【作者】 周敏；

【导师】 李煌；

【作者基本信息】 南京大学 ， 口腔医学， 2014， 硕士

【摘要】 第一部分Salubrinal抑制ERS反应对周期性牵张刺激诱导的大鼠,髁突软骨细胞凋亡保护作用的研究[目的]研究Salubrinal对周期性牵张刺激诱导下的ERS介导的原代大鼠髁突软骨细胞凋亡的保护作用。[方法]1.选取3周龄SD大鼠,取髁突软骨进行原代培养。对第三代髁突软骨细胞进行相关实验。2.周期性张应变刺激(cyclic strain, CS)为20%,0.5Hz,24h矩形波刺激。Salubrinal+CS组则需用40mol/L Salubrinal预处理30 mmin后再予以同样的周期性张应变刺激。实验分为三组：对照组、周期性张应变刺激组(CS组)、Salubrinal+CS组。采用流式细胞技术检测细胞凋亡率；采用RT-qPCR检测ERS标志分子GRP78、GRP94、CHOP、Caspase-12的mRNA水平改变,Western印迹法检测GRP78、GRP94、CHOP、Caspase-12蛋白水平表达改变。[结果]1.20%周期性张应变刺激处理大鼠髁突软骨细胞24小时后,细胞凋亡率明显上升(P<0.05); Salubrinal+CS组细胞凋亡率较CS组显著降低(P<0.05)。2.20%周期性张应变刺激诱导大鼠髁突软骨细胞的ERS凋亡相关分子CHOP、 Caspase-12 mRNA水平表达升高,Caspase-12蛋白水平升高：Salubrinal可以逆转这些变化。[结论]1.20%周期性张应变刺激力可以诱导大鼠髁突软骨细胞发生凋亡,ERS凋亡蛋白表达增加。2. Salubrinal可以通过抑制大鼠髁突软骨细胞发生ERS,对20%周期性张应变刺激诱导的凋亡起保护作用。第二部分Salubrinal抑制ERS反应对低氧刺激诱导的大鼠髁突软骨细胞凋亡保护作用的研究[目的]研究ERS抑制剂Salubrinal对低氧诱导刺激下大鼠髁突软骨细胞(所有的软骨细胞都特指大鼠髁突软骨细胞)凋亡的保护作用。[方法]1.体内实验部分：选取6周龄SD大鼠,戴用特殊设计的矫形器(专利号：201120210396.4)对其髁突软骨施加压力刺激,单纯加力组作用时间7天,Salubrinal预处理组则在加力关节腔局部注射Salubrinal。不戴矫形器的SD大鼠作为对照组,免疫组化检测加力后髁突关节软骨组织的缺氧诱导因子Hif-1α的表达改变。2.体外实验部分：选取3周龄SD大鼠,取髁突软骨进行原代细胞培养,所有实验均使用P3代髁突软骨细胞。3.实验分为三组：对照组、低氧刺激组、Salubrinal+低氧刺激组,其中低氧刺激为5%低氧24h和1%低氧30h两组设计,Salubrinal的预处理需在低氧刺激前30min开始；流式细胞术检测各组的细胞凋亡率。4.根据流式结果选取合适的低氧刺激强度处理细胞。采用RT-qPCR检测各组细胞的ERS标志分子GRP78、GRP94、CHOP、Caspase-12mRNA表达的改变,Western印迹法检测各组细胞的ERS标志分子GRP78、GRP94、CHOP、Caspase-12及Hif-1α蛋白表达的改变。[结果]1.大鼠髁突软骨在压应力刺激作用下,缺氧诱导因子Hif-la的表达水平明显上升(P<0.05), Salubrinl预处理组较单纯加力组Hif-la的表达水平明显降低(P<0.05)。2.5%低氧刺激24h后,大鼠髁突软骨细胞凋亡率无明显差异(P>0.05)； 1%低氧刺激30h,大鼠髁突软骨细胞凋亡上升,Salubrinal可缓解低氧刺激造成的大鼠髁突软骨细胞凋亡。3.正常培养条件Hif-1α不表达,1%低氧刺激30h后Hif-1α仪蛋白表达,且Salubrinal预处理降低了缺氧引起的Hif-1α表达升高(P<0.05)。1%低氧刺激30h后,大鼠髁突软骨细胞中GRP78、GRP94、CHOP、Caspase-12的mRNA和蛋白表达水平显著性升高(P<0.05)。Salubrinal+1%低氧组软骨细胞GRP78、GRP94、CHOP、Caspase-12的mRNA和蛋白水平上的表达较1%低氧组显著降低(P<0.05)。[结论]1.大鼠髁突关节软骨在压应力刺激作用下处于缺氧状态。2.1%低氧刺激作用30h可以诱导大鼠髁突软骨细胞发生ERS,从而导致凋亡。3. Salubrinal可以通过抑制大鼠髁突软骨细胞发生ERS,对体内外低氧刺激诱导的凋亡起保护作用。更多还原

【Abstract】 Part 1:The study on protective effects of Salubrinal on mandibular condylar chondrocytes apoptosis induced by cyclic strainObjective:To investigate the protective effects of Salubrinal on mandibular condylar chondrocytes (All chondrocytes are specific to rat mandibular condylar chondrocytes) apoptosis induced by cyclic strain.Methods:1. Chondrocytes from 3-week-old Sprague-Dawley rats were isolated and cultured in vitro. We used third-generation chondrocytes in order to maintain their original characteristics.2. Chondrocytes were subjected to 20% elongation of cyclic strain for 24 hours (rectangular wave) or with a combination of 40mmol/L Salubrinal for 30 minutes. Chondrocytes were divided into three groups:Control group,20% cyclic strain group, Salubrinal+20% cyclic strain group. Flow cytometer was used to detect cell apoptosis after 24h treatment. The expression of GRP78, GRP94, CHOP, Caspase-12 was detected by real-time quantitive PCR and Western Blot.Results:1. Chondrocytes apoptosis increased (P<0.05) after 24 hours treatment of 20% cyclic strain. Pretreatment of Salubrinal could decrease chondrocytes apoptosis induced by cyclic strain (P<0.05).2.20% Cyclic strain increased the expression of CHOP, Caspase-12 on transcription level and Caspase-12 expressionon protein translation level in chondrocytes. Salubrinal+20% Cyclic strain down regulated their expression.Conclusion:1.20% Cyclic strain triggered chondrocytes apoptosis through ER stress.2. Salubrinal protected chondrocytes from apoptosis induced by 20% cyclic strain through down regulated ER stress signaling proteins.Part 2:The study on protective effects of Salubrinal on mandibular condylar chondrocytes apoptosis induced by hypoxiaObjective:To investigate the protective effects of Salubrinal on mandibular condylar chondrocytes apoptosis induced by hypoxia.Methods:1. In vivo experiment:we used an established animal model for loading compressive mechanical stress to mandibular condylar cartilage of 6-week-old Sprague-Dawley rats for 7 days, Salubrinal pretreatment was local intra-articular injection. Control group SD rats do not wear any equipment. Detect the expression of hypoxia-inducible factor-1α of mandibular condylar cartilage through immunohistochemistry.2. Invitro experiment:Mandibular Condylar chondrocytes from 3-week-old Sprague-Dawley rats were isolated and cultured in vitro. We used third-generation mandibular condylar chondrocytes in order to maintain their original characteristics.3. Chondrocytes were divided into three groups:Control group, Hypoxia group, Salubrinal+hypoxia group. For hypoxia treatment, cells were cultured in a hypoxia chamber flushed with 5% O2 and 5% CO2 with balance of 90% nitrogen at 37℃ for 24h or 1% O2 and 5% CO2 with balance of 94% nitrogen for 30h. Control cells were placed in a 5% CO2 and 95% air incubator (20% O2) at 37℃. Salubrinal pretreatment (40 mmol/L) should be 30 mins prior to hypoxia culture. Cell apoptosis was detected by flow cytometry.4. Select the appropriate hypoxia stimulation magnitude of chondrocytes according to the results of flow cytometry.The expression of GRP78, GRP94, CHOP, Caspase-12 was detected by real-time quantitive PCR and Western Blot. The expression of Hif-1 a was also detected by Western Blot.Results:1. The expression of hypoxia-inducible factor-1a (Hif-1 a) in chondrocytes was increased after applying compressive mechanical stress to mandibular condylar cartilage(P<0.05). Pretreatment of Salubrinal could decreased the expression of Hif-1 a.2.5% 02,24h hypoxia did not induce chondrocytes apoptosis while 1% 02,30h hypoxia induced chondrocytes apoptosis(P<0.05). Pretreatment of Salubrinal could decrease chondrocytes apoptosis induced by hypoxia (1% 02,30h).3. Under normoxia (20% 02) condition, Hif-1 a was not expressed.1% 02,30h hypoxia induced Hif-1 a protein expression and Salubrinal pretreatment would reduce the increase of hypoxia-induced Hif-1 a expression (P<0.05).1% 02,30h hypoxia increased the expression of GRP78, GRP94, CHOP, Caspase-12 on both transcription and protein translation level in chondrocytes. Salubrinal+1% 02,30h hypoxia down regulated their expression.Conclusion:1. Condyle cartilage of SD rats under compressive mechanical stress for 7 days was in hypoxia state.2. Hypoxia (1%,30h) triggered chondrocytes apoptosis through ER stress.3. Salubrinal protected chondrocytes from apoptosis induced by hypoxia (1%,30h) through down regulated ER stress signaling pathway.更多还原