负载肿瘤细胞裂解物的髓样与浆细胞样树突状细胞复合疫苗体外抗肺癌效应研究

Anti-lung cancer effect of myeloid and plasmacytoid dendritic cell combined vaccines loaded with tumor cell lysates in vitro

目的探讨负载热处理Lewis肺癌细胞裂解物的小鼠髓样树突状细胞(mDC)与浆细胞样树突状细胞(pDC)复合疫苗治疗肺癌的可行性。方法利用重组小鼠fms样酪氨酸激酶受体3配体体外诱导骨髓细胞,磁珠分选mDC和pDC,将热处理Lewis肺癌细胞裂解物分别加入mDC、pDC和mDC+pDC(比例为1∶1),比较各组刺激淋巴细胞增殖及诱导T细胞在体外杀伤肿瘤细胞的效应;利用流式细胞仪检测各组免疫表型CD80、CD86、CD40和主要组织相容性复合体Ⅱ类(MHC-Ⅱ)分子的变化,酶联免疫吸附试验检测细胞因子白细胞介素6(IL-6)、IL-12和肿瘤坏死因子α(TNF-α)的分泌情况。结果在负载热处理肿瘤细胞裂解物后,mDC+pDC组(mDC∶pDC=1∶1)刺激淋巴细胞增殖的能力(10.80±0.66)均高于相同细胞数量下单独培养的mDC组和pDC组(分别为8.63±0.65和7.10±0.46,均P<0.05)。在效应细胞:靶细胞(E∶T)=10∶1的条件下,负载热处理肿瘤细胞裂解物的mDC+pDC组细胞杀伤率(31.68%±2.93%)均高于相同细胞数量下单独培养的mDC组和pDC组(分别为17.44%±0.97%和10.29%±1.33%,均P<0.05);在E∶T=20∶1的条件下,mDC+pDC组的细胞杀伤率(54.77%±3.28%)均高于相同细胞数量下单独培养的mDC组和pDC组(分别为35.25%±1.51%和15.52%±0.73%,均P<0.05);在E∶T=40∶1的条件下,mDC+pDC组的细胞杀伤率(73.01%±0.91%)均高于相同细胞数量下单独培养的mDC组和pDC组(分别为51.36%±0.58%和22.65%±1.28%,均P<0.05)。随着E∶T比例增高,各组细胞的杀伤率也随之增高。负载热处理Lewis肺癌细胞裂解物的mDC+pDC组表面CD80、CD86、CD40和MHC-Ⅱ分子的平均荧光强度,均高于同等培养条件下相同细胞数量的mDC组和pDC组。在负载热处理肿瘤细胞裂解物条件下,mDC+pDC组、mDC组和pDC组细胞中IL-6水平分别为(586.67±52.52)pg/ml、(323.33±67.14)pg/ml和(166.67±16.07)pg/ml,IL-12水平分别为(2 568.75±119.24)pg/ml、(2 156.25±120.55)pg/ml和(672.92±31.46)pg/ml,TNF-α水平分别为(789.33±48.08)pg/ml、(584.89±116.49)pg/ml和(291.56±40.73)pg/ml;mDC+pDC组细胞中IL-6、IL-12和TNF-α水平均高于相同细胞数量单独培养的mDC组和pDC组(均P<0.05)。结论负载热处理Lewis肺癌细胞裂解物的小鼠pDC与mDC复合疫苗,在体外诱导T淋巴细胞增殖及体外抗肿瘤效应上存在协同作用,且这种协同抗肿瘤作用与共刺激分子表达上调和细胞因子分泌增多等有关。

Objective To investigate the feasibility of myeloid and plasmacytoid dendritic cell combined vaccines loaded with heat-treated Lewis lung cancer cell lysates for treatment of lung cancer in mice.Methods Bone marrow cells were induced by the recombinant mouse fms-like tyrosine kinase receptor 3 ligand(rmFlt3-L)in vitro,myeloid dendritic cells(mDC)and plasmacytoid dendritic cells(pDC)were separated by magnetic beads.The mDC,pDC,and mDC∶pDC=1∶1 were stimulated with heat-treated Lewis lung cancer cell lysates,respectively.The effects of each group on stimulating of lymphocyte proliferation and inducing of T cell to kill tumor cells in vitro were compared.The alternations of the immunophenotypes of CD80,CD86,CD40 and major histocompatibility complexⅡ(MHC-Ⅱ)were detected by flow cytometry.The secretion of cytokines including interlukin-12(IL-12),interlukin-6(IL-6),and tumor necrosis factorα(TNF-α)were detected by enzyme-linked immunosorbent assay(ELISA).Results The lymphocyte proliferation in mice stimulated with mDC+pDC group loaded with heat-treated Lewis lung cancer cell lysates was 10.80±0.66,significantly higher than 8.63±0.65 of mDC group and 7.10±0.46 pDC group under the same culture conditions,respectively(P<0.05).When the ratio of effector cells:target cells(E∶T)was 10∶1,the killing rate of the mDC+pDC group loaded with heat-treated tumor cell lysate was 31.68%±2.93%,significantly higher than 17.44%±0.97%of mDC group and 10.29%±1.33%of pDC group,respectively(P<0.05).When the ratio of E∶T was 20∶1,the killing rate of the mDC+pDC group loaded with heat-treated tumor cell lysate was 54.77%±3.28%,significantly higher than 35.25%±1.51%of mDC group and 15.52%±0.73%of pDC group,respectively(P<0.05).When the ratio of E∶T was 40∶1,the killing rate of the mDC+pDC group loaded with heat-treated tumor cell lysate was 73.01%±0.91%,significantly higher than 51.36%±0.58%of mDC group and 22.65%±1.28%of pDC group,respectively(P<0.05).With the rate of E∶T increased,the killing rate also increased.The mean fluorescence intensities of surface molecules including CD80,CD86,CD40 and MHC-Ⅱof mDC:pDC=1 group pulsed with heat-treated Lewis lung cancer cell lysates were higher than those of mDC group and pDC group.The IL-6 cytokine concentrations of mDC+pDC group,mDC group and pDC group loaded with heat-treated Lewis lung cancer cell lysates were(586.67±52.52)pg/ml,(323.33±67.14)pg/ml and(166.67±16.07)pg/ml,respectively.The concentrations of IL-12 in each group were(2 568.75±119.24)pg/ml,(2 156.25±120.55)pg/ml and(672.92±31.46)pg/ml,respectively.The concentrations of TNF-αin each group were(789.33±48.08)pg/ml,(584.89±116.49)pg/ml and(291.56±40.73)pg/ml,respectively.The concentrations of IL-6,IL-12 and TNF-αsecreted by mDC+pDC group were much higher than those of mDC group and pDC group under the same culture conditions(P<0.05).Conclusions The mDCs and pDCs combined vaccines pulsed with heat-treated Lewis lung cancer cell lysates have synergistic effects on inducing of T lymphocyte proliferation and killing tumor cells in vitro.This synergistic anti-tumor effect is related with up-regulation of co-stimulatory molecules and increased secretion of cytokines.