本文關(guān)鍵詞：多西紫杉醇和人參皂甙對宮頸癌疫苗HPV16 L2E6E7的佐劑作用　出處：《浙江大學》2016年博士論文　論文類型：學位論文

  更多相關(guān)文章： 多西紫杉醇 人參皂甙 HPV16 L2E6E7融合蛋白疫苗 佐劑 抗腫瘤疫苗

【摘要】：宮頸癌是威脅女性健康的第二大惡性腫瘤。研究表明其發(fā)生與人乳頭狀病毒(Human papillomavirus, HPV)感染密切相關(guān)。因此,有可能以通過預防和治療HPV感染來防治宮頸癌。目前國外已有預防HPV感染的疫苗問世。HPV16 L2E6E7是我國目前正在研制的一種HPV融合蛋白疫苗(簡稱HPV疫苗),目前正在申報臨床試驗。像其它蛋白疫苗一樣,該融合蛋白疫苗尚存在免疫原性差的缺點。在疫苗中添加佐劑是增強疫苗免疫效果的常用方法。本論文擬研究在HPV疫苗中添加多西紫杉醇和人參皂甙對該疫苗免疫效果的影響,為臨床應用提供參考。1多西紫杉醇對HPV疫苗的免疫佐劑作用目的：探究多西紫杉醇對HPV疫苗抗腫瘤免疫的增強作用。實驗一：在-1天給36只C57BL/6小鼠接種TC-1腫瘤細胞(104個/只)后,將小鼠隨機分成6組：(1)生理鹽水組,(2)疫苗組(120 μg HPV),(3)多西紫杉醇(200 μg)對照組,(4)多西紫杉醇佐劑1組(120μg HPV+100μg多西紫杉醇),(5)多西紫杉醇佐劑2組(120μgHPV+200μg多西紫杉醇),(6)多西紫杉醇佐劑3組(120 μg HPV+400μg多西紫杉醇)。于第0、3和7天在小鼠后腿肌肉處先后注射免疫三次。在21天收集小鼠瘤塊用于檢測抑瘤率、收集血清用于檢測IgG抗體及其亞類和脾細胞用于檢測E749-56抗原刺激產(chǎn)生的分泌IFN-y和IL-10的淋巴細胞數(shù)、Treg細胞數(shù)以及CTL細胞活性。實驗二：分組和處理同實驗一。每隔3天測量腫瘤大小并觀察記錄小鼠存活情況。結(jié)果：多西紫杉醇顯著抑制了小鼠體內(nèi)腫瘤的生長,延長了荷瘤小鼠存活時間,提高了HPV疫苗在荷瘤小鼠體內(nèi)誘導的特異性抗體及其亞類水平和CTL細胞對TC-1腫瘤細胞的殺傷能力,減少了脾細胞中Treg細胞的數(shù)量,上調(diào)了分泌E749-56特異性IFN-y的淋巴細胞數(shù),而對分泌IL-10的淋巴細胞數(shù)影響較小。2人參皂甙Rg1對HPV疫苗的免疫佐劑作用目的：探究人參皂甙Rg1對HPV疫苗的免疫增強作用。方法：將64只小鼠隨機分成8組：(1)生理鹽水組,(2)Rg1(100μg)對照組,(3)疫苗低劑量(10μgHPV)組,(4)疫苗高劑量(100μgHPV)組,(5)Rg1佐劑1組(10μgHPV+25μgRg1), (6) Rg1佐劑2組(10μgHPV+50μgRg1), (7) Rg1佐劑3組(10μgHPV+100 μg Rg1)組,(8)Rgl佐劑4組(10μgHPV+200μgRg1)。于第0、3和7天在小鼠后腿肌肉處先后注射免疫三次。在第21天采集小鼠血清用于檢測IgG及其亞類水平,收集脾細胞檢測刀豆蛋白(ConA)和脂多糖(LPS)誘導的淋巴細胞增殖活性,E749-56抗原刺激產(chǎn)生的IFN-y分泌細胞數(shù)、淋巴細胞表達IL-4和IL-12 mRNA水平。結(jié)果：Rg1能顯著提高小鼠體內(nèi)抗HPV特異性抗體及其亞類水平,增加分泌E749-56特異性IFN-γ的淋巴細胞數(shù),提高淋巴細胞對LPS和ConA刺激的刺激指數(shù)以及細胞因子IL-4和IL-12 mRNA表達水平。3人參皂甙Rg3對HPV疫苗的免疫佐劑作用目的：探究人參皂甙Rg3對HPV疫苗的免疫增強作用。方法：將36只C57BL/6小鼠隨機分成6組,(1)生理鹽水組,(2) Rg3 (50對照組,(3)疫苗組(120μgHPV), (4) Rg3佐劑1組(120μgHPV+25μgRg3), (5) Rg3佐劑2組(120μHPV+50μRg3)組,(6)Rg3佐劑3組(120 μg HPV+100 μg Rg3)于第0、3和7天在小鼠后腿肌肉處先后注射免疫三次。在21天收集血清用于檢測IgG抗體；收集脾細胞用于檢測分泌E749-56特異性IFN-γ的淋巴細胞數(shù)。結(jié)果Rg3顯著提高了正常小鼠體內(nèi)抗HPV特異性抗體水平和增加分泌E749-56特異性IFN-γ淋巴細胞數(shù)。4人參皂甙Rg3和多西紫杉醇配伍不增加對HPV疫苗的佐劑作用目的：探究人參皂甙Rg3和多西紫杉醇配伍對HPV疫苗抗腫瘤免疫的增強作用。實驗一：在-1天將C57BL/6小鼠接種TC-1腫瘤細胞(104個／只),然后隨機分成8組：(1)生理鹽水組,(2) Rg3 (100μg)對照組,(3)多西紫杉醇(200 gg)對照組,(4)配伍對照組(100μg Rg3+200 μg多西紫杉醇),(5)疫苗(120μg HPV)組,(6)Rg3佐劑組(120μg HPV+100μgRg3),(7)多西紫杉醇佐劑組(120μgHPV+200μg多西紫杉醇),(8)配伍佐劑組(120 μg HPV +100μgRg3+200μ多西紫杉醇)。于第0、3和7天在小鼠后腿肌肉處先后注射免疫三次。在21天收集小鼠血清用于檢測IgG抗體及其亞類；收集小鼠脾細胞用于檢測分泌E749-56特異性IFN-y的淋巴細胞數(shù)；收集小鼠瘤塊用于計算抑瘤率。實驗二：分組和處理同實驗一,每隔3天測量腫瘤大小并觀察記錄小鼠存活情況。結(jié)果：多西紫杉醇和Rg3配伍與單獨使用多西紫杉醇作為佐劑相比,在延長荷瘤小鼠的存活時間、提高特異性抗體水平和增加分泌IFN-y的淋巴細胞數(shù)方面,無顯著差異,說明Rg3不增加多西紫杉醇的佐劑作用。5多西紫杉醇對細胞免疫的影響目的： 研究多西紫杉醇對細胞免疫的影響。方法：將20只C57BL/6小鼠分別在0,3,7天進行肌肉注射120μg HPV疫苗。在21天收集脾臟、周圍淋巴結(jié)內(nèi)的淋巴細胞用于檢測T細胞的殺傷活性和Fas、FasL、M6PR、Perform、GzB以及Caspase-3 mRNA的表達情況。結(jié)果：(1)與單獨處理CTL細胞或者TC-1細胞相比,多西紫杉醇同時處理這兩種細胞后,CTL對TC-1細胞的殺傷作用最強,且呈濃度依賴關(guān)系；(2)多西紫杉醇能顯著增強TC-1細胞中Fas、M6PR和Caspase-3基因和T細胞中FasL和GzB mRNA的表達水平。6多西紫杉醇作為HPV疫苗佐劑對腫瘤微環(huán)境的影響目的： 研究多西紫杉醇對腫瘤微環(huán)境的影響。方法： 在-1天給40只C57BL/6小鼠接種TC-1腫瘤細胞(104個/只)后,將小鼠隨機分成4組：(1)生理鹽水組,(2)多西紫杉醇(200μg)對照組,(3)疫苗組(120μg HPV),(4)多西紫杉醇佐劑組(120μHPV+200μ多西紫杉醇)。于第0、3和7天在小鼠后腿肌肉處先后注射免疫三次。在21天收集小鼠瘤塊用于檢測treg細胞數(shù)量和IL-10、IFN-γ、VEGF以及STAT-3 mRNA的表達水平。結(jié)果：多西紫杉醇降低了瘤塊中Treg細胞的數(shù)量以及IL-10、VEGF和STAT-3 mRNA的表達水平,增加了IFN-γ mRNA的表達水平。綜上所述,多西紫杉醇對HPV疫苗具有顯著佐劑作用,可顯著提高HPV疫苗對荷瘤小鼠的抗腫瘤能力,延長荷瘤小鼠存活時間；人參皂甙Rg1、Rg3對HPV疫苗具有佐劑作用,增強HPV疫苗誘導的免疫應答水平；人參皂甙Rg3和多西紫杉醇配伍與多西紫杉醇單獨應用比較對HPV疫苗誘導的免疫應答無顯著差異,說明Rg3不增加多西紫杉醇的佐劑作用；多西紫杉醇的佐劑作用可能與其能增強細胞免疫功能和改善腫瘤微環(huán)境有關(guān)。本論文研究結(jié)果對于采用多西紫杉醇和人參皂甙作為佐劑提高HPV疫苗的免疫效果具有重要意義。
[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鑲跨槫緇嗚優(yōu)(104涓紡鍙，

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