本文選題：成骨不全 + 內(nèi)質(zhì)網(wǎng)應(yīng)激��； 參考：《濟(jì)南大學(xué)》2016年碩士論文

【摘要】：成骨不全(osteogenesis imperfecta,OI),又稱脆骨病,是一種結(jié)締組織異�？蛇z傳疾病,臨床表現(xiàn)包括:多發(fā)性骨折及長(zhǎng)期骨折引起的骨骼畸形,脊柱側(cè)彎,牙本質(zhì)發(fā)育不全,藍(lán)鞏膜,皮膚彈性下降和關(guān)節(jié)松弛等。成骨不全是遺傳性罕見(jiàn)疾病,兒童中的發(fā)病率不到萬(wàn)分之一,約90%的患者因?yàn)镃OL1A1和COL1A2基因突變,導(dǎo)致膠原蛋白合成數(shù)量及結(jié)構(gòu)異常引起。突變蛋白堆積在細(xì)胞內(nèi)質(zhì)網(wǎng)中引發(fā)內(nèi)質(zhì)網(wǎng)應(yīng)激(ER stress),內(nèi)質(zhì)網(wǎng)應(yīng)激通過(guò)細(xì)胞自噬(Autophogy)對(duì)異常的膠原分子和前膠原蛋白進(jìn)行降解,同時(shí)通過(guò)多種途徑促進(jìn)正常膠原蛋白的產(chǎn)生。研究目的:1)驗(yàn)證成骨不全患者原代細(xì)胞中內(nèi)質(zhì)網(wǎng)應(yīng)激和細(xì)胞自噬的存在。2)初步探索內(nèi)質(zhì)網(wǎng)介導(dǎo)的細(xì)胞自噬對(duì)膠原蛋白合成的影響,為該病的治療尋找新思路。研究方法:1)采集成骨不全患者和正常人骨、皮膚組織樣本和臨床信息。2)成骨/成纖維細(xì)胞原代培養(yǎng)并提取細(xì)胞RNA和全蛋白3)通過(guò)RT-q PCR,在基因水平進(jìn)行內(nèi)質(zhì)網(wǎng)應(yīng)激和細(xì)胞自噬的驗(yàn)證。4)通過(guò)免疫印記方法,在蛋白水平進(jìn)行內(nèi)質(zhì)網(wǎng)應(yīng)激和細(xì)胞自噬的驗(yàn)證5)通過(guò)透射電鏡(50萬(wàn)倍)觀察,應(yīng)激細(xì)胞自噬過(guò)程的形態(tài)學(xué)表現(xiàn)。6)探索優(yōu)化膠原蛋白的提取方法。7)利用氯喹等試劑,探索細(xì)胞自噬的信號(hào)通路。8)通過(guò)對(duì)膠原蛋白合成量的分析,判斷抑制劑和激動(dòng)劑對(duì)信號(hào)通路的影響。實(shí)驗(yàn)結(jié)果:1)組織處理及成纖維細(xì)胞培養(yǎng)成骨不全患者的皮膚和骨組織經(jīng)處理后于細(xì)胞培養(yǎng)瓶?jī)?nèi)進(jìn)行貼壁培養(yǎng),在含有10%胎牛血清、1%青鏈霉素的DMEM培養(yǎng)基中,24h內(nèi)開(kāi)始貼壁,貼壁效果良好。2)內(nèi)質(zhì)網(wǎng)應(yīng)激驗(yàn)證試驗(yàn)與對(duì)照組相比,成骨不全患者原代成纖維細(xì)胞中提取的核酸分析結(jié)果顯示,內(nèi)質(zhì)網(wǎng)應(yīng)激相關(guān)基因Bip上調(diào)1.5-2.5倍,GRP94上調(diào)2.1-3.3倍,ERp72上調(diào)1.38-2.4倍,Hsp47上調(diào)1.6-2.7倍。內(nèi)質(zhì)網(wǎng)應(yīng)激相關(guān)蛋白,Hsp47實(shí)驗(yàn)組條帶明顯,對(duì)照組無(wú)明顯條帶;Bip、Grp94、Erp72實(shí)驗(yàn)組蛋白表達(dá)量分別是對(duì)照組7.56倍、7.34倍和3.54倍。3)細(xì)胞自噬驗(yàn)證試驗(yàn)與對(duì)照組相比,實(shí)驗(yàn)組細(xì)胞自噬相關(guān)基因p62上調(diào)2.2-2.4倍,LC3上調(diào)1.7-3.2倍。利用免疫酶標(biāo)技術(shù)分析蛋白表達(dá)量差異,結(jié)果顯示,對(duì)照組只表達(dá)LC3I型蛋白,實(shí)驗(yàn)組同時(shí)表達(dá)LC3I型和LC3II型蛋白,表達(dá)量是對(duì)照組3.08倍。4)自噬小泡觀察透射電鏡觀察結(jié)果,成骨不全患者原代體細(xì)胞中出現(xiàn)了自噬小泡。5)膠原蛋白提取方法的探索實(shí)驗(yàn)結(jié)果表明從細(xì)胞中和培養(yǎng)液中提取的膠原蛋白,最適的胃蛋白酶消化時(shí)間為5小時(shí),作用濃度1mg/m L。培養(yǎng)液中抗壞血酸濃度在0.1~0.15m M之間,β-氨基丙腈濃度達(dá)到0.1m M時(shí),連續(xù)培養(yǎng)96h以上膠原蛋提取量最理想。6)自噬抑制劑和激活劑對(duì)成骨不全成纖維細(xì)胞生長(zhǎng)影響初步結(jié)果發(fā)現(xiàn),成骨不全成纖維細(xì)胞自噬通路部分抑制后,有利于細(xì)胞的存活,自噬加強(qiáng)后細(xì)胞快速死亡。7)細(xì)胞自噬對(duì)膠原蛋白合成的影響實(shí)驗(yàn)結(jié)果表明,在成骨不全患者體細(xì)胞中,自噬激動(dòng)劑明顯抑制膠原蛋白的合成,自噬抑制劑可以提高膠原蛋白亞基和成熟膠原的合成。結(jié)論:通過(guò)一系列的實(shí)驗(yàn)證實(shí),成骨不全患者的成纖維細(xì)胞中存在明顯的內(nèi)質(zhì)網(wǎng)應(yīng)激現(xiàn)象,與膠原蛋白修飾折疊相關(guān)的分子伴侶表達(dá)量明顯提高,以促進(jìn)膠原蛋白的正常折疊和產(chǎn)生。同時(shí),由于蛋白的積聚,引發(fā)內(nèi)質(zhì)網(wǎng)應(yīng)激,啟動(dòng)細(xì)胞自噬,降解異常膠原蛋白,緩解應(yīng)激壓力。在成骨不全患者成纖維細(xì)胞中,細(xì)胞自噬信號(hào)通路受多種抑制劑和激動(dòng)劑調(diào)控,實(shí)驗(yàn)結(jié)果顯示,細(xì)胞自噬部分抑制后,有利于細(xì)胞的生存及膠原蛋白的合成量,本結(jié)果可能為成骨不全治療方法的探索提供了新的思路。
[Abstract]:Osteogenesis imperfecta (OI), also known as crisp bone disease, is an abnormal and hereditary disease of connective tissue. Clinical manifestations include bone malformation caused by multiple fractures and long-term fractures, scoliosis, dentin dysplasia, blue sclera, skin elastic descending and joint relaxation. Osteogenesis incompetence is a rare hereditary disease in children. The incidence of the disease is less than 1/10000, and about 90% of the patients with COL1A1 and COL1A2 mutations cause the number and structural abnormalities of collagen synthesis. The mutant protein accumulates in the endoplasmic reticulum and induces endoplasmic reticulum stress (ER stress), and endoplasmic reticulum stress reduces the abnormal collagen molecules and procollagen through cell autophagy (Autophogy). Solution, and promote the production of normal collagen through a variety of ways. Objective: 1) to verify the existence of endoplasmic reticulum stress and the existence of autophagy in the primary cells of the patients with osteogenesis incompetence (.2) to explore the effect of endoplasmic reticulum mediated autophagy on collagen synthesis, and to find new ideas for the treatment of this disease. Methods: 1) acquisition of osteogenesis incompetence. Patients and normal human bone, skin tissue samples and clinical information.2) osteogenesis / fibroblast culture and extraction of cell RNA and total protein 3) through RT-q PCR, endoplasmic reticulum stress and cell autophagy at gene level,.4) through immuno imprinting methods, endoplasmic reticulum stress and cell autophagy at protein level 5) through penetration Electron microscopy (500 thousand times) observation, morphological expression of autophagy in stress cells.6) explore optimization of collagen extraction method.7) using chloroquine and other reagents to explore cell autophagy signaling pathway.8) through the analysis of collagen synthesis to determine the effect of inhibitors and agonists on the signaling pathway. Experimental results: 1) tissue processing and fibrinolysis The skin and bone tissue of the patients with osteogenesis in the cell culture were treated in the cell culture bottle after treatment. In the DMEM medium containing 10% fetal bovine serum and 1% penicillin, the wall was adhered to the wall in 24h and the effect was good.2). The endoplasmic reticulum stress test was compared with the control group, and the primary fibroblasts of the patients with osteogenesis were extracted from the control group. The results of nucleic acid analysis showed that endoplasmic reticulum stress related gene Bip up regulation 1.5-2.5 times, GRP94 up regulation 2.1-3.3 times, ERp72 up 1.38-2.4 times up regulation, Hsp47 up-regulated 1.6-2.7 times. The stress related protein of endoplasmic reticulum, Hsp47 experimental group was obvious, the control group had no obvious band, Bip, Grp94, Erp72 experimental group protein expression was 7.56 times, 7.34 times and 3. of the control group, respectively. 54 times.3) the autophagy test was compared with the control group. The autophagy related gene p62 was up to up 2.2-2.4 times and the LC3 was up to 1.7-3.2 times. The result showed that the control group only expressed the LC3I protein, the experimental group expressed the LC3I and LC3II protein at the same time, and the expression was 3.08 times.4 in the control group. The experimental results of autophagic vesicles observation transmission electron microscope, the method of collagen extraction from autophagic vesicles.5 in the primary somatic cells of the patients with osteogenesis imperfection, the experimental results of collagen extraction showed that the optimum pepsin digestion time was 5 hours, and the concentration of ascorbic acid in the 1mg/m L. culture solution was the concentration of the collagen extracted from the cells and the medium. Between 0.1~0.15m M, when the concentration of beta aminopropanonitrile reaches 0.1M M, the optimal extraction of collagen eggs above 96h is the most ideal.6) the effects of autophagy inhibitors and activators on the growth of osteoblast fibroblasts are preliminary found that the autophagic pathway of the osteoblast fibroblasts is partially suppressed after the autophagic pathway is suppressed and the autophagy strengthens the cells. The effect of autophagy on collagen synthesis by rapid death.7) experimental results show that autophagy agonists significantly inhibit the synthesis of collagen in the somatic cells of patients with osteogenesis incomplete, and autophagy inhibitors can improve the synthesis of collagen subunits and mature collagen. Conclusion: a series of experiments have proved that the fibroblasts of the patients with osteogenesis have been proved to be fibroblasts. There is an obvious endoplasmic reticulum stress phenomenon in the cells. The expression of molecular chaperone associated with collagen modification is obviously improved to promote the normal folding and production of collagen. At the same time, the accumulation of protein causes endoplasmic reticulum stress, autophagy of cells, degradation of abnormal collagen and stress stress. In fibroblasts, the autophagy signaling pathway is regulated by a variety of inhibitors and agonists. The experimental results show that the inhibition of autophagy is beneficial to cell survival and collagen synthesis. This result may provide a new way of thinking for the exploration of osteogenesis incomplete therapy.
【學(xué)位授予單位】：濟(jì)南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：R681.1

【參考文獻(xiàn)】

相關(guān)期刊論文 前2條

1 辛毅;李娜;黃益民;崔巍;劉颯;許秀芳;張兆光;;人臍帶間充質(zhì)干細(xì)胞的原代培養(yǎng)及多向分化潛能的研究[J];細(xì)胞與分子免疫學(xué)雜志;2013年10期

2 鄧力,陳槐卿,鄭虎,彭雪梅;去卵巢山羊成骨細(xì)胞的體外培養(yǎng)[J];生物醫(yī)學(xué)工程學(xué)雜志;1999年03期

，

本文編號(hào)：2039057