【作者】 黄裕；

【导师】 唐良萏；

【作者基本信息】 重庆医科大学 ， 妇产科学， 2004， 硕士

【摘要】 目的：通过研究宫颈癌患者外周血单核细胞来源的树突状细胞(dendritic cells, DCs)体外诱导、扩增方法以及生物学特性,进一步在体外研究DC在宫颈癌免疫中的作用，为DC疫苗将来应用于临床提供理论依据。方法：1．宫颈癌患者外周血树突状细胞的体外扩增和鉴定 从宫颈癌患者外周血中分离出单核细胞,在rhGM-CSF、rhIL-4和TNF-α作用下培养7-10天,通过形态学观察、FACS分析(检测DC表面特异性标记CD1α及DC功能相关分子HLA-DR、CD80的表达)和混合淋巴细胞反应,从形态学和结构特征、表面特异性标记及生物学活性等方面鉴定所获细胞是典型的、有活力的树突状细胞。2．负载抗原树突状细胞诱导T细胞增殖效应 制备宫颈癌患者癌组织肿瘤细胞抗原,取同一患者外周血,按前述方法诱导扩增出DC。同时以细胞因子IL-2刺激外周血分离来的异体淋巴细胞,得到T细胞。将抗原致敏DC后与T细胞共同培养,MTT法检测不同时间段抗原负载的DC刺激同源异体淋巴细胞的增殖效应。3．宫颈癌患者外周血树突状细胞抗宫颈癌免疫 负载抗原DC体<WP=8>外激活初始T淋巴细胞,产生肿瘤特异性CTL,MTT法检测比较负载抗原DC与未负载抗原DC诱导的CTL对Hela细胞和SKOV3细胞不同的杀伤效应,同时测定单纯抗原刺激下的T淋巴细胞对以上两种肿瘤细胞的非特异性杀伤作用。结果：1．宫颈癌患者外周血单核细胞来源DC的鉴定和生物学特性 联合应用细胞因子GM-CSF、IL-4和TNF-α,刺激宫颈癌患者外周血分离而来的单核细胞,在37℃、5%CO2条件下,培养7天即可诱导出典型的DC。经光学显微镜和透射电镜观察显示,所诱导的细胞具有典型DC的形态学和结构特征；流式细胞仪分析证实所获细胞表达DC特异性表面标记CD1α,同时表达功能相关分子HLA-DR、CD80；混合淋巴细胞反应的检测表明,该细胞具有较强的刺激同种T淋巴细胞增殖的能力,具有典型DC的基本生物学特性。2．负载抗原DC诱导T细胞增殖效应 分别测定DC与T淋巴细胞共同培养24h、48h、72h、96h后的吸光度,计算刺激指数,可见负载肿瘤细胞抗原的DC能有效促进T淋巴细胞的增殖生长,与对照T淋巴细胞组和DC组比较,存在显著性差异(P<0.05)。3．宫颈癌患者外周血DC抗宫颈癌免疫 被宫颈癌细胞抗原致敏的DC,激活同源异体T淋巴细胞,生成肿瘤抗原特异性CTL,对宫颈癌Hela细胞产生高效而特异的杀伤作用(56.410%),对卵巢癌SKOV3细胞仅有一定的杀伤作用(24.901%)；而未致敏DC激活的CTL对以上两种<WP=9>肿瘤细胞只有很低的杀伤作用,分别为10.256%和20.151%。单纯肿瘤抗原刺激下的T淋巴细胞,对Hela和SKOV3细胞产生的只是非特异性杀伤作用,其效率仅为1.865%及15.613%。结论：1．宫颈癌患者外周血单核细胞在细胞因子GM-CSF、IL-4和TNF-α的联合刺激诱导下,可以分化为典型的树突状细胞,具有典型形态学和结构特点,表达特异性表面分子,能够刺激T淋巴细胞的增殖,在抗肿瘤免疫中具有重要作用。2．源自宫颈癌患者外周血的DC,负载宫颈癌抗原后,激活产生的CTL能选择性特异性杀伤宫颈癌Hela细胞,而对卵巢癌SKOV3细胞的杀伤率较低,说明DC具有很强的抗原递呈功能。3．来自宫颈癌患者的DC诱导激活得到抗原特异性CTL,在体外能产生较强的特异性免疫反应,为DC疫苗治疗宫颈癌患者应用于临床的有效性和可行性提供了理论依据。更多还原

【Abstract】 Objective To study the methods of induction, biological character of dendritic cells(DCs) which derived from the peripheral blood of patients with cervical cancer, and further to study the anti-tumor effect activated by DCs in vitro. It will provide the experimental foundation for clinical immunotherapy of DCs.Methods1．The induction and biological character of DCs derived from human monocytes Monocytes isolated from the peripheral blood of patients with cervical cancer are cultured at 37℃ in humidified 5% CO2 in air in medium with rhGM-CSF,rhIL-4 and TNF-α for 7-10 days. DCs from human monocytes are analyzed on morphology, cellar structure under optics and electronic microscope; and then phenotype of DCs are detected <WP=11>by FACS analysis including specific CD1α, and function-related molecules HLA-DR, CD80. Stronger allo-MLR is one of DCs’ characteristics, it is performed by using MTT method to observe the ability of DCs stimulating lymphocytes proliferation.2．The ability of DCs loaded with tumor cell antigen stimulating lymphocytes proliferation In order to study the ability of DCs which loaded with antigen stimulating lymphocytes proliferation, monocytes isolated from the peripheral blood of patients with cervical cancer are cultured in medium with rhGM-CSF, rhIL-4 and TNF-α. At the same time, got the tumor cell antigen from cervical tumor tissue of the same patient. Gain T lymphocytes by using cytokine IL-2 to stimulate antologous lymphocytes. Then cultured the tumor antigen, DCs and T lymphocytes all together, the proliferation of T lymphocytes in the different days are detected respectively by using MTT method.3．DCs and anti-cervical cancer immunity Autologous lymphocytes proliferation activated by induced DCs loaded with tumor cell antigen is detected by using MTT method to observe the presenting role of DCs. To detect the specific cytotoxic effect activated by DCs on Hela and SKOV3 cells, CTL activity is observed by counting the killed Hela and SKOV3 cells in vitro. <WP=12>ResultThe identification of DCs and its biological character Typical DCs can be induced when monocytes are cultured in medium with rhM-CSF,rhIL-4 and TNF-α for 7 days. It shows typical morphology and cellular structure of dendritic cell under optics and electronic microscope. It highly express the specific cellular phenotype CD1α and other APC-related functional molecules CD80 and HLA-DR. The detection of allo-MLR shows that DCs can stimulate strong T lymphocytes proliferation.The ability of DCs loaded with tumor cell antigen stimulating lymphocytes proliferation The optical densities of 24h,48h,72h,96h are detected respectively, the stimulating indexs show that DCs loaded with tumor cell antigen can stimulating strong antologous lymphocyte proliferation, it is more effective than control’s(P<0.05).DCs anti-cervical cancer immunity DCs loaded with tumor cell antigen can generate strong specific CTL that kill the target cells—Hela cells(56.410%), SKOV3 cells(24.901%), while CTLs induced by DCs which cultured without antigen kill the target cells weakly(10.256% and 20.151% respectively).T lymphocytes stimulated by tumor antigen only generate non-specific immunoreaction, kill rates are 1.865% and 15.613%. <WP=13>ConclusionCulturing human monocytes in medium with rhGM-CSF、rhIL-4 and TNF-α can induce typical DCs. It shows potent antigen presenting role to exogenous antigen.DCs from the peripheral blood of patients with cervical cancer pulsed by tumor cell antigen not only strongly stimulate autologous lymphocytes proliferation, but also play an important role in anti-tumor immunity.DCs loaded with tumor cell antigen derived from patient monocytes can generate specific CTLs that have potent cytotoxic effect on cervical cancer Hela target cells. According to yhe experimental result, we put forward the theory on the treatment of cervical cancer by the use of DCs vaccine.更多还原