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  本文選題：椎間盤退變 + 髓核組織　； 參考：《第三軍醫(yī)大學》2011年碩士論文

【摘要】：目的: 探討髓核細胞凋亡程度與BNIP3表達的關系。為深入了解髓核細胞凋亡的機制提供實驗依據(jù)。 方法: 兔退變椎間盤髓核組織BNIP3表達檢測:健康3月齡雄性新西蘭大白兔40只,體重(2.3±0.2)kg,隨機分為對照組(n=10)及實驗組(n=20)。實驗組大白兔采用針刺L3、4、L4、5及L5、6椎間盤制備椎間盤退變模型。于術后4、8周實驗組各取10只大白兔、對照組各取5只大白兔,通過MRI檢查評價手術椎間盤退變情況,采用組織學觀察和TUNEL法檢查兔椎間盤髓核組織中凋亡細胞,用免疫組織化學染色法檢測兔椎間盤髓核細胞BNIP3的表達。 體外缺氧條件下髓核細胞凋亡與BNIP3表達檢測:采用組織塊法原代培養(yǎng)兔NPCs,取一代細胞分別在正常氧和1%氧濃度下培養(yǎng)24、48、72小時,應用臺盼藍染色、流式細胞術及TUNEL染色觀察缺氧對細胞凋亡的影響;采用免疫組化及RT-PCR觀察BNIP3在缺氧條件下培養(yǎng)的NPCs中的表達情況. 結果: 兔退變椎間盤髓核組織BNIP3表達檢測: (1) MRI檢查示實驗組未手術對照節(jié)段(L_(1、2)、L_(2、3)及L_(6、7))椎間盤均正常,術后4、8周手術節(jié)段椎間盤髓核信號強度呈逐漸降低趨勢。 (2)組織學觀察及TUNEL檢查示對照組椎間盤組織中髓核細胞密度高,偶見極少量散在的凋亡細胞存在。實驗組術后4周、8周時椎間盤組織內髓核細胞密度逐漸降低,存在較多的凋亡細胞。 (3)對照組椎間盤組織髓核細胞中無BNIP3蛋白表達;實驗組術后4周、8周椎間盤組織髓核細胞中BNIP3蛋白表達逐漸上調。 體外缺氧條件下髓核細胞凋亡與BNIP3表達檢測: 正常氧條件下BNIP3沒有表達,缺氧條件下隨著培養(yǎng)時間的延續(xù),NPCs凋亡率不斷增加BNIP3表達不斷增強。 結論: (1)椎間盤的退變與髓核組織中細胞密度下降有關;(2)細胞凋亡是椎間盤髓核細胞減少的原因之一;(3)缺氧條件下能誘導NPCs凋亡增加;(4)BNIP3參與了椎間盤髓核細胞凋亡。
[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 鹵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.
【學位授予單位】：第三軍醫(yī)大學
【學位級別】：碩士
【學位授予年份】：2011
【分類號】：R363

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