K-ras突变多肽负载树突状细胞诱导抗胰腺癌特异性免疫

目的 探讨K-ras(12-Val)突变多肽负载树突状细胞(DC),诱导胰腺癌特异性免疫的可行性.方法 采用未成熟Dc体外负载K-ras多肽(YKLVVVGAV)后诱导成熟,抗K-ras(12-Val)单抗检测突变抗原位点表达后与T淋巴细胞混合扩增特异性细胞毒性T淋巴细胞(CTL),MTT法测定CTL对胰腺癌细胞和正常细胞的免疫杀伤活性.建市裸鼠胰腺癌移植瘤模型,分别予K-ras特异性CTL肿瘤瘤内及尾静脉注射,观察不同治疗途径对肿瘤生长的抑制效果.结果 负载K-ras(12-Val)多肽的DC成熟后表达突变抗原位点并有效地刺激自体CD8+:(44.8±2.1)%]和同种异体T细胞活化;诱导的特异性CTL对胰腺癌细胞杀伤率按效靶比(10:1、20:1、50:1)分别为(21.2±1.9)%、(32.4±2.1)%、(45.7±5.3)%,杀伤效率均显著高于非特异性激活的T细胞治疗组(均P<0.05),对正常组织无损伤(均P>0.05).裸鼠K-ras特异性CTL治疗后8 d,瘤内治疗组肿瘤体积(68±13)mm3即明显低于对照组(87±14)mm3及IL-2非特异性治疗组(79±19)mm3(均P<0.05),K-ras特异性CTL治疗可以显著提高裸鼠的生存率(P<0.05).免疫组化染色证实K-ras特异性CTL具有体内迁移到肿瘤病灶的能力.结论 利用K-ras(12-Val)突变多肽体外负载树突状细胞,能诱导有效的抗胰腺癌特异性免疫反应.

Specific immune against pancreatic cancer induced by dendritic cells pulsed with mutant K-ras peptide

Objective To investigate whether dendritic cells(DCs)pulsed with mutant K-ras peptide(12-Val)can induce efficiently specific anti-tumor immune response aganist pancreatic cancer.Methods Immature DCs were isolated from the pefipheral blood of a volunteer and were pulsed with synthesized mutant K-ras peptide(YKLVVVGAV).When the DCs were matured the expression rate of Kras antigen epitope on the DC's surface was detected by monoantibody(K-ras-12-Val).Autogeneic and homologous T cells were mixed with the mutant K-ras peptide-pulsed DCs.Human pancreatic cancer cells of the line Patu8988 were mixed with cytotoxic T lymphocytes(CTLs)cultured for 5 days.and the killing eriects of the CTLs on the ceils was assessed by MTY method.Patu8988 cells were iniected subcutaneously into nude mice.cancer cells were obtained from the tumor masses and injected subcutaneously into other nude mice to establish mice models of pancreatic cancer.Then 32 mice with pancreatic cancer were randomly divided into 4 equal groups:control group.K-ras specific CTL intra-tumor injection group,CTL caudal vein injection group,and IL-2 activated non-specific CTL intra-tumor injection group.The tumor size was measure regularly.Immunohistochemistry was used to detect the pathological analysis of the transplanted tumors.Results The mutational epitope(K-ras-12-Val)induced by mutant peptide could be foand on the DCs' suIface efficiently.After co-incubation with the mature DCs pulsed with tumor antigen the autogeneic T cells were activated.the CD8+T cells accounted for(44.8±2.1)%.Without damage the normaI tissue cells,the killing rate of activated K-ras specific CTLs to the tumor cell when Lhe ratios of CTL:Patu8988 cells were 10:1,20:1,and 50:1 were(21.2±1.9)%,(32.4±2.1)%,and(45.7±5.3)% respectively.alJ while the killing efficiency significantly superior to those of the non-specific activated Tlymphocyte(all P<0.05).Eight days after CTL iniection into the nude mice the tumor size of the intratumor injection group was(68±13)mm3,significantly smaller than those of the control group and IL-2 activated non-specific CTL intra-tumor injection group(87±14)mm3 and(79±19)mm3,both P<0.05].The survival rates of the nude mice of the K-ras specific CTL intra-tumor injection group,CTL caudal vein injection group.and IL-2 activated non-specific CTL intra-tumor injection group were all significantly higher than that of the contml group(all P<0.05),and the survival rate ofthe K-ras specific CTL intra-tumor injection group was significantly higher than that of the IL-2 activated non-specific CTL intra.tumor injection group(P<0.05).Immunohistochemieal staining confirmed that K-ras specific CTL had the ability to move toward tumor.Conclusion DCs pulsed with mutant K-ras peptide(12-Val)induces specific anti-tumor immune response in pancreatic cancer efficiently.