以同种异基因型胶质瘤细胞及其裂解物与同基因型胶质瘤细胞裂解物作为免疫原的混合疫苗治疗大鼠胶质瘤

发布时间：2018-02-13 17:08

本文关键词： 胶质瘤免疫治疗疫苗　出处：《西南医科大学》2017年硕士论文　论文类型：学位论文

【摘要】：目的:本研究探讨以同种异基因型的C6胶质瘤细胞及其裂解物与同基因型的9L胶质瘤细胞裂解物作为免疫原的疫苗对大鼠胶质瘤的治疗作用,以及经该疫苗治疗后大鼠胶质瘤组织的侵袭力变化情况,以期为胶质瘤的免疫治疗提供更多的可行性方案。方法:(1)采用立体定向技术,将1×10~6/10μl个9L胶质瘤细胞悬液注入Fisher 344大鼠右侧尾状核区,建立9L/Fisher 344大鼠胶质瘤原位模型。(2)接种后的大鼠随机分为三组:空白组,二十万单位治疗组,三十万单位治疗组,每组各10只。其中空白组大鼠不接受治疗;二十万单位治疗组大鼠皮下注射以C6细胞(2×105个)、C6细胞裂解物(含2×105个细胞)及9L细胞裂解物(含2×105个细胞)作为免疫原的疫苗;三十万单位治疗组大鼠皮下注射以C6细胞(3×105个)、C6细胞裂解物(含3×105个细胞)及9L细胞裂解物(含3×105个细胞)作为免疫原的疫苗。所有治疗均于接种9L细胞后第1天进行。(3)以50天为大鼠生存观察期,记录其生存状况及生存时间。于接种9L细胞后第10、20、30、40、50天对所有大鼠行MRI检查,动态观察大鼠胶质瘤体积变化情况。接种9L细胞后第20天,各组随机处死1只大鼠行HE染色及免疫组化Podoplanin、Fascin染色,以观察大鼠胶质瘤组织病理及侵袭能力改变情况。结果:(1)以50天为大鼠生存观察期,空白组的生存率为0;二十万单位治疗组大鼠的生存率为89%;三十万单位治疗组大鼠的生存率为100%。治疗组大鼠生存时间比空白组显著延长,差异有统计学意义(P0.05);二十万单位治疗组与三十万单位治疗组之间比较,差异无统计学意义(P0.05)。(2)所有大鼠增强MRI检查均可见右侧尾状核区明显强化,本次实验制模成功率为100%。MRI动态检查发现,随瘤龄增加,空白组大鼠胶质瘤呈现持续增大,而治疗组大鼠胶质瘤呈现先增大后减小的生长趋势。接种9L细胞第20天时,计算各组肿瘤体积:空白组大鼠胶质瘤体积为(451.25±9.36)mm3,二十万单位治疗组大鼠胶质瘤体积为(122.52±22.30)mm3,三十万单位治疗组大鼠胶质瘤体积为(112.06±17.97)mm3。单因素方差分析证明空白组大鼠胶质瘤体积明显大于治疗组,差异具有统计学意义(P0.05);二十万单位治疗组与三十万单位治疗组之间比较,差异无统计意义(P0.05)。(3)HE染色可见,空白组大鼠胶质瘤细胞密度高,排列紧密,呈巢状,部分区域伴有出血、坏死及新生血管;治疗组大鼠脑胶质瘤组织的肿瘤细胞密度稍低,典型巢状排列及新生血管少见。免疫组化Podoplanin及Fascin染色可见,空白组大鼠Podoplanin及Fascin呈强阳性表达,而在治疗组中,Podoplanin及Fascin表达均降低,且Podoplanin染色的降低程度比Fascin更加显著;二十万单位治疗组与三十万单位治疗组之间相比较,均能观察到少量的胶质瘤细胞表达Podoplanin及Fascin,两者之间无明显差异。结论:本研究成功建立9L/Fisher 344大鼠脑胶质瘤模型,并于接种后第1天予以同种异基因型的C6胶质瘤细胞及其裂解物与同基因型的9L胶质瘤细胞裂解物作为免疫原的疫苗进行治疗。实验结果证明了该疫苗能够打破荷瘤机体对自身肿瘤的免疫耐受,诱导荷瘤机体的抗肿瘤免疫反应,防止免疫逃避的发生,延长荷瘤大鼠生存时间并降低胶质瘤组织侵袭力。
[Abstract]:Objective: To study the 9L glioma cell lysis with allogeneic type C6 glioma cells and lysates with the same genotype as vaccine immunogens on rat glioma treatment, as well as by the vaccine after treatment of rat glioma tissue invasion force change situation, in order to provide feasibility of more for immunotherapy of glioma. Methods: (1) by stereotactic technique, 1 x 10~6/10 L 9L glioma cell suspension was injected into the right caudate nucleus of 344 Fisher rats, 344 rats to establish 9L/Fisher glioma orthotopic model. (2) the rats were randomly divided into after the three groups: blank control group, treatment group two hundred thousand units, three hundred thousand units in the treatment group, 10 rats in each group. The rats in the blank group without treatment; two hundred thousand treated rats by subcutaneous injection of C6 cells (2 * 105), C6 cell lysates (containing 2 x 105 cells) and 9L cell Lysates (containing 2 x 105 cells) as vaccine immunogens; three hundred thousand treated rats by subcutaneous injection of C6 cells (3 * 105), C6 cell lysates (containing 3 x 105 cells) and 9L cell lysates (containing 3 x 105 cells) as vaccine immunogens all treatment was performed in first days after inoculation of 9L cells. (3) for a 50 day rat survival was observed, recorded the survival time in 10,20,30,40,50 days after inoculation of 9L cells of all rats were examined with MRI, the dynamic observation of rat glioma volume changes. Twentieth days were inoculated with 9L cells, 1 rats in each group were sacrificed for HE staining and immunohistochemical staining to observe Podoplanin, Fascin, glioma invasion and pathological changes in rats. Results: (1) to 50 days for the survival of rats after the observation period, the survival rate of control group was 0; two hundred thousand in the treatment group mice survival rate was 89%; thirty Survival million units of rats in the treatment group was 100%. treatment group rats survival time was significantly longer than that in control group, the difference was statistically significant (P0.05); two hundred thousand units and three hundred thousand units in treatment group between the treatment groups, the difference was not statistically significant (P0.05). (2) all rats enhanced MRI examination showed the right caudate nuclear area enhancement, the experiment system of the success rate of model was found for the 100%.MRI dynamic inspection, with the increase of tumor age, blank group rat glioma has continued to increase, while the treatment group rat glioma growth showed a trend of first increasing and then decreasing. 9L cells were inoculated twentieth days, tumor volume were calculated: blank group the glioma volume (451.25 + 9.36) mm3, two hundred thousand units of volume treatment of glioma rats for (122.52 + 22.30) mm3, three hundred thousand units of volume treatment of glioma rats for (112.06 + 17.97) mm3. single factor variance analysis show that the blank group The rat glioma volume was significantly greater than the treatment group, the difference was statistically significant (P0.05); two hundred thousand units and three hundred thousand units in treatment group between the treatment groups, the difference was not statistically significant (P0.05). (3) HE staining: blank group rat glioma cells with high density, arranged closely, nests, part area with hemorrhage, necrosis and tumor angiogenesis; treatment group rat brain glioma tumor cell density was low, the typical nests and neovascularization rare. Immunohistochemical staining of Podoplanin and Fascin, the rats in the blank group Podoplanin and Fascin showed strong positive expression in the treatment group, the expression of Podoplanin and Fascin decreased, and Podoplanin staining decreased more significantly than Fascin; two hundred thousand unit and three hundred thousand unit treatment group between the treatment groups were compared and observed in a small number of glioma cells expressed Podoplanin and Fascin, both No significant difference. Conclusion: This study successfully established 9L/Fisher rat brain glioma model 344, 9L glioma cell lysis and allogeneic type in first days after inoculation of C6 glioma cells and lysates with the same genotype as vaccine immunogens for treatment. The experimental results show that the the vaccine can break tumor immune tolerance to tumor, induce antitumor immune response of tumor bearing mice, prevent immune escape, prolong the survival time of tumor bearing rats and reduce glioma invasiveness.

【学位授予单位】：西南医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R739.41

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