HIF-1诱导BNIP3在人胶质瘤A172细胞系中促凋亡作用的实验研究

发布时间：2019-05-08 20:40

【摘要】：目的检测缺氧环境下人胶质瘤A172细胞系中HIF-1、BNIP3的表达及两者之间的关系，并探讨其对胶质瘤细胞的抑制增殖和促凋亡作用。方法1.培养人胶质母细胞瘤A172细胞系并制作缺氧箱模型，并将其随机分为A组(空白对照组)、B组（缺氧组）、C组（缺氧+HIF-1激动剂）、D组（缺氧+HIF-1激动剂+BNIP-3抑制剂）共4组，相差显微镜观察各组细胞在24h、48h、72h时间点的细胞形态学变化，MTT法检测各组细胞增殖抑制情况。2. Tunel荧光一步法检测各组A172细胞凋亡和增殖。3.运用细胞免疫组化方法检测各组细胞中HIF-1与BNIP-3的蛋白表达情况。结果1.相差显微镜观察：与A组比较，B、C、D三组细胞存活减少，增殖受到抑制，以B、C两组为著。MTT法：B、C、D组细胞增殖受到抑制，以B、C两组为著。2.Tunel荧光法检测：与A组相比，B、C两组细胞存活增殖差异有显著统计学意义（P0.05）；与B组比较，C组细胞存活增殖差异有统计学意义（P0.05）；与C组比较，D组细胞存活增殖差异有统计学意义(P0.05)；3.免疫组化检测：(1)蛋白HIF-1表达：与A组比较，B、C、D三组HIF-1的表达明显增高，差异具有统计学意义（P0.05）；与B组比较，C、D两组中HIF-1的表达增高，差异有统计学意义（P0.05）。(2)蛋白BNIP-3表达：D组BNIP-3表达与B、C两组比较明显减少，差异有统计学意义（P0.05），A、B、C、D四组各时间点BNIP-3表达与HIF-1表达呈正相关，具有统计学意义。结论1.人胶质瘤A172细胞系在缺氧环境下增殖可受到抑制，细胞发生凋亡；2.HIF-1在缺氧环境下表达增高，，并可诱导BNIP-3的生成，两者呈正相关关系；3.HIF-1可诱导A172细胞发生凋亡，可能的机制可能与其诱导BNIP-3的生成有关。
[Abstract]:Aim to investigate the expression of HIF-1,BNIP3 in human glioma cell line A172 under hypoxia and its relationship, and to explore its inhibitory effect on proliferation and apoptosis of human glioma cell line A172. Method 1. Human glioblastoma cell line A172 was cultured and hypoxia box model was made. They were randomly divided into group A (blank control group), B) (hypoxia group), C group (hypoxia HIF-1 agonist). Group D (hypoxia HIF-1 agonist BNIP-3 inhibitor) was divided into 4 groups. Phase contrast microscope was used to observe the morphological changes of the cells at 24 h, 48 h, 72 h, and MTT assay was used to detect the inhibition of cell proliferation. 2. Detection of apoptosis and proliferation of A172 cells by Tunel fluorescence one-step method. 3. The protein expression of HIF-1 and BNIP-3 in the cells of each group was detected by immunocytochemistry. Outcome 1. Phase contrast microscope observation: compared with group A, three groups B, C, D had a decrease in survival and proliferation, especially in B and C groups. MTT method: B, C, D group cell proliferation was inhibited, B, C, D groups were inhibited, B, C, D group cell proliferation was inhibited, B, C, D group were inhibited by B, C, D. 2.Tunel fluorescence assay: compared with group A, there was significant difference in cell survival and proliferation between group B and group C (P0.05). Compared with group B, group C had significant difference in cell survival and proliferation (P0.05); group D had significant difference in cell survival and proliferation compared with group C (P0.05); Immunohistochemistry: (1) the expression of protein HIF-1: compared with group A, the expression of HIF-1 in group B, C and D was significantly higher than that in group A (P0.05). Compared with group B, the expression of HIF-1 in group C and D was significantly higher than that in group B (P0.05). (2). The expression of BNIP-3 in group D was significantly lower than that in group B and C (P < 0.05). The difference was statistically significant (P0.05). There was a positive correlation between BNIP-3 expression and HIF-1 expression at each time point in A, B, C, D groups. Conclusion 1. The proliferation and apoptosis of human glioma cell line A172 were inhibited in hypoxia environment, and the expression of 2.HIF-1 was increased in hypoxia environment, and the production of BNIP-3 was induced, and there was a positive correlation between them. 3.HIF-1 can induce apoptosis of A172 cells, and the possible mechanism may be related to the induction of BNIP-3 production.
【学位授予单位】：遵义医学院
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R739.41

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