榄香烯抑制人晶状体上皮细胞增殖的蛋白质组研究

Study on proteomics of inhibitory effects of elemene on proliferation of human lens epithelial cell

目的探讨中药单体榄香烯对碱性成纤维细胞生长因子（rhbFGF）诱导的人晶状体上皮细胞（HLE-B3）增殖的抑制作用及其蛋白质组学规律。方法实验研究。共分为3组：正常组、rhbFGF组及榄香烯组。正常组为培养的HLE-B3，rhbFGF组加入终浓度为10μg／LrhbFGF，榄香烯组加入终浓度为10μg/L rhbFGF和80mg／L榄香烯。24h后四甲基偶氮唑蓝法检测榄香烯对HLE-B3增殖的抑制作用；蛋白质芯片联合表面增强激光解析离子化飞行时问质谱技术检测分析榄香烯作用于HLE-B3后蛋白质表达谱的改变、寻求差异蛋白。采用单因素方差分析，并通过Dunnett-t检验进行正常组、rhbFGF组、榄香烯组吸光度值的两两比较；对每个质荷比峰值做Kruskal-Wallis秩和检验，采用Nemenyi检验对正常组、rhbFGF组、榄香烯组中每个特定质荷比的蛋白质峰值进行两两比较。结果（1）四甲基偶氮唑蓝法检测显示：rhbFGF组HLE—B3吸光度A值（0．599±0．053）比正常组（0．409±0．042）显著升高，榄香烯组HLE—B3吸光度A值（0．450±0．061）比rhbFGF组显著降低，抑制率达24．90％，差异有统计学意义（F=28．886，P=0．000）。（2）用rhbFGF诱导HLE-B3增殖后，出现了5个差异表达的蛋白点，其中质荷比为8093和9516的2个蛋白点表达上调，质荷比为5361、9666及13767的3个蛋白点表达下调；榄香烯作用于增殖的HLE-B3后，出现10个差异表达的蛋白点，其中质荷比为2487、4392、8566及11600的4个蛋白点表达上调，质荷比为3679、4826、6861、9516、9557和9672的6个蛋白点表达下调。结论榄香烯能有效抑制rhbFGF诱导的HLE-B3增殖，质荷比为9516的蛋白点可能是榄香烯抑制rhbFGF诱导的HLE-B3增殖的作用靶点。

Objective To investigate the inhibitory effects of natural medicinal monomer elemene （Ele） on proliferation of human lens epithelial cells B3 （HLE-B3） inducing by recombinant human basic fibroblast growth factor（rhbFGF） and to pursue the proteomics regularity of the inhibitory effects of Ele on proliferation of HLE-B3. Methods Experimental study. This study is divided into three group： control group,rhbFGF group and Ele group. Using 10 μg/L rhbFGF to induce proliferation of HLE-B3. Proliferative HLE-B3 were incubated with 80 mg/L Ele in CO2 incubator for 24 hours. Then the inhibitory effects of Ele on proliferation of HLE-B3 was detected by methyl thiazolyl tetrazolium （MTT）. The change of expressions of all protein in HLE-B3 was assayed and analyzed by protein array and surface-enhanced laser desorption ionization time of flight mass spectrometry （SELDI-TOF-MS） proteomics technology. Results MTT test showed that the A values of rhbFGF（0. 599 ±0. 053） group were higher than that of control group（0. 409 ±0. 042） remarkably. The A values of Ele group （0. 450 ±0. 061 ） decreased obviously compared to rhbFGF group, the inhibition rates were 24.90% （ F = 28. 886, P = 0. 000）. Five different protein spots were obtained in proliferative HLE-B3 induced by rhbFGF. The expressions were up-regulated in two of the five protein spots at the ratios of mass/charge （m/z） of 8093 and 9516, while the expressions were downregulated in three of the five protein spots at m/z of 5361,9666 and 13 767. Ten different protein spots were obtained in HLE-B3 incubated with Ele. The expressions were up-regulated in four of the ten protein spots at m/z of 2487, 4392, 8566 and 11 600, while the expressions were down-regulated in six of the ten protein spots at m/z of 3679, 4826, 6861, 9516, 9557 and 9672. Conclusions Ele could effectively inhibit HLE-B3 proliferation induced by rhbFGF. The protein spot at m/z of 9516 might be the target of proliferative inhibition in HLE-B3 by Ele.