摘要
背景：利用转基因技术诱导皮肤真皮成纤维细胞分化为特殊器官组织功能的前驱细胞具有重要意义。目的：观察肌肉转录调节因子（myogenic determination，MyoD）基因转染对大鼠真皮成纤维细胞生物学特性的影响。设计、时间及地点：单一样本观察，细胞基因工程学实验，于2005-01／2007—02在同济大学附属东方医院心胸外科和细胞治疗室完成。材料：清洁级雄性SD大鼠6只，购自上海西普尔．必凯实验动物有限公司。Ⅱ型胶原蛋白酶为GIBCO公司产品，稻瘟菌素粉剂为美国Promega公司产品，慢病毒载体pLenti6／V5-DEST为Invitrogen公司产品，BrdU为美国Sigma公司产品。方法：无菌条件下切取大鼠腹侧全层皮肤，采用200IU／mL的Ⅱ型胶原蛋白酶在4℃下解离大鼠皮肤真皮组织，组织块培养法获取贴壁的真皮成纤维细胞，待生长近融合状态时传代扩增。取传至第3-6代细胞，利用携带MyoD cDNA的真核表达载体pLenti6／V5-DEST-MyoD进行转染，经终浓度为50mg／L稻瘟菌素筛选后，挑选转基因单克隆细胞传代培养，向细胞培养液中加入10μmol／L BrdU于37℃条件下孵育48h。主要观察指标：细胞形态学观察，免疫细胞化学染色鉴定，体外BrdU标记情况。结果：体外分离培养的真皮成纤维细胞纯度高，形态良好，生长增殖功能正常，转染后经稻瘟菌素筛选2周即可见克隆形成。光镜下真皮成纤维细胞免疫细胞化学染色后波形蛋白呈阳性表达，转染MyoD基因后肌动蛋白亦呈阳性表达。10μmol／LBrdU孵育48h的标记率〉98％，且连续传代5次后仍可检测到BrdU阳性细胞。结论：真核表达载体成功介导MyoD基因转染体外分离培养的大鼠真皮成纤维细胞，并诱导其转化为具有潜在功能的类骨骼肌细胞，可在体外稳定生长扩增。
BACKGROUND： It is of significance to induce differentiation of dermal fibroblasts into precursor cells of special organ tissues using transgenic technology. OBJECTIVE： To observe effects of transfection with myogenic determination gene （MyoD cDNA） on biological characteristics of rat dermal fibroblasts. DESIGN, TIME AND SETTING： The single sample observation, cell genetic engineering experiment was performed at the Department of Cardiothoracic Surgery and Room of Cell Therapy, East Hospital Affiliated to Tongji University between January 2005 and February 2007. MATERIALS： Six clean male Sprague Dawley rats were purchased from Xipuer-Bikai, China. Collagenase type Ⅱ （Gibco, USA）, Blasticidin （Promega, USA）, chronic viral vector pLenti6/V5-DEST （Invitrogen, USA）, and bromodeoxyuridine （BrdU） （Sigma, USA） were used in this study. METHODS： Full-thickness skins were sterily obtained from rats. Rat dermis was dissociated using 200 IU/mL collagenase type Ⅱ at 4 ℃. Adhered dermal fibroblasts were collected by tissue block culture method. When ceils were confluence, cells were passaged. At the third to sixth passage, MyoD cDNA was transduced into dermal fibroblasts using the eukaryotic expression vector pLenti6/V5-DEST-MyoD, and the cells were incubated in medium containing 50 mg/L blasticidin for screening. Transgenic monoclone cells were picked for passage, and then incubated in medium containing 10 μmol/L BrdU for 48 hours at 37 ℃. MAIN OUTCOME MEASURES： The morphologic characters of the converted cells were examined, and BrdU labeling irnmunocytochemistry ex vivo. RESULTS： In vitro cultured dermal fibroblasts had high purity, satisfactory morphologic characters and proliferation function. Dermal fibroblast clones were formed at 2 weeks by blasticidin screening after transduction. Immunocytochemical staining showed vimentin was positive in cultured dermal fibroblasts under an optical microscope; The expression of myogenic alpha-actin （muscle-specific protein） was observed in the transfected ceils, and incubation of the cells with BrdU at 10 μmol/L and for an optimal length of 48 hours appeared to achieve the highest labeling index, both of which exceeded 98%, with the labeling identifiable in five consecutive passages ex vivo. CONCLUSION： Dermal fibroblasts can be converted after MyoD cDNA transduced using eukaryotic expression vector, and can be induced to transformation into skeletal muscle-like cells, which acquired cells can be cultured in stable condition ex vivo.
出处
《中国组织工程研究与临床康复》
CAS
CSCD
北大核心
2008年第43期8524-8528,共5页
Journal of Clinical Rehabilitative Tissue Engineering Research
基金
国家自然科学基金(30471719)
上海市教委曙光计划(04SG25)
上海市东方医院科研项目(DF2007Q07)~~
