心肌损伤修复实验指标:MyoD基因重组腺病毒载体的构建及鉴定(英文)  

Experimental index of plerosis of myocardial injury: Construction and identification of a recombinant adenoviral vector carrying MyoD gene

作　　者：周秀娟[1] 黄峻[1] 姚堃[2] 马春玲[2] 周锋[2] 

机构地区：[1]南京医科大学第一附属医院心脏科,江苏省南京市210029 [2]南京医科大学微生物学与免疫学系,江苏省南京市210029

出　　处：《中国临床康复》2006年第12期174-176,共3页Chinese Journal of Clinical Rehabilitation

基　　金：国家自然科学基金资助项目(30370575)~~

摘　　要：背景:MyoD基因是肌肉转录因子家族成员之一,转染MyoD基因可以启动肌分化过程,使非肌细胞转变为肌细胞。MyoD基因仅在骨骼肌表达,基于心肌细胞和骨骼肌细胞有相同的收缩装置,可以设想将外源MyoD基因诱导坏死心肌局部的成纤维细胞向具收缩功能的骨骼肌细胞转化,可能会成为临床治疗心衰的又一种方法。目的:构建和鉴定MyoD基因重组腺病毒载体,为研究MyoD对心肌损伤的修复作用提供试验基础。设计:单一样本实验。单位:南京医科大学第一附属医院心脏科。材料:实验于2004-09/2005-09在南京医科大学微生物学与免疫学实验室完成。以MyoD基因和非复制型腺病毒表达载体为研究对象。方法:从pEMSV-MyoD质粒上用聚合酶链反应法扩增出MyoDcDNA片段,再通过聚合酶链反应使MyoD基因加上CACC序列接头,经过定向拓扑克隆使目的基因连接到转移载体上,再通过LR酶促反应,将目的基因转移到腺病毒表达载体DNA上,获得MyoD基因重组的腺病毒DNA,用脂质体转染法转染HEK293A细胞,包装扩增出MyoD基因重组的腺病毒。主要观察指标:聚合酶链反应鉴定和DNA测序来证实MyoDcDNA片段大小和序列的正确性,并测定病毒滴度。结果:经聚合酶链反应鉴定和DNA测序证实MyoD基因重组腺病毒含大小正确的目的片段,其DNA序列正确。病毒滴度为1.3×1011pfu/mL。结论:成功构建了MyoD基因重组腺病毒载体。BACKGROUND： MyoD gene is one of family members of muscle transcription factors. Transfection MyoD gene can switch on the procedure of differentiation of muscles, and transit non-muscle cells into muscle cells. The MyoD gene only expresses in skeletal muscles. Based on the same contractive structure in myocardial cells and skeletal muscle cells, it is imagined that the conversion from exogenous MyoD gene-induced fibroblast in local myocardium into skeletal muscle cells that had contractive function may become another method in the treatment of congestive heart failure on clinic. OBJECTIVE： To construct and identify a recombinant adenoviral vector carrying MyoD gene for further studies on the recovery function of MyoD gene in myocardial injury. DESIGN： Single sample experiment. SETTING： Department of Cardiology, First Affiliated Hospital, Nanjing Medical University. MATERIALS： The experiment was conducted at the Laboratory of Microbiology and Immunology, Nanjing Medical University between September 2004 and September 2005. MyoD gene and non-replicating form expressive. vector of adenovirus were taken as research materials. METHODS： MyoD cDNA fragments were extracted from plasmids pEMSV-MyoD with polymerase chain reaction （PCR）, and PCR was used to clone the whole-length gene of MyoD. After adding CACC sequence at 5＇ end, MyoD gene was cloned by orient topology into transfer ventor, pENTR/D-TOPO. Objective gene was transferred into adenoviral expression vector DNA via pENTR/D-TOPO vector. The recombinant adenoviral vectors transfected into HEK293A cells by using lipofectamine were packaged and amplified. MAIN OUTCOME MEASURES： Evaluation of PCR and DNA sequencing were used for confirming the size of segment and correctness of rank of MyoD cDNA and detecting the titre of virus. RESULTS： MyoD recombinant adenovirus contained target segment with precise length confirmed by PCR and DNA sequence that was correct. The titre of virus was 1.3×10^11 pfu/mL. CONCLUSION： The recombinant adenoviral vect

关 键 词：肌D蛋白质/遗传学 腺病毒科/遗传学 

分 类 号：R394[医药卫生—医学遗传学]