兔髓核细胞凋亡与BNIP3表达及意义

发布时间：2018-07-10 05:06

本文选题：椎间盘退变 + 髓核组织　；参考：《第三军医大学》2011年硕士论文

【摘要】：目的: 探讨髓核细胞凋亡程度与BNIP3表达的关系。为深入了解髓核细胞凋亡的机制提供实验依据。方法: 兔退变椎间盘髓核组织BNIP3表达检测:健康3月龄雄性新西兰大白兔40只,体重(2.3±0.2)kg,随机分为对照组(n=10)及实验组(n=20)。实验组大白兔采用针刺L3、4、L4、5及L5、6椎间盘制备椎间盘退变模型。于术后4、8周实验组各取10只大白兔、对照组各取5只大白兔,通过MRI检查评价手术椎间盘退变情况,采用组织学观察和TUNEL法检查兔椎间盘髓核组织中凋亡细胞,用免疫组织化学染色法检测兔椎间盘髓核细胞BNIP3的表达。体外缺氧条件下髓核细胞凋亡与BNIP3表达检测:采用组织块法原代培养兔NPCs,取一代细胞分别在正常氧和1%氧浓度下培养24、48、72小时,应用台盼蓝染色、流式细胞术及TUNEL染色观察缺氧对细胞凋亡的影响;采用免疫组化及RT-PCR观察BNIP3在缺氧条件下培养的NPCs中的表达情况. 结果: 兔退变椎间盘髓核组织BNIP3表达检测: (1) MRI检查示实验组未手术对照节段(L_(1、2)、L_(2、3)及L_(6、7))椎间盘均正常,术后4、8周手术节段椎间盘髓核信号强度呈逐渐降低趋势。 (2)组织学观察及TUNEL检查示对照组椎间盘组织中髓核细胞密度高,偶见极少量散在的凋亡细胞存在。实验组术后4周、8周时椎间盘组织内髓核细胞密度逐渐降低,存在较多的凋亡细胞。 (3)对照组椎间盘组织髓核细胞中无BNIP3蛋白表达;实验组术后4周、8周椎间盘组织髓核细胞中BNIP3蛋白表达逐渐上调。体外缺氧条件下髓核细胞凋亡与BNIP3表达检测: 正常氧条件下BNIP3没有表达,缺氧条件下随着培养时间的延续,NPCs凋亡率不断增加BNIP3表达不断增强。结论: (1)椎间盘的退变与髓核组织中细胞密度下降有关;(2)细胞凋亡是椎间盘髓核细胞减少的原因之一;(3)缺氧条件下能诱导NPCs凋亡增加;(4)BNIP3参与了椎间盘髓核细胞凋亡。
[Abstract]:Objective: to investigate the relationship between apoptosis of nucleus pulposus cells and expression of BNIP3. To provide experimental evidence for further understanding the mechanism of nucleus pulposus apoptosis. Methods: the expression of BNIP3 in the nucleus pulposus of degenerative intervertebral disc of rabbits was detected: 40 healthy 3-month-old male New Zealand white rabbits with body weight of (2.3 卤0.2) kg were randomly divided into control group (n = 10) and experimental group (n = 20). The degenerative model of intervertebral disc was established by acupuncture of L _ 3, L _ 3, L _ 4, L _ 4 and L _ (5), in the experimental group. Ten rabbits were taken from experimental group and 5 rabbits from control group at 4 weeks and 8 weeks after operation. The degeneration of intervertebral disc was evaluated by MRI, and the apoptotic cells in nucleus pulposus were examined by histological observation and Tunel method. The expression of BNIP3 in nucleus pulposus cells of rabbit intervertebral disc was detected by immunohistochemical staining. Apoptosis of nucleus pulposus cells and expression of BNIP3 in hypoxia in vitro: NPCs were cultured in primary culture with tissue block method. The cells were cultured at normal oxygen concentration and 1% oxygen concentration for 24 ~ 48 ~ 72 hours, respectively, and were stained with Trypan blue. Flow cytometry and Tunel staining were used to observe the effect of hypoxia on apoptosis and the expression of BNIP3 in NPCs cultured under hypoxia was observed by immunohistochemistry and RT-PCR. Results: the expression of BNIP3 in the degenerative nucleus pulposus of rabbits was detected: (1) MRI showed that the intervertebral discs of the unoperated control segments (L1F2 and L6F7) were normal in the experimental group. The signal intensity of nucleus pulposus decreased gradually 4 weeks after operation. (2) histological observation and Tunel showed that the density of nucleus pulposus cells in the control group was high, and a few apoptosis cells were occasionally found. In the experimental group, the density of nucleus pulposus cells gradually decreased and there were more apoptotic cells in the intervertebral disc tissue at 4 weeks and 8 weeks after operation. (3) there was no expression of BNIP3 protein in the nucleus pulposus cells of the control group. The expression of BNIP3 protein in nucleus pulposus cells of the experimental group increased gradually 4 weeks and 8 weeks after operation. Apoptosis and expression of BNIP3 in nucleus pulposus cells under hypoxia in vitro: there was no expression of BNIP3 in normal oxygen condition, and the expression of BNIP3 increased with the extension of culture time. Conclusion: (1) the degeneration of intervertebral disc is related to the decrease of cell density in nucleus pulposus; (2) apoptosis is one of the reasons for the decrease of nucleus pulposus cells in intervertebral disc; (3) apoptosis of NPCs can be induced by hypoxia; (4) BNIP3 is involved in intervertebral. Apoptosis of nucleus pulposus disk cells.
【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R363

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