【作者】 肖安涛；

【导师】 董长垣；

【作者基本信息】 武汉大学 ， 病原生物学， 2004， 硕士

【摘要】 本研究室在对蓝舌病毒中国湖北株-3（BTV-HbC₃）长期的研究过程中，注意到BTV-HbC₃可以在HeLa细胞，一种来自于人类宫颈癌的细胞，中良好地增殖，而后进一步发现其可以感染其他一些恶性肿瘤细胞。这个现象激发了我们对于一种新的癌症治疗方法的想象：肿瘤的病毒疗法。我们进行了几个实验，以探索BTV-HbC₃是否具备成为一种溶癌病毒的潜能。 分别以BTV-HbC₃感染几株细胞，包括人胚肺成纤维细胞HEL，人宫颈癌细胞系HeLa，人肺腺癌细胞系SPC-A-1，人星形胶质瘤细胞U251，小鼠星形胶质瘤细胞C6。这样，就能够观察BTV-HbC₃引起的细胞病变效应，以考察是否所有的受试细胞都对这种病毒敏感。 在各株受试细胞上滴定BTV-HbC₃的组织培养半数感染量TCID₅₀，应用双向免疫扩散实验来检测是否所有感染BTV-HbC₃的受试细胞中都有BTV-HbC₃抗原出现等方法，考察了受试细胞中子代病毒的增殖能力，借以鉴别BTV-HbC₃对正常细胞和肿瘤细胞的感染性的差别。 为了证实双向免疫扩散实验结果的可靠性，我们又应用逆转录聚合酶链反应RT-PCR来探测BTV-HbC₃在这些细胞中的复制状况，并借助透射电子显微镜，观察了受试细胞超微结构的形态学改变，清楚地了解BTV-HbC₃与细胞相互作用的形态学和形态发生学后果。 试验结果表明：BTV-HbC₃能够导致除了人胚肺成纤维细胞之外的所有细胞发生细胞病变效应；在抗-BTV-HbC₃免疫血清和除了人胚肺成纤维细胞之外的所有细胞裂解液之间出现了抗原抗体复合物免疫沉淀线；逆转录聚合酶链反应检测到除了人胚肺成纤维细胞之外的所有受试细胞中的BTV-HbC₃特异性核酸序列。 但是，我们发现，尽管BTV-HbC₃能够在所有的受试肿瘤细胞中复制，但是BTV-HbC₃对所有受试细胞的感染性并不相同。 这些实验结果中，一个很重要的指标是透射电子显微镜观察结果。几乎每一株感染BTV-HbC₃的肿瘤细胞内都能产生数量不等的蓝舌病毒粒子，有些细胞中大量的病毒颗粒排列成晶格状；以及较多病毒空衣壳。在C6细胞中，我们观察到一些特别的现象：细胞染色质浓缩，在细胞核周边聚集，并且伴随有大小不等的胞浆内空泡和胞浆浓缩，显示了细胞凋亡特征。电镜图像明显地揭示出：（1）上述受试肿瘤细胞均产生了CPE；（2）受试肿瘤细胞出现的CPE分为两类，一种是病毒大量增殖，细胞裂解死亡；另一种是病毒有一定量增殖，并诱导肿瘤细胞凋亡。 根据以上的结果，我们可以得到一个初步的结论，就是BTV－HbC₃不感染正常人胚肺成纤维细胞，但是它可以选择性地在肿瘤细胞中增殖并使之产生不同程度的细胞裂解效应或诱导细胞凋亡，最终杀死肿瘤细胞。这些细胞中，人肺腺癌细胞系SPC一A一1对BTV一HbC3有最高的敏感性。实验显示BTV一HbC3具有靶向性杀死某些肿瘤细胞的潜能。尽管其机制尚有待我们进一步深入研究，但这些资料为把BTV一HbC3发展为一种可能的溶癌病毒提供了初步的实验室依据更多还原

【Abstract】 In the long time duration of studying on Bluetongue Virus of Hubei China strain 3, BTV-HbC3, We noted that BTV-HbC3 could be propagated well in HeLa cell line, a cell line derived from human cervical carcinoma, and later, we found it can even infect some other cancer cells. This inspired our imaginations of a therapeutic method for carcinoma: virotherapy of carcinoma. So, here we proceeded several experiments to explore whether BTV-HbC3 has the potential of becoming an oncolytic virus.First, by using BTV-HbC3 to infect some human and animal cancer cells including human lung cancer SPC-A-1 cells, human cervical carcinoma HeLa cells, human astrocytoma U251 cells, mouse-derived astrocytoma C6 cells and a normal cell strain: human embryonic lung cells separately, we could observe the cytopathic effects induced by BTV-HbC3 to investigate if all the tested cells are susceptible to the virus.At the same time, we titrated the TCID50 of BTV-HbC3 on all the tested cells, and used double immunodiffusing test in order to detect if there are BTV-HbC3 antigens in all the cells infected with BTV-HbC3, so that, we could investigate the propagation ability of progeny virus in these cell lines. Through these methods, we can distinguish if there are some differences between the infectivity of BTV-HbC3 to normal and to tumor cells.To improve the reliability of the results of double immunodiffusing test, Reverse transcription-PCR was used to detect the proliferations of BTV-HbC3 in these cells. We also used transmission electron microscope to observe the ultra structure morphology changes of the tested cells. Through this, we can very clearly to see the morphology sequent and morphgenesis sequent of interaction between the virus and all the tested cells.The results show: BTV-HbC3 could induce cytopathic effects on all the cells except human embryonic lung cells; double immunodiffusing test result could clearly display the Immunoprecipitation of antibody-antigen compounds between all the cells except human embryonic lung cells and anti- BTV-HbC3. Reverse transcription-PCR also can detect the specific nucleic acid sequence of BTV-HbC3 in all the cells except human embryonic lung cells.But, we found that, though BTV-HbC3 can replicate in all the tested tumor cells, the infectivity of it are not all the same to them.The most important result of these experiments is brought by transmission electron microscope. The photos show that almost every tumor cell strainsiiiinfected with BTV-HbC3 could produce different amount of bluetongue virus particles. In some cells, numerous virus particles arranged like crystals with many vacuum virus capsids. In C6 cell strains we observed some special images: condensed chromatin at the nuclear margin with many cytoplasmic vacuoles in different size. These images show the characters of cell apoptosis.These EM images clearly discovered that: (1) all the tested tumor cells came into being CPE; (2) the CPE can be divided into two kinds: The first one is those viruses propagate in a large amount and lyse tumor cells. The other is that viruses propagate to some degree and induce tumor cells’ apoptosis.According to above results, we could primarily draw a conclusion that BTV-HbC does not infect normal human embryonic lung cells, but it can selectively replicate in tumor cells and induce different degree of cytopathic effects or induce cell apoptosis, and finally, kill all the tested tumor cells. Among all the tested cells, human lung cancer SPC-A-1 cells have the highest sensitivity to BTV-HbC.These experiments showed that BTV-HbC has the potential of targeted killing some kinds of tumor cells. Though the mechanism needs further study, these data can still provide the laboratory basis for approaching the possibility of BTV-HbC as an oncolytic virus.更多还原