Objective To investigate the anti-tumor efficiency of cytotoxic T-lymphocyte (CTL) induced by activated B lymphocyte after hepatocellular carcinoma (HCC) alpha fetoprotein (AFP) mRNA transfection.Methods B lymphocytes were fractionated,purified and activated by recombinant human soluble sCD40L.PGEM4Z/AFP/A64-EGFP plasmid was established in vitro,mixed with polymerase T7RNA,and then transcribed into AFP mRNA with Poly (A) sequence.B lymphocytes electrotransfected with AFP mRNA were in the experimental group,B lymphocytes electrotransfected with GAPDH mRNA were in the negative control group,and untreated B lymphocytes were in the blank control group.The expressions of antigen-presenting cell (APC)markers (CD19,CD20,CD21,CD40,CD80,CD83) and major histocompatibility complex (MHC) of the 3 groups were detected.B lymphocytes of the 3 groups were cultured with T lymphocytes at ratios of 1∶40,1 ∶ 20,1∶10 and 1∶5 to induce and ampify CTL,and then the absorbance values were detected to evaluate the proliferation ability of T lymphocytes.The killing activity of CTL was investigated with HCC cell line SMMC7721 as the target cells.All data were analyzed using the paired t test,one-way analysis of variance or Tamhane's T2 test.Results The expressions of CD19,CD20,CD21,CD40,CD80 and CD83 of the experimental group were 74 ± 11,78 ±8,80 ± 10,90 ± 11,82 ± 6,56 ± 5,which were significantly higher than 51 ± 5,60 ± 7,53 ± 5,73 ± 8,50 ± 5 and 49 ± 6 of the negative control group,and 46 ± 3,54 ± 5,41 ± 3,56 ± 5,52 ± 6 and 21 ± 4 of the blank control group (t =5.302,4.812,7.627,5.932,9.142,7.813; 11.581,7.036,13.592,12.873,9.235,14.619,P ＜ 0.01).The proliferation of CTL of the experimental group was significantly higher than that in the negative control group and blank control group (t =18.203,23.714,15.062,9.417 ; 16.833,19.392,13.871,6.592,P ＜0.01).When the T lymphocytes were mixed with the HCC cell line SMMC7721 at the ratios of 40∶ 1,20∶1and 10∶1,the killing rates of HCC cells by CTL of the exprimental group were 43％ 4％,32％ ± 4％ and 22％ ±3％,which were significantly higher than 15％ ± 5％,7％ ± 3％ and 6％ ±2％ of the negative control group,and 7％±3％,8％±3％ and 9％±4％ of the blank control group (t =9.141,13.272,11.901; 14.372,12.835,9.507,P ＜ 0.01).Conclusion Activated B lymphocytes after HCC AFP mRNA transfection may effectively induce CTL to kill HCC cells.%目的 研究肝癌细胞AFP mRNA转染活化的B淋巴细胞诱导细胞毒性T淋巴细胞的抗肿瘤作用.方法 分离、纯化B淋巴细胞,重组人可溶性CIM0配体(sCD40L)活化人外周血B淋巴细胞；构建PGEM4Z/AFP/A64-EGFP质粒,加入T7RNA聚合酶,转录具有PolyA尾端的AFP mRNA；将提取的AFPmRNA电转染B淋巴细胞作为实验组,选取GAPDH mRNA转染者作为阴性对照组,未转染的B淋巴细胞作为空白对照组.检测各组B淋巴细胞表面抗原提呈细胞标记分子(CD19、CD20、CD21、CD40、CD80、CD83)及主要组织相容性抗原的表达情况；将3组B淋巴细胞与T淋巴细胞分别按1∶40,1∶20,1∶10和1∶5的比例混合培养、诱导、扩增抗原激活T淋巴细胞并测定吸光度值以检测T淋巴细胞增殖能力；以T淋巴细胞作为效应细胞,肝癌细胞系SMMC7721为靶细胞,检测T淋巴细胞对肝癌细胞的杀伤活性.两两比较采用配对t检验,多组比较采用单因素方差分析,方差不齐时采用Tamhane's T2检验.结果 实验组B淋巴细胞表面标志分子CD19、CD20、CD21、CD40、CD80和CD83的表达水平分别为74±11、78 ±8、80±10、90±11、82±6、56±5,显著高于阴性对照组的51±5、60±7、53±5、73±8、50±5、49±6,以及空白对照组的46±3、54±5、41±3、56±5、52±6、21 ±4(t =5.302、4.812、7.627、5.932、9.142、7.813,11.581、7.036、13.592、12.873、9.235、14.619,P＜0.01).实验组吸光度值显著高于阴性对照组和空白对照组(t=18.203、23.714、15.062、9.417,16.833、19.392、13.871、6.592,P＜0.01).当T淋巴细胞与肝癌细胞系SMMC7721按照40∶1、20∶1和10∶1的比例混合后,实验组B淋巴细胞诱导产生的T淋巴细胞对肝癌细胞的杀伤率分别为43％±4％、32％±4％和22％±3％,显著高于阴性对照组的15％±5％、7％±3％和6％±2％,以及空白对照组的7％±3％、8％±3％和9％±4％(t=9.141、13.272、11.901,14.372、12.835、9.507,P＜0.01).结论 肝癌细胞AFP mRNA转染的B淋巴细胞可有效诱导T淋巴细胞杀伤肝癌细胞.
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