慢病毒介导MMP-3shRNA和Fas-siRNA体外转染人退变髓核细胞的生物学效应

发布时间：2018-06-20 22:56

  本文选题：慢病毒 + MMP-3　； 参考：《青岛大学》2017年硕士论文

【摘要】：目的:通过慢病毒介导MMP-3shRNA和Fas-siRNA双基因体外转染人退变髓核细胞,探讨单基因、双基因联合转染后对人退变髓核细胞功能的影响。方法:a.体外培养人退变髓核细胞,对其进行台盼蓝染色、甲苯胺蓝染色,观察细胞活力以及细胞形态;b.分别根据人MMP-3基因与Fas基因的mRNA序列合成不同的MMP-3 shRNA与Fas-siRNA序列,与酶切后的慢载体链接并转入感受态293T细胞,对慢病毒进行包装。c.慢病毒感染退变髓核细胞,按照处理方式不同,本实验分为5组,分别为:A-空白对照组,B-AAV阴性对照组,C-AAV-MMP-3shRNA组,D-AAV-FassiRNA组,E-AAV-FassiRNA-MMP3shRNA组;d.转染72小时后CCK8测定各组之间的细胞增殖情况;72小时通过荧光定量PCR检测转染髓核细胞mRNA水平变化(MMP-3、Fas以及Ⅱ型胶原、蛋白多糖);96小时后通过Western Blot检测髓核细胞内蛋白水平变化(Ⅱ型胶原蛋白、蛋白多糖)。结果:a.细胞形态学进行观察发现初消化的成人退变髓核细胞贴壁时间约为4-5天,初贴壁时的形态表现为椭圆形、梭形、多角形,形状不规则,胞质向外伸突并随着时间延长逐渐伸长。经10-14 d以后,90%细胞呈融合状态,形状类似漩涡状或火焰状的细胞团,胞质丰富且带有一定折光性,细胞核较大轮廓清楚,呈卵圆形核,有大约1-3个核仁。经过传代后,第一代细胞之间的粘附性较原代变差,细胞形态变为长梭形,这与之前的研究相一致原代髓核细胞经甲苯胺蓝染色后呈短梭形、多角形等不规则形状,胞质均呈蓝色,细胞核位于细胞中央或偏向一侧细胞膜,颜色较周围胞质深。b.退变髓核细胞原代培养期间存活率在97%以上,第1代培养期间存活率在92-96%之间,第二代仍能维持90%左右,以后细胞存活率逐渐下降。c.72小时后用荧光显微镜检测重组慢病毒侵染人退变髓核细胞后的GFP、BFP表达情况,结果显示各组退变髓核细胞均已达到很高的转染效果。d.与空白对照组相比较,Fas干扰组、MMP3干扰组及MMP3+Fas双基因共转组Fas、MMP-3、II型胶原、蛋白多糖的mRNA表达量增加,且双基因组效果优于单基因组,与空白对照组相比较,Fas干扰组、MMP3干扰组及MMP3+Fas双基因共转组髓核细胞的蛋白多糖和Ⅱ型胶原蛋白表达量增高,且双基因组效果优于单基因组,以上结果差异均具有统计学意义(P0.05)。结论:1.通过基因沉默技术干扰髓核细胞内Fas、MMP-3基因的表达,可以有效抑制髓核细胞的凋亡,双基因共转染具有协同作用。2.慢病毒介导的RNAi可以显著抑制人退变髓核细胞MMP-3、Fas的表达,增加人退变髓核细胞外基质表达量,且双基因具有协同效应。
[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.
【学位授予单位】：青岛大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R681.5

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