肝癌细胞甲胎蛋白mRNA转染活化的B淋巴细胞诱导细胞毒性T淋巴细胞的抗肿瘤效应

Anti tumor efficiency of cytotoxic T lymphocyte induced by activated B lymphocyte after hepatocellular carcinoma alpha fetoprotein mRNA transfection

    摘要
  • 摘要: 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AFP mRNA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∶〖KG-*3/5〗40，1∶〖KG-*3/5〗20，1∶〖KG-*3/5〗10和1∶〖KG-*3/5〗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〖WTBX〗t〖WTBZ〗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。

     

    Abstract: 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 〖WTBX〗in vitro,〖WTBZ〗 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∶ ±4% of the blank control group (〖WTBX〗t=9.141, 13.272, 11.901; 14.372, 12.835, 9.507, P〖WTBZ〗<0.01).
    Conclusion Activated B lymphocytes after HCC AFP mRNA transfection may effectively induce CTL to kill HCC cells.

     

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