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目的:通过体外杀伤试验,研究人肝癌特异性甲胎蛋白hAFP(137-145)短肽段修饰树突细胞(DC)后诱导的细胞毒性T淋巴细胞(CTL)对人肝癌细胞的特异杀伤效应,并与完整hAFP蛋白的杀伤效应相比较。方法:选取健康人外周血单个核细胞,体外诱导成熟DC;构建hAFP表达载体,在BL21细菌中异丙基硫代半乳糖苷(IPTG)诱导原核表达后纯化,与合成的hAFP(137-145)短肽段分别修饰诱导DC细胞,体外刺激细胞毒性T细胞(CTL),通过流式细胞仪法和细胞毒性检测试剂盒法检测修饰后的DC所诱导的CTL在体外对肝癌细胞株的杀伤作用;构建荷瘤裸鼠人HepG2肝癌模型,检测该肽段修饰DC后诱导的CTL细胞在裸鼠过继体内实验中对人肝癌转移瘤的杀伤作用。结果:成功构建了pET-hAFP表达载体,原核表达并纯化后用Western blot检测到hAFP蛋白的表达;hAFP蛋白和hAFP(137-145)短肽段均能在体外修饰和诱导DC细胞的增殖和成熟;hAFP蛋白和hAFP(137-145)短肽段修饰DC体外均能诱导特异性CTL,并通过体外实验验证了其对于肝癌细胞的杀伤效应,其中hAFP(137-145)短肽段诱导CTL的能力和所诱导CTL对于肝癌细胞的杀伤能力相对全长hAFP蛋白较强;动物实验证实,该hAFP(137-145)肽段修饰DC后诱导的CTL细胞在荷瘤裸鼠过继体内实验中对人肝癌转移瘤有明显的杀伤作用,杀伤效率优于完整hAFP蛋白。结论:hAFP蛋白和hAFP(137-145)短肽段修饰DC均具有单独体外诱导特异性CTL的作用,体外实验和动物实验均证实此CTL有特异地杀伤HegG2肝癌细胞的作用;hAFP(137-145)短肽段诱导CTL效率和杀伤肿瘤细胞效率均优于完整hAFP蛋白。
OBJECTIVE: To study the specific killing effect of cytotoxic T lymphocytes (CTLs) induced by dendritic cells (DCs) modified by human hepatoma-specific alpha-fetoprotein hAFP (137-145) on human hepatocellular carcinoma cells in vitro , Compared with the killing effect of intact hAFP protein. Methods: Human peripheral blood mononuclear cells (PBMCs) were selected to induce mature DCs in vitro. HAFP expression vector was constructed. The prokaryotic expression of isopropylthiogalactopyranoside (IPTG) in BL21 was purified and compared with the synthetic hAFP (137-145 ) Short peptide fragments were modified to induce DC cells stimulated in vitro cytotoxic T lymphocytes (CTL), by flow cytometry and cytotoxicity detection kit method to detect modified DC-induced CTL in vitro killing of liver cancer cell lines To construct the HepG2 hepatocellular carcinoma model in nude mice and detect the killing effect of CTL cells induced by the peptide modified DC on human hepatoma metastasis in nude mice. Results: The pET-hAFP expression vector was successfully constructed and expressed in E. coli. The expression of hAFP protein was detected by Western blot. Both hAFP and hAFP (137-145) short peptides could modify and induce the proliferation of DCs in vitro and in vitro. Mature; hAFP protein and hAFP (137-145) short peptide modified DC can induce specific CTL in vitro and its killing effect on liver cancer cells was verified by in vitro experiments, in which hAFP (137-145) short peptide induced CTL And the ability of induced CTL to kill hepatoma cells is stronger than the full-length hAFP protein. Animal experiments confirmed that the CTL cells induced by the hAFP (137-145) peptide modification of DC in adoptive in vivo experiments in nude mice Human hepatoma metastases have obvious killing effect, killing efficiency is better than the complete hAFP protein. CONCLUSION: Both hAFP and hAFP (137-145) modified DCs can induce specific CTL in vitro. Both in vitro and in vivo experiments demonstrated that CTL could specifically kill HegG2 hepatocarcinoma cells. HAFP (137- 145) short peptides induced CTL efficiency and killing tumor cells were better than the efficiency of the complete hAFP protein.