RNA干扰TERT基因表达对大鼠脊髓损伤后瘢痕形成的实验研究

发布时间：2018-05-31 04:03

本文选题：端粒酶逆转录酶 + RNA干扰技术　；参考：《新疆医科大学》2015年硕士论文

【摘要】：目的：利用星型胶质细胞逆转录酶基因的慢病毒载体,转染星型胶质细胞及作用于大鼠脊髓损伤区,观察其对星型胶质细胞生长的影响及损伤区胶质瘢痕的形成。方法：体外培养大鼠脊髓源星型胶质细胞,用逆转录酶基因的慢病毒载体转染星型胶质细胞；取60只SD大鼠,随机分成4组：分别为实验组；阴性对照组；模型组；正常组。利用改良Allen's重物坠落法建立脊髓损伤动物模型,分别于造模成功后的3d、1w、2w、4w、6w,5个时间点取脊髓标本,利用免疫印迹(western-blot)法检钡GFAP、PCR-ELISA法检测端粒酶的表达及免疫组化技术观察胶质瘢痕的形成。结果：培养的细胞经鉴定为星型胶质细胞,TERT基因慢病毒载体对星型胶质细胞的转染率达85%-90%；且转染后生长态势出现明显改变,在转染后的24-48小时细胞凋亡达50%-60%。阴性对照组及空白组生长态势无明显改变。对脊髓损伤区瘢痕形成的影响,不同治疗方式其BBB评分结果存在差异(P0.01),且各对照组与干扰组均有统计学意义(P0.001)。干扰组大鼠损伤脊髓GFAP表达低于其余各组,且逐渐减少,与阴性对照组及空白组比较均有统计学差异(P0.001),阴性及空白对照则增多,两者间无差异(P0.05)；端粒酶的表达在干扰组中降低,与其他各组比较均有统计差异(P0.001)。标记NF-200荧光的,TERT基因慢病毒载体干扰组及正常组其荧光表达明显高于阴性对照组及空白组。结论：星型胶质细胞体外培养可获成功,靶向TERT基因的慢病毒载体体外转染星型胶质细胞对其生长态势有明显影响,并可加速凋亡。作用于大鼠脊髓损伤区的TERT慢病毒载体对脊髓损伤的修复有促进作用,可以减少损伤区胶质瘢痕的形成,为脊髓损伤的治疗研究提供进一步实验依据。
[Abstract]:Aim: to investigate the effects of galactocyte reverse transcriptase gene lentivirus vector on the growth of astrocytes and the formation of glial scar in the injured area of spinal cord in rats. Methods: rat spinal cord astrocytes were cultured in vitro and transfected with lentivirus vector of reverse transcriptase gene. Sixty SD rats were randomly divided into four groups: experimental group, negative control group, model group and normal group. An animal model of spinal cord injury was established by modified Allen's weight fall method. The spinal cord specimens were collected at 5 time points at 3 days, 1 week, 2 weeks, 4 weeks and 6 weeks after the establishment of the model. The expression of telomerase and the formation of glial scar were detected by Western blot- blot assay with PCR-ELISA. Results: the cultured cells were identified as stellate glial cell tert gene lentivirus vector transfection rate of 85-90, and after transfection, the growth trend was obviously changed, after 24-48 hours after transfection, the apoptosis reached 50-60%. The growth state of negative control group and blank group had no obvious change. The effect of different treatment methods on scar formation in spinal cord injury area was significantly different (P 0.01), and there was significant difference between the control group and the interference group (P 0.001). The expression of GFAP in the injured spinal cord in the interference group was lower than that in the other groups, and gradually decreased. Compared with the negative control group and the blank group, the expression of GFAP in the injured spinal cord was significantly lower than that in the negative control group and the blank control group, but there was no difference between the two groups, and the expression of telomerase was decreased in the interference group. Compared with other groups, there was statistical difference (P 0.001). The fluorescence expression of NF-200 labeled lentivirus vector interference group and normal group was significantly higher than that of negative control group and blank group. Conclusion: the cultured astrocytes can be successfully cultured in vitro. The lentivirus vector targeting TERT gene has a significant effect on the growth of astrocytes and can accelerate the apoptosis of astrocytes. The TERT lentivirus vector acting on the spinal cord injury area can promote the repair of spinal cord injury, reduce the formation of glial scar in the injured area, and provide further experimental evidence for the treatment of spinal cord injury.
【学位授予单位】：新疆医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R651.2

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