仙台病毒缺损干扰颗粒抗大鼠脑胶质瘤初步研究

发布时间：2018-03-11 13:21

本文选题：仙台病毒Tianjin株　切入点：缺损干扰颗粒　出处：《天津医科大学》2009年硕士论文　论文类型：学位论文

【摘要】：目的：鉴于仙台病毒Tianjin株对成年人的低致病性,以及先前的实验表明仙台病毒其它基因型对肿瘤有抑制作用。本课题拟通过仙台病毒Tianjin株缺损干扰颗粒对肿瘤细胞的生物学影响,探索不必外源基因的表达载体和不必具有直接溶瘤能力的病毒,利用仙台病毒Tianjin株缺损干扰颗粒辅助治疗肿瘤的方法。并且初步研究其治疗肿瘤的机理,以期为肿瘤治疗提供新的思路。方法：1.敏感细胞的筛选：向所选定的癌细胞株:A549、MEL526、Libr、C6、 HT2、SW480、A498接种活仙台病毒Tianjin株,通过细胞CPE、细胞成活率、细胞凋亡、细胞培养液TCID50、PCR等形态学、生物学和分子生物学方法确定对该病毒敏感的细胞系。2.仙台病毒Tianjin株缺损干扰颗粒的制备：仙台病毒Tianjin株通过鸡胚培养法扩增,超速离心纯化仙台病毒Tianjin株活病毒,超速离心蔗糖密度梯度分离缺损干扰颗粒,提纯出的缺损干扰颗粒使用鸡胚培养法验证是否能够继续增殖,如果不能增殖则为仙台病毒Tianjin株缺损干扰颗粒。3.使用仙台病毒Tianjin株缺损干扰颗粒按不同浓度接种DC细胞,检测DC细胞产生IL-6、TNF-α、IFN-α等有抗肿瘤作用的细胞因子的量,验证仙台病毒Tianjin株缺损干扰颗粒刺激DC细胞抗肿瘤的能力。4.使用确定出的敏感细胞系接种适宜的动物,制作荷瘤动物模型。肿瘤内接种仙台病毒Tianjin株缺损干扰颗粒,通过直接测量肿瘤大小、细胞因子检测、PCR、免疫组化等方法评估仙台病毒Tianjin株缺损干扰颗粒治疗肿瘤的可行性,并初步确定治疗的机理。结果：大鼠脑胶质瘤细胞C6株对仙台病毒Tianjin株最具高度敏感性,病毒TCID50达到11.24+0.08.其它被测试细胞株均未达到此值,一般在0-3之间。由此确定实验动物为大鼠,品种为Wistar大鼠。在促进DC细胞体外增值实验中,与对照组比较,仙台病毒Tianjin株缺损干扰颗粒可以刺激DC细胞产生抗癌作用的细胞因子,IL-6、TNF-α、IFN-α有明显升高(均为P0.001)。在大鼠体内实验中,与对照组比较,仙台病毒Tianjin株缺损干扰颗粒可以刺激正常大鼠高表达有抗癌作用的细胞因子,IL-6、TNF-α、IFN-α有明显升高(均为P0.001)。大鼠脑瘤模型造模失败。结论：大鼠脑胶质瘤细胞C6株对仙台病毒Tianjin株高度敏感。仙台病毒Tianjin株接种C6细胞后可引起细胞明显病变,促进凋亡,并且作用持续。仙台病毒Tianjin株缺损干扰颗粒可以促进树突细胞成熟,高表达抗癌物质,有助于消除肿瘤。仙台病毒Tianjin株缺损干扰颗粒可以刺激正常大鼠高表达有抗癌作用的细胞因子,对大鼠脑胶质瘤应该有潜在的治疗作用。但是大鼠荷瘤失败,对活体肿瘤的直接作用效果未得到论证,有待进一步研究。
[Abstract]:Objective: in view of the low pathogenicity of Sendai virus Tianjin strain to adults, Other genotypes of Sendai virus (Sendai virus) have inhibitory effect on tumor. This study aims to study the biological effects of Sendai virus Tianjin strain defect interference granules on tumor cells. To explore the method of adjuvant therapy of Sendai virus Tianjin strain defect interference granules without the expression vector of exogenous gene and virus without direct tumor lysis ability, and to preliminarily study the mechanism of tumor treatment. In order to provide new ideas for tumor treatment. Methods: 1. Screening of sensitive cells: inoculation of Sendai virus Tianjin strain with selected cancer cell lines: cell line: A549MEL526LibrC6, HT2OSW480A498. Morphology of Sendai virus Tianjin strain was obtained by cell CPE, cell survival rate, cell apoptosis, TCID50 PCR, and so on. Biological and molecular biological methods to determine the sensitive cell line of Sendai virus. 2. Preparation of defective interfering particles of Sendai virus Tianjin strain: Sendai virus Tianjin strain was amplified by chicken embryo culture and purified by ultracentrifugation, and the live virus of Sendai virus Tianjin strain was purified by ultracentrifugation. The defect interference granules were separated by ultracentrifugation sucrose density gradient. The purified defective interference granules were tested by chicken embryo culture method to see if they could continue to proliferate. If Sendai virus Tianjin strain is not proliferative, it is the defect interfering particle of Sendai virus Tianjin strain. DC cells are inoculated with different concentration of Sendai virus Tianjin strain defect interference granules to detect the amount of cytokines produced by DC cells such as IL-6 TNF- 伪 IFN- 伪, and so on, which have anti-tumor effect, such as IL-6, TNF- 伪, IFN- 伪, and so on. To test the ability of Sendai virus Tianjin strain to stimulate DC cells to resist tumor. 4. To establish a tumor-bearing animal model by inoculating the identified sensitive cell lines with appropriate animal models. The tumor was inoculated with Sendai virus Tianjin strain defect interference particles. The feasibility of Sendai virus (Sendai virus) Tianjin strain defect interference granules in the treatment of tumor was evaluated by direct measurement of tumor size, detection of cytokines and immunohistochemical method, and the mechanism of treatment was preliminarily determined. Results: the C6 strain of glioma cells was most sensitive to Sendai virus Tianjin strain, the virus TCID50 reached 11.240.08.The other tested cell lines did not reach this value, generally in the range of 0-3. The experimental animals were identified as rats. Sendai virus Tianjin strain defect interference granule can stimulate DC cells to produce anticancer effect, the cytokine, IL-6, TNF- 伪 and IFN- 伪 (all are P0.001), are significantly increased in the experiment of promoting DC cell proliferation in vitro, compared with the control group, in vivo, Sendai virus strain Tianjin strain can stimulate DC cells to produce anti-cancer effect. Compared with the control group, the Sendai virus Tianjin strain defect interference granule could stimulate the high expression of IL-6 TNF- 伪 and IFN- 伪 in normal rats (all P0.001). The rat brain tumor model failed. Conclusion: C6 strain of rat brain glioma cells is highly sensitive to Sendai virus Tianjin strain. Sendai virus Tianjin strain inoculated with C6 cells can cause obvious cytopathic changes and promote apoptosis. Sendai virus Tianjin strain defect interference granule can promote dendritic cell maturation and high expression of anticancer substances. Sendai virus Tianjin strain can stimulate high expression of anti-cancer cytokines in normal rats, which should have potential therapeutic effect on glioma in rats. The direct effect on tumor in vivo has not been demonstrated and further research is needed.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R373;R739.41

【相似文献】