRNA expression of ChAT and nNOS in NSCs group compared with the NS group and the differences had statistical significance.Conclusions: Neural stem cells can survive and differentiate into neurons and glial cells after transplanted into the colonic myenteron of aganglionic mice and partially regulate the neuromuscular modulation of colon. This provides the experimental basis for further studies of neural stem cell transplantation therapy for HD.更多还原