發(fā)布時間：2018-06-20 22:56

  本文選題：慢病毒 + MMP-3��； 參考：《青島大學(xué)》2017年碩士論文

【摘要】：目的:通過慢病毒介導(dǎo)MMP-3shRNA和Fas-siRNA雙基因體外轉(zhuǎn)染人退變髓核細胞,探討單基因、雙基因聯(lián)合轉(zhuǎn)染后對人退變髓核細胞功能的影響。方法:a.體外培養(yǎng)人退變髓核細胞,對其進行臺盼藍染色、甲苯胺藍染色,觀察細胞活力以及細胞形態(tài);b.分別根據(jù)人MMP-3基因與Fas基因的mRNA序列合成不同的MMP-3 shRNA與Fas-siRNA序列,與酶切后的慢載體鏈接并轉(zhuǎn)入感受態(tài)293T細胞,對慢病毒進行包裝。c.慢病毒感染退變髓核細胞,按照處理方式不同,本實驗分為5組,分別為:A-空白對照組,B-AAV陰性對照組,C-AAV-MMP-3shRNA組,D-AAV-FassiRNA組,E-AAV-FassiRNA-MMP3shRNA組;d.轉(zhuǎn)染72小時后CCK8測定各組之間的細胞增殖情況;72小時通過熒光定量PCR檢測轉(zhuǎn)染髓核細胞mRNA水平變化(MMP-3、Fas以及Ⅱ型膠原、蛋白多糖);96小時后通過Western Blot檢測髓核細胞內(nèi)蛋白水平變化(Ⅱ型膠原蛋白、蛋白多糖)。結(jié)果:a.細胞形態(tài)學(xué)進行觀察發(fā)現(xiàn)初消化的成人退變髓核細胞貼壁時間約為4-5天,初貼壁時的形態(tài)表現(xiàn)為橢圓形、梭形、多角形,形狀不規(guī)則,胞質(zhì)向外伸突并隨著時間延長逐漸伸長。經(jīng)10-14 d以后,90%細胞呈融合狀態(tài),形狀類似漩渦狀或火焰狀的細胞團,胞質(zhì)豐富且?guī)в幸欢ㄕ酃庑?細胞核較大輪廓清楚,呈卵圓形核,有大約1-3個核仁。經(jīng)過傳代后,第一代細胞之間的粘附性較原代變差,細胞形態(tài)變?yōu)殚L梭形,這與之前的研究相一致原代髓核細胞經(jīng)甲苯胺藍染色后呈短梭形、多角形等不規(guī)則形狀,胞質(zhì)均呈藍色,細胞核位于細胞中央或偏向一側(cè)細胞膜,顏色較周圍胞質(zhì)深。b.退變髓核細胞原代培養(yǎng)期間存活率在97%以上,第1代培養(yǎng)期間存活率在92-96%之間,第二代仍能維持90%左右,以后細胞存活率逐漸下降。c.72小時后用熒光顯微鏡檢測重組慢病毒侵染人退變髓核細胞后的GFP、BFP表達情況,結(jié)果顯示各組退變髓核細胞均已達到很高的轉(zhuǎn)染效果。d.與空白對照組相比較,Fas干擾組、MMP3干擾組及MMP3+Fas雙基因共轉(zhuǎn)組Fas、MMP-3、II型膠原、蛋白多糖的mRNA表達量增加,且雙基因組效果優(yōu)于單基因組,與空白對照組相比較,Fas干擾組、MMP3干擾組及MMP3+Fas雙基因共轉(zhuǎn)組髓核細胞的蛋白多糖和Ⅱ型膠原蛋白表達量增高,且雙基因組效果優(yōu)于單基因組,以上結(jié)果差異均具有統(tǒng)計學(xué)意義(P0.05)。結(jié)論:1.通過基因沉默技術(shù)干擾髓核細胞內(nèi)Fas、MMP-3基因的表達,可以有效抑制髓核細胞的凋亡,雙基因共轉(zhuǎn)染具有協(xié)同作用。2.慢病毒介導(dǎo)的RNAi可以顯著抑制人退變髓核細胞MMP-3、Fas的表達,增加人退變髓核細胞外基質(zhì)表達量,且雙基因具有協(xié)同效應(yīng)。
[Abstract]:Objective: To investigate the effect of single gene and double gene combined transfection on the function of human degeneration medullary cells through transfection of MMP-3shRNA and Fas-siRNA double genes mediated by lentivirus and double gene in vitro. Methods: A. cultured human degeneration medullary cells in vitro, trypan blue staining, toluidine blue staining, observation of cell viability and cell morphology. B., based on the mRNA sequence of the human MMP-3 gene and the Fas gene, synthesized different MMP-3 shRNA and Fas-siRNA sequences, linked with the slow vector after the enzyme cut and transferred to the sensory 293T cells, and packed the lentivirus to the degenerative nucleus pulposus cells of the lentivirus. According to the different treatment methods, the experiment was divided into 5 groups: A- blank control group and B-AAV, respectively. Negative control group, group C-AAV-MMP-3shRNA, group D-AAV-FassiRNA and E-AAV-FassiRNA-MMP3shRNA group; CCK8 was used to determine the cell proliferation between each group after 72 hours of D. transfection, and 72 hours by fluorescence quantitative PCR to detect the changes of mRNA level in transfected nucleus pulposus cells (MMP-3, Fas, type II gluin and proteoglycan); and 96 hours later, the nucleus pulposus was detected by Western Blot. Changes in the level of intracellular protein (type II collagen, proteoglycan). Results: the morphological observation of A. cells found that the adherent time of the first digested adult degenerative nucleus pulposus cells was about 4-5 days, and the shape of the initial adherence was elliptical, spindle, polygon, irregular shape, the cytoplasm outstretched and extended with the time extension. 10-14 D After that, the 90% cells were fused, shaped like whirlpool or flamellike cell masses, rich in cytoplasm and with a certain refraction. The nucleus of the nucleus was clear, the nucleus was oval and about 1-3 nucleolus. After passage, the adhesion between the first generation cells was worse than that of the original, and the cell morphology changed into a long shuttle form. The original nucleus pulposus cells were stained with short spindle shape, polygon and other irregular shape, and the cytoplasm was blue. The nucleus was located in the central cell or on one side of the cell membrane. The survival rate was more than 97% during the primary culture of.B. degenerative nucleus pulposus cells compared with the surrounding cytoplasm, and the survival rate of the first generation was between the second generations during the first generation culture. The GFP and BFP expression of recombinant lentivirus infected human degeneration nucleus pulposus cells were detected by the fluorescence microscope after.C.72 hours. The results showed that all the degenerative nucleus pulposus cells had reached a high transfection effect,.D. was compared with the blank control group, Fas interference group, MMP3 interference group and MMP3+Fas The mRNA expression of Fas, MMP-3, II type collagen and proteoglycan increased in the double gene co rotation group, and the effect of the double genome was better than that of the single genome. Compared with the blank control group, the expression of proteoglycan and type II collagen protein in the nucleus pulposus cells of Fas interference group, MMP3 interference group and MMP3+Fas double gene co transfer group increased, and the effect of the double genome was better than that of the single gene. The differences in the above results were statistically significant (P0.05). Conclusion: 1. the expression of Fas and MMP-3 gene can effectively inhibit the apoptosis of nucleus pulposus cells through gene silencing technique, which can inhibit the expression of MMP-3 and Fas in human degeneration medullary cells by CO transfection of.2. lentivirus mediated RNAi. The expression of extracellular matrix in nucleus pulposus was degenerated, and the double gene had synergistic effect.
【學(xué)位授予單位】：青島大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R681.5

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本文編號：2045994