轻链型淀粉样变中轻链致心肌毒性的机制研究

发布时间：2018-02-01 11:12

本文关键词： 轻链型淀粉样变心肌毒性发病机制　出处：《北京协和医学院》2017年博士论文　论文类型：学位论文

【摘要】：背景和目的轻链型淀粉样变(AL)是一种肿瘤性浆细胞产生的轻链沉积在各个器官导致其功能不全的血液疾病。如无有效的治疗,患者的生存期仅有1～2年。心脏受累程度是决定AL患者预后的重要因素。但是,AL中轻链致心肌毒性的机制还不清楚。另外,AL轻链(AL-LC)的来源受限也是困扰研究者们的一个重要问题,目前AL-LC的主要来源是从患者尿液中提取,存在获取AL-LC总量有限及LC的异质性等诸多问题,而从原核系统表达AL-LC则导致其翻译后修饰缺乏。因此,建立稳定表达AL-LC的真核表达平台也至关重要。材料和方法通过构建pcDNA3.1重组质粒,转入293T细胞体外表达IGLV1-44来源的AL心肌毒性轻链,建立AL轻链的真核系统表达平台。将合成的AL轻链作用于心肌细胞,并设置多发性骨髓瘤轻链(MM-LC)对照及空白对照,研究轻链与H9C2心肌细胞的相互作用。首先,通过免疫荧光激光共聚焦定位检测轻链与心肌细胞的定位关系。其次,用流式细胞术检测心肌细胞的凋亡率及活性氧自由基(ROS)水平。再次,用Western Blot分析心肌细胞蛋白表达水平的变化,探究AL轻链心肌毒性可能涉及的分子通路。结果转入pcDNA3.1重组质粒的293T细胞稳定合成表达AL轻链,AL轻链的真核系统表达平台成功建立。AL与MM轻链均可进入心肌细胞,但AL轻链明显多于MM轻链,且能在细胞外间质形成沉积,包绕心肌细胞。然而,AL轻链与MM轻链均不与线粒体共定位。将体外合成的AL轻链作用于心肌细胞引起细胞凋亡,使用浓度为40μg/ml、60μg/ml及80μg/ml的AL轻链作用心肌细胞24h后的凋亡率分别为10%、18%和26%,相比MM轻链对照及空白对照(～5%)明显升高。利用DCFH-DA检测心肌细胞ROS水平,60μg/ml AL轻链作用2h及4h后心肌细胞的MFI值分别为13018.39±338.37及16766.86±29.47,其中4h组显著高于对照组水平(MM 组:13191.70±409.04,空白对照 12917.81±614.69)。同时,在作用6h或12h时,AL组的凋亡标志物c-casp3水平均可检测到显著升高。分子通路方面,AL轻链处理心肌细胞6h及12h后,p-p38 MAPK水平较MM组及空白对照升高,且下游的PP2A水平在6h时明显升高,12h时回到对照水平,但NF-κB、p-MEK1/2及JNK水平AL组与对照无明显差别。另外,AL轻链可引起心肌细胞p-AMPK及下游Fox03水平下降,但激活AMPK并不能逆转心肌细胞的凋亡,或FoxO3表达水平的上升。结论我们成功建立了 AL轻链的真核系统表达平台,并验证了其表达的AL轻链能诱导心肌细胞凋亡,且符合AL心肌毒性轻链的定位特点,这为后续AL轻链心肌毒性机制研究提供了良好的平台。其次,AL轻链与MM轻链均可进入心肌细胞,但不与线粒体共定位。再次,AL轻链可导致心肌细胞p-p38MAPK及AMPK等信号分子水平的变化,但激活AMPK并不能逆转心肌细胞凋亡。
[Abstract]:Background and objective Light chain amyloidosis (ALL) is a blood disease caused by the deposition of light chain in various organs, such as no effective treatment. The survival time is only 1 ~ 2 years. The degree of cardiac involvement is an important factor to determine the prognosis of AL patients. However, the mechanism of myocardial toxicity caused by light chain in AL is not clear. The limited source of ALL-LC is also an important problem for researchers. At present, the main source of AL-LC is extracted from the urine of patients. There are many problems such as the limited amount of AL-LC and the heterogeneity of LC, and the expression of AL-LC from prokaryotic system leads to the lack of post-translational modification. It is also important to establish a eukaryotic expression platform for the stable expression of AL-LC. Materials and methods were used to construct the recombinant plasmid of pcDNA3.1. Transfered into 293T cells to express acute myocardial toxicity light chain derived from IGLV1-44 in vitro, the eukaryotic expression platform of AL light chain was established, and the synthesized AL light chain was acted on cardiac myocytes. The interaction between light chain and H9C2 cardiomyocytes was studied with multiple myeloma light chain (MM-LC) control and blank control. Immunofluorescence laser confocal localization was used to detect the localization relationship between light chain and cardiomyocytes. Secondly, flow cytometry was used to detect the apoptosis rate of cardiomyocytes and the level of reactive oxygen species (Ros). The changes of protein expression in cardiomyocytes were analyzed by Western Blot. Results 293T cells transfected into pcDNA3.1 recombinant plasmid synthesized and expressed AL light chain stably. The eukaryotic expression platform of light chain of AL was successfully established. Both light chain of AL and MM could enter into cardiomyocytes, but light chain of AL was much more than light chain of MM, and could form deposition in the extracellular interstitial. However, the light chain of AL and MM were not co-located with mitochondria. The light chain of AL synthesized in vitro induced the apoptosis of cardiomyocytes at a concentration of 40 渭 g / ml. The apoptosis rates of myocardial cells exposed to 60 渭 g / ml and 80 渭 g / ml AL light chain for 24 hours were 10 ~ 18% and 26%, respectively. Compared with MM light chain control and blank control, the level of ROS in cardiomyocytes was detected by DCFH-DA. The MFI values of 60 渭 g / ml AL light chain were 13018.39 卤338.37 and 16766.86 卤29.47, respectively. 4 h group was significantly higher than that of control group (13191.70 卤409.04) and blank control group (12917.81 卤614.69). The level of apoptotic marker c-casp3 in AL group was significantly increased at 6 or 12 h after treatment with ALL light chain for 6 h and 12 h. The level of p-p38 MAPK was higher than that of MM group and control group, and the downstream PP2A level was significantly increased at 6h and 12h, but NF- 魏 B. There was no significant difference in p-MEK1 / 2 and JNK levels between AL group and control group. In addition, ALL light chain could induce the decrease of p-AMPK and downstream Fox03 levels in cardiomyocytes. However, activation of AMPK could not reverse the apoptosis of cardiomyocytes or increase the expression of FoxO3. Conclusion We have successfully established the eukaryotic expression platform of light chain of AL. It is verified that the expressed AL light chain can induce apoptosis of myocardial cells and accord with the localization characteristics of acute myocardial toxicity light chain, which provides a good platform for further study of myocardial toxicity mechanism of AL light chain. Secondly. Al light chain and MM light chain can enter cardiomyocytes, but not co-located with mitochondria. Furthermore, AL light chain can cause changes of signal molecules such as p-p38 MAPK and AMPK in cardiac myocytes. However, activation of AMPK could not reverse cardiomyocyte apoptosis.
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R597.2

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