【摘要】： 第一部分：新生小鼠神經干細胞分離培養(yǎng)、鑒定及分化 目的：探討從新生小鼠大腦皮質分離培養(yǎng)出神經干細胞并在體外大量擴增的方法，為進一步研究神經干細胞移植治療先天性巨結腸癥提供可靠的細胞供體。 方法：用機械吹打法從新生小鼠大腦皮質分離出神經干細胞，臺酚藍計數活細胞，應用添加B27、bFGF和EGF的無血清培養(yǎng)基進行原代及傳代培養(yǎng)，MTT法測定神經干細胞增殖情況，取原代培養(yǎng)形成的神經球采用有限稀釋法進行神經干細胞的單克隆培養(yǎng)，并將所獲得的單克隆細胞傳代培養(yǎng)。利用10％胎牛血清自然誘導神經干細胞分化，利用添加NGF的培養(yǎng)基研究神經干細胞向膽堿能神經元定向分化，倒置顯微鏡下觀察其分化情況。運用SABC免疫細胞化學技術對原代、傳2代及單克隆神經球行Nestin抗原檢測，鑒定神經干細胞；對自然分化后的細胞行NF-200、GFAP和MBP檢測，鑒定分化細胞的類型，并計算各型細胞的陽性率；對NGF定向誘導后的細胞行ChAT檢測，并計算各組ChAT陽性細胞率。 結果：原代培養(yǎng)成功得到懸浮生長的細胞球克隆，免疫組化檢測顯示該細胞克隆Nestin抗原表達強陽性，傳代后可得到具有相同生物學特性的細胞群。通過有限稀釋法可以培養(yǎng)出單克隆來源的細胞群，且該單克隆細胞同樣Nestin抗原表達強陽性。原代、傳代及單克隆來源的細胞群均具有持續(xù)增殖的能力，經胎牛血清誘導后均可分化為NF-200、GFAP和MBP表達陽性的神經元、星形膠質細胞和少突膠質細胞。NGF定向誘導可顯著提高分化細胞中ChAT陽性細胞率(15．48％)，與FBS組(4．49％)相比差異有統(tǒng)計學意義。 結論：利用無血清培養(yǎng)技術成功從新生小鼠大腦皮質分離培養(yǎng)出神經干細胞，并通過單克隆培養(yǎng)獲得大量純化可作為細胞移植供體的神經干細胞，NGF在體外培養(yǎng)環(huán)境中可顯著提高神經干細胞向膽堿能神經元分化的比例。 第二部分：JetPEI介導GDNF及EDNRB共轉染神經干細胞實驗研究 目的：探討神經干細胞轉染的新方法，并觀察轉染后目的基因在神經干細胞內的表達情況，為聯合基因導入神經干細胞移植治療先天性巨結腸癥奠定實驗基礎。 方法：原代培養(yǎng)新生小鼠大腦皮質源性神經干細胞，運用JetPEI轉染試劑將目的基因GDNF和EDNRB共轉染至神經干細胞內，免疫熒光顯微鏡觀察、流式細胞儀檢測綠色熒光蛋白(GFP)表達情況，測定轉染效率，RT-PCR檢測目的基因mRNA表達情況。 結果：成功培養(yǎng)擴增出可用于基因轉染的神經干細胞，，轉染后24小時即可在免疫熒光顯微鏡下觀察到GFP的表達，流式細胞儀檢測顯示24、48、72小時轉染效率分別為17．56％、26．38％，27．53％。RT-PCR顯示目的基因在神經干細胞內成功表達，48和72小時mRNA表達量較高。 結論：運用JetPEI成功將目的基因轉染至神經干細胞內，且目的基因可以在神經干細胞內有效表達，為相關神經相關性疾病的基因治療奠定了實驗基礎。 第三部分：小鼠去神經節(jié)巨結腸模型的構建及鑒定 目的：探索建立適于神經干細胞移植的巨結腸動物模型的方法，并觀察研究該模型的的病理組織學特征。 方法：90只雄性昆明小鼠隨機分為正常對照組、生理鹽水組(NS組)和苯扎氯銨組(BAC組)3組，BAC組以0．5％苯扎氯銨(BAC)處理降結腸漿膜層15min，NS組以生理鹽水代替，正常對照組不做任何處理。術后通過大體解剖觀察各組對象結腸變化，HE染色觀察處理段結腸壁組織學改變，計數每mm腸管神經元數目。乙酰膽堿酯酶組織化學染色及NF-200免疫組織化學染色檢測肌間神經叢消除情況。RT-PCR檢測NF-200、GFAP、ChAT、nNOS mRNA表達水平。 結果：大體解剖見正常對照組無異常；NS組腹腔內有輕微粘連，無腸腔狹窄；BAC組處理段結腸狹窄梗阻，近段結腸大量糞便堆積，呈不同程度的擴張。組織學檢測見正常對照組及NS處理組結腸壁腸肌層排列有序，粘膜層及粘膜下層無損傷，腸神經節(jié)存在。BAC組粘膜及粘膜下層無明顯病理性改變，平滑肌層增厚，肌間神經元數目明顯減少，與正常對照組和NS組相比差異有統(tǒng)計學意義。乙酰膽堿酯酶組織化學染色見正常對照組及NS組腸肌間及部分粘膜下神經元及神經纖維染為棕黃色，BAC組AChE表達明顯降低，腸肌間無陽性表達，粘膜下可見輕微著色。免疫組織化學染色顯示BAC組肌間NF-200表達陰性，正常對照組及NS組NF-200表達陽性。半定量RT-PCR檢測顯示BAC組NF-200、GFAP、ChAT、nNOS mRNA表達量均明顯下調，與其它兩組相比差異有統(tǒng)計學意義。 結論：運用0．5％苯扎氯銨成功選擇性去除了小鼠結腸肌間神經叢，構建成與先天性巨結腸癥具有相似病理特征的小鼠巨結腸模型，為下一步的神經干細胞移植治療先天性巨結腸癥奠定實驗基礎。 第四部分：神經干細胞在巨結腸小鼠結腸壁內存活分化研究 目的：研究神經干細胞在去神經節(jié)小鼠結腸壁內的存活分化情況，探討神經干細胞移植治療結腸無神經節(jié)細胞癥的可行性。 方法：0．5％苯扎氯銨(BAC)處理8周齡昆明小鼠結腸漿膜層選擇性去除結腸壁神經節(jié)制作巨結腸模型，原代培養(yǎng)新生小鼠大腦皮質來源神經干細胞，Hoechst33342標記傳代純化后的神經干細胞。運用微量注射器將標記后的神經干細胞移植入模型鼠病變腸段，分別于術后第7、14、21、28天行大體觀察，HE染色，免疫組織熒光檢測，RT-PCR檢測，觀察小鼠生物學特性和神經干細胞存活分化情況。 結果：原代培養(yǎng)神經干細胞Nestin表達陽性，體外培養(yǎng)可分化為神經元和神經膠質細胞。BAC處理后，HE染色及免疫組織化學染色顯示小鼠結腸肌間神經從消失。神經干細胞移植后各觀測時間點可見Hoechst33342標記陽性細胞，免疫組織熒光檢測顯示NSCs組術后第7天結腸壁存在Nestin表達陽性細胞，21天后可見NSE及GFAP表達陽性細胞，NS組有少量陽性細胞，神經元計數顯示NSCs組神經元平均數目為137．50個／mm，明顯高于NS組，差異有統(tǒng)計學意義。NSCs組ChAT、nNOS mRNA相對表達量明顯高于NS組，差異有統(tǒng)計學意義。 結論：移植后的神經干細胞可以在去神經節(jié)小鼠結腸壁內存活并分化為神經元及膠質細胞，部分恢復腸道神經的調節(jié)作用，為神經干細胞移植治療先天性巨結腸癥提供了實驗依據。
[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.
【學位授予單位】：華中科技大學
【學位級別】：博士
【學位授予年份】：2009
【分類號】：R329

【參考文獻】

相關期刊論文 前3條

1 李鵬飛;王春芳;;骨髓基質細胞對共培養(yǎng)條件下的脊髓源性神經干細胞分化為膽堿能神經元的誘導[J];解剖學雜志;2006年06期

2 李鵬飛;王春芳;;胚胎大鼠脊髓神經干細胞體外培養(yǎng)與定向分化為膽堿能神經元的研究[J];神經解剖學雜志;2007年06期

3 盧蓉;黃丹平;黃冰;高楠;王智崇;葛堅;;Hoechst 33342標記恒河猴皮膚干細胞向結膜上皮細胞分化[J];中山大學學報(醫(yī)學科學版);2007年03期

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