[Establishment and identification of human cervical cancer cell line C33A stably expressing human papillomavirus type 58 E6E7 fusion gene].

The study was performed to construct a human cervical cancer cell line C33A which can stably express HPV58E6E7 fusion gene. Firstly, C33A cells were transfected with the recombinant lentivirus LV-HPV58E6E7 which contained HPV58E6E7 fusion gene, and the stably transfected cells (LV-HPV58E6E7/C33A) were screened out by flow cytometry. MTT was used to observe the growth of LV-HPV58E6E7/C33A cells and flow cytometry was carried out to detect the cell cycle. LV-HPV58E6E7/C33A cells were inoculated into the left armpits of nude mice. Then, the transcription and expression of HPV58E6E7 fusion gene was detected by qRT-PCR and Western blot, respectively. The results showed that HPV58E6E7 fusion gene can promote the proliferation of C33A cells. HPV58E6E7 fusion gene can be stably transcripted and expressed in vaccinated nude mice. The conclusion indicated that we successfully established a cervical cancer cell line LV-HPV58E6E7/C33A which can stably express HPV58E6E7 fusion gene. This cell line will provide an antigen cell line for the immune effect detection of HPV58 therapeutic vaccine.为构建稳定表达人乳头瘤病毒（HPV）58 型 E6E7 融合基因的人宫颈癌 C33A 细胞系，将实验前期构建好并实现真核表达的重组慢病毒颗粒 LV-HPV58E6E7 转染入 HPV（–）的人宫颈癌 C33A 细胞，经流式细胞仪分选出稳定转染的阳性克隆，利用四唑盐比色（MTT）法检测转染后细胞的生长情况以及流式细胞术检测细胞周期，并将稳定表达 HPV58E6E7 融合基因的 C33A 细胞 LV-HPV58E6E7/C33A 接种于裸鼠左腋下成瘤，用荧光定量 PCR（qRT-PCR）、Western blot 检测瘤组织中 HPV58 型 E6E7 融合基因的转录和表达。结果显示 HPV58E6E7 融合基因可促进 C33A 细胞的增殖；LV-HPV58E6E7/C33A 细胞株能在裸鼠体内稳定转录及表达 HPV58 型 E6E7 融合基因。这表明我们成功建立了能稳定表达 HPV58E6E7 融合基因的人宫颈癌 C33A 细胞系 LV-HPV58E6E7/C33A，为 HPV58 型治疗性疫苗的免疫效果检测提供了抗原细胞来源。.