【作者】 程春霞；

【导师】 薛敏；

【作者基本信息】 中南大学 ， 妇产科学， 2012， 博士

【摘要】 Poly Ⅰ:C诱导负载HPV E744-62多肽脐血DCs体外抗宫颈癌的免疫效应研究目的：观察Poly Ⅰ:C诱导负载HPV E744-62多肽的人脐血DCs体外抗宫颈癌的免疫效应。方法：采集健康足月妊娠孕妇的脐带血,密度梯度离心法分离获得脐血单个核细胞,取贴壁细胞体外经rhGM-CSF、rhIL-4诱导培养7天获DCs。分别用Poly Ⅰ:C、HPV16E744-62多肽、Poly Ⅰ:C联合HPVl6E744-62多肽来刺激DCs,设未经抗原负载的DCs作为对照组。观察细胞形态,收集培养9天的各组DCs,采用流式细胞仪检测DCs相关表型：CDl α、HLA-DR、CD40和CD83;ELISA法检测DCs上清中IL-12、IL-10的含量,混合淋巴细胞反应测定各组DCs体外刺激T细胞增殖的能力,ELISA法检测T细胞培养上清液中的IFN-γ含量,MTT法检测CTL对宫颈癌细胞系CaSki和肝癌细胞系HepG2的杀伤效应。结果：各组DCs均可诱导分化为一定数量的具有典型形态和表型的DCs。以HPV16E744₆2多肽联合Poly Ⅰ:C组最高。与未经抗原负载的DCs相比,其他各组抗原刺激的DCs数量、表型CD1α、HLA-DR、CD40和CD83表达率、分泌IL-12的能力、刺激同种异体T细胞增殖能力、分泌IFN-Y水平及激活的CTL对CaSki细胞杀伤力均较高,尤其是以HPV16E744-62多肽联合Poly Ⅰ:C组效果最明显,同时其分泌IL-10水平亦最低。而此类CTL对HepG2细胞无特异性杀伤效应。结论：负载HPV16E744-62多肽的脐血DCs体外可诱导特异性杀伤宫颈癌细胞的免疫效应,Poly Ⅰ:C作为免疫佐剂可增强抗肿瘤免疫反应。第二章Poly Ⅰ:C诱导负载HPV E744-62多肽脐血DCs体内抗宫颈癌的免疫效应研究。目的：观察Poly Ⅰ:C诱导负载HPV E744-62多肽的人脐血DCs对T细胞免疫重建荷人宫颈癌裸鼠的肿瘤生长的影响。方法：对裸鼠经尾静脉射入宫颈癌患者T淋巴细胞进行细胞免疫重建,24h后皮下注射人宫颈癌细胞系CaSki细胞,建立荷瘤裸鼠模型。荷瘤1周后根据注射疫苗不同将裸鼠分为5组：皮下注射各组DCs疫苗：Ctrl组（未负载抗原的脐血DCs）、E组（负载HPV16E744-62多肽的脐血DCs）、P组（单独PolyⅠ:C刺激脐血DCs）、E+P组（HPVE744-62多肽联合PolyⅠ:C诱导脐血DCs）,NS组（设皮下注射生理盐水作为空白对照组）。2周后再次接种疫苗强化1次。观察荷瘤裸鼠一般生物学特性、成瘤率、成瘤潜伏期、肿瘤大小及其生存期。荷瘤后35天检测裸鼠血中人CD3+、CD4+、CD8+T细胞比率及血中人IFN-γ水平。结果：裸鼠成瘤率为100%,成瘤潜伏期为5-8天。各组均可检测到人CD3+/CD4+、CD3-/CD8+T细胞,说明裸鼠T细胞免疫重建成功。与空白对照比较,其他各组裸鼠血中人CD3+/CD4+、CD3+/CD8+T细胞阳性率及IFN-γ水平均有升高,且有较高的抑瘤率和较长的生存时间,尤其以E+P组最为明显。结论：PolyⅠ:C诱导负载HPV16E744-62多肽的脐血DCs可以在人T细胞免疫重建荷人宫颈癌裸鼠模型体内诱发出抗肿瘤免疫反应。更多还原

【Abstract】 Part Ⅰ Study on the effect of anti-cervical cancer immunity induced by cord blood-derived dendritic cells loaded with HPV E744-62peptide and activated by Poly Ⅰ:C in vitro.Objective:To investigate the effect of anti-cervical cancer immunity induced by cord blood-derived dendritic cells loaded with HPV E744-62peptide and activated by Poly Ⅰ:C in vitro.Methods:Cord blood was collected from healthy term delivery mother.Cord blood mononuclear cells were isolated by density centrifugation. The adherent fraction was cultured in presence of rhGM-CSF/IL-4for7days. DCs were activated by HPV E744,62peptide, Poly Ⅰ:C, and HPV E744.62peptide combined with Poly I:C, respectively, with DCs without loading antigen as control group. The yield of mature DCs was calculated and the morphological changes of the cultured cells were observed by light microscopy. Surface expressions of CD1a, HLA-DR, CD40and CD83were analysized by flow cytometry analysis. IL-12and IL-10levels of DCs were detected using ELISA method. The ability of DCs to stimulate the proliferation of lymphocytes was determined by mixed lymphocyte reaction. IFN-γ level of T cells was detected using ELISA method. MTT assay was employed to test the inhibition rates of tumor specific cytotoxic T lymphocyte on human cervical cancer cell line CaSki and human hepatocarcinoma cell line HepG2.Results:A certain amount of DCs with typical morphology and phenotype were harvested from all groups. Antigen-loaded DCs had higher expression of CD1a, HLA-DR, CD40, and CD83, ability of inducing proliferation of allogeneic T cells, secretion of IL-12from DCs, secretion of IFN-γ from T cells, specific antitumor effect of CTL on CaSki cervical cancer cells, and lower secretion of IL-10, than that of DCs without loading antigen, especially in group of DCs activated by HPV16E744-62peptide combined with Poly Ⅰ:C, while CTL had no specific antitumor effect to HepG2.Conclusion:It can be induced the specific anti-cervical cancer immunity by cord blood-derived dendritic cells loaded with HPV E744-62peptide in vitro. Poly Ⅰ:C might act as a adjuvant to enhance antitumor effect. Part II Study on the effect of anti-cervical cancer immunity in vivo induced by cord blood-derived dendritic cells loaded with HPV E744-62peptide and activated by Poly I:C in vivo.Objective:To evaluate the effect of anti-cervical cancer immunity induced by cord blood-derived dendritic cells loaded with HPV E744.62peptide and activated by poly I:C in bearing human cervical cancer nude mice with immune reconstitution of human T cells.Methods:Cell immune reconstitution of nude mice was established by tail vein injection of T cells from cervical cancer patients. Then24hours later, those nude mice were injected subcutaneously with CaSki cells to establish bearing human cervical cancer model. And one week later, those nude mice were inoculated with different vaccines by subcutaneously injection, and randomly divided into5groups according to the type of vaccines:group Ctrl （vaccinated with cord blood-derived dendritic cells loaded without any antigen）, group E （vaccinated with cord blood-derived dendritic cells loaded with HPV E744-62peptide）, group P （vaccinated with cord blood-derived DCs activated by poly Ⅰ:C）, group E+P （vaccinated with cord blood-derived DCs activated by HPV E744-62peptide and poly I:C）, and group NS （vaccinated with NS, as control group）. The second injection was given two weeks interval. Biological behavior of nude mice, tumorigenecity, latent period, tumor volume, and the survival period of nude mice were observed. At35days after nude mice bearing human cervical cancer, the percentage of human CD3+, CD4+, CD8+T cells in mice blood and the level of IFN-γ in mice serum were detected.Results:The tumorigenecity was100%and the latent period is5to8days. Human CD3+/CD4+, CD3+/CD8+T cells could detected in all mice blood, indicating that T cell immune reconstitution of nude mice was established successfully. Compared with group NS, others groups could elevate the percentage of human CD3+/CD4+and CD3+/CD8+cells, increase IFN-γ production, inhibit the tumor growth, and prolong survival period of nude mice, especially in group E+P. Conclusion:Anti-tumor immune response could be induced by cord blood-derived dendritic cells loaded with HPV E744₆2peptide and activated by poly Ⅰ:C in bearing human cervical cancer nude mice model with immune reconstitution of human T cells.更多还原