摘要
目的以猿猴空泡病毒40大T抗原基因（simian virus 40 large tumor antigen，SV40Tag）转染SD大鼠牙囊细胞（rats’dental follicle cells，rDFC）构建无限增殖化rDFC，以期为牙周组织工程研究提供稳定的细胞来源。方法分离SD大鼠下颌第一磨牙牙胚，组织块法分离培养原代rDFC，免疫组织化学技术鉴定细胞来源。利用脂质体介导含有SV40Tag的质粒pSSR69-pAmpho转染293细胞，取病毒上清液感染rDFC，潮霉素筛选，阳性克隆扩大培养（无限增殖化组）；以未转染细胞作对照组，观察细胞形态，计算细胞群体倍增时间，绘制细胞生长曲线和倍增曲线。反转录聚合酶链反应（reverse transcription polymerase chain reaction，RT—PCR）检测rDFC中SV40Tag基因的表达，蛋白质印迹法检测端粒酶表达，计算平板克隆形成率，行软琼脂克隆形成实验及细胞致瘤性实验。对无限增殖化组和对照组细胞群体倍增时间采用两独立样本t检验，对两组细胞平板克隆形成率采用两独立样本率χ2检验。结果无限增殖化rDFC形态与正常rDFC形态无明显差别。无限增殖化rDFC中SV40Tag RT—PCR结果显示在357bp处有一条特异扩增条带，未转染细胞未见条带，无限增殖化rDFC端粒酶表达显著高于正常细胞。两组rDFC生长曲线无明显差异，倍增曲线显示无限增殖化组rDFC保持了较强的增殖能力。无限增殖化rDFC平板克隆形成率[34％（33／96）]高于对照组[22％（21／96）]，但差异无统计学意义（χ2=3．71，P〉0．05），软琼脂内不能形成克隆；两组细胞致瘤性实验均未见肿瘤形成。结论SV40Tag基因无限增殖化rDFC能在体外长期培养并多次传代，具有相对稳定的增殖特性和功能状态，为良性转化，有望进一步应用于牙周组织工程研究。
Objective To construct SD rat immortalized dental follicle cells （rDFC） induced by simian virus 40 large tumor antigen（SV40Tag） gene to provide a reliable cell source for periodontal tissue engineering research. Methods The rDFC was isolated by tissue mass method combined with enzyme digestion method and evaluated by immunohistochemistry. Cell293 were transfected with plasmid pSSR69/ pAmpho containing SV40Tag gene by mediating liposome. Normal rDFC were infected with virus-contained supernate and the successfully transfected cell lines were screened with hygromycin, and positive clones were cultured. While non-transfected cells served as negative controls, the cell morphology was observed, the proliferation characteristics was evaluated by calculating cell population. The expression of SV40Tag gene and telomerase in cells was detected by reverse transcription polymerase chain reaction （RT-PCR） and Western blotting respectively. The biological property of immortalized rDFC was assessed with calculating formation rate of flat cloning, soft agar colony formation test and tumor-forming test. Results Morphology of immortalized rDFC was not different from that of normal rDFC. The RT-PCR results of SV40Tag revealed amplification band at 357 bp, while no band was seen in the normal cells. The expression of telomerase in immortalized rDFC was higher than that in normal rDFC. The two groups had no significant difference in growth curves, but the immortalized rDFC exhibited stronger proliferative activity. No significant differences of formation rate in fiat cloning were observed between the immortalized rDFC [ 34% （33/96） ] and normal rDFC at passage four [22% （21/96） ] （χ2 =3.71, P 〉0.05）. No cell cloning was seen in soft agar and the tumor formation was not observed in nude mice. Conclusions The rDFC induced by SV40Tag gene could be cultured and passaged in vitro, which retained the stable proliferation and differentiation characteristics and could be used for periodontal tissue engineering res
出处
《中华口腔医学杂志》
CAS
CSCD
北大核心
2012年第10期631-636,共6页
Chinese Journal of Stomatology
基金
国家自然科学基金（30870754）
重庆市第二批高等学校优秀人才支持计划[渝教人2010（72）]
关键词
牙囊
猴病毒40
组织工程
大鼠
无限增殖化
Dental sac
Simian virus 40
Tissue engineering
Rats
Immortalization
