本文關鍵詞： 輕鏈型淀粉樣變 心肌毒性 發(fā)病機制　出處：《北京協(xié)和醫(yī)學院》2017年博士論文　論文類型：學位論文

【摘要】：背景和目的輕鏈型淀粉樣變(AL)是一種腫瘤性漿細胞產生的輕鏈沉積在各個器官導致其功能不全的血液疾病。如無有效的治療,患者的生存期僅有1～2年。心臟受累程度是決定AL患者預后的重要因素。但是,AL中輕鏈致心肌毒性的機制還不清楚。另外,AL輕鏈(AL-LC)的來源受限也是困擾研究者們的一個重要問題,目前AL-LC的主要來源是從患者尿液中提取,存在獲取AL-LC總量有限及LC的異質性等諸多問題,而從原核系統(tǒng)表達AL-LC則導致其翻譯后修飾缺乏。因此,建立穩(wěn)定表達AL-LC的真核表達平臺也至關重要。材料和方法通過構建pcDNA3.1重組質粒,轉入293T細胞體外表達IGLV1-44來源的AL心肌毒性輕鏈,建立AL輕鏈的真核系統(tǒng)表達平臺。將合成的AL輕鏈作用于心肌細胞,并設置多發(fā)性骨髓瘤輕鏈(MM-LC)對照及空白對照,研究輕鏈與H9C2心肌細胞的相互作用。首先,通過免疫熒光激光共聚焦定位檢測輕鏈與心肌細胞的定位關系。其次,用流式細胞術檢測心肌細胞的凋亡率及活性氧自由基(ROS)水平。再次,用Western Blot分析心肌細胞蛋白表達水平的變化,探究AL輕鏈心肌毒性可能涉及的分子通路。結果轉入pcDNA3.1重組質粒的293T細胞穩(wěn)定合成表達AL輕鏈,AL輕鏈的真核系統(tǒng)表達平臺成功建立。AL與MM輕鏈均可進入心肌細胞,但AL輕鏈明顯多于MM輕鏈,且能在細胞外間質形成沉積,包繞心肌細胞。然而,AL輕鏈與MM輕鏈均不與線粒體共定位。將體外合成的AL輕鏈作用于心肌細胞引起細胞凋亡,使用濃度為40μg/ml、60μg/ml及80μg/ml的AL輕鏈作用心肌細胞24h后的凋亡率分別為10%、18%和26%,相比MM輕鏈對照及空白對照(～5%)明顯升高。利用DCFH-DA檢測心肌細胞ROS水平,60μg/ml AL輕鏈作用2h及4h后心肌細胞的MFI值分別為13018.39±338.37及16766.86±29.47,其中4h組顯著高于對照組水平(MM 組:13191.70±409.04,空白對照 12917.81±614.69)。同時,在作用6h或12h時,AL組的凋亡標志物c-casp3水平均可檢測到顯著升高。分子通路方面,AL輕鏈處理心肌細胞6h及12h后,p-p38 MAPK水平較MM組及空白對照升高,且下游的PP2A水平在6h時明顯升高,12h時回到對照水平,但NF-κB、p-MEK1/2及JNK水平AL組與對照無明顯差別。另外,AL輕鏈可引起心肌細胞p-AMPK及下游Fox03水平下降,但激活AMPK并不能逆轉心肌細胞的凋亡,或FoxO3表達水平的上升。結論我們成功建立了 AL輕鏈的真核系統(tǒng)表達平臺,并驗證了其表達的AL輕鏈能誘導心肌細胞凋亡,且符合AL心肌毒性輕鏈的定位特點,這為后續(xù)AL輕鏈心肌毒性機制研究提供了良好的平臺。其次,AL輕鏈與MM輕鏈均可進入心肌細胞,但不與線粒體共定位。再次,AL輕鏈可導致心肌細胞p-p38MAPK及AMPK等信號分子水平的變化,但激活AMPK并不能逆轉心肌細胞凋亡。
[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 鹵338.37 and 16766.86 鹵29.47, respectively. 4 h group was significantly higher than that of control group (13191.70 鹵409.04) and blank control group (12917.81 鹵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.
【學位授予單位】：北京協(xié)和醫(yī)學院
【學位級別】：博士
【學位授予年份】：2017
【分類號】：R597.2

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本文編號：1481720