【摘要】： 第一部分神經(jīng)干細(xì)胞的培養(yǎng)和鑒定 目的:掌握神經(jīng)干細(xì)胞培養(yǎng)技術(shù),為進(jìn)一步實(shí)驗(yàn)提供可供移植的神經(jīng)干細(xì)胞。方法:孕12-14天SD大鼠胚胎數(shù)只,分離獲得大腦皮層和海馬區(qū)腦組織,機(jī)械吹打制成單細(xì)胞懸液;離心后用神經(jīng)干細(xì)胞培養(yǎng)液重懸細(xì)胞,按照1×10~6個(gè)細(xì)胞/ml密度接種于培養(yǎng)瓶中;定期觀察,每2-3天半定量換液一次,7-10天傳代一次。取培養(yǎng)的細(xì)胞行神經(jīng)干細(xì)胞特異性抗原Nestin鑒定、增殖活性鑒定及分化能力鑒定。 結(jié)果:培養(yǎng)的神經(jīng)干細(xì)胞聚集成細(xì)胞球以懸浮的方式生長(zhǎng),培養(yǎng)7-10天后細(xì)胞球形態(tài)致密,大小較均一,傳代后仍然可得到大量的次代細(xì)胞球。培養(yǎng)細(xì)胞的Nestin及BrdU檢測(cè)呈陽(yáng)性,貼壁分化后可分化為β-Tublin或GFAP陽(yáng)性細(xì)胞。 結(jié)論:此部分實(shí)驗(yàn)培養(yǎng)的細(xì)胞具有自我復(fù)制更新的能力和多向分化的潛能,是符合移植實(shí)驗(yàn)要求的神經(jīng)干細(xì)胞。 第二部分神經(jīng)干細(xì)胞體外分化抗原表達(dá)時(shí)程的研究 目的:研究體外神經(jīng)干細(xì)胞向神經(jīng)元分化過(guò)程中標(biāo)志性抗原的表達(dá)情況,為進(jìn)一步研究神經(jīng)干細(xì)胞體外增殖分化影響因素以及進(jìn)行體內(nèi)移植的研究奠定基礎(chǔ)。 方法:取12～14天胎鼠大腦皮層,體外培養(yǎng)神經(jīng)干細(xì)胞;于第1次傳代后第3天將培養(yǎng)的神經(jīng)干細(xì)胞置于含有分化培養(yǎng)液的培養(yǎng)皿中進(jìn)行分化;于不同的分化時(shí)間點(diǎn)(1、4、7、10和14天)采用免疫細(xì)胞化學(xué)方法檢測(cè)Nestin、SOX2、DCX、TuJ1、NeuN各抗原的表達(dá)情況。 結(jié)果:Nestin和SOX2在開(kāi)始時(shí)表達(dá)量非常近似,幾乎在所有的細(xì)胞兩者都同時(shí)表達(dá),隨時(shí)間的推移,兩者的表達(dá)量都逐漸減少,但SOX2的表達(dá)時(shí)間相對(duì)Nestin而言持續(xù)更長(zhǎng);DCX表達(dá)量在分化1周內(nèi)較高且變化不大,隨后開(kāi)始下降,至分化14天時(shí)僅有少數(shù)細(xì)胞呈DCX陽(yáng)性;TuJ1在分化7天時(shí)就已經(jīng)有少部分表達(dá),隨后其表達(dá)量逐漸增多;分化14天時(shí)有38.27%的細(xì)胞表達(dá)TuJ1;在貼壁分化的一周內(nèi)未見(jiàn)NeuN陽(yáng)性細(xì)胞出現(xiàn),但至分化14天時(shí),已有NeuN陽(yáng)性細(xì)胞占21.11%;在貼壁分化第1天時(shí)就有大量的GFAP陽(yáng)性細(xì)胞,其表達(dá)量隨分化時(shí)間延長(zhǎng)而逐漸減少,但下降趨勢(shì)較Nestin緩和,至分化14天時(shí)仍有大量的GFAP陽(yáng)性細(xì)胞。 結(jié)論:神經(jīng)干細(xì)胞體外分化過(guò)程中抗原的表達(dá)是一個(gè)相互交錯(cuò)的過(guò)程,未分化狀態(tài)標(biāo)志性抗原表達(dá)的減少與分化狀態(tài)標(biāo)志性抗原表達(dá)的增加互相對(duì)應(yīng)。 第三部分運(yùn)動(dòng)訓(xùn)練在缺血性腦梗死大鼠神經(jīng)干細(xì)胞移植治療中的作用 目的:探討運(yùn)動(dòng)訓(xùn)練對(duì)缺血性腦梗死大鼠神經(jīng)干細(xì)胞移植后神經(jīng)功能和缺血腦區(qū)超微結(jié)構(gòu)改變的影響。 方法:建立Sprague-Dawley大鼠大腦中動(dòng)脈缺血/再灌注模型為實(shí)驗(yàn)對(duì)象,隨機(jī)分為腦缺血對(duì)照組(對(duì)照組)、電動(dòng)跑臺(tái)訓(xùn)練組(運(yùn)動(dòng)組)、神經(jīng)干細(xì)胞移植組(移植組)、神經(jīng)干細(xì)胞移植聯(lián)合運(yùn)動(dòng)訓(xùn)練組(聯(lián)合組),每組6或10只。術(shù)后第5天時(shí)將超順磁氧化鐵(super paramagnetic iron oxide,SPIO)標(biāo)記的神經(jīng)干細(xì)胞移植到缺血側(cè)紋狀體區(qū)。術(shù)后第6天開(kāi)始,運(yùn)動(dòng)組和聯(lián)合組大鼠給予定量的電動(dòng)跑臺(tái)運(yùn)動(dòng)訓(xùn)練,4周運(yùn)動(dòng)訓(xùn)練期間所有4組大鼠均進(jìn)行定期的神經(jīng)功能評(píng)估。運(yùn)動(dòng)4周后處死大鼠,透射電鏡觀察SPIO標(biāo)記的神經(jīng)干細(xì)胞在宿主腦內(nèi)存活遷徙以及宿主腦區(qū)超微結(jié)構(gòu)變化情況。 結(jié)果:與對(duì)照組比較,運(yùn)動(dòng)組和聯(lián)合組的神經(jīng)功能評(píng)分在運(yùn)動(dòng)訓(xùn)練2周后,差異有統(tǒng)計(jì)學(xué)意義(P＜0.05,P＜0.01);移植組差異無(wú)統(tǒng)計(jì)學(xué)意義(P＞0.05)。聯(lián)合組可見(jiàn)SPIO標(biāo)記的神經(jīng)干細(xì)胞密度較大,遷徙也相對(duì)廣泛,宿主缺血腦區(qū)超微結(jié)構(gòu)優(yōu)于其他各組。 結(jié)論:運(yùn)動(dòng)訓(xùn)練可以促進(jìn)神經(jīng)干細(xì)胞移植對(duì)腦梗死大鼠的治療作用,但其具體的機(jī)制還有待進(jìn)一步研究。
[Abstract]:Part 1 cultivation and identification of neural stem cells
AIM: To master the culture technology of neural stem cells and provide transplantable neural stem cells for further experiments. Methods: The cerebral cortex and hippocampus were isolated from several embryos of SD rats from 12 to 14 days gestation, and the single cell suspension was made by mechanical blowing. The cultured cells were identified by Nestin, proliferation activity and differentiation ability.
Results: The cultured neural stem cells grew in suspension. After 7-10 days of culture, the cells were compact and uniform in size. After passage, a large number of secondary cell spheres could be obtained. Nestin and BrdU of cultured cells were positive, and they could differentiate into beta-Tublin or GFAP positive cells after adherent differentiation.
CONCLUSION: The cells cultured in this part of the experiment have the ability of self-replication and self-renewal and the potential of multi-differentiation. They are neural stem cells that meet the experimental requirements of transplantation.
The second part is about the time course of antigen expression in neural stem cells in vitro.
AIM: To study the expression of marker antigens during the differentiation of neural stem cells into neurons in vitro, so as to lay a foundation for further study on the factors influencing the proliferation and differentiation of neural stem cells in vitro and the study of transplantation in vivo.
Methods: Nestin, SOX2, DCX, TuJ1 and NeuN antigens were detected by immunocytochemistry at different time points (1, 4, 7, 10 and 14 days). Situation.
Results: The expression levels of Nestin and SOX2 were very similar at the beginning, and almost all cells expressed both at the same time. Over time, the expression of SOX2 decreased gradually, but the expression time of SOX2 lasted longer than that of Nestin. The expression of DCX was higher and unchanged during the first week of differentiation, then began to decline, and only at the 14th day of differentiation. A few cells were DCX-positive; TuJ1 was expressed in a small part at 7 days of differentiation, and then gradually increased; 38.27% of the cells expressed TuJ1 at 14 days of differentiation; no NeuN-positive cells were found within one week of adherent differentiation, but 21.11% of the cells were NeuN-positive at 14 days of differentiation; and a large number of GFA-positive cells were found on the first day of adherent differentiation. The expression of P-positive cells decreased gradually with the prolongation of differentiation time, but the downward trend was milder than that of Nestin. There were still a large number of GFAP-positive cells at the 14th day of differentiation.
CONCLUSION: The expression of antigens in neural stem cells during differentiation in vitro is an intertwined process, and the decrease of expression of undifferentiated marker antigens corresponds to the increase of expression of differentiated marker antigens.
The effect of third part exercise training on neural stem cell transplantation in rats with ischemic cerebral infarction
Objective: To investigate the effects of exercise training on nerve function and ultrastructure of ischemic brain after neural stem cell transplantation in rats with ischemic cerebral infarction.
METHODS: Sprague-Dawley rats were randomly divided into cerebral ischemia control group (control group), treadmill training group (exercise group), neural stem cell transplantation group (transplantation group), neural stem cell transplantation combined with exercise training group (combined group), 6 or 10 rats in each group. The super paramagnetic iron oxide (SPIO) labeled neural stem cells were transplanted into the ischemic striatum. The rats in the exercise group and the combined group were given quantitative treadmill exercise training on the 6th day after operation. All the rats in the four groups were assessed periodically during the exercise training period. The rats were sacrificed after 4 weeks of exercise, and the transmission electron microscopy was used. The migration of SPIO labeled neural stem cells in the host brain and the ultrastructural changes of the host brain were observed.
Results: Compared with the control group, the neurological function scores of the exercise group and the combined group were significantly different after 2 weeks of exercise training (P < 0.05, P < 0.01); there was no significant difference between the transplantation group and the control group (P > 0.05). Each group.
Conclusion: Exercise training can promote the therapeutic effect of neural stem cell transplantation on rats with cerebral infarction, but its specific mechanism needs further study.
【學(xué)位授予單位】：復(fù)旦大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2009
【分類(lèi)號(hào)】：R329

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本文編號(hào)：2202119