内质网应激诱导的细胞自噬在成骨不全胶原蛋白形成中的作用

发布时间：2018-06-19 07:09

本文选题：成骨不全 + 内质网应激　；参考：《济南大学》2016年硕士论文

【摘要】：成骨不全(osteogenesis imperfecta,OI),又称脆骨病,是一种结缔组织异常可遗传疾病,临床表现包括:多发性骨折及长期骨折引起的骨骼畸形,脊柱侧弯,牙本质发育不全,蓝巩膜,皮肤弹性下降和关节松弛等。成骨不全是遗传性罕见疾病,儿童中的发病率不到万分之一,约90%的患者因为COL1A1和COL1A2基因突变,导致胶原蛋白合成数量及结构异常引起。突变蛋白堆积在细胞内质网中引发内质网应激(ER stress),内质网应激通过细胞自噬(Autophogy)对异常的胶原分子和前胶原蛋白进行降解,同时通过多种途径促进正常胶原蛋白的产生。研究目的:1)验证成骨不全患者原代细胞中内质网应激和细胞自噬的存在。2)初步探索内质网介导的细胞自噬对胶原蛋白合成的影响,为该病的治疗寻找新思路。研究方法:1)采集成骨不全患者和正常人骨、皮肤组织样本和临床信息。2)成骨/成纤维细胞原代培养并提取细胞RNA和全蛋白3)通过RT-q PCR,在基因水平进行内质网应激和细胞自噬的验证。4)通过免疫印记方法,在蛋白水平进行内质网应激和细胞自噬的验证5)通过透射电镜(50万倍)观察,应激细胞自噬过程的形态学表现。6)探索优化胶原蛋白的提取方法。7)利用氯喹等试剂,探索细胞自噬的信号通路。8)通过对胶原蛋白合成量的分析,判断抑制剂和激动剂对信号通路的影响。实验结果:1)组织处理及成纤维细胞培养成骨不全患者的皮肤和骨组织经处理后于细胞培养瓶内进行贴壁培养,在含有10%胎牛血清、1%青链霉素的DMEM培养基中,24h内开始贴壁,贴壁效果良好。2)内质网应激验证试验与对照组相比,成骨不全患者原代成纤维细胞中提取的核酸分析结果显示,内质网应激相关基因Bip上调1.5-2.5倍,GRP94上调2.1-3.3倍,ERp72上调1.38-2.4倍,Hsp47上调1.6-2.7倍。内质网应激相关蛋白,Hsp47实验组条带明显,对照组无明显条带;Bip、Grp94、Erp72实验组蛋白表达量分别是对照组7.56倍、7.34倍和3.54倍。3)细胞自噬验证试验与对照组相比,实验组细胞自噬相关基因p62上调2.2-2.4倍,LC3上调1.7-3.2倍。利用免疫酶标技术分析蛋白表达量差异,结果显示,对照组只表达LC3I型蛋白,实验组同时表达LC3I型和LC3II型蛋白,表达量是对照组3.08倍。4)自噬小泡观察透射电镜观察结果,成骨不全患者原代体细胞中出现了自噬小泡。5)胶原蛋白提取方法的探索实验结果表明从细胞中和培养液中提取的胶原蛋白,最适的胃蛋白酶消化时间为5小时,作用浓度1mg/m L。培养液中抗坏血酸浓度在0.1~0.15m M之间,β-氨基丙腈浓度达到0.1m M时,连续培养96h以上胶原蛋提取量最理想。6)自噬抑制剂和激活剂对成骨不全成纤维细胞生长影响初步结果发现,成骨不全成纤维细胞自噬通路部分抑制后,有利于细胞的存活,自噬加强后细胞快速死亡。7)细胞自噬对胶原蛋白合成的影响实验结果表明,在成骨不全患者体细胞中,自噬激动剂明显抑制胶原蛋白的合成,自噬抑制剂可以提高胶原蛋白亚基和成熟胶原的合成。结论:通过一系列的实验证实,成骨不全患者的成纤维细胞中存在明显的内质网应激现象,与胶原蛋白修饰折叠相关的分子伴侣表达量明显提高,以促进胶原蛋白的正常折叠和产生。同时,由于蛋白的积聚,引发内质网应激,启动细胞自噬,降解异常胶原蛋白,缓解应激压力。在成骨不全患者成纤维细胞中,细胞自噬信号通路受多种抑制剂和激动剂调控,实验结果显示,细胞自噬部分抑制后,有利于细胞的生存及胶原蛋白的合成量,本结果可能为成骨不全治疗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.
【学位授予单位】：济南大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R681.1

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