凝血酶敏感蛋白1对人肝癌细胞株HCCLM3生长和黏附的作用

Influence of thrombospondin-1 on the growth and adhesion of human hepatoma carcinoma cell line HCCLM3

目的:观察凝血酶敏感蛋白1对人肝癌细胞株HCCLM3的生长抑制和黏附的作用。方法:实验于2006-05/2007-01在河南省高等学校省级开放实验室完成。①采用浓度为2×107L-1的HCCLM3对数生长期单细胞悬液接种培养后,采用MTT法检测凝血酶敏感蛋白1对HCCLM3的生长抑制作用。②对数生长期的HCCLM3细胞株接种于纤黏连蛋白覆盖的96孔板后,按照处理因素分组:空白对照组、凝血酶敏感蛋白1组(加入凝血酶敏感蛋白1蛋白40mg/L)、CD36阻断组和CD47阻断组[先加入对应的单抗(浓度5mg/L),在体积分数为0.05CO2,37℃温箱孵育30min使单抗与细胞充分结合后再加入凝血酶敏感蛋白1]。培养72h后MTT方法检测凝血酶敏感蛋白1对细胞黏附力的作用,染色后直接计数黏附细胞。结果:①凝血酶敏感蛋白1对HCCLM3细胞的生长抑制率随着浓度的提高而增加,40mg/L与50mg/L相比抑制作用差异无显著性。抑制率随着时间的延长而增加,在72h抑制作用最明显。②黏附力的检测显示:凝血酶敏感蛋白1组,CD36阻断组,CD47阻断组细胞黏附力均显著低于对照组(P<0.05),但凝血酶敏感蛋白1组,CD36阻断组,CD47阻断组细胞黏附力差异无显著性(P>0.05)。结论:凝血酶敏感蛋白1对HCCLM3的生长有抑制作用,在一定范围内呈剂量依赖性和时间依赖性。凝血酶敏感蛋白1可抑制人肝癌细胞株HCCLM3与细胞外基质的黏附能力,作用途径可能与受体CD36,CD47无关。

AIM： To observe the effect of thrombospondin-1 （TSP-1） on the growth and adhesion of human hepatoma carcinoma cell line HCCLM3. METHODS： The experiment was performed in the Open Laboratory at Henan Universities from May 2006 to January 2007. （1）The inhibitory effect of TSP-1 on HCCLM3 growth at logarithmic growth phase （2×10^7 L^-1） was detected by MTT test. （2）HCCLM3 growth at logarithmic growth phase were seeded onto 96-well plate covered with fibronectin, and divided into blank control group, TSP-1 group （40 mg/L TSP-1）, CD36-blocked group and CD47-blocked group, which were incubated in incubator with 0.05 volume fraction CO2 at 37 ℃ for 30 minutes after adding 5 mg/L corresponding monoclonal antibody, following by TSP-1. Seventy-two hours later, the effect of TSP-1 on cell adhesion was detected by MTT test and cell counting. RESULTS： （1）The inhibitory effect of TSP-1 on HCCLM3 growth was enhanced with the increased concentration and time. There was no significantly difference between 40 mg/L and 50 mg/L. The maximum effect was at 72 hour. （2）The cellular adhesion in experimental groups was significantly reduced compared with that in control group （P 〈 0.05）. There was no difference in cellular adhesion among TSP-1 group, CD47-blocked group and CD36-blocked group （P 〉 0.05）. CONCLUSION： The inhibition of TSP-1 to the growth of HCCLM3 cell depends on the dose and time. TSP-1 could inhibit the adhesion to extracellular matrix of HCCLM3, which may be not related with CD36 and CD47.