小鼠神經(jīng)干細胞移植治療去神經(jīng)節(jié)巨結(jié)腸實驗研究
發(fā)布時間：2019-05-22 08:21
【摘要】： 第一部分：新生小鼠神經(jīng)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(zhì)分離培養(yǎng)出神經(jīng)干細胞并在體外大量擴增的方法，為進一步研究神經(jīng)干細胞移植治療先天性巨結(jié)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(zhì)分離出神經(jīng)干細胞，臺酚藍計數(shù)活細胞，應(yīng)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(jīng)干細胞增殖情況，取原代培養(yǎng)形成的神經(jīng)球采用有限稀釋法進行神經(jīng)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(dǎo)神經(jīng)干細胞分化，利用添加NGF的培養(yǎng)基研究神經(jīng)干細胞向膽堿能神經(jīng)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(xué)技術(shù)對原代、傳2代及單克隆神經(jīng)球行Nestin抗原檢測，鑒定神經(jīng)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(dǎo)后的細胞行ChAT檢測，并計算各組ChAT陽性細胞率。 結(jié)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(xué)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(jīng)胎牛血清誘導(dǎo)后均可分化為NF-200、GFAP和MBP表達陽性的神經(jīng)元、星形膠質(zhì)細胞和少突膠質(zhì)細胞。NGF定向誘導(dǎo)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(xué)意義。 結(jié)論：利用無血清培養(yǎng)技術(shù)成功從新生小鼠大腦皮質(zhì)分離培養(yǎng)出神經(jīng)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(jīng)干細胞，NGF在體外培養(yǎng)環(huán)境中可顯著提高神經(jīng)干細胞向膽堿能神經(jīng)元分化的比例。 第二部分：JetPEI介導(dǎo)GDNF及EDNRB共轉(zhuǎn)染神經(jīng)干細胞實驗研究 目的：探討神經(jīng)干細胞轉(zhuǎn)染的新方法，并觀察轉(zhuǎn)染后目的基因在神經(jīng)干細胞內(nèi)的表達情況，為聯(lián)合基因?qū)肷窠?jīng)干細胞移植治療先天性巨結(jié)腸癥奠定實驗基礎(chǔ)。 方法：原代培養(yǎng)新生小鼠大腦皮質(zhì)源性神經(jīng)干細胞，運用JetPEI轉(zhuǎn)染試劑將目的基因GDNF和EDNRB共轉(zhuǎn)染至神經(jīng)干細胞內(nèi)，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(zhuǎn)染效率，RT-PCR檢測目的基因mRNA表達情況。 結(jié)果：成功培養(yǎng)擴增出可用于基因轉(zhuǎn)染的神經(jīng)干細胞，，轉(zhuǎn)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(zhuǎn)染效率分別為17．56％、26．38％，27．53％。RT-PCR顯示目的基因在神經(jīng)干細胞內(nèi)成功表達，48和72小時mRNA表達量較高。 結(jié)論：運用JetPEI成功將目的基因轉(zhuǎn)染至神經(jīng)干細胞內(nèi)，且目的基因可以在神經(jīng)干細胞內(nèi)有效表達，為相關(guān)神經(jīng)相關(guān)性疾病的基因治療奠定了實驗基礎(chǔ)。 第三部分：小鼠去神經(jīng)節(jié)巨結(jié)腸模型的構(gòu)建及鑒定 目的：探索建立適于神經(jīng)干細胞移植的巨結(jié)腸動物模型的方法，并觀察研究該模型的的病理組織學(xué)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(jié)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(shù)后通過大體解剖觀察各組對象結(jié)腸變化，HE染色觀察處理段結(jié)腸壁組織學(xué)改變，計數(shù)每mm腸管神經(jīng)元數(shù)目。乙酰膽堿酯酶組織化學(xué)染色及NF-200免疫組織化學(xué)染色檢測肌間神經(jīng)叢消除情況。RT-PCR檢測NF-200、GFAP、ChAT、nNOS mRNA表達水平。 結(jié)果：大體解剖見正常對照組無異常；NS組腹腔內(nèi)有輕微粘連，無腸腔狹窄；BAC組處理段結(jié)腸狹窄梗阻，近段結(jié)腸大量糞便堆積，呈不同程度的擴張。組織學(xué)檢測見正常對照組及NS處理組結(jié)腸壁腸肌層排列有序，粘膜層及粘膜下層無損傷，腸神經(jīng)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(jīng)元數(shù)目明顯減少，與正常對照組和NS組相比差異有統(tǒng)計學(xué)意義。乙酰膽堿酯酶組織化學(xué)染色見正常對照組及NS組腸肌間及部分粘膜下神經(jīng)元及神經(jīng)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(xué)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(diào)，與其它兩組相比差異有統(tǒng)計學(xué)意義。 結(jié)論：運用0．5％苯扎氯銨成功選擇性去除了小鼠結(jié)腸肌間神經(jīng)叢，構(gòu)建成與先天性巨結(jié)腸癥具有相似病理特征的小鼠巨結(jié)腸模型，為下一步的神經(jīng)干細胞移植治療先天性巨結(jié)腸癥奠定實驗基礎(chǔ)。 第四部分：神經(jīng)干細胞在巨結(jié)腸小鼠結(jié)腸壁內(nèi)存活分化研究 目的：研究神經(jīng)干細胞在去神經(jīng)節(jié)小鼠結(jié)腸壁內(nèi)的存活分化情況，探討神經(jīng)干細胞移植治療結(jié)腸無神經(jīng)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(jié)腸漿膜層選擇性去除結(jié)腸壁神經(jīng)節(jié)制作巨結(jié)腸模型，原代培養(yǎng)新生小鼠大腦皮質(zhì)來源神經(jīng)干細胞，Hoechst33342標(biāo)記傳代純化后的神經(jīng)干細胞。運用微量注射器將標(biāo)記后的神經(jīng)干細胞移植入模型鼠病變腸段，分別于術(shù)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(xué)特性和神經(jīng)干細胞存活分化情況。 結(jié)果：原代培養(yǎng)神經(jīng)干細胞Nestin表達陽性，體外培養(yǎng)可分化為神經(jīng)元和神經(jīng)膠質(zhì)細胞。BAC處理后，HE染色及免疫組織化學(xué)染色顯示小鼠結(jié)腸肌間神經(jīng)從消失。神經(jīng)干細胞移植后各觀測時間點可見Hoechst33342標(biāo)記陽性細胞，免疫組織熒光檢測顯示NSCs組術(shù)后第7天結(jié)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(jīng)元計數(shù)顯示NSCs組神經(jīng)元平均數(shù)目為137．50個／mm，明顯高于NS組，差異有統(tǒng)計學(xué)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(xué)意義。 結(jié)論：移植后的神經(jīng)干細胞可以在去神經(jīng)節(jié)小鼠結(jié)腸壁內(nèi)存活并分化為神經(jīng)元及膠質(zhì)細胞，部分恢復(fù)腸道神經(jīng)的調(diào)節(jié)作用，為神經(jīng)干細胞移植治療先天性巨結(jié)腸癥提供了實驗依據(jù)。
[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(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2009
【分類號】：R329
本文編號：2482801
[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(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2009
【分類號】：R329
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相關(guān)期刊論文 前3條
1 李鵬飛;王春芳;;骨髓基質(zhì)細胞對共培養(yǎng)條件下的脊髓源性神經(jīng)干細胞分化為膽堿能神經(jīng)元的誘導(dǎo)[J];解剖學(xué)雜志;2006年06期
2 李鵬飛;王春芳;;胚胎大鼠脊髓神經(jīng)干細胞體外培養(yǎng)與定向分化為膽堿能神經(jīng)元的研究[J];神經(jīng)解剖學(xué)雜志;2007年06期
3 盧蓉;黃丹平;黃冰;高楠;王智崇;葛堅;;Hoechst 33342標(biāo)記恒河猴皮膚干細胞向結(jié)膜上皮細胞分化[J];中山大學(xué)學(xué)報(醫(yī)學(xué)科學(xué)版);2007年03期
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