光化学内噬对腺病毒感染人喉鳞状细胞癌细胞转染效率的影响

Effect of photochemical internalization on the transfection efficiency of adenovirus infection of human laryngeal squamous carcinoma

目的研究光敏剂内消旋四苯基卟啉二磺酸盐酸盐(meso-tetraphenylporphine disulphonic acid dihydrochoride,TPPS2a)诱导的光化学内噬作用(photo-chemical internalization,PCI)对腺病毒(adenovirus,Ad)感染人喉鳞状细胞癌Hep-2细胞(简称Hep-2细胞)转染效率的影响。方法利用前期构建的带有人端粒酶逆转录酶启动子(human telomerase reversetranscriptase,hTERT)以及增强型绿色荧光蛋白(enhancedgreen fluorescent protein,eGFP)的Ad载体(Ad-hTERT-eGFP)感染Hep-2细胞,同时给予PCI于细胞,通过荧光显微镜检测和流式细胞分析技术,研究Ad滴度、TPPS2a及光照时间等因素对Hep-2细胞中eGFP表达效率的影响;Westernblot法检测Hep-2细胞柯萨奇腺病毒受体(coxsackieadenovirus receptor,CAR)蛋白的表达量。结果 PCI处理后的Hep-2细胞eGFP表达率明显升高,随着光照时间的延长及病毒滴度的增大,eGFP表达效率也随之升高。Westernblot检测Hep-2细胞CAR蛋白表达水平较低,且与eGFP表达效率呈正相关。结论 PCI在一定程度上能提高Ad对Hep-2细胞的转染效率,Ad对Hep-2细胞的转染效率较低可能与CAR表达量较低有关。

OBJECTIVE The aim of this study is toshow the differences of transfection efficiency between Photochemical internalization（PCI）-treatment group and non PCI-treatment group,which was induced by photosensitizer meso-Tetraphenylporphine disulphonic acid dihydrochoride（TPPS 2a）after adenovirus（Ad） infection of human laryngeal squamous carcinoma Hep-2 cells.METHODS The adenovirus vector Ad-hTERTeGFP which contained human telomerase reverse transcriptase promoter（hTERT）and enhanced green fluorescent protein（eGFP）was used to infect Hep-2 cells.Meanwhile,PCI was added to Hep-2 cells.Then,fluorescence microscopy and flow cytometry were used to test the expression efficiency of eGFP which was influenced by several factors such as MOI,TPPS 2a and illumination time.Western blot analysis was used to detect the dose of coxsackie adenovirus receptor（CAR） expression of Hep-2 cells.RESULTS The expression of eGFP in Hep-2 cells was significantly increased after PCI treatment.With the extension of exposure time,the expression efficiency of eGFP was also increased.With the virus titer increased,the expression efficiency of eGFP was increased too.CAR protein expression was lower in Hep-2 cells detected by Western blot and that had positively correlated with eGFP expression efficiency.CONCLUSION To a certain extent,PCI can enhance the transfection efficiency of adenovirus on human laryngeal carcinoma Hep-2 cells.The lower transfection efficiency of adenovirus on Hep-2 cells may be related to low CAR expression.