DC荷载α-GalCer联合肿瘤特异性CTL对小鼠Heps肝癌移植瘤生长的影响

doi:10.3760/cma.j.issn.1673-422X.2016.11.002

摘要/Abstract

摘要： 目的探讨树突状细胞（DC）荷载α-半乳糖神经酰胺（α-GalCer）联合肿瘤特异性细胞毒T淋巴细胞（CTL）对小鼠Heps肝癌移植瘤生长的影响。方法诱导扩增小鼠骨髓来源的DC细胞和T淋巴细胞，使其成为具有肿瘤特异性的CTL；DC细胞体外荷载αGalCer。建立Heps肝癌移植瘤模型，采用随机数字表法将模型鼠分为4组（n=9）：对照组（静脉给予生理盐水）、CTL组、DC荷载αGalCer组、DC细胞荷载αGalCer联合CTL组。干预2周后取肿瘤组织，称取各瘤体重量，计算抑瘤率。免疫组织化学法和Western blotting检测各组移植瘤组织内Bax和Bcl2的表达水平。结果CTL组、DC荷载αGalCer组和联合治疗组的平均瘤重分别为（1.07±0.15）g、（1.11±0.17）g、（0.79±0.14）g，均低于对照组（1.69±0.23）g，差异有统计学意义（t=14.176，P=0.023；t=12.351，P=0.034；t=18.672，P=0.000）。联合治疗组的平均瘤重低于CTL组及DC荷载αGalCer组，差异有统计学意义（t=15.236，P=0.012；t=11.176，P=0.037）。联合治疗组（53.25%）的抑瘤率高于CTL组（36.69%）和DC荷载αGalCer组（34.32%），差异有统计学意义（P=0.034；P=0.021）。免疫组织化学检测发现，CTL组、DC荷载αGalCer组及联合治疗组移植瘤内Bax阳性细胞数量分别为35.83±0.75、33.67±0.82、41.17±1.17，与对照组（21.67±2.16）相比，差异有统计学意义（t=-13.789，P=0.002；t=-15.116，P=0.001；t=-17.452，P=0.000）；联合治疗组移植瘤内Bax阳性细胞数与CTL组和DC荷载αGalCer组相比，差异有统计学意义（t=-7.730，P=0.009；t=-5.872，P=0.011）。CTL组、DC荷载αGalCer组及联合治疗组移植瘤内Bcl2阳性细胞数量分别为30.83±0.75、31.67±1.03、25.00±0.89，与对照组相比（38.67±1.21），差异有统计学意义（t=9.234，P=0.007；t=11.738，P=0.003；t=20.608，P=0.000）；联合治疗组移植瘤内Bcl2阳性细胞数与CTL组和DC荷载αGalCer组相比，差异有统计学意义（t=11.952，P=0.003；t=12.223，P=0.002）。Western boltting检测结果显示CTL组、DC荷载αGalCer组和联合治疗组移植瘤内Bax的表达水平分别为0.46±0.01、0.42±0.03、0.55±0.01，与对照组（0.31±0.02）相比，差异有统计学意义（t=1.035，P=0.032；t=1.124，P=0.027；t=1.425，P=0.010）；联合治疗组移植瘤内Bax表达水平与CTL组和DC荷载αGalCer组相比，差异有统计学意义（t=1.305，P=0.013；t=1.421，P=0.010）。CTL组、DC荷载αGalCer组和联合治疗组移植瘤内Bcl2的表达水平分别为0.34±0.03、0.33±0.02、0.24±0.01，与对照组相比（0.46±0.01），差异有统计学意义（t=-1.123，P=0.025；t=-1.061，P=0.031；t=1.278，P=0.014）；联合治疗组移植瘤内Bcl2阳性细胞与CTL组和DC荷载αGalCer组相比，差异有统计学意义（t=1.160，P=0.021；t=1.219，P=0.015）。结论DC荷载αGalCer与肿瘤特异性CTL联合应用对小鼠Heps肝癌移植瘤具有协同杀伤作用。

关键词: 树突细胞, T淋巴细胞, 肝肿瘤, 自然杀伤细胞, &alpha, -半乳糖神经酰胺

Abstract: ObjectiveTo investigate the effects of dendritic cells (DCs) loading alphaGalactosylceramide (αGalCer) combined with tumor specific cytotoxic T lymphocytes (CTLs) on the growth of transplanted Heps hepatoma in mice. MethodsWe induced the augmentation of the DC cells and T lymphocyte derived from the mice bone marrow, and enabled them to be specific CTLs. DC cells loaded αGalCer in vitro. First we established a Heps liver cancer xenograft model, then divided the model mice into 4 groups by random number table method (n=9): control group (intravenous injection with physiological saline), CTL group, DC loading αGalcer group and DC loading αGalcer combined with CTLs group. After 2 weeks of intervention, we extracted the tumor tissue, weighed the tumor and calculated the inhibition rate of tumor. The expressions of Bax/Bcl2 cells in groups of transplanted tumor tissues were detected using immunohistochemistry and Western blotting. ResultsThe average tumor weight of CTL group, DC loading αGalCer group and combined treatment group were (1.07±0.15)g, (1.11±0.17)g, (0.79±0.14)g, respectively. All of them were lower than that of control group (1.69±0.23)g, with significant differences (t=14.176, P=0.023; t=12.351, P=0.034; t=18.672, P=0.000). The average tumor weight of combined treatment group was lower than those of the CTL group and DC loading αGalCer group, with significant differences (t=15.236, P=0.012; t=11.176, P=0.037). Compared to the CTL group (36.69%) and DC loading αGalCer group (34.32%), the combined treatment group had a higher tumor inhibition rate (53.25%; P=0.034, P=0.021). Immunohistochemical assay showed that the numbers of Baxpositive cells in CTL group, DC loading αGalCer group and combined treatment group were 35.83±0.75, 33.67±0.82, 41.17±1.17 respectively, and compared with the control group (21.67±2.16), the differences were statistically significant (t=-13.789, P=0.002; t=-15.116, P=0.001; t=-17.452, P=0.000). The numbers of Baxpositive cells in combined treatment group were different with CTL group and DC loading αGalCer group (t=-7.730, P=0.009; t=-5.872, P=0.011). The numbers of Bcl2positive cells in CTL group, DC loading αGalCer group and combined treatment group were 30.83±0.75, 31.67±1.03, 25.00±0.89, and compared with the control group (38.67±1.21), the differences were statistically significant (t=9.234, P=0.007; t=11.738, P=0.003; t=20.608, P=0.000). The numbers of Bcl2positive cells in combined treatment group were different with CTL group and DC loading αGalCer group (t=11.952, P=0.003; t=12.223, P=0.002). Western blotting test results showed that the expression levels of Bax in CTL group, DC loading αGalCer group and combined treatment group were 0.46±0.01, 0.42±0.03, 0.55±0.01, and compared with the control group (0.31±0.02), the differences were statistically significant (t=1.035, P=0.032; t=1.124, P=0.027; t=1.425, P=0.010). The expression level of Bax in combined treatment group was different with CTL group and DC loading αGalCer group (t=1.305, P=0.013; t=1.421, P=0.010). The positive expressions of Bcl2 in CTL group, DC loading αGalCer group and combined treatment group were 0.34±0.03, 0.33±0.02, 0.24±0.01, and compared with the control group (0.46±0.01), the differences were statistically significant (t=-1.123, P=0.025; t=-1.061, P=0.031; t=1.278, P=0.014); the positive expression level of Bcl2 in combined treatment group was different with CTL group and DC loading αGalCer group (t=1.160, P=0.021; t=1.219, P=0.015). ConclusionIt has synergistic killing effect on transplanted Heps hepatoma in mice using DC loading αGalCer combined with the tumor specific CTL.

Key words: Dendritic cells, T-lymphocytes, Liver neoplasms, Natural killer T cells, alpha-Galactosylceramide

王鹏，栾智勇，刘军权，杭敏，潘进，张南征. DC荷载α-GalCer联合肿瘤特异性CTL对小鼠Heps肝癌移植瘤生长的影响[J]. 国际肿瘤学杂志, 2016, 43(11): 806-811.

Wang Peng, Luan Zhiyong, Liu Junquan, Hang Min， Pan Jin, Zhang Nanzheng. Effects of DC loaded α-GalCer combined with tumor specific cytotoxic T lymphocytes on the growth of transplanted Heps hepatoma in mice[J]. Journal of International Oncology, 2016, 43(11): 806-811.

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