NIBV对原代鸡肾细胞生长及相关基因表达的影响

发布时间：2018-03-03 07:51

本文选题：肾型传染性支气管炎病毒　切入点：鸡肾细胞　出处：《江西农业大学》2016年硕士论文　论文类型：学位论文

【摘要】：为研究NIBV对鸡肾细胞活性,抗氧化功能以及与细胞凋亡相关基因表达量等的影响,探讨NIBV的致病机理。本实验通过已建立的鸡肾细胞体外培养模型,用剂量为半数组织感染量的NIBV病毒感染鸡肾细胞,检测NIBV病毒在肾细胞内的动态复制变化;通过MTT四唑盐法和乳酸脱氢酶测定试剂盒检测NIBV病毒对肾细胞活性和细胞膜的损伤情况,用试剂盒测定细胞和细胞培养液中超氧化物歧化酶和黄嘌呤氧化酶活性、丙二醛和尿酸含量;SYBR-PCR检测原代鸡肾细胞内XOD,Bak1,Bcl2,P53,Caspase-3,DDIT3的m RNA相对基因表达量变化。结果表明:感染NIBV后,鸡肾细胞的形态和结构发生明显变化,病毒组细胞活力极显著下降(P0.01),细胞内的病毒粒子数量先升高后下降,在36 hpi达到最高;与对照组相比,病毒组细胞LDH含量、XOD活性、UA浓度、上清液SOD活性和胞内MDA浓度均极显著升高(P0.01);胞内SOD活性在72 hpi极显著下降(P0.01);荧光定量PCR结果显示病毒组细胞XOD,Bak1,Bcl2,P53,Caspase-3 mRNA基因表达量较对照组都极显著升高(P0.01),病毒组Bcl2上游调控基因DDIT3的mRNA基因表达量较对照组极显著下降(P0.01)。说明,肾型传染性支气管炎病毒感染抑制原代鸡肾细胞的增殖,同时引起细胞黄嘌呤氧化酶活性上升和抗氧化功能下降,NIBV病毒感染影响鸡肾细胞凋亡相关基因的表达。
[Abstract]:In order to study the effects of NIBV on the activity, antioxidant function and expression of apoptosis-related genes of chicken kidney cells, and to explore the pathogenesis of NIBV, we established a model of chicken renal cell culture in vitro. Chicken renal cells were infected with NIBV virus with a dose of 50% tissue infection. The dynamic replication of NIBV virus in renal cells was detected. MTT tetrazolium salt assay and lactate dehydrogenase assay were used to detect the damage of NIBV virus to renal cells and cell membrane. The activities of superoxide dismutase (SOD) and xanthine oxidase (xanthine oxidase) in cell and cell culture medium were measured by the kit. Malondialdehyde (MDA) and uric acid (uric acid) were detected by SYBR-PCR in primary chicken renal cells. The expression of m RNA in primary chicken kidney cells was detected by Bcl2P53Caspase-3 caspase-3 DDIT3. The results showed that the morphology and structure of chicken renal cells changed significantly after NIBV infection. The activity of virus group cells decreased significantly (P 0.01), and the number of virus particles increased first, then decreased, and reached the highest level at 36 hpi. Compared with the control group, the concentration of LDH activity and UA in virus group cells were increased. The activity of SOD and the concentration of intracellular MDA in supernatant were significantly higher than those in control group, and the intracellular SOD activity decreased significantly at 72 hpi. The results of fluorescence quantitative PCR showed that the expression of Caspase-3 mRNA gene in XODN Bak1 Bcl2P5Caspase-3 cells was significantly higher than that in control group, and the expression of Caspase-3 mRNA in virus group Bcl2 was significantly higher than that in control group. The expression of mRNA gene in DDIT3 was significantly lower than that in the control group (P 0.01). The infection of renal infectious bronchitis virus inhibited the proliferation of primary chicken renal cells and caused the increase of xanthine oxidase activity and the decrease of antioxidant function. The infection of NIBV virus affected the expression of apoptosis-related genes in chicken kidney cells.
【学位授予单位】：江西农业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S858.31

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