【摘要】：第一部分體外實(shí)驗(yàn)鈣離子依賴(lài)的內(nèi)質(zhì)網(wǎng)應(yīng)激信號(hào)通路在大鼠髁突軟骨細(xì)胞中的體外研究[目的]研究鈣離子依賴(lài)的ERS信號(hào)通路在體外培養(yǎng)大鼠髁突軟骨細(xì)胞中的作用。[方法]選取3周齡的SD大鼠,取顳下頜關(guān)節(jié)髁突軟骨進(jìn)行原代培養(yǎng),采用第三代髁突軟骨細(xì)胞進(jìn)行實(shí)驗(yàn)。用常見(jiàn)的ERS誘導(dǎo)劑4 μg/ml衣霉素(Tunicamycin, Tm)處理大鼠髁突軟骨細(xì)胞,或用鈣離子通道受體阻斷劑2-APB、Rya,2-APB+Rya預(yù)處理30min后再加入4 μg/ml Tm,處理24h后收集細(xì)胞。實(shí)驗(yàn)分為：空白對(duì)照組、Tm組、Tm+2APB組、Tm+Rya組、Tm+2APB+Rya組。采用Fura-2/AM熒光染料進(jìn)行胞漿內(nèi)鈣離子染色,然后用熒光光譜儀檢測(cè)細(xì)胞內(nèi)鈣離子濃度的變化；流式細(xì)胞儀檢測(cè)細(xì)胞凋亡率；采用RT-qPCR和Western Blot檢測(cè)GRP78.GRP94、 CHOP、Caspase-12內(nèi)質(zhì)網(wǎng)應(yīng)激標(biāo)志分子在nRNA和蛋白水平的表達(dá)改變。[結(jié)果]1.Tm處理大鼠髁突軟骨細(xì)胞24小時(shí)后,細(xì)胞鈣離子濃度顯著升高(P0.05)。經(jīng)鈣離子通道受體阻斷劑預(yù)處理后,細(xì)胞內(nèi)鈣離子濃度顯著降低(Tm+2APB+Rya組Tm+Rya組Tm+2APB組),但仍高于空白對(duì)照組。而單純用鈣離子通道受體阻斷劑處理后與空白對(duì)照組無(wú)顯著差異。2.Tm處理大鼠髁突軟骨細(xì)胞24小時(shí)后,細(xì)胞有明顯凋亡增加(P0.01),而經(jīng)鈣離子通道受體阻斷劑預(yù)處理后,凋亡顯著減少(P0.01),與空白對(duì)照組無(wú)顯著差異。3.Tm處理大鼠髁突軟骨細(xì)胞24小時(shí)后,ERS標(biāo)志分子GRP78、GRP94、CHOP、 Caspase-12 mRNA及其蛋白水平上表達(dá)升高。鈣離子通道受體阻斷劑可以抑制Tm誘導(dǎo)的大鼠髁突軟骨細(xì)胞(GRP78、GRP94、CHOP、Caspase-12 mRNA及其蛋白水平上表達(dá)升高,除Caspase-12,均以2APB+Rya效果佳。[結(jié)論]1.Tm可以誘導(dǎo)大鼠髁突軟骨細(xì)胞發(fā)生ERS,導(dǎo)致細(xì)胞凋亡。2.Tm可以使大鼠髁突軟骨細(xì)胞內(nèi)鈣離子濃度升高。3.鈣離子通道受體阻斷劑可以抑制大鼠髁突軟骨細(xì)胞發(fā)生ERS,降低相應(yīng)的內(nèi)質(zhì)網(wǎng)應(yīng)激蛋白分子表達(dá),從而對(duì)Tm誘導(dǎo)的大鼠髁突軟骨細(xì)胞的凋亡起保護(hù)作用。第二部分體內(nèi)實(shí)驗(yàn)鈣離子依賴(lài)的內(nèi)質(zhì)網(wǎng)應(yīng)激信號(hào)通路在壓力刺激下大鼠髁突軟骨病理變化過(guò)程中的作用及其機(jī)制研究[目的]觀察鈣離子阻斷劑及壓力作用下大鼠髁突軟骨組織形態(tài)學(xué)、超微結(jié)構(gòu)以及凋亡活性變化,以及ERS相關(guān)蛋白GRP78、GRP94、CHOP、Caspase-12表達(dá)的改變,探討鈣離子信號(hào)依賴(lài)的內(nèi)質(zhì)網(wǎng)信號(hào)通路在大鼠髁突軟骨病理變化過(guò)程中的作用及其機(jī)制。[方法]選擇6周齡SD雄性大鼠90只,用本課題組自主設(shè)計(jì)的顳下頜關(guān)節(jié)壓應(yīng)力加載的動(dòng)物模型裝置(專(zhuān)利號(hào)：201120210396.4),對(duì)大鼠髁突軟骨施加向上、向后的80g/側(cè)超負(fù)荷壓應(yīng)力刺激誘導(dǎo)大鼠髁突軟骨發(fā)生內(nèi)質(zhì)網(wǎng)應(yīng)激,設(shè)為空白對(duì)照組、MF(mechanical force)組、MF+2APB組、MF+Rya組、MF+2APB+Rya組,加力時(shí)間為3天和7天組。采用GENMED組織鈣離子濃度比色法定量檢測(cè)大鼠髁突軟骨組織鈣離子濃度的變化；蘇木素-伊紅(HE)染色觀察大鼠髁突軟骨層厚度及形態(tài)學(xué)的變化；透射電鏡觀察大鼠髁突軟骨細(xì)胞內(nèi)質(zhì)網(wǎng)超微結(jié)構(gòu)的變化；TUNEL熒光染色觀察大鼠髁突軟骨細(xì)胞凋亡的變化；免疫組化(immunohistochemistry, IHC)檢測(cè)內(nèi)質(zhì)網(wǎng)應(yīng)激標(biāo)志分子GRP78、GRP94. Caspase-12的表達(dá)改變。[結(jié)果]壓力加載后大鼠髁突軟骨中的鈣離子濃度升高,隨時(shí)間的延長(zhǎng),濃度逐漸增加。在重力加載下,HE切片染色顯示大鼠髁突軟骨層厚度變薄,髁突軟骨細(xì)胞出現(xiàn)內(nèi)質(zhì)網(wǎng)擴(kuò)張和空泡性改變；細(xì)胞凋亡在3天組出現(xiàn)增加。IHC結(jié)果顯示大鼠髁突軟骨細(xì)胞內(nèi)內(nèi)質(zhì)網(wǎng)應(yīng)激標(biāo)志分子GRP78、GRP94, Caspase-12的表達(dá)明顯升高。而當(dāng)關(guān)節(jié)腔注射鈣離子通道受體阻斷劑后,可以抑制上述情況的發(fā)生,且2-APB+Rya組要依次優(yōu)于Rya組和2-APB組,單純加藥組與空白對(duì)照在上述各項(xiàng)中無(wú)顯著差異。[結(jié)論]鈣離子依賴(lài)的內(nèi)質(zhì)網(wǎng)應(yīng)激信號(hào)通路在大鼠髁突軟骨病理變化過(guò)程起著重要作用,鈣離子通道受體阻斷劑可以降低髁突軟骨組織鈣離子濃度,緩解大鼠髁突軟骨的變薄效應(yīng),抑制大鼠髁突軟骨細(xì)胞發(fā)生凋亡。鈣離子通道受體阻斷劑可以通過(guò)降低大鼠髁突細(xì)胞內(nèi)ERS標(biāo)志分子的表達(dá)來(lái)緩解持續(xù)的內(nèi)質(zhì)網(wǎng)應(yīng)激反應(yīng),從而對(duì)大鼠髁突軟骨起著保護(hù)作用。
[Abstract]:The role of the calcium-ion-dependent ERS signal pathway in the in vitro culture of the condylar cartilage cells was studied in the first part in vitro. [Methods] Three-week-old SD rats were selected for primary culture of the mandibular joint condylar cartilage, and the third-generation condylar chondrocytes were used for the experiment. The cells of the condylar cartilage of the rat were treated with 4. mu. g/ ml of tunicamycin (Tm), or 4. m u.g/ ml of Tm was added after the pretreatment with the calcium channel receptor blocker 2-APB, Rya,2-APB + Rya for 30 min, and the cells were collected after 24 h treatment. The experiment was divided into two groups: blank control group, Tm group, Tm + 2APB group, Tm + Rya group, Tm + 2APB + Rya group. Intracellular calcium ion was stained with a Fura-2/ AM fluorescent dye, then the change of intracellular calcium ion concentration was detected by a fluorescence spectrometer, and the cell apoptosis rate was detected by flow cytometry. RT-qPCR and Western Blot were used to detect the changes in the expression of nRNA and protein in GRP78. GRP94, CHOP and Caspase-12 endoplasmic reticulum stress marker. [Results] 1. After 24 hours of treatment of the condylar cartilage cells of the rat, the concentration of calcium in the cells increased significantly (P0.05). After the pretreatment of the calcium channel receptor blocker, the intracellular calcium ion concentration decreased significantly (Tm + 2APB + Rya group, Tm + Rya group, Tm + 2APB group), but was still higher than that of the blank control group. 2. After 24 hours of treatment of the condylar cartilage cells of the rat, the apoptosis of the cells increased significantly (P0.01), and the apoptosis was significantly reduced after the pretreatment with the calcium channel receptor blocker (P0.01). There was no significant difference between the control group and the blank control group. After 24 hours of treatment of the condylar cartilage cells of the rat, the expression of the ERS marker molecules GRP78, GRP94, CHOP, Caspase-12 mRNA and its protein level was increased. The calcium channel receptor blocking agent can inhibit the expression of the expression of the mRNA and its protein of the condylar cartilage of the rat (GRP78, GRP94, CHOP, Caspase-12) induced by the Tm, except the Caspase-12, which has the best effect of 2 APB + Rya. [Conclusion] 1. Tm can induce the formation of ERS of the condylar cartilage cells of the rat, resulting in the apoptosis of the cells. The calcium channel receptor blocking agent can inhibit the generation of ERS of the condylar cartilage cells of the rat, and reduce the expression of the corresponding endoplasmic reticulum stress protein so as to protect the apoptosis of the Tm-induced rat condylar cartilage cells. In the second part, the effect of calcium ion-dependent endoplasmic reticulum stress signal pathway on the pathological changes of the condylar cartilage of the rat under pressure stimulation and its mechanism were studied.[Objective] To observe the morphology of the condylar cartilage of the rat under the action of calcium ion blocking agent and pressure. The changes of ultrastructure and apoptosis activity, as well as the changes of the expression of ERS-related protein GRP78, GRP94, CHOP, and Caspase-12, discussed the role of endoplasmic reticulum signaling pathway dependent on calcium ion signal in the pathological changes of condylar cartilage in rats and its mechanism. [Methods] 90 male rats of 6-week-old SD were selected, and the animal model of the mandibular joint stress loading (Patent No.: 201120210396.4), which was designed by the research group, was applied to the condylar cartilage of the rat. The endoplasmic reticulum stress in the condylar cartilage of the rat was induced by stress stimulation of 80 g/ side, which was set as the blank control group, the MF (mechanical force) group, the MF + 2 APB group, the MF + Rya group, the MF + 2 APB + Rya group, and the time of application was 3 days and 7 days. The changes of the calcium ion concentration in the condylar cartilage of the rat were measured by using the GENMED tissue calcium ion concentration colorimetric method. The changes of the thickness and morphology of the condylar cartilage were observed by hematoxylin-eosin (HE) staining, and the ultrastructure of the endoplasmic reticulum of the condylar cartilage was observed by transmission electron microscopy. TUNEL (TUNEL) fluorescence staining was used to observe the changes of the apoptosis of the condylar cartilage cells in the rat. The expression of the endoplasmic reticulum stress marker (GRP78, GRP94) was detected by immunohistochemistry (IHC). The expression of Caspase-12 was changed. [Results] The concentration of calcium in the condylar cartilage of the rat after pressure loading was increased, and the concentration was gradually increased with the time. Under gravity loading, HE section staining showed that the thickness of the condylar cartilage layer of the rat was thin, the endoplasmic reticulum expansion and the vacuolation of the condylar cartilage cells were changed, and the apoptosis of the cells increased in the 3-day group. IHC results show that the expression of endoplasmic reticulum stress marker molecule GRP78, GRP94 and Caspase-12 in the condylar cartilage cell of the rat is obviously increased. The 2-APB + Rya group was superior to the Rya group and the 2-APB group, and the simple treatment group and the blank control group had no significant difference in the above-mentioned items. [Conclusion] The calcium ion-dependent endoplasmic reticulum stress signal pathway plays an important role in the process of the pathological changes of the condylar cartilage of the rat, and the calcium channel receptor blocking agent can reduce the calcium ion concentration of the condylar cartilage tissue and relieve the thinning effect of the condylar cartilage of the rat. So as to inhibit the apoptosis of the condylar cartilage cells of the rat. The calcium channel receptor blocking agent can relieve the sustained endoplasmic reticulum stress response by reducing the expression of the ERS marker molecules in the condylar cells of the rat, thereby protecting the condylar cartilage of the rat.
【學(xué)位授予單位】：南京大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類(lèi)號(hào)】：R782

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相關(guān)期刊論文 前1條

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本文編號(hào)：2443775