【作者】 赵飞兰；

【导师】 罗国容；

【作者基本信息】 广西医科大学 ， 人体解剖与组织胚胎学， 2010， 博士

【摘要】 目的：(1)了解动力素(kinectin)作为肝癌相关抗原在肝癌细胞株BEL-7404中mRNA的表达情况,评价kinectin作为肝癌抗原分子标记以及肝癌免疫治疗特异性靶位的可能性。(2)运用体外基因工程的方法表达、分离纯化kinectin-MBP重组融合蛋白、SMP-30-MBP重组融合蛋白(SMP-30为课题组前期已深入研究的另一肝癌相关抗原,考虑实验室有库存蛋白,本实验顺带比较检测其杀伤效应),探讨kinectin、SMP-30以融合蛋白形式致敏DC后,能否刺激自体T淋巴细胞转化为CTL以及CTL对BEL-7404肝癌细胞有无杀伤作用。(3)免疫组化的方法检测kinectin融合蛋白致敏的DC刺激T细胞亚群活化情况,初步探讨以DC为基础的kinectin瘤苗在肝癌的临床应用价值及其生物学机制。方法：(1)运用RT-PCR技术检测国产肝癌细胞株BEL-7404 kinectin基因(D2选择剪接区)mRNA的表达,并与肝癌组织、相应癌旁肝组织、正常肝组织比较,了解其表达情况。(2)运用体外蛋白重组技术表达、分离纯化kinectin-MBP融合蛋白、SMP-30-MBP融合蛋白,并与正常人外周血树突状细胞(Dendritic cell,DC)孵育；ELISA检测kinectin-MBP致敏组DC培养上清液IL-12、IFN-γ分泌量；尼龙毛柱分离自体T淋巴细胞后,以致敏DC诱导自体T淋巴细胞为细胞毒性T淋巴细胞(CTL),MTT法检测各组T淋巴细胞增殖情况,LDH 4 h释放法检测CTL对BEL-7404肝癌细胞的细胞毒作用；(3)SupervisionTM,HRP试剂盒免疫组化法检测kinectin-MBP孵育DC组刺激增殖的T细胞中CD4+、CD8+T细胞亚群活化情况,初步判断以DC为基础的kinectin瘤苗在肝细胞癌的生物学作用及机制。结果：(1)kinectin(D2选择剪接区)mRNA在BEL-7404肝癌细胞株及两例肝癌组织中呈较强阳性表达,相应癌旁肝组织则呈弱阳性,一例正常肝组织未见表达。(2)运用体外重组蛋白技术成功表达、分离纯化出kinectin-MBP, SMP-30-MBP, SDS-PAGE电泳检测分子量与预期分子量相吻合。(3)DC培养、T淋巴细胞增殖、杀伤实验结果：PBMC经rhGM-CSF、rhIL-4诱导培养1周后,出现典型的DC形态；结合FCM细胞表型检测鉴定为DC；DC经Kinectin-MBP致敏后,IL-12、IFN-γ分泌量明显高于MBP孵育组和未致敏DC组(P均＜0.05)；经kinectin-MBP、SMP-30-MBP融合蛋白致敏的DC刺激自体T细胞增殖的能力显著高于MBP孵育DC及非致敏DC组(P均＜0.05)；刺激细胞(DC)占效应细胞(T细胞)比例越大,则增殖能力越强；经kinectin-MBP、SMP-30-MBP融合蛋白致敏的DC诱导的CTL对BEL-7404肝癌细胞均具有较高的杀伤率,在效靶比为25：1时杀伤率达最高峰(分别为65.00±1.47%；64.40±1.40%)。(4)免疫组化检测结果：Kinectin-MBP蛋白致敏DC刺激的T细胞组CD8+T细胞明显高于MBP孵育DC刺激的T细胞组、非致敏DC刺激组及普通培养T细胞组(P均＜0.05)；各组CD4值之间、CD4／CD8值之间比较,差异无统计学意义(P值均＞0.05)。结论：kinectin基因(D2选择剪接区)在BEL-7404肝癌细胞株中表达呈较强阳性,具有作为肝癌诊断的辅助分子标记、肝癌免疫治疗特异性靶位的潜在价值。体外蛋白重组技术可成功表达、分离纯化出Kinectin-MBP融合蛋白；经Kinectin-MBP、SMP-30-MBP融合蛋白致敏的DC能有效刺激自体T淋巴细胞的增殖,且可诱导T细胞转化为CTL,后者对BEL-7404肝癌细胞具有较强的细胞毒活性。考虑课到SMP-30的表达特点以及课题组前期实验结果,SMP-30在正常肝组织亦存在阳性表达,故SMP-30能否作为肝癌疫苗尚有待深入研究；但是,结合我们的实验结果,目的蛋白Kinectin-MBP作为肝癌相关抗原可通过致敏DC发挥比较明显的抗肿瘤功能,有望开发为肝癌生物治疗的新疫苗。更多还原

【Abstract】 Objective:(1) To investigate the mRNA expression of kinectin in hepatocellular carcinoma(HCC) cell line BEL-7404, and evaluate the possibility of immunotherapy as a molecular marker of diagnosis, as well as a liver-specific target for treatment. (2) Kinectin-MBP,SMP-30-MBP recombinant fusion protein were expressed and purified by using genetic engineering methods in vitro respectively. Investigate the ability of pulsed DC with hepatocellular carcinoma-associated antigen kinectin-MBP, SMP-30-MBP to stimulate autologous T lymphocytes into CTL, and also detect its cytotoxicity on BEL-7404. (3) preliminarily explore the clinicial application of kinectin in HCC and its role of its biological mechanism.Methods:(1) With reverse transcripts polymerase chain reaction(RT-PCR), the kienctin mRNA level of BEL-7404 HCC cell Line, hepatocellular carcinoma tissue, corresponding adjacent non-HCC tissue and normal liver tissue were analyzed. (2) Kinectin-MBP, SMP-30-MBP fusion protein were expressed and purified by means of recombinant protein technology in vitro,and were incubated with human peripheral blood dendritic cells (Dendritic cell,DC),then to induce cytotoxic T lymphocyte (CTL) with pulsed DC.Then cytotoxicity of CTL against BEL-7404 hepatoma cell was assayed by LDH 4 hours release.(3) Detect the proliferation of T cells CD4+, CD8+T cell subset with SupervisionTM, HRP immunohistochemistry, and to determine the role of kinectin in HCC.Results:(1)The mRNA of Kinectin have a strong expression in BEL-7404 cell line and HCC tissue,low expression in corresponding adjacent non-HCC tissue and normal liver tissue.Successfully expressed and purified kinectin-MBP, SMP-30-MBP by used of recombinant protein technology in vitro, the molecular weight are consistent with the expected molecular weight by electrophoresis.(2) The results of DC culture, T lymphocyte proliferation, CTL’s cytotoxicity toward target cell BEL-7404:PBMCs presented typical morphologic characteristics of DCs after induced by rhGM-CSF and rhIL-4 for 7 days.Comparing with the other two groups, the level of CIL-12 and CIFN-yraised significantly in culture medium supernatants of kinectin-MBP treated group (p<0.05); The proliferation capacity of autologous T cell cultured with the kinectin-MBP, SMP-30-MBP fusion protein-pulsed DC were significantly higher than MBP-pulsed, non-treated cultured DC group (p<0.05). The greater of the ratio of stimulated cells (DC) accounts of effector cells (T cells), the stronger the proliferation ability; higher anti-tumor activity were found by DC-induced The CTL of the BEL-7404 hepatoma cells in kinectin, SMP-30 fusion protein-pulsed groups, the highest killing rate was found when the ratio of effector:target was 25:1 (65.00±1.47%; 64.40±1.40% respectively).(3) The results of immunohistochemistry:the number of CD8 positive T-cell increased remarkably in kinectin-MBP-DC-T cell group, compared with the MBP-DC-T cell group, DC-T cells group and normal cultured T cells(p<0.05). Furthermore, there are no significant difference of the number of CD4 positive T-cells, CD4/CD8 among of these four group (P> 0.05).Conclusion:The mRNA level of kinectin (D2 alternative splicing area) showed strong positive expression in the hepatoma cell lines BEL-7404 and HCC tissues. And it can be used as molecular markers for early diagnosis and possible targets for immunotherapy of human HCC. The DCs be pulsed with kinectin, SMP-30 fusion protein can stimulate T lymphocyte proliferation effectively; the pulsed DC can also induce CTL successfully, and have strong cytotoxic activity toward the BEL-7404.The DCs be pulsed of kinectin, fusion protein have a marked anti-tumor function, they are expected to develop the new vaccines for HCC biological therapy.更多还原