本文選題：脊髓損傷 + 神經(jīng)干細胞��； 參考：《山西醫(yī)科大學》2015年碩士論文

【摘要】：目的:1.使用Alexa Fluor R Red檢測RNAi MAX對神經(jīng)干細胞的轉(zhuǎn)染效率,確定最終的轉(zhuǎn)染效率最高的時間。2.觀察mi R-31對神經(jīng)干細胞向運動神經(jīng)元分化時所起作用,研究mi R-31和神經(jīng)干細胞之間的相互關(guān)系。方法:第一部分:RNAi MAX對神經(jīng)干細胞的影響及轉(zhuǎn)染效率的測定首先從胎鼠體內(nèi)獲得脊髓源神經(jīng)干細胞,體外分離培養(yǎng)并鑒定。當干細胞傳至3代后,接種于鋪設多聚賴氨酸處理過蓋玻片的六孔板中,分為不加RNAi MAX組和加RNAi MAX兩組,通過0d、1d、2d和3d的連續(xù)觀察,研究RNAi MAX對神經(jīng)干細胞的影響作用。并繼續(xù)將細胞分為1d、2d和3d三組,轉(zhuǎn)染Alexa Fluor R Red觀察RNAi MAX對神經(jīng)干細胞的轉(zhuǎn)染效率。當達到相應的時間點時,取出細胞并用多聚甲醛固定,之后將細胞位于熒光顯微鏡下觀察,計算發(fā)出紅色熒光細胞和總細胞數(shù)之比確定RNAi MAX對神經(jīng)干細胞的轉(zhuǎn)染效率。第二部分:mi R-31對神經(jīng)干細胞的誘導作用將細胞分為干擾組、干擾對照組、過表達組、過表達對照組和對照組,轉(zhuǎn)染相應試劑后觀察細胞的形態(tài)學變化,并通過Ch AT的免疫細胞化學染色觀察膽堿能神經(jīng)元的分化情況。提取RNA后檢測mi R-31的誘導是否成功,并反轉(zhuǎn)錄為c DNA后通過熒光定量PCR檢測下游基因在mi R-31的表達變化后的變化情況。通過以上分析mi R-31在神經(jīng)干細胞干性維持方面的作用。結(jié)果:第一部分:當RNAi MAX加入到神經(jīng)干細胞后,細胞開始死亡。通過在時間節(jié)點鏡下觀察可知,在第一天時轉(zhuǎn)染效率是20%-30%,第二天時轉(zhuǎn)染效率是40%-55%,當達到第三天時,轉(zhuǎn)染效率為60%-75%。說明Alexa Fluor R Red會隨著時間的推移增加轉(zhuǎn)染的細胞數(shù),提高細胞的轉(zhuǎn)染效率。第二部分:轉(zhuǎn)染mi R-31的mimic和inhibitor后,細胞的形態(tài)和分子層面都發(fā)生了變化。形態(tài)學方面可以看出,轉(zhuǎn)染mi R-31的抑制劑后,細胞的形態(tài)更接近于運動神經(jīng)元,細胞呈長梭形,在免疫細胞化學染色中可以看出Ch AT的表達升高,細胞熒光呈強陽性;而在轉(zhuǎn)染mi R-31過表達試劑后,細胞貼壁后形態(tài)變化不明顯,突起伸出不多,Ch AT的表達明顯降低,熒光強度明顯弱于抑制組。micro RNA的反轉(zhuǎn)錄產(chǎn)物熒光定量PCR結(jié)果顯示干擾組的表達為0.867,干擾對照組的表達為2.406,過表達組為1.022×106,過表達對照組為1.506×105,說明細胞轉(zhuǎn)染成功。而下游基因也有明顯變化。在干擾組中表達明顯升高的有hb9和stmn1,表達降低的有Nestin、lats2、Rhobtb和SATB2。過表達組明顯升高的有Nestin、hb9、lats2和SATB2,表達降低的基因有Rhobtb。結(jié)論:1.RNAi MAX對神經(jīng)干性毒性較大,會造成大量的細胞死亡,在實驗時需加大細胞的用量。通過轉(zhuǎn)染效率的測定,RNAi MAX在第三天的轉(zhuǎn)染效率最高。2.mi R-31高表達對神經(jīng)干細胞有很好的干性維持作用,抑制時神經(jīng)干細胞向運動神經(jīng)元的分化增加。
[Abstract]:Purpose 1. Alexa Fluor R Red was used to detect the transfection efficiency of neural stem cells (NSCs) by RNAi MAX. To observe the effect of mi R-31 on the differentiation of neural stem cells into motor neurons, and to study the relationship between mi R-31 and neural stem cells. Methods: in the first part, the effect of MAX on neural stem cells and its transfection efficiency were measured. Firstly, spinal cord derived neural stem cells were obtained from fetal mice and isolated and identified in vitro. When the stem cells were transferred to the third passage, the cells were inoculated into the six hole plates treated with poly-lysine and divided into two groups: no RNAi MAX group and RNAi MAX group. The effect of RNAi MAX on neural stem cells was studied by observing the effects of RNAi MAX on neural stem cells for 2 days and 3 days. The cells were divided into three groups: 1 d, 2 d and 3 d. The transfection efficiency of RNAi MAX on neural stem cells was observed by Alexa Fluor R Red. When the cells reached the corresponding time point, the cells were fixed with paraformaldehyde, then the cells were observed under fluorescence microscope, and the ratio of red fluorescent cells to total cells was calculated to determine the transfection efficiency of RNAi MAX to neural stem cells. The second part: the inductive effect of: mi R-31 on neural stem cells: interference group, interference control group, overexpression group, overexpression control group and control group. The morphological changes of the cells were observed after transfection of corresponding reagents. The differentiation of cholinergic neurons was observed by immunocytochemical staining of chat. After extraction of RNA, the induction of miR-31 was detected, and the change of downstream gene expression was detected by fluorescence quantitative PCR after reverse transcription into c DNA. The role of mi R-31 in dry maintenance of neural stem cells was analyzed. Results: the first part: when RNAi MAX was added to neural stem cells, the cells began to die. The transfection efficiency was 20-30on the first day, 40-55on the second day, and 60-75on the third day. The results showed that Alexa Fluor R Red could increase the number of transfected cells and increase the transfection efficiency with time. The second part: after transfection of mimic and inhibitor of miR-31, the morphology and molecular level of the cells were changed. Morphological analysis showed that after transfection with the inhibitor of miR-31, the morphology of the cells was more similar to that of the motoneurons and the cells were fusiform. In the immunocytochemical staining, the expression of chat was increased and the fluorescence of the cells was strongly positive. However, after transfection of mi R-31 overexpression reagent, the morphological changes of the cells were not obvious, and the expression of Chat, which was not protruded and protruded, was significantly decreased after the transfection of mi R-31 overexpression reagent. The fluorescence intensity was significantly weaker than that in the inhibition group. The fluorescence quantitative PCR showed that the expression of the reverse transcription product was 0.867 in the interference group, 2.406 in the interference control group, 1.022 脳 10 6 in the overexpression group and 1.506 脳 10 5 in the over-expression control group, which indicated that the cell transfection was successful. And the downstream gene also has the obvious change. In the interference group, the expression of hb9 and stmn1 increased significantly, while the expression of nestinnlats2rhobtb and SATB2 decreased. In the overexpression group, Nestinhb9 lats2 and SATB2 were significantly increased, and Rhobtb was significantly decreased in the overexpression group. Conclusion: 1. RNAi MAX is more toxic to the nerve and can cause a large number of cell death, so the amount of cells should be increased in the experiment. The transfection efficiency of RNAi MAX was the highest on the third day. The high expression of 2.mi R-31 had a good dry maintenance effect on neural stem cells, and the differentiation of neural stem cells into motor neurons increased during inhibition.
【學位授予單位】：山西醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：R651.2

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本文編號：1854105