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

【摘要】：目的: 探討髓核細(xì)胞凋亡程度與BNIP3表達(dá)的關(guān)系。為深入了解髓核細(xì)胞凋亡的機(jī)制提供實(shí)驗(yàn)依據(jù)。 方法: 兔退變椎間盤(pán)髓核組織BNIP3表達(dá)檢測(cè):健康3月齡雄性新西蘭大白兔40只,體重(2.3±0.2)kg,隨機(jī)分為對(duì)照組(n=10)及實(shí)驗(yàn)組(n=20)。實(shí)驗(yàn)組大白兔采用針刺L3、4、L4、5及L5、6椎間盤(pán)制備椎間盤(pán)退變模型。于術(shù)后4、8周實(shí)驗(yàn)組各取10只大白兔、對(duì)照組各取5只大白兔,通過(guò)MRI檢查評(píng)價(jià)手術(shù)椎間盤(pán)退變情況,采用組織學(xué)觀察和TUNEL法檢查兔椎間盤(pán)髓核組織中凋亡細(xì)胞,用免疫組織化學(xué)染色法檢測(cè)兔椎間盤(pán)髓核細(xì)胞BNIP3的表達(dá)。 體外缺氧條件下髓核細(xì)胞凋亡與BNIP3表達(dá)檢測(cè):采用組織塊法原代培養(yǎng)兔NPCs,取一代細(xì)胞分別在正常氧和1%氧濃度下培養(yǎng)24、48、72小時(shí),應(yīng)用臺(tái)盼藍(lán)染色、流式細(xì)胞術(shù)及TUNEL染色觀察缺氧對(duì)細(xì)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(dá)情況. 結(jié)果: 兔退變椎間盤(pán)髓核組織BNIP3表達(dá)檢測(cè): (1) MRI檢查示實(shí)驗(yàn)組未手術(shù)對(duì)照節(jié)段(L_(1、2)、L_(2、3)及L_(6、7))椎間盤(pán)均正常,術(shù)后4、8周手術(shù)節(jié)段椎間盤(pán)髓核信號(hào)強(qiáng)度呈逐漸降低趨勢(shì)。 (2)組織學(xué)觀察及TUNEL檢查示對(duì)照組椎間盤(pán)組織中髓核細(xì)胞密度高,偶見(jiàn)極少量散在的凋亡細(xì)胞存在。實(shí)驗(yàn)組術(shù)后4周、8周時(shí)椎間盤(pán)組織內(nèi)髓核細(xì)胞密度逐漸降低,存在較多的凋亡細(xì)胞。 (3)對(duì)照組椎間盤(pán)組織髓核細(xì)胞中無(wú)BNIP3蛋白表達(dá);實(shí)驗(yàn)組術(shù)后4周、8周椎間盤(pán)組織髓核細(xì)胞中BNIP3蛋白表達(dá)逐漸上調(diào)。 體外缺氧條件下髓核細(xì)胞凋亡與BNIP3表達(dá)檢測(cè): 正常氧條件下BNIP3沒(méi)有表達(dá),缺氧條件下隨著培養(yǎng)時(shí)間的延續(xù),NPCs凋亡率不斷增加BNIP3表達(dá)不斷增強(qiáng)。 結(jié)論: (1)椎間盤(pán)的退變與髓核組織中細(xì)胞密度下降有關(guān);(2)細(xì)胞凋亡是椎間盤(pán)髓核細(xì)胞減少的原因之一;(3)缺氧條件下能誘導(dǎo)NPCs凋亡增加;(4)BNIP3參與了椎間盤(pán)髓核細(xì)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(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類(lèi)號(hào)】：R363

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