脂肪组织释放细胞因子脂联素的球状结构域对人乳腺癌细胞MDA-MB-231生长抑制及诱导凋亡的效应

Globular domain of adiponectin mediates antiproliferative and apoptotic responses in human breast cancer cell MDA-MB-231

目的:脂联素作为一种脂肪组织释放的细胞因子,具有抗糖尿病、抗动脉粥样硬化等生物学功能,脂联素球状结构域(gapM1)为其重要的功能结构域。最近研究发现,脂联素对恶性肿瘤细胞有一定的抑制作用。实验拟进一步讨论gapM1对人乳腺癌细胞MDA-MB-231的生长抑制作用及诱导凋亡作用。方法:实验于2005-03/2006-03在复旦大学上海医学院病理实验室完成。①实验材料:gapM1购自R&D公司。②实验过程:选用人乳腺癌MDA-MB-231细胞进行体外培养,不同剂量的gapM1(0,0.5,2,8mg/L)处理细胞48h后,显微镜进行形态学观察。③评估:采用BrdU掺入法检测细胞增殖情况;采用TdT介导的dUTP末端标记法原位观察癌细胞凋亡,并计算平均凋亡指数;流式细胞仪测定细胞周期和凋亡率。结果:①倒置显微镜观察结果:不同剂量的gapM1作用MDA-MB-231细胞后,未见明显的细胞形态学改变,但细胞收缩、脱落,细胞增殖明显受到抑制,且随浓度的增大,细胞数量减少。②BrdU掺入法检测结果:不同剂量的gapM1对细胞生长均有一定的抑制作用,且同一处理时相点各组间比较差异均有显著性(P<0.05)。③TUNEL法检测结果:不同剂量的gapM1作用于MDA-MB-231细胞48h,0.5,2,8mg/L组细胞的凋亡率高于0mg/L组(P<0.05)。④流式细胞仪分析结果:gapM1作用MDA-MB-231细胞48h,G0～G1期细胞增多,S期细胞显著减少,细胞周期分布特点与药物浓度有关,随浓度增大,变化更加明显。同时可观察到细胞凋亡峰,细胞凋亡率也呈剂量依赖趋势。结论:gapM1对人乳腺癌MDA-MB-231细胞有明显的抑制增殖和诱导凋亡作用。

AIM： Adiponectin, a kind of cytokine released by adipose tissue, has the biological functions to resist diabetes and artherosclerosis. Globular domain of adiponectin （gapM1） is an important functional domain of adiponectin. Recent studies have suggested that adiponectin inhibit malignant tumor cells to certain extent. This study investigated the effects of gapM l on human breast cancer cell line MDA-MB-231. METHODS： The experiment was conducted in the pathological laboratory of Fudan University Shanghai Medical College from March 2005 to March 2006. （1）MDA-MB-231 cells were in vitro incubated with increasing concentration of gapM1 （0, 0.5, 2, and 8mg/L, R＆D company） for 48 hours. The morphous was observed by microscopy. （2）The change in proliferation was determined by BrdU incorporation, and apoptotic cells were detected by TUNEL method. Mean apoptosis index was calculated and the distribution of cell cycles were examined with flow cytometry. RESULTS： （1）Inverted microscopic observation showed there was no typical morphological change in MDA-MB-231 cell incubated with increasing concentration of gapM l, but more detached cells appeared. Moreover, cell number was decreased with the increase in gapMl concentration. （2）BrdU incorporation assay suggested that the BrdU incorporation rate declined significantly and there were statistically differences among different groups at the same time （P 〈 0.05）. （3）TUNEL method indicated that after 48 hours of treatment with gapM l, the apoptosis rate of cancer cells in 0.5, 2, 8 mg/L groups were significantly increased compared with 0 mg/L group （P 〈 0.05）. （4）Flow cytometry analysis showed that gapMl induced an increase in the cell cycle in G0-G1 phase, and an arrest in S phase. Cell cycle distribution was correlated with drug concentration. Meanwhile, the enhanced G1-S phase of cell cycle arrest was accompanied with the enhanced apoptotic peak. CONCLUSION： gapM 1 has the capabilities of inhibiting proliferation and inducing apoptosis in MDA-MB-231 cells in vitro.