目的通过体外、体内实验探讨肿瘤细胞柯萨奇腺病毒受体(coxsackieadenovirusreceptor,CAR)表达水平与腺病毒转导效率的关系,为腺病毒相关的生物制剂在临床个体化应用提供实验依据。方法将载有绿色荧光蛋白的Ad5型腺病毒(Ad-GFP)以100MOI、200MOI分别感染人食管癌细胞系KYSE510、KYSE150、EC9706、人宫颈癌细胞系HeLa、人卵巢癌细胞系SKOV3、人肝癌细胞系HepG2和人肺癌细胞系A549,感染后48h通过流式细胞术检测Ad-GFP对不同细胞系的转导效率。采用Westernblotting方法检测这些细胞中CAR的表达水平。将HeLa、A549、SKOV3、EC9706细胞分别接种于裸鼠腋下,接种细胞数分别为3×106、3×106、3×106、2×106,建立裸鼠移植瘤模型。待肿瘤长径达5~7mm时,在裸鼠移植瘤内注射Ad-GFP,每次1×109PFU,间隔48h注射第2次。第2次注射后48h处死裸鼠,剖取瘤组织,荧光显微镜观察冰冻切片中GFP的表达情况以判定腺病毒在瘤体内的转导效率,同时用免疫组化法检测瘤组织内的CAR表达水平。结果200MOIAd-GFP感染A549、HeLa、HepG2、KYSE150细胞48h后分别有92.67%、89.31%、84.98%、74.59%的细胞表达GFP;而SKOV3、KYSE510、EC9706细胞中腺病毒的转导效率明显降低,GFP阳性率分别为30.06%、27.40%、18.93%;各种细胞的CAR蛋白表达水平与腺病毒的转导效率呈正相关。注射Ad-GFP的裸鼠移植瘤组织中可见HeLa、A549瘤组织内有明显的点状绿色荧光,而SKOV3、EC9706瘤组织内表达GFP的细胞数明显少于前两种瘤组织;HeLa、A549裸鼠移植瘤组织内大多数瘤细胞高表达CAR(),SKOV3、EC9706移植瘤组织内CAR表达水平较低(+或-),表明瘤体内CAR表达水平与Ad-GFP的转导效率也呈正相关。结论体内外实验均显示肿瘤细胞的CAR表达水平与5型腺病毒转导效率密切相关。肿瘤患者治疗前检测组织中CAR表达水平有助于规范腺病毒载体的基因治疗药物的个体化使用。 Objective To investigate the relationship between the expression level of coxsackieadenovirus receptor (CAR) and the transduction efficiency of adenovirus in vitro and in vivo, and to provide experimental evidence for the clinical application of adenovirus-related biological agents. Methods Human Adenovirus Ad5 (Ad-GFP) with green fluorescent protein was infected with 100MOI and 200MOI respectively and infected human esophageal cancer cell lines KYSE510, KYSE150, EC9706, human cervical cancer cell line HeLa, human ovarian cancer cell line SKOV3, and human liver cancer. The cell line HepG2 and the human lung cancer cell line A549 were examined for the transduction efficiency of Ad-GFP to different cell lines by flow cytometry 48 h after infection. The expression of CAR in these cells was detected by Western blotting. HeLa, A549, SKOV3, and EC9706 cells were inoculated into the axilla of nude mice, and the number of inoculated cells was 3×106, 3×106, 3×106, and 2×106, respectively, and a nude mouse transplanted tumor model was established. When the length of the tumor reached 5~7mm, Ad-GFP was injected into the nude mice transplanted with 1*109 PFU each time, and the second injection was performed at intervals of 48 hours. The nude mice were sacrificed 48 h after the second injection and the tumor tissues were dissected out. The expression of GFP in frozen sections was observed under fluorescence microscope to determine the transduction efficiency of adenovirus in tumors. Meanwhile, the expression of CAR in tumor tissues was detected by immunohistochemistry. Level. Results After 48 hours of 200MOIAd-GFP infection in A549, HeLa, HepG2, and KYSE150 cells, 92.67%, 89.31%, 84.98%, and 74.59% of the cells expressed GFP, respectively. The transduction efficiency of adenovirus was significantly decreased in SKOV3, KYSE510, and EC9706 cells. The positive rates of GFP were 30.06%, 27.40%, and 18.93%, respectively; the expression of CAR protein in various cells was positively correlated with the transduction efficiency of adenovirus. In HeLa and A549 tumors, Ad-GFP-injected xenograft tumors exhibited significant spot-like green fluorescence, while the number of GFP-expressing cells in SKOV3 and EC9706 tumors was significantly less than that of the first two tumors; HeLa, A549 The majority of tumor cells in nude mice transplanted with high expression of CAR (), the expression level of CAR in SKOV3, EC9706 transplanted tumor tissue was low (+ or -), indicating that the expression of CAR in the tumor and the transduction efficiency of Ad-GFP also positive Related. Conclusion Both in vitro and in vivo experiments have shown that the expression level of CAR in tumor cells is closely related to the transduction efficiency of type 5 adenovirus. Detecting the expression level of CAR in tissues before treatment of tumor patients helps to regulate the individual use of adenovirus vector gene therapy drugs.