【作者】 顾兴华；

【导师】 王克强；

【作者基本信息】 复旦大学 ， 人体解剖与组织胚胎学， 2007， 博士

【摘要】 Neuregulin（NRG）家族的基因有四个，其中NRG-1在神经、心脏和乳腺等系统或组织中发挥重要的作用。rhNRG-1（recombinant human NRG-1）激活心肌细胞上的ErbB2／4受体后，能改善心梗大鼠的心功能，但其机制尚不够清楚。本课题通过基因芯片筛选，并经过Real time PCR、Western blot验证后，发现rhNRG-1能增加一个预测的基因的表达：Similar to Myosin light chain kinase 2，skeletal／cardiac muscle（MLCK2）（GenBank accession number XM₂14645）。采用Northern、Western blot和Immunofluorescence等方法，证明该基因特异性分布于心肌细胞，并进一步证明它能使mlc-2v（Myosin light chain-2，ventricular）磷酸化，说明它是心肌特异性的MLCK，干扰其RNA表达后，rhNRG-1恢复受损心肌细胞的作用消失。初步证明rhNRG-1能增加心肌细胞特异性MLCK的表达，从而改善心功能。本课题共分五个部分：第一部分rhNRG-1能改善心梗大鼠的心功能。结扎大鼠冠状动脉前降支，建立心梗模型，根据EF值（左室射血分数）随机分为rhNRG-1治疗组、赋形剂对照组，连续静脉滴注rhNRG-1（10μg／kg／2hx7天）或同等体积赋形剂，于用药前、后分别进行心超检测。同步设立假手术组：开胸但不结扎冠状动脉，也不给予任何药物。结果：用药后rhNRG-1组LVEDD（左室舒张末内径）、LVEDS（左室收缩末内径）、EF、FS（左室缩短分数）分别为9.46±0.20mm、7.62±0.23mm、44.7±1.9％、19.8±1.1％，与赋形剂组相比，LVEDD、LVEDS分别下降了9.1％、18.8％，而EF、FS分别升高了62.5％、75.1％（P＜0.01，n＞10）。证明rhNRG-1能改善心梗大鼠的心功能。第二部分大鼠心肌基因芯片检测及相关验证表明：rhNRG-1能使Similar RLCK表达增加。1．基因芯片检测：选取第一部分实验动物的左室非梗区，每组取3只，独立提取RNA，共9份RNA样品，分别与9张基因芯片杂交（Rat Genome 230 2.0 Arrays, Affymetrix）。结果提示，rhNRG-1能增加一个预测基因的表达：Similar to Myosin light chain kinase 2, skeletal/cardiac muscle（MLCK2）（GenBank accession number XM₂14645）。当假手术组标化为1.000时，赋形剂组为0.677，而rhNRG-1组为1.652，rhNRG-1／赋形剂比值为2.441，有统计学差异（P＜0.01，n=3）。2．Real time PCR和Western blot验证了基因芯片的结果：Similar MLCK N端1400bp是特异性的片断，故选取1018-1317片断，采用Real time PCR验证了其基因变化。选用487-885片断，构建Similar MLCK-GST／pGEX-2T质粒，表达similar MLCK（487-885）-GST融合蛋白，免疫兔子获得高效价抗体，进行Western blot验证了其蛋白水平的变化。结果与基因芯片的结果相吻合，证明rhNRG-1确实增加了该基因的表达。第三部分大鼠Similar MLCK特异性分布于心肌细胞1．Northern blot：选取Similar MLCK 238-885片断作为探针，用α-³²PdCTP标记后，与大鼠多种组织northern杂交膜进行杂交，结果只在心肌组织中有单一的条带（约4.3kb）。2．Western blot：采用第二部分试验中制备的similar MLCK抗体，与大鼠多种组织蛋白进行杂交，结果发现similar MLCK只在心肌组织中表达（约86kd）。3．Immunofluorescence：新生大鼠原代心肌细胞未纯化培养后免疫荧光染色发现：similar MLCK只分布于心肌细胞，smooth MLCK分布于心肌组织内的平滑肌细胞。成年大鼠心脏冰冻切片免疫荧光显示：similar MLCK分布于心肌细胞，而smooth MLCK分布于血管组织。综合上述结果，可以证实：similar MLCK特异性分布于心肌细胞。第四部分大鼠Similar MLCK能使MLC-2v磷酸化，是心肌特异性MLCK。构建大鼠cardiac MLCK／pCDNA3表达质粒，转染cos7细胞后，收集细胞裂解液作为cardiac MLCK的来源，加入体外反应体系(含有MLC-2V，以及Ca²⁺，CaM，ATP等物质)，观察mlc-2v的磷酸化。结果表明：Cardiac MLCK能使mlc-2v磷酸化且必须依赖Ca、CaM的存在。Cardiac MLCK用量相同时，随着作用时间的延长，mlc-2v磷酸化水平越高，20min时能检测到磷酸化，100min时基本达到饱和。在相同作用时间下，随着Cardiac MLCK用量的增加，mlc-2v磷酸化水平越高。作用时间相同、Cardiac MLCK用量一样时，不同温度下mlc-2v磷酸化无明显变化。特异性分布于心肌细胞的Similar MLCK能使MLC-2v磷酸化，且有明显的量效和时间关系，证实了similar MLCK是心肌特异性MLCK。第五部分rhNRG-1通过增加cardiac MLCK的表达而改善心功能。1．筛选有效的cardiac MLCK RNA表达干扰片段：针对cardiac MLCK CDS区156-174（GCTACAGTGTGTGTGCCAA）等片断，构建RNAiⅠ，Ⅱ，Ⅲ质粒（pRNAT-CMLCK-Ⅰ，Ⅱ，Ⅲ），然后分别与cardiac MLCK／pCDNA3表达质粒，共同转染cos7细胞，用Western blot检测cardiac MLCK的变化。结果：RNAiⅠ，Ⅱ，Ⅲ质粒分别使cardiac MLCK的表达下降80％，50％，20％，提示RNAiⅠ质粒干扰效果最好。2．rhNRG-1通过增加cardiac MLCK的表达而改善心肌细胞的结构：干扰或对照质粒转进心肌细胞后，会有GFP绿色荧光，借助α-actinin染色能显示心肌细胞的横纹结构，通过图像分析系统将横纹紊乱的面积分为不同等级：Area＜25％，25％≤area＜50％，50％≤area＜75％，area≥75％，统计各级细胞数量，分析比较各组横纹结构的变化。结果：正常、正常+RNAiⅠ、无血清+对照质粒、无血清+对照质粒+rhNRG-1、无血清+rhNRG-1+RNAiⅠ组内，紊乱面积≥50％的细胞分别为2％、80％、60％、28％、80％。说明cardiac MLCK的表达受干扰后，正常细胞的横纹排列紊乱；无血清培养能使心肌细胞横纹排列紊乱，rhNRG-1能恢复紊乱的横纹；Cardiac MLCK表达被干扰后，rhNRG-1恢复紊乱横纹的作用消失。说明rhNRG-1可通过增加cardiac MLCK的表达而恢复心肌细胞的结构，进而改善心功能。结论：1．Similar MLCK（GenBank accession number XM₂14645）可称作Cardiac specific MlCK，这是一个新的MLCK的发现。2．rhNRG-1能改善心梗大鼠的心功能。3．rhNRG-1能使Cardiac specific MLCK表达增加。4．Cardiac MLCK表达被干扰后，rhNRG-1恢复受损心肌细胞的作用消失。5．rhNRG-1增加cardiac MLCK的表达，从而改善心肌收缩功能。潜在价值和创新点：1．Similar MLCK（GenBank accession number XM₂14645）可称作Cardiac specific MlCK，这是一个新的MLCK的发现，可作为新的药物靶点，筛选有效的增强心肌收缩的药物。2．rhNRG-1能改善心梗大鼠的心功能，并增加Cardiac specific MLCK的表达。3．rhNRG-1恢复受损心肌的作用必须依赖Cardiac MLCK。更多还原

【Abstract】 The ErbB family of receptor tyrosine kinases and their ligands, neuregulins（NRGs）, are critical involved not only in cardiac development but also in themaintenance of structural and functional integrity of the adult heart, rhNRG-1（recombinant human NRG-1）improves cardiac function and survival in models ofischemic, dilated, and viral cardiomyopathy with the ErbB-activation. But themechanism is still unknown.Genechips, followed by confirmation via Real time PCR and Western blot, wereused to study the mechanism. The main result was that rhNRG-1 can increase theexpression of a predict gene: Similar to Myosin light chain kinase 2, skeletal/cardiacmuscle （MLCK2） （GenBank accession number XM₂14645）, which was proved to berat’s cardiac specific MLCK by Northern blot, Western blot, Immunofluorescence andmlc-2v-p detection in this thesis, rhNRG-1 can recovery the disorderedcardiomyocytes’ sarcomeres, but the RNA interfering of cardiac MLCK can disturbthe recovery. So, rhNRG-1 improves cardiac function of infracted rats by increasingthe expression of cardiac specific myosin light chain kinase.There are five parts in this thesis.PartⅠrhNRG-1 improves the cardiac function of infarcted rats. Rat’s left ventricle was infarcted by ligation of the left anterior descending coronaryartery （LAD）. Sham operated animals were similarly treated except that the suturearound the LAD was left untied, rhNRG-1（10μg/kg/2h x 7days, ivgtt.） or vehicle wasadministered by implanted osmotic minipumps. Sham-operated animals received notreatment. Echocardiography was performed before and after treatments. Results:LVEDD, LVEDS decreased 9.1%, 18.8%, EF, FS increased 62.5%, 75.1%（P＜0.01,n＞10） after treated with rhNRG-1, compared with vehicle. RhNRG-1 improvescardiac function of infarcted rats obviously.PartⅡrhNRG-1 increases the expression of similar MLCK, discovered byGenechip and confirmed by Real time PCR and Western blot.The RNA samples in different groups were extracted from the non-infarction area ofleft ventricle coming from the first part experiment, reversed to cDNA, labeled cRNA,hybridized with Rat Genome 230 2.0 Arrays （Affymetrix）. Result: rhNRG-1 increased the expression of a predict gene: Similar to Myosin light chain kinase 2,skeletal/cardiac muscle （MLCK2）（GenBank accession number XM₂14645）. Thevalues in sham, vehicle and rhNRG-1 groups were 1.000, 0.677 and 1.652 separately,rhNRG-1/vehicle was 2.441（P＜0.01, n=3）.This result was confirmed by Real time PCR and Western blot. The 1400bp in the Nterminal of similar MLCK is the specific segment decided by NCBI blast. So, thespecific segment （1018-1317） was amplified in the Real time PCR; Similar MLCK（487-885）-GST fusion protein, coming from the Similar MLCK-GST/pGEX-2Tplasmid, was used to immune rabbit, get specific antibody of similar MLCK, andconfirm the Genechip’s result by Western blot and so on.PartⅢSimilar MLCK distributes in the cardiomyocytes specifically.The probe of similar MLCK （238-885）, labeled withα-³²PdCTP, was hybridized withrat’s multiple tissue northern blots, the single band （about 4.3kb） was shown on theheart tissue lane. Similar MLCK （about 86kd） was only expressed in the heart tissue,confirmed by multiple tissue western blots in which the similar MLCK specificalantibody, produced in the second part experiment, was used.The cultured neonatal rat cardiomyocytes and the frozen slides of adult rat heart werestained withα-actinin, smooth muscle actin, similar MLCK and smooth MLCKantibodies. Result: similar MLCK limited in the cardiomyocytes, smooth MLCKlocated in the smooth muscle within the heart tissure. Similar MLCK and smoothMLCK were distributed on cardiomyocyte and vascular separately.PartⅣSimilar MLCK, which can phosphorylate the MLC-2v, is a cardiacspecific MLCK.Cos7 cells were transferred with cardiac MLCK /pCDNA3, lysised, mixed withMLC-2v, Ca2+,CaM, ATP and so on. The phosphorylation of MLC-2v was detectedby western blot to confirm that similar MLCK is a cardiac specific MLCK.Result: Cardiac MLCK phosphorylated the mlc-2v dependent on the Ca2+, CaM. Thereaction times longer, the dose of cardiac MLCK larger, the phosphorylation ofmlc-2v was stronger, but the temperature had no evident effect on this reaction. Thesimilar MLCK, distributed on the cardiomyocytes specifically, can phosphorylate themlc-2v, so similar MLCK is proved to be a cardiac specific MLCK.PartⅤrhNRG-1 benefits cardiomyocytes by increasing the expression ofcardiac MLCK. RNAiⅠ,Ⅱ,Ⅲplasmids were co-transferred into cos7 with cardiac MLCK/pCDNA3 plasmid separately, detect the cardiac MLCK by western blot. Result: RNAiⅠ,Ⅱ,Ⅲplasmids decreased the expression of cardiac MLCK about 80%, 50%, 20%respectively. RNAiⅠwas the best one and chosen to do the followed expriment.Cultured rat neonatal cardiomyocytes were transferred with control or RNAiⅠplasmids, and divided into five groups. The sarcomere arrangements of transferredcells, in which green fluor can be seen because the expression of GFP, were observedbyα-actinin staining. The disordered sarcomeres can be divided into four grades byimaging analysis system. The cell numbers in different grades were calculated. Result:In the normal, normal+RNAiⅠ, serum free+control, serum free+rhNRG-1+control,and serum free+rhNRG-1+RNAiⅠgroups, the percent of cells, whose disorderedareas≥50%, were 2%、80%、60%、28%、80% separately. The sarcomeres of normalcardiomyocytes were disturbed when the RNA of cardiac MLCK was silenced.rhNRG-1 can recover the disordered sarcomere with serum free culture, but thisfunction disappeared when cardiac MLCK RNA was interfered. So, rhNRG-1 benefitscardiomyocytes by increasing the expression of cardiac MLCK.Conclusions:1. Similar MLCK （GenBank accession number XM₂14645） is a cardiac specificMLCK.2. rhNRG-1 improves cardiac function of infarcted rats.3. rhNRG-1 increases the expression of cardiac specific MLCK.4. The function of rhNRG-1 （recovering the damaged cardiaomyocytes） disappearedwhen the RNA of cardiac MLCK was interfered.5. rhNRG-1 benefits cardiomyocytes by increasing the expression of cardiac MLCK.The potential application and novelty of this project:1. Similar MLCK （GenBank accession number XM₂14645） is a cardiac specificMLCK, potential original medical target for the new drugs about enhancing thecardiac contraction.2. rhNRG-1 improves cardiac function of infracted rats and increases the expressionof cardiac specific MLCK.3. rhNRG-1 benefits the damaged cardiomyocytes depending on the expression ofcardiac MLCK.更多还原