纳秒脉冲消融肝癌对小鼠肝脏区域免疫细胞的影响
Nanosecond pulse ablation of hepatocellular carcinoma effect on the immune cells in the liver region of mice
目的 探讨纳秒脉冲消融原发性肝癌对小鼠肝脏局部免疫细胞的影响.方法 将40只C57BL-6J小鼠按每组10只随机分为空白对照组、肿瘤组、手术切除组及纳秒脉冲组.对肿瘤组、手术切除组及纳秒脉冲组小鼠肝左叶注入Hepa 1-6细胞建立原位肝癌模型.在建模后第7天对手术切除组行肝左叶切除术,对纳秒脉冲组行纳秒脉冲治疗.治疗后第7天处死所有小鼠,对空白对照组小鼠肝、肿瘤组小鼠肝左叶病灶、纳秒脉冲组脉冲区域及4组肝右叶正常肝组织分别用流式检测仪检测CD3+T、CD4+T、CD8+T、调节性T细胞(Treg)、髓源性抑制细胞(MDSC)、自然杀伤细胞(NK)、B细胞,计算CD4+T与CD8+T的比值.结果 在空白对照组小鼠肝左叶、肿瘤组小鼠肝左叶病灶、纳秒脉冲组脉冲区域中,CD4+T细胞表达空白对照组>纳秒脉冲组>肿瘤组[(25.77±3.76)%比(15.72±2.70)%比(12.68±3.13)%,P<0.05];CD8+T细胞肿瘤组>空白对照组>纳秒脉冲组[(14.01±2.75)%比(13.99±1.41)%比(8.42±2.21)%,P<0.05];CD4+T与CD8+T比值纳秒脉冲组>空白对照组>肿瘤组[(1.90±0.17)比(1.86±0.32)比(0.93±0.21),P<0.05];B细胞纳秒脉冲组>空白对照组>肿瘤组[(47.65±3.77)%比(33.74±3.91)%比(15.94±6.10)%,P<0.05];MDSC细胞肿瘤组>纳秒脉冲组>空白对照组[(18.49±2.74)%比(8.41±3.05)%比(2.15±0.69)%,P<0.05];而CD3+T细胞、NK细胞及Treg细胞在上述3组中差异均无统计学意义(均P>0.05).在4组肝右叶正常肝组织中CD4+T与CD8+T的比值空白对照组>纳秒脉冲组>手术切除组>肿瘤组[(1.86±0.32)比(1.85±0.43)比(1.52±0.16)比(1.36±0.29),P<0.05];B细胞纳秒脉冲组>手术切除组>空白对照组>肿瘤组[(46.85±8.30)%比(34.23±6.17)%比(33.74±3.91)%比(27.64±2.20)%,P<0.05];Treg细胞肿瘤组>手术切除组>纳秒脉冲组>空白对照组[(26.34±6.23)%比(7.01±2.04)%比(3.63±1.59)%比(3.19±1.50)%,P<0.05];MDSC在肿瘤组>手术切除组>纳秒脉冲组>空白对照组[(12.22±2.02)%比(5.00±0.73)%比(2.87±0.96)%比(2.15±0.69)%,P<0.05];而CD3+T、CD4+T、CD8+T、NK细胞在上述四组间差异均无统计学意义(均P>0.05).结论 纳秒脉冲消融原发性肝癌会激起消融区域及其他肝叶的免疫应答,这有可能是纳秒脉冲引起的机体抗肿瘤免疫效应.
更多Objective To analyze the changes of local immune cells in liver of mice caused by nanosecond pulse therapy for hepatocellular carcinoma. Methods Forty C57BL-6J of mice were randomly divided into four groups:negative control group ( n=10 ) , tumor group ( n=10 ) , surgical resection group (n=10) and nanosecond pulse group (n=10). Hepa 1-6 cells were injected into the left hepatic lobe of mice in tumor group, resection group and nanosecond pulse group to construct the orthotopic xenograft tumor model. Left hepatic lobectomy was performed in the surgical excision group and nanosecond pulse was performed in the nanosecond pulse group 7 days after the construction. All mice were sacrificed 7 days after the treatment. CD3+ was detected by flow cytometry in the left hepatic lobe lesion, the nanosecond pulse group and the normal liver tissue of the right hepatic lobe in the liver and tumor groups of the blank control group. T, CD4+T, CD8+T, regulatory T cells (Treg), myeloid-derived suppressor cells (MDSC), natural killer cells (NK), B cells, and the ratio of CD4+T to CD8+T. Results In the blank control group, the tumor group the number of lesion in the mice and the pulse area of the nanosecond pulse group CD4+T cells in blank control group (normal liver) >nanosecond pulse group >tumor group [(25. 77 ± 3. 76)% vs. (15. 72 ± 2. 70)% vs. (12. 68 ± 3. 13)%, P<0. 05]; CD8+T cell tumor group>blank control group>nanosecond pulse group [(14. 01 ± 2. 75)% vs. (13. 99 ± 1. 41)% vs. (8. 42 ± 2. 21)%, P<0. 05]. The ratio of CD4+T to CD8+T in nanosecond pulse group > blank control group > tumor group [ ( 1. 90 ± 0. 17) vs. (1. 86 ± 0. 32) vs. (0. 93 ± 0. 21), P<0. 05];B cell nanosecond pulse group> blank control group > tumor group [(47. 65 ± 3. 77)% vs. (33. 74 ± 3. 91)% vs. (15. 94 ± 6. 10)%, P<0. 05];MDSC cell tumor group > nanosecond pulse group > blank control group [(18. 49 ± 2. 74)% vs. (8. 41 ± 3. 05)% vs. (2. 15 ± 0. 69)%, P<0. 05]. However, CD3+T cells, NK cells and Treg cells showed no statistical significance among the three groups (all P>0. 05). Normal liver tissue in right lobe of liver in 4 groups the ratio of CD4+T to CD8+T in blank control group >nanosecond pulse group >surgical resection group >tumor group [(1. 86 ± 0. 32) vs. (1. 85 ± 0. 43) vs. (1. 52 ± 0. 16) vs. (1. 36 ± 0. 29), P<0. 05]; B cell nanosecond pulse group >surgical resection group >blank control group > Tumor group [(46. 85 ± 8. 30)% vs. (34. 23 ± 6. 17)% vs. (33. 74 ± 3. 91)% vs. (27. 64 ± 2. 20)%, P<0. 05];Treg cell tumor group >resection group>nanosecond pulse group>blank control group [(26. 34 ± 6. 23)%vs. (7. 01 ± 2. 04)% vs. (3. 63 ± 1. 59)% vs. (3. 19 ± 1. 50)% , P<0. 05]; MDSC in tumor group>resection group > nanosecond pulse group > blank control group [ ( 12. 22 ± 2. 02 )% vs. ( 5. 00 ± 0. 73)% vs. (2. 87 ± 0. 96)% vs. (2. 15 ± 0. 69)%,P <0. 05]. However, there were no statistically significant differences in CD3+T, CD4+T, CD8+T and NK cells among the four groups ( all P >0. 05 ) . Conclusion Nanosecond pulse ablation of primary hepatocellular carcinoma of mice can induce immune response in ablation area and other hepatic lobes, which may be due to the anti-tumor immunity induced by nanosecond pulse.
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