卡波氏肉瘤相关疱疹病毒感染神经元细胞SH-SY5Y的特点研究

The characteristics of neuronal cells SH-SY5Y infected by kaposi's sarcoma associated herpesvirus

为检测卡波氏肉瘤相关疱疹病毒(Kaposi's sarcoma associated herpesvirus,KSHV)是否能够直接感染神经元SH-SY5Y细胞,分析病毒相关基因的表达水平和细胞增殖情况等特点,为进一步研究KSHV感染在中枢神经系统疾病中的作用提供依据。采用佛波酯(12-O-Tetradecanoylphorbol 13-acetate,TPA)从BCBL1细胞中诱导并提取有感染性的KSHV病毒,感染神经元细胞SH-SY5Y,48 h后显微镜下观察细胞形态变化。提取感染细胞的RNA,通过RT-PCR技术检测裂解态的ORF26和潜伏态的潜伏相关核抗原(Latency Associated Nuclear Antigen,LANA)的m RNA表达水平,通过免疫荧光检测LANA蛋白的表达水平,验证KSHV的成功感染,并在感染后1、3、5、7 d进行细胞计数,与未感染的细胞做比较,以检测KSHV感染对细胞增殖能力的影响。结果显示,显微镜下观察KSHV感染与未感染的SH-SY5Y细胞形态,见未感染的SH-SY5Y细胞呈梭形聚团生长,感染KSHV后,细胞变大,形态变圆,倾向于分散生长。RT-PCR检测发现ORF26和LANA在KSHV感染的SH-SY5Y细胞中均有表达。由此可知,免疫荧光检测到LANA蛋白在细胞中也有表达,这些结果确定了KSHV可以成功感染SH-SY5Y细胞,并可同时表达潜伏态和裂解态的病毒基因。细胞计数结果表明KSHV感染促进了SH-SY5Y细胞的增殖能力。

To detect whether Kaposi＇s sarcoma associated herpesvirus （KSHV） can directly infect neuronal cells SH-SY5Y or not, the virus gene expression level and cell proliferation characters was analyzed. KSHV virus was induced and extracted from BCBLI cells by phorbol ester （12-O-Tetradecanoylphorbol 13-acetate, TPA）, and then the neuronal ceils were infected by KSHV. The morphological changes of the cells were observed after 48h, and the RNA of the infected ceils was extracted. RT-PCR was used to detect the mRNA expression level of lytic ORF26 and Latent Associated Nuclear Antigen （LANA）. The LANA protein was detected by indirect immunofluorescence （IFA） to verify the infection with KSHV. The KSHV infected and non-infected cell number was counted at day 1, day 3, day 5 and day 7 to investigate the variation of cell proliferation ability after KSHV infection. Under the microscope, non-infected SH-SY5Y cells were spindle-shaped cluster growth. After infection with KSHV, the cells became larger, morphological rounded, and tend to scatter. The expression of ORF26 and LANA were detected in KSHV infected SH-SY5Y cells by RT-PCR. The results of IFA revealed that LANA protein was also expressed in infected cells. These results confirmed that KSHV could successfully infect SH-SY5Y cells. Cell count results indicated that KSHV could increase the proliferation ability of SH-SY5Y cells. It can be concluded that KSHV can successfully infect neuronal cells and express both lytic and latent genes. KSHV can increase the proliferation ability of SH-SYSY ceils.