【摘要】：目的：神經(jīng)干細胞是修復中樞神經(jīng)系統(tǒng)損傷具有發(fā)展前景的工具細胞，但在體外培養(yǎng)易出現(xiàn)細胞衰老，嚴重影響移植效果。最新研究表明Bmi1基因是維持干細胞增殖所必須的一種重要基因，在生理情況下能保持特異靶基因處于穩(wěn)定的被抑制狀態(tài)，起到維持細胞存活、生長的重要功能。本研究擬構建Bmi1特異性siRNA慢病毒載體并探討其對人類胚胎紋狀體來源神經(jīng)干細胞增殖的調控作用，為神經(jīng)干細胞臨床應用修復中樞神經(jīng)系統(tǒng)損傷提供理論依據(jù)。 方法：本實驗首先從8-12周自然流產胎兒的大腦皮層區(qū)中有效分離出神經(jīng)干細胞并在含有絲分裂原的無血清培養(yǎng)基中進行傳代培養(yǎng)，通過特異標記物染色（Nestin）以及將神經(jīng)干細胞向神經(jīng)元（Tuj1）和星形膠質細胞（GFAP）分化來鑒定其為神經(jīng)干細胞，將神經(jīng)干細胞分成三組進行實驗，即Bmi1轉染組、GFP空病毒轉染組和未經(jīng)病毒轉染組。其次，構建Bmi1的慢病毒載體，進行RNA干擾，檢測慢病毒轉染效率，并通過熒光實時定量PCR的方法檢測Bmi1基因的干擾后的表達情況。再次，在有效干擾Bmi1基因后，通過BrdU摻入率檢測細胞的增生能力的改變情況。最后，用β半乳糖苷酶檢測細胞的衰老情況。通過以上數(shù)據(jù)比較三組神經(jīng)干細胞的增殖能力以及衰老情況。 結果：(1)經(jīng)Nestin、Tuj1、GFAP染色后證明實驗所取細胞為神經(jīng)干細胞。(2)在轉染48h后進行顯微鏡下觀察，發(fā)現(xiàn)兩病毒組轉染組的神經(jīng)干細胞均帶有綠色熒光，并且轉染效率都超過了90％，對比轉染后0h的Bmi-1基因的相對轉錄水平(99.6±8．25)％，48、72、168h的Bmi-1基因的相對轉錄水平均下降，數(shù)值分別為：(35.6±9.67)％、(26.4±12.05)％和(35.6±10.42)％，差異有統(tǒng)計學意義。(3)檢測不同時間點的Bmi1干擾后細胞的增殖情況，，不同時間點的Bmi1干擾組和GFP對照空病毒干擾組的BrdU摻入率比值分別為1.00±0.27、0．58±0．27、0．54±0.23、0.55±0.25，單因素方差分析后，48h、72h、168h與0h相比BrdU摻入率均下降，差異有統(tǒng)計學意義(P0．01)。(4)Bmi1干擾組和GFP-對照空病毒組神經(jīng)干細胞的衰老率分別為(59．53±15．38)％、(41．76±12．45)％，兩組神經(jīng)干細胞的衰老率之間差異有統(tǒng)計學意義(P0．01)。經(jīng)過Bmi1干擾一周后，神經(jīng)干細胞的增殖能力發(fā)生了顯著的下降，隨之而來的是神經(jīng)干細胞衰老數(shù)量發(fā)生顯著的增加。 結論：本實驗成功分離培養(yǎng)了人胚胎紋狀體來源神經(jīng)干細胞，構建Bmi1的慢病毒干擾載體轉染效率高。Bmi1基因是神經(jīng)干細胞保持增殖活性狀態(tài)關鍵調控基因，使神經(jīng)干細胞長期保持增殖活性從而滿足腦修復重建的需要。
[Abstract]:Objective: neural stem cells (NSCs) are promising tool cells for repairing central nervous system injury, but cell senescence is easy to occur in vitro culture, which seriously affects the transplantation effect. New research shows that Bmi1 gene is an important gene necessary to maintain stem cell proliferation. It can keep the specific target gene in a stable and inhibited state under physiological conditions and play an important role in maintaining cell survival and growth. The aim of this study was to construct a Bmi1 specific siRNA lentivirus vector and to investigate its regulatory effect on the proliferation of neural stem cells derived from human embryonic striatum, and to provide a theoretical basis for the clinical application of neural stem cells to repair central nervous system injury. Methods: neural stem cells (NSCs) were isolated from the cortical region of spontaneous abortion fetus at 8-12 weeks and cultured in serum-free medium containing mitogen. Neural stem cells were identified as neural stem cells by staining (Nestin) and differentiating neural stem cells into neurons (Tuj1) and astrocytes (GFAP). Neural stem cells were divided into three groups: Bmi1 transfection group. GFP empty virus transfection group and no virus transfection group. Secondly, the lentivirus vector of Bmi1 was constructed, the RNA interference was carried out, the efficiency of lentivirus transfection was detected, and the expression of Bmi1 gene after interference was detected by real-time quantitative PCR. Thirdly, after effectively interfering with Bmi1 gene, BrdU incorporation rate was used to detect the change of cell proliferation ability. Finally, 尾-galactosidase was used to detect cell senescence. The proliferative ability and senescence of three groups of neural stem cells were compared with the above data. Results: (1) after Nestin,Tuj1,GFAP staining, the cells were identified as neural stem cells. (2) after 48 hours of transfection, the neural stem cells in the two groups were observed under microscope. The relative transcription level of Bmi-1 gene at 0 h after transfection was (99.6 鹵8.25)%, and the relative transcription level of Bmi-1 gene at 48 h after transfection was (35.6 鹵9.67)%. The difference was statistically significant between (26.4 鹵12.05)% and (35.6 鹵10.42)%. (3) the proliferation of cells after Bmi1 interference at different time points was detected. The ratios of BrdU incorporation rates in Bmi1 interference group and GFP control group were 1.00 鹵0.27 鹵0.27 鹵0.57 鹵0.54 鹵0.23 鹵0.55 鹵0.25, respectively. After univariate ANOVA, the incorporation rate of BrdU decreased at 48h / 72h / 168h compared with 0h respectively. The senescence rates of neural stem cells in Bmi1 interference group and GFP- control group were (59.53 鹵15.38)% and (41.76 鹵12.45)%, respectively. There was significant difference in senescence rate of neural stem cells between the two groups (P 0.01). After a week of Bmi1 interference, the proliferation of neural stem cells decreased significantly, followed by a significant increase in the number of neural stem cells aging. Conclusion: neural stem cells derived from human embryonic striatum were isolated and cultured successfully in this experiment. The lentivirus interference vector of Bmi1 was constructed with high transfection efficiency. Bmi1 gene is the key regulation gene of neural stem cells to maintain proliferative activity. Neural stem cells to maintain long-term proliferative activity to meet the need for brain repair and reconstruction.
【學位授予單位】：大連醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R363

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