【作者】 万亚峰；

【导师】 易继林；

【作者基本信息】 华中科技大学 ， 外科学， 2010， 博士

【摘要】 目的①本研究拟构建AFP真核表达载体,体外转染CD40L活化的B淋巴细胞,制备AFP-B细胞瘤苗。诱导针对AFP特异性CTL免疫作用。初步探讨肿瘤非特异性抗原AFP作为靶点用于肝癌免疫治疗的可行性。②利用肝癌细胞株Hepal-6总RNA转染CD40L活化的小鼠B细胞,探讨其诱导特异性细胞毒性T淋巴细胞(CTL)抗肿瘤免疫作用。方法①利用密度梯度离心法从小鼠脾脏淋巴细胞悬液分离、纯化T、B淋巴细胞,并在CD40L和rmIL-4联合作用下活化B细胞。并利用流式细胞仪分析B淋巴细胞表面标志及主要组织相容性抗原的表达情况。②利用RT-PCR克隆出目的基因AFPcDNA,定向插入pGEM4Z/A64质粒,从而制备成pGEM4Z/AFP/A64质粒。并经酶切,电泳鉴定。利用SpeI限制性内切酶使pGEM4Z/AFP/A64质粒进行线性化,在T7RNA聚合酶作用下,体外转录出AFPmRNA。③随后将AFPmRNA转染入CD40L活化的B细胞,观察转染前后B细胞的表型变化。同时将转染的B淋巴细胞刺激T淋巴细胞诱导、扩增CTL,检测CTL杀伤活性及分泌IFN-γ的水平。④提取肝癌细胞Hepal-6总RNA,然后转染活化B细胞,检测转染后各组B细胞表面APC标记及主要组织相容性抗原的表达情况。转染的B淋巴细胞刺激T淋巴细胞,诱导、扩增CTL,检测CTL杀伤活性及分泌IFN-γ的水平。结果①CD40L和rmIL-4活化的B细胞表面分子CD40、CD86、CD80、H-2Kb及I-Ab表达流式检测阳性细胞数明显高于对照组,两组相比较p<0.05。活化的B细胞刺激T细胞的所测得光密度值高于对照组,两者比较p<0.05。②成功构建AFP真核表达载体,酶切、电泳鉴定可见一清晰条带位于1800bp左右,与目的基因1818bp相符。利用Western blot方法可检测出有70kD左右的特异性蛋白条带,与目的基因蛋白分子量70kD相符。③转染AFPmRNA的B细胞表面组织相容性分子及共刺激分子分别高于未转染组,两者比较(P<0.01)。转染活化的B细胞刺激T细胞增殖能力高于未转染组(P<0.01)。转染的B细胞诱导的特异性杀伤率高于未转染组,两者比较(P<0.01)。转染的B细胞刺激的T细胞产生的CTL分泌INF-γ的水平高于对照组(P<0.01)。④提取的Hepal-6总RNA经琼脂糖凝胶电泳显示有28S、18S和5S三条带。转染总RNA的B细胞其表面组织相容性分子及共刺激分子表达高于小鼠肝细胞RNA组,两者相比较p<0.05；也明显高于脂质体对照组及空白对照组,与其相比较p<0.01。转染的B淋巴细胞刺激T细胞所测OD值高于对照组,与其比较有统计学意义。转染的B淋巴细胞诱导的特异性杀伤率高于对照组,两组比较(p<0.05)。转染的B细胞刺激产生CTL分泌INF-γ的水平显著高于对照组,两组相比较p<0.05。结论①AFPmRNA转染活化的B淋巴细胞在体外能诱导产生针对AFP特异性CTL反应。②以肝癌细胞Hepal-6总RNA转染的B淋巴细胞,也可有效诱导CTL杀伤肝癌细胞。为肝癌治疗提供了两种可能的新思路。更多还原

【Abstract】 ObjectivesFirst, to construct AFP eukaryotic expression vector, then transcribe AFPmRNA and transfect it into B lymphocytes activated by CD40L in vitro for preparation of AFP-B cell vaccine. And induce specific CTL immunity against AFP.then discuss preliminary the feasibility of immunotherapy of hepatocellular carcinoma as a target for AFP.Second, Total RNA of Hepal-6,a hepatocelluar carcinoma cell line,were introduced into mouse B lymphocytes activated by CD40L,then study the antitumor effectivity of B lymphocytes in the context of inducing cytotoxic T lymphocytes (CTLs).MethodsFirst, T and B lymphocytes were collected, isolated and purfied from mouse spleen lymphocytes by density gradient centrifugation method. B cells were initially activated by CD40L and rmIL-4. The expression of B cell markers and major histocomability complex (MHC) on cell surface were detected by FCM.Second, the goal gene AFPcDNA was cloned by RT-PCR. Insert cloned AFPcDNA into the plasmid pGEM4Z/A64 in the direction to create the plasmid pGEM4Z/AFP/A64. It was determined by enzyme digestion and electrophoretic analysis. Linearization of pGEM4Z/AFP/A64 with SpeI, then followed by in vitro transcription AFPmRNA with T7 RNA polymerase.Third, AFPmRNA was transfected into activated B lymphocytes. The expression of B cell surface markers was determined.CTL was obtained by stimulating T lymphocytes with transfected B lymphocytesd, and then the killing activity of CTL and the IFN-r secretion were quantified. Last, Total RNA was extracted from Hepal-6, and then it was transfected into B lymphocytes. The expressions of antigen presenting cell markers and major histocomability complex on cell surface were detected, and then the killing activity of CTL and the IFN-r secretion were quantified.ResultsFirst, The MHC and co-stimulating molecules(CD40,CD86,CD80,H-2Kb,I-Ab) expression of B lymphocytes activated by CD40L and rmIL-4 were higher than unactivated B cells(p<0.05). OD value of B cell activated stimulator T cell was higher than unactivated B cell group (p<0.05).Second, the vector pGEM4Z/AFP/A64 was constructed, and it was determined by enzyme digestion and electrophoretic analysis that a clear band at 1800bp position was showed, consistenting with the target gene 1818bp. A 70.0kD specific protein band was detected by Western blot; consistenting with molecular weight of the target protein.Third, the MHC and co-stimulating molecules expression of B lymphocytes transfected by AFPmRNA were higher than untransfected B cells, comparison between the two groups (p<0.01). OD value of transfected B cell stimulator T cell was higher than untransfected B cell group(p<0.05).The killing activity and IFN-y secretion of CTL after stimulation of AFPmRNA-transfected B lymphocyte was more significant than control group, comparison between the two groups (p<0.01).Last, Electrophoresis of the whole RNA extracted from Hepal-6 cells showed that 28s,18s and 5s bands. The MHC and co-stimulating molecules expression increased significantly higher than other groups after total RNA transfected, comparison with the concrol groups p<0.05. OD value of B lymphocyte transfected by total RNA stimulator T cell was higher than control groups.The killing activity and IFN-y secretion of CTL after stimulation of RNA-transfected B lymphocyte was more significant than control group (p<0.05). ConclusionFirst, B lymphocytes loaded with AFPmRNA can induce AFP-specific CTL in vitro.Second, HCC RNA-transfected B lymphocyte is capable of inducing anti-tumor effects through CTL response. It may present two potential pathways for future HCC treatment.更多还原