摘要
目的:构建表达钙黏蛋白-11(Cadherin-11)的重组腺病毒载体,并观察其对大鼠牙髓细胞生物学特性的影响。方法 :PCR扩增Cadherin-11,在引物中引入NheⅠ和HindⅢ酶切位点,构建pDC316-mCMV-EGFP-Cadherin-11重组腺病毒穿梭载体。用含目的基因的重组质粒转染293T细胞,获得含目的基因的高滴度腺病毒。原代培养大鼠牙髓细胞,并用pDC316-mCMVEGFP-Cadherin-11重组腺病毒进行转染。采用免疫细胞化学法和RT-PCR法分别检测目的基因Cadherin-11的表达。结果:以pDC316-mCMV-EGFP-Cadherin-11转染大鼠牙髓细胞后,Cadherin-11mRNA和蛋白均为阳性表达(均P<0.05),且感染复数(MOI)为50时病毒转染效率最高。携带Cadherin-11目的基因的大鼠牙髓细胞生长速度明显高于阴性对照组和空白对照组(P<0.05)。结论:构建重组腺病毒载体pDC316-mCMV-EGFP-Cadherin-11可高效转染大鼠牙髓细胞,并稳定表达Cadherin-11基因;过表达Cadherin-11对大鼠牙髓细胞生长具有一定的促进作用。
Objective： To construct a recombinant adenovirus expressing Cadherin-11 and explore the effect of Cadherin-11 on the biological characteristics of rat dental pulp cells （rDPCs）. Methods： Cadherin-11 fragments was subcloned by PCR from pMD18-T-Cadherin-11 plasmid and inserted into the Nhe Ⅰ/Hind Ⅲ sites of pDC-316-mCMV-EGFP. The recombinant vector was packed in the 293T cells to obtain high titer recombinant adenovirus. Primary rDPCs were infected with the recombinant adenovirus and the expressions of Cadherin-11 were detected by reverse transcription-polymerase chain reaction （RT-PCR） and immunocytochemistry. Results： Recombinant adenoviral vector pDC316-mCMV-EGFP-Cadherin-11 was successfully constructed, and Cadherin-11 was stably expression in rDPCs using the adenovirus packed from this vector. The rDPCs overexpressing Cadherin-11 grew faster than the negative control and blank control cells （ P 〈0.05）. Conclusion： Cadherin-11 was stably expressed in rDPCs using recombinant adenovirus packed from vector pDC316-mCMV-EGFP-Cadherin-11 and it was a useful tool for further study on the function of Cadherin-11. Cadherin-11 overexpression could promote the growth of rDPCs.
作者
陆彦玲
陈婉
赵文青
蔡捷
吴煜
Lu Yanling Chen Wan Zhao Wenqing Cai Jie Wu Yu(Guangxi Medical University, Nanning 530021, China School of Stomatology, Guangxi Medical University, Nanning 530021, China)
出处
《广西医科大学学报》
CAS
2017年第2期168-172,共5页
Journal of Guangxi Medical University
基金
国家自然科学基金资助项目(No.81660182)
广西自然科学基金资助项目(No.2011GXNSFA018216)
广西医疗卫生适宜技术研究与开发基金资助项目(No.S201305-02)
