人釉原蛋白基因转染牙周膜细胞的实验研究

Experimental study on human periodontal ligament cells transfected with human amelogenin gene

目的:构建含有人釉原蛋白(human amelogenin,hAm)基因的慢病毒载体质粒,用重组慢病毒感染人牙周膜细胞(human periodontal ligament cells,hPDLCs),初步探讨釉原蛋白基因修饰种子细胞用于牙周组织工程修复的可行性。方法:采用RT-PCR方法获取hAm编码基因,构建慢病毒载体质粒FUAmW,重组质粒经双酶切及测序鉴定。聚乙烯亚胺(polytheylenimine,PEI)法三质粒共转染293T细胞,获取重组慢病毒FUAmW FUGW,感染hPDLCs,应用荧光显微镜观察绿色荧光蛋白(green fluorescene protein,GFP)表达,流式细胞仪(flow cytometer,FCM)检测慢病毒载体转染效率。通过RT-PCR检测hPDLC中hAm基因的表达。结果:测序证实,RT-PCR产物序列与Genebank公布的Am编码序列一致,双酶切证实目的片段插入重组质粒。293T细胞及hPDLCs感染72h后,荧光显微镜下可见绿色荧光。FCM测得GFP感染2种细胞的阳性率分别为69.46%和33.99%。RT-PCR证实重组慢病毒FUAmW感染的细胞能表达hAm基因。结论:成功构建含有人釉原蛋白基因的慢病毒载体质粒,经293T细胞包装,得到重组慢病毒可感染牙周膜细胞。

PURPOSE： To construct the recomhinant lentiviral vector of human amelogenin gene, infect human periodontal ligament cells with the recomhinant lentivirus,and evaluate the feasihility of applying modified PDLCs as seeds for a further periodontal reconstruction. METHODS： The mature peptide of hAm cDNA was cloned and linked into the vector plasmid , the recombinant plasmid FUAmW was confirmed by double enzyme digestion and sequence analysis. Recombinant lentivirus was prepared from 293T cells by polytheylenimine （PEI）-mediated transient cotransfection. The hPDLCs and 293T cells were infected with the generated lentivirus. The infection efficiency was analysed by detection of green fluorescence protein （GFP） with fluorescent microscope and flow cytometer 72 hours later. The expression of hAm gene was detected by reverse transcription polymerase chain reaction （RT-PCR）. RESULTS： The sequence of inserted fragment in recombinant plasmid was identical to the hAm sequence reported in Genebank. Green fluorescence was visible under fluorescent microscope, FCM assay showed that positive percentage was 69.46% and 33.99% in 293T and hPDLCs, respectively. The targeted gene was obtained in the experimental groups by RT-PCR. CONCLUSIONS ： The recombinan lentiviral vector of hAm gene is constructed successfully and it could be transfected into cultured hPDLCs. hAm gene and seed cells may be used for further study in the fields periodontal tissue engineering.