低强度脉冲超声增强人外周血淋巴细胞增殖及抗癌活性的实验研究

Enhancement of proliferation and anti-tumor activity of human peripheral blood lymphocytes by low-intensity pulsed ultrasound:a experimental study

目的探讨低强度脉冲超声(LIPUS)对人外周血淋巴细胞(HPBL)增殖和抗肿瘤效应的影响。方法采用Ficoll梯度离心法分离HPBL;将LIUPS强度0设为对照组,10 mW/cm^2组、20 mW/cm^2组、30 mW/cm^2组、40 mW/cm^2组、50 mW/cm^2组为刺激组刺激HPBL增殖,每天一次,每次刺激10 min,连续刺激2 d。CCK-8法检测各组细胞增殖活性;酶联免疫吸附法检测抗肿瘤因子[γ干扰素(INF-γ)、白介素2(IL-2)、肿瘤坏死因子α(TNF-α)、粒细胞-巨噬细胞集落刺激因子(GM-CSF)]表达情况;采用共培养HPBL与人肺巨细胞癌95D细胞检测其抗肿瘤活性。结果 LIPUS刺激HPBL2 d共2次后,30 mW/cm^2组的细胞增殖能力增强,与对照组和其他强度刺激组比较,细胞数量和细胞团块数量均增多。CCK-8检测结果显示,30 mW/cm^2组光密度值与对照组、20 mW/cm^2组、40 mW/cm^2组、50 mW/cm^2组比较差异均有统计学意义(P=0.032、0.027、0.027、0.001)。抗肿瘤因子检测显示,30 mW/cm^2组INF-γ、IL-2、TNF-α和GM-CSF分别为(39.66±8.30)ng/L、(619.84±125.94)ρg/ml、(461.29±14.08)ng/L、(108.11±2.07)ng/L,均高于对照组[(24.40±6.84)ng/L、(279.56±128.18)ρg/ml、(417.17±21.44)ng/L和(96.53±1.56)ng/L]和50 mW/cm^2组[(16.52±0.87)ng/L、(141.00±93.71)ρg/ml、(171.96±9.46)ng/L、(72.56±8.95)ng/L],差异均有统计学意义(均P<0.01)。共培养实验显示,30 mW/cm^2组人肺巨细胞癌95D细胞杀伤率为51.11%±5.21%,显著高于对照组(42.86%±0.55%)和50 mW/cm^2组(42.00%±3.67%),差异均有统计学意义(均P<0.05)。结论 LIPUS刺激可提高HPBL的增殖活性和杀伤率。

ObjectiveTo investigate the effect of low intensity pulsed ultrasound(LIPUS)on the proliferation and anti-tumor effect of human peripheral blood lymphocytes(HPBL).MethodsFicoll gradient centrifugation was used to separate HPBL.LIUPS of 0 intensity was set as the control group,and the stimulation group was 10 mW/cm^2,20 mW/cm^2,30 mW/cm^2,40 mW/cm^2,and 50 mW/cm^2,respectively,then HPBL proliferation was stimulated by stimulation groups for 10 min/d for two consecutive days.The proliferation activity of the cells was measured by the CCK-8 assay.The expressions of anti-tumor factors interferon-γ(INF-γ),interleukin 2(IL-2),tumor necrosis factor α(TNF-α)and granulocyte-macrophage colony stimulating factor(GM-CSF) in the supernatant of culture medium were detected by the ELISA assay. The anti-tumor activity was detected by co-culture of HPBL and human lung giant cell carcinoma cells(95 D).ResultsAter 2 d stimuli,30 mW/cm^2 group significantly promoted HPBL proliferation compared with the control group and other stimulation groups after stimulated for two consecutive days(P<0.01).The results of CCK-8 showed that the optical density of 30 mW/cm^2 group was significantly different from that of the control group,20 mW/cm^2 group,40 mW/cm^2 group and 50 mW/cm^2 group,respectively(P=0.032,0.027,0.027,0.001).Anti-tumor factor detection showed that the levels of INF-γ,IL-2,TNF-αand GM-CSF in 30 mW/cm^2 group were(39.66±8.30)ng/L,(619.84±125.94)ρg/ml,(461.29±14.08)ng/L,(108.11±2.07)ng/L,respectively,which were higher than those in the control group[(24.40±6.84)ng/L,(279.56±128.18)ρg/ml,(417.17±21.44)ng/L and(96.53±1.56)ng/L]and 50 mW/cm^2 group[(16.52±0.87)ng/L,(141.00±93.71)ρg/ml,(171.96±9.46)ng/L,(72.56±8.95)ng/L],and the differences were statistically significant(all P<0.01).Co-culture experiment showed that the killing rate of 95 D cells in 30 mW/cm^2 group was 51.11%±5.21%,which was significantly higher than that in thecontrol group(42.86%±0.55%)and 50 mW/cm^2 group(42.00%±3.67%),there were significant(both P<0.05).ConclusionThe proliferation activity and killing rate of HPBL were increased after LIPUS stimulation.