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学科主题: 外科学
题名:
内质网应激对软骨细胞生存状态和自噬的影响
作者: 吴浩
答辩日期: 2016-05-11
导师: 曹永平
专业: 外科学
授予单位: 北京大学
授予地点: 北京大学第一临床医学院
学位: 博士
关键词: 骨关节炎 ; 软骨细胞 ; 内质网应激 ; 自噬 ; 凋亡 ; 衣霉素
其他题名: The change of chondrocytes viability and autophagy activity after endoplasmic reticulum stress
分类号: R681.3
摘要:

目的:探究在衣霉素诱导的内质网应激模型中,内质网应激反应对软骨细胞生存状态的影响及其对软骨细胞自噬有无诱导作用。

方法:1)取4-6周龄雄性SD大鼠,手术获取关节软骨,体外分离培养关节软骨细胞,应用Ⅱ型胶原免疫荧光测定鉴定软骨细胞;2)应用衣霉素刺激关节软骨细胞,建立软骨细胞内质网应激模型。分别应用CCK-8及Annexin V-FITC流式法测定低剂量(0.5μg/ml)和高剂量(10μg/ml)衣霉素作用下大鼠关节软骨细胞的细胞增殖活性及细胞凋亡情况。3)应用Western-Blot技术测定衣霉素作用下软骨细胞内质网应激反应相关蛋白(GRP78、CHOP)和自噬相关蛋白(Beclin-1、LC3B)的表达情况,并应用透射电镜技术观察衣霉素作用下软骨细胞内质网、自噬体等超微结构的变化情况,初步明确衣霉素对内质网应激反应和软骨细胞自噬作用的影响;4) 应用siRNA转染技术沉默软骨细胞GRP78基因,Western-Blot法测定衣霉素作用下正常软骨细胞和转染后的软骨细胞自噬相关蛋白(Beclin-1、LC3B)表达的差异,并应用透射电镜观察两组细胞自噬体形成的差异,进一步明确内质网应激反应对细胞自噬的影响;5)应用3-MA预处理大鼠软骨细胞抑制其自噬作用后,Annexin V-FITC流式法测定衣霉素作用48hr后3-MA预处理的细胞和正常细胞凋亡率有无差异,明确软骨细胞自噬作用对内质网应激诱导的细胞凋亡是否具有保护作用。

结果:1)大鼠关节软骨细胞在显微镜下呈梭形或多角形,免疫荧光染色显示培养的软骨细胞Ⅱ型胶原呈阳性表达;2)在衣霉素作用下,大鼠关节软骨细胞增殖活性下降、细胞凋亡比例增加,且衣霉素的这种作用随着作用剂量、时间的增加而更加显著;3)在低剂量(0.5μg/ml)衣霉素作用下,软骨细胞内质网应激反应相关蛋白GRP78、CHOP和自噬相关蛋白Beclin-1自加药后12hr表达显著增加(P<0.05),LC3B-Ⅰ在24hr表达显著增加(P<0.05)并在48hr开始显著转化为LC3B-Ⅱ(P<0.05);在高剂量(10μg/ml)衣霉素作用下,软骨细胞内质网应激相关蛋白GRP78、CHOP分别自12hr和6hr表达显著增加(P<0.05),自噬相关蛋白Beclin-1、LC3B-Ⅰ自12hr表达显著增加(P<0.05),LC3B-Ⅱ自24hr表达显著升高(P<0.05);透射电镜观察软骨细胞发现,随着衣霉素作用时间的延长,软骨细胞内质网腔逐渐扩张、自噬体形成增多;由此可见,衣霉素诱导软骨细胞内质网应激反应增强的同时还伴有自噬作用的增强;4)应用GRP78特异性siRNA转染软骨细胞,大鼠关节软骨细胞GRP78的表达显著下降,转染效率超过90%;应用低剂量(0.5μg/ml)衣霉素分别诱导正常细胞和转染细胞,转染细胞自噬相关蛋白Beclin-1、LC3B-Ⅱ较正常细胞显著下降(P<0.05),透射电镜观察转染细胞内自噬体明显减少,表明衣霉素作用导致的软骨细胞自噬增强是由内质网应激反应介导的;5)经低剂量(0.5μg/ml)衣霉素作用48hr,应用3-MA预处理的软骨细胞较正常软骨细胞凋亡显著增加(P<0.05),表明自噬作用对内质网应激诱导的细胞凋亡起保护作用;

结论:衣霉素能够诱导软骨细胞内质网应激反应的增强,持续的内质网应激反应会导致软骨细胞凋亡的增加。同时,内质网应激反应增强可以诱导软骨细胞自噬的激活,而自噬作用对内质网应激诱导的细胞凋亡具有缓解作用。

英文摘要:

Objective: This study was aimed at exploring the effect of endoplasmic reticulum (ER) stress on chondrocytes viability and autophagy activity.

Methods: 1) Rat chondrocytes were isolates from 4-6week old Sprague-Dawley male rats and cultured in vitro. The chondrocytes were identified through Immunofluorescent staining of collagen type Ⅱ. 2) The ER stress of chondrocytes was induced by tunicamycin in the study. Under the treatment of low-dose (0.5μg/ml) and high-dose(10μg/ml) tunicamycin, the viability of chondrocytes was determined using the CCK-8 assay. To further determine the cytotoxicity of tunicamycin on chondrocytes, AnnexinV-FITC flow cytometry were used to quantify apoptosis. 3) To investigate the Unfolded protein reaction (UPR) level and autophagy activity of chondrocytes under the treatment of tunicamycin, GRP78, CHOP, Beclin-1 and LC3B were measured by Western-Blot analysis. Technique of Electron Microscope (TEM) scanning was also used to access the autophagosome formation of chondrocytes under the tunicamycin treatment. 4) To further evaluate the effect of ER stress on autophagy of chondrocytes, the UPR was suppressed by small interfering RNA (siRNA) targeting GRP78, an UPR-essential gene. The autophagy activity of GRP78-knockout chondrocytes were assessed by Western-Blot analysis (Beclin-1, LC3B-Ⅱ) and TEM scan. 5) To evaluate the effect of autophagy on apoptosis of chondrocytes induced by tunicamycin, 3-methyl adenine (3-MA) was used to inhibit autophagy activity. Following tunicamycin stimulation, the apoptosis of chondrocytes pretreated with 3-MA was quantified using AnnexinV-FITC flow cytometry.

Results: 1) Rat chondrocytes appeared multi –angular or fusiform, immunofluorescent

staining showed that cultured chondrocytes had a positive expression of collagen type Ⅱ. 2) Following 72hr treatment of tunicamycin, the viability of chondrocytes decreased significantly, and the rate of apoptosis chondrocytes increased. These results also indicated tunicamycin induced chondrocytes apoptosis in a time and dose dependent manner. 3) Under the low-dose (0.5μg/ml) treatment of tunicamycin, Western-Blot analysis showed the protein levels of GRP78, CHOP and Beclin-1 significantly increased following 12hr treatment (P<0.05), and then LC3B-Ⅰincreased significantly at 24hr (P<0.05) with converting to LC3B-Ⅱ at 48hr (P<0.05). Under the high-dose treatment of tunicamycin, GRP78 and CHOP significantly increased during 6-12hr treatment (P<0.05). Following, the protein level of Beclin-1 and LC3B-Ⅰ increased at 12hr (P<0.05), and LC3B-Ⅱ level raised at 24hr (P<0.05). TEM scanning showed the swelling of endoplasmic reticulum and a large number of pre‑autophagosomal structures (PAS) or autophagosomes formation under tunicamycin treatment. These observations suggested tunicamycin can induce UPR and autophagy activation in chondrocytes. 4) With the 24hr stimulation of tunicamycin, the GRP78 level of chondrocytes transfected with GRP78-siRNA decreased significantly (P<0.05). In the meantime, Western-Blot analysis showed Beclin-1 and LC3B-Ⅱ also decreased significantly (P<0.05) in GRP78-siRNA transfecting chondrocytes compared with normal chondrocytes, and TEM scanning exhibited that the number of PAS or autophagosomes decreased in GRP78-knockout chondrocytes. These changes suggested that the activation of autophagy following tunicamycin stimulation was induced by the unfolded protein reaction. 5) Following the 48hr low-dose (0.5μg/ml) treatment of tunicamycin, the apoptosis ratio of chondrocytes pre-treated with 3-MA was much lower than the normal chondrocytes (P<0.05). This result confirmed the protective effect of autophagy in chondrocytes exposure to tunicamycin.

Conclusion: The present study revealed that the treatment of tunicamycin leads to activation of UPR in chondrocytes, and persistent UPR induces the apoptosis of chondrocytes. The UPR induced by tunicamycin also result in the activation of autophagy in chondrocytes, which is an adaptive response to protect chondrocytes from apoptosis.

语种: 中文
相关网址: 查看原文
内容类型: 学位论文
URI标识: http://ir.bjmu.edu.cn/handle/400002259/125263
Appears in Collections:北京大学第一临床医学院_学位论文

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作者单位: 北京大学第一临床医学院

Recommended Citation:
吴浩. 内质网应激对软骨细胞生存状态和自噬的影响[D]. 北京大学第一临床医学院. 北京大学. 2016.
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