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目的:比较AFP抗原以不同方式负载DCs对后者生物学功能的影响及体外刺激CTL对肝癌Hep G2细胞的杀伤作用。方法:分离健康供者外周血单个核细胞培养DC,分别用(A)人工合成AFP抗原肽、(B)Hep G2细胞裂解物、(C)Hep G2细胞分泌的外泌体(tumor exosome,T-exo)、(D)AFP抗原的重组腺相关病毒(recombinant adeno-associated virus expressingα-fetoprotein antigen,r AAV/AFP)致敏DC前体细胞及(E)未负载抗原的DC作为对照,经GM-CSF、IL-4及LPS联合诱导DCs分化成熟。流式细胞术检测r AAV/AFP病毒感染效率、各组DCs表型及其剌激初始T细胞的增殖效应,7-ADD/CFSE双染法流式细胞术检测各组DCs诱导CTL对AFP阳性Hep G2细胞的杀伤作用。结果:几种抗原负载方式均可诱导DCs成熟、促进CTL增殖及特异性识别并杀伤Hep G2细胞,但r AAV/AFP感染DCs后,其CD83、CD86、ICAM-1、CD58、CD40分子表达水平明显高于对照组(P<0.05),r AAV/AFP+DC组和Hep G2-Texo+DC组对Hep G2细胞的杀伤作用均分别显著优于其他抗原负载(AFP/peptide+DC、Hep G2 lysate+DC)组[(44.92±4.12)%vs(28.42±3.29)%、(24.28±1.79)%;(41.40±2.87)%vs(28.42±3.29)%、(24.28±1.79)%;均P<0.05)]。结论:r AAV/AFP高效感染DCs后能有效刺激初始T细胞增殖,并增强CTL对AFP阳性靶细胞的杀伤活性,而负载Texo的DCs也能诱导显著的抗肝癌效应,上述结果为基于DCs的肝癌疫苗的研发提供新的思路。
OBJECTIVE: To compare the effects of AFP antigen loaded DCs on the biological functions of the latter and the cytotoxicity of CTL on Hep G2 cells in vitro. Methods: DCs from peripheral blood mononuclear cells of healthy donors were isolated and cultured. AFP antigen (A), AFP peptide (B), lysate of Hep G2 cells, (C) exosome secreted by Hep G2 cells -exo), (D) DC precursor cells sensitized with recombinant adeno-associated virus positive α-fetoprotein antigen (AAV / AFP) and (E) antigen-free DCs were used as controls, -CSF, IL-4 and LPS induced differentiation and maturation of DCs. Flow cytometry was used to detect the infection efficiency of rAAV / AFP virus, the phenotype of DCs in each group and the proliferative effect of initial T cells stimulated by 7-ADD / CFSE double staining. Hep G2 cell killing effect. Results: Several antigen loading methods could induce the maturation of DCs, promote the proliferation of CTLs and specifically recognize and kill Hep G2 cells. However, the expression of CD83, CD86, ICAM-1, CD58 and CD40 in DCs infected with AAV / AFP (P <0.05). The killing effect of rAAV / AFP + DC group and Hep G2-Texo + DC group on Hep G2 cells were significantly better than that of the other antigens (AFP / peptide + DC, Hep G2 (44.22 ± 3.29)%, (24.28 ± 1.79)%; (41.40 ± 2.87)% vs (28.42 ± 3.29)%, (24.28 ± 1.79)%, respectively; P <0.05)]. CONCLUSION: r AAV / AFP can effectively stimulate the proliferation of naive T cells and enhance the cytotoxic activity of CTL on AFP-positive target cells, while DCs loaded with Texo can induce significant anti-hepatocellular carcinoma effect. The above results are based on DCs HCC vaccine research and development provide new ideas.