多西紫杉醇和人参皂甙对宫颈癌疫苗HPV16 L2E6E7的佐剂作用

发布时间：2018-01-01 03:29

  本文关键词：多西紫杉醇和人参皂甙对宫颈癌疫苗HPV16 L2E6E7的佐剂作用　出处：《浙江大学》2016年博士论文　论文类型：学位论文

  更多相关文章： 多西紫杉醇 人参皂甙 HPV16 L2E6E7融合蛋白疫苗 佐剂 抗肿瘤疫苗

【摘要】：宫颈癌是威胁女性健康的第二大恶性肿瘤。研究表明其发生与人乳头状病毒(Human papillomavirus, HPV)感染密切相关。因此,有可能以通过预防和治疗HPV感染来防治宫颈癌。目前国外已有预防HPV感染的疫苗问世。HPV16 L2E6E7是我国目前正在研制的一种HPV融合蛋白疫苗(简称HPV疫苗),目前正在申报临床试验。像其它蛋白疫苗一样,该融合蛋白疫苗尚存在免疫原性差的缺点。在疫苗中添加佐剂是增强疫苗免疫效果的常用方法。本论文拟研究在HPV疫苗中添加多西紫杉醇和人参皂甙对该疫苗免疫效果的影响,为临床应用提供参考。1多西紫杉醇对HPV疫苗的免疫佐剂作用目的：探究多西紫杉醇对HPV疫苗抗肿瘤免疫的增强作用。实验一：在-1天给36只C57BL/6小鼠接种TC-1肿瘤细胞(104个/只)后,将小鼠随机分成6组：(1)生理盐水组,(2)疫苗组(120 μg HPV),(3)多西紫杉醇(200 μg)对照组,(4)多西紫杉醇佐剂1组(120μg HPV+100μg多西紫杉醇),(5)多西紫杉醇佐剂2组(120μgHPV+200μg多西紫杉醇),(6)多西紫杉醇佐剂3组(120 μg HPV+400μg多西紫杉醇)。于第0、3和7天在小鼠后腿肌肉处先后注射免疫三次。在21天收集小鼠瘤块用于检测抑瘤率、收集血清用于检测IgG抗体及其亚类和脾细胞用于检测E749-56抗原刺激产生的分泌IFN-y和IL-10的淋巴细胞数、Treg细胞数以及CTL细胞活性。实验二：分组和处理同实验一。每隔3天测量肿瘤大小并观察记录小鼠存活情况。结果：多西紫杉醇显著抑制了小鼠体内肿瘤的生长,延长了荷瘤小鼠存活时间,提高了HPV疫苗在荷瘤小鼠体内诱导的特异性抗体及其亚类水平和CTL细胞对TC-1肿瘤细胞的杀伤能力,减少了脾细胞中Treg细胞的数量,上调了分泌E749-56特异性IFN-y的淋巴细胞数,而对分泌IL-10的淋巴细胞数影响较小。2人参皂甙Rg1对HPV疫苗的免疫佐剂作用目的：探究人参皂甙Rg1对HPV疫苗的免疫增强作用。方法：将64只小鼠随机分成8组：(1)生理盐水组,(2)Rg1(100μg)对照组,(3)疫苗低剂量(10μgHPV)组,(4)疫苗高剂量(100μgHPV)组,(5)Rg1佐剂1组(10μgHPV+25μgRg1), (6) Rg1佐剂2组(10μgHPV+50μgRg1), (7) Rg1佐剂3组(10μgHPV+100 μg Rg1)组,(8)Rgl佐剂4组(10μgHPV+200μgRg1)。于第0、3和7天在小鼠后腿肌肉处先后注射免疫三次。在第21天采集小鼠血清用于检测IgG及其亚类水平,收集脾细胞检测刀豆蛋白(ConA)和脂多糖(LPS)诱导的淋巴细胞增殖活性,E749-56抗原刺激产生的IFN-y分泌细胞数、淋巴细胞表达IL-4和IL-12 mRNA水平。结果：Rg1能显著提高小鼠体内抗HPV特异性抗体及其亚类水平,增加分泌E749-56特异性IFN-γ的淋巴细胞数,提高淋巴细胞对LPS和ConA刺激的刺激指数以及细胞因子IL-4和IL-12 mRNA表达水平。3人参皂甙Rg3对HPV疫苗的免疫佐剂作用目的：探究人参皂甙Rg3对HPV疫苗的免疫增强作用。方法：将36只C57BL/6小鼠随机分成6组,(1)生理盐水组,(2) Rg3 (50对照组,(3)疫苗组(120μgHPV), (4) Rg3佐剂1组(120μgHPV+25μgRg3), (5) Rg3佐剂2组(120μHPV+50μRg3)组,(6)Rg3佐剂3组(120 μg HPV+100 μg Rg3)于第0、3和7天在小鼠后腿肌肉处先后注射免疫三次。在21天收集血清用于检测IgG抗体；收集脾细胞用于检测分泌E749-56特异性IFN-γ的淋巴细胞数。结果Rg3显著提高了正常小鼠体内抗HPV特异性抗体水平和增加分泌E749-56特异性IFN-γ淋巴细胞数。4人参皂甙Rg3和多西紫杉醇配伍不增加对HPV疫苗的佐剂作用目的：探究人参皂甙Rg3和多西紫杉醇配伍对HPV疫苗抗肿瘤免疫的增强作用。实验一：在-1天将C57BL/6小鼠接种TC-1肿瘤细胞(104个／只),然后随机分成8组：(1)生理盐水组,(2) Rg3 (100μg)对照组,(3)多西紫杉醇(200 gg)对照组,(4)配伍对照组(100μg Rg3+200 μg多西紫杉醇),(5)疫苗(120μg HPV)组,(6)Rg3佐剂组(120μg HPV+100μgRg3),(7)多西紫杉醇佐剂组(120μgHPV+200μg多西紫杉醇),(8)配伍佐剂组(120 μg HPV +100μgRg3+200μ多西紫杉醇)。于第0、3和7天在小鼠后腿肌肉处先后注射免疫三次。在21天收集小鼠血清用于检测IgG抗体及其亚类；收集小鼠脾细胞用于检测分泌E749-56特异性IFN-y的淋巴细胞数；收集小鼠瘤块用于计算抑瘤率。实验二：分组和处理同实验一,每隔3天测量肿瘤大小并观察记录小鼠存活情况。结果：多西紫杉醇和Rg3配伍与单独使用多西紫杉醇作为佐剂相比,在延长荷瘤小鼠的存活时间、提高特异性抗体水平和增加分泌IFN-y的淋巴细胞数方面,无显著差异,说明Rg3不增加多西紫杉醇的佐剂作用。5多西紫杉醇对细胞免疫的影响目的： 研究多西紫杉醇对细胞免疫的影响。方法：将20只C57BL/6小鼠分别在0,3,7天进行肌肉注射120μg HPV疫苗。在21天收集脾脏、周围淋巴结内的淋巴细胞用于检测T细胞的杀伤活性和Fas、FasL、M6PR、Perform、GzB以及Caspase-3 mRNA的表达情况。结果：(1)与单独处理CTL细胞或者TC-1细胞相比,多西紫杉醇同时处理这两种细胞后,CTL对TC-1细胞的杀伤作用最强,且呈浓度依赖关系；(2)多西紫杉醇能显著增强TC-1细胞中Fas、M6PR和Caspase-3基因和T细胞中FasL和GzB mRNA的表达水平。6多西紫杉醇作为HPV疫苗佐剂对肿瘤微环境的影响目的： 研究多西紫杉醇对肿瘤微环境的影响。方法： 在-1天给40只C57BL/6小鼠接种TC-1肿瘤细胞(104个/只)后,将小鼠随机分成4组：(1)生理盐水组,(2)多西紫杉醇(200μg)对照组,(3)疫苗组(120μg HPV),(4)多西紫杉醇佐剂组(120μHPV+200μ多西紫杉醇)。于第0、3和7天在小鼠后腿肌肉处先后注射免疫三次。在21天收集小鼠瘤块用于检测treg细胞数量和IL-10、IFN-γ、VEGF以及STAT-3 mRNA的表达水平。结果：多西紫杉醇降低了瘤块中Treg细胞的数量以及IL-10、VEGF和STAT-3 mRNA的表达水平,增加了IFN-γ mRNA的表达水平。综上所述,多西紫杉醇对HPV疫苗具有显著佐剂作用,可显著提高HPV疫苗对荷瘤小鼠的抗肿瘤能力,延长荷瘤小鼠存活时间；人参皂甙Rg1、Rg3对HPV疫苗具有佐剂作用,增强HPV疫苗诱导的免疫应答水平；人参皂甙Rg3和多西紫杉醇配伍与多西紫杉醇单独应用比较对HPV疫苗诱导的免疫应答无显著差异,说明Rg3不增加多西紫杉醇的佐剂作用；多西紫杉醇的佐剂作用可能与其能增强细胞免疫功能和改善肿瘤微环境有关。本论文研究结果对于采用多西紫杉醇和人参皂甙作为佐剂提高HPV疫苗的免疫效果具有重要意义。
[Abstract]:Cervical cancer is a threat to women's health second. Research shows that the incidence of malignant tumors and human papilloma virus (Human, papillomavirus, HPV) infection are closely related. Therefore, it is possible to the prevention and treatment of HPV infection to prevent cervical cancer. Currently there is a vaccine to prevent HPV infection was.HPV16 L2E6E7 is a HPV currently the development of China's fusion protein vaccine (HPV vaccine), is currently being reported in clinical trials. Like other protein vaccine, the fusion protein vaccine still has the disadvantages of poor immunogenicity. Add in vaccine adjuvant is a common method to enhance the immune effect of vaccine. This study intends to add in the HPV vaccine and effect of docetaxel effect of Ginsenoside on the immune effect of the vaccine, provide the adjuvant effect of docetaxel on HPV vaccine.1 reference to clinical application: the study of docetaxel on HPV disease Enhancement of seedling anti-tumor immunity. Experiment one: to 36 C57BL/6 mice inoculated with TC-1 tumor cells in -1 days (104 /), the mice were randomly divided into 6 groups: normal saline group (1), (2) the vaccine group (120 g HPV), docetaxel (200 (3) g) control group, (4) 1 groups of docetaxel adjuvant (120 g HPV+100 g (docetaxel), 2 (5) of docetaxel adjuvant group 120 gHPV+200 g docetaxel), docetaxel (6) adjuvant group 3 (120 g HPV+400 g in 0,3 and docetaxel). The 7 day hind legs in mice the muscle has injected three times. In 21 days were collected for detecting the tumor inhibition rate, serum was collected for detection of IgG antibody and its subtypes and spleen cell number for the secretion of IFN-y and IL-10 to detect E749-56 antigen stimulation, Treg cells and CTL cell activity. Experiment two: grouping and processing the same the measured every 3 days. The amount of tumor size were observed in mice. Results: Docetaxel significantly inhibited tumor growth in mice, prolong the survival time of mice with tumor, improve the killing effect of HPV vaccine on TC-1 tumor cells in specific antibody induced in mice and its subclass levels and reduce the number of CTL cells, Treg cells spleen cells, increase the number of lymphocytes secreted E749-56 specific IFN-y, and the number of secretion of IL-10 immune adjuvant effect of ginsenoside Rg1 on the small.2 HPV vaccine Objective: To explore the effect of ginsenoside Rg1 to enhance the immune function of HPV vaccine. Methods: 64 mice were randomly divided into 8 groups: (1) the normal saline group, (2) Rg1 (100 g) control group, (3) low dose vaccine (10 gHPV) group, (4) vaccine in high dose (100 gHPV) group, (5) Rg1 adjuvant group 1 (10 gHPV+ 25 gRg1), 2 (6) Rg1 adjuvant group (10 gHPV+ 50 gRg1 (7), 3 (10) Rg1 adjuvant group gHPV+100 g Rg1 group), Rgl group (8) adjuvant 4 (10 gHPV+200 gRg1). At 0,3 and 7 days in the mouse hind leg muscle has injected three times. On the twenty-first day mice serum was collected for detecting IgG and subclass levels, spleen cells were collected to detect concanavalin A (ConA) and lipopolysaccharide (LPS) induced lymphocyte proliferation, E749-56 antigen stimulated secretion of IFN-y cells, the expression of IL-4 and IL-12 lymphocytes mRNA level. Results: Rg1 can significantly improve the mice anti HPV specific antibodies and subclass levels, increasing the number of lymphocytes the secretion of E749-56 specific IFN- gamma, LPS and ConA to improve lymphocyte stimulation index and cytokines IL-4 and IL-12 expression level of.3 mRNA adjuvant effect of ginsenoside Rg3 on HPV vaccine Objective: To explore the effect of ginsenoside Rg3 to enhance the immune function of HPV vaccine. Method: 36 C57BL/6 mice were randomly divided into 6 groups, normal saline group (1), (2) Rg3 (50 control group, (3) the vaccine group (120 gHPV), Rg3 group (4) adjuvant 1 (120 gHPV+25 gRg3), Rg3 (5) 2 groups (120 adjuvant HPV+50 Rg3 (Rg3) group, 6) adjuvant 3 groups (120 g HPV+100 g Rg3) at 0,3 and 7 days in the mouse hind leg muscle has injected three times. For the detection of IgG antibody in the serum was collected 21 days; spleen cells collected for detection of specific E749-56 lymphocytes secrete IFN- gamma. Results Rg3 significantly increased in normal mice anti HPV specific antibody level and increase the secretion of E749-56 specific IFN- gamma.4 lymphocyte count of ginsenoside Rg3 and docetaxel combination does not increase the adjuvant effect of HPV vaccine Objective: to strengthen the role of ginsenoside Rg3 and docetaxel combination on the antitumor immunity of HPV vaccine. Experiment one: in -1 day C57BL/6 mice were inoculated with T C-1鑲跨槫缁嗚優(104涓�锛忓彛�

本文编号：1362754