课题背景：脂联素和其球状结构域（gapM1）虽然是仅由脂肪细胞分泌的脂肪细胞因子，但是在肥胖者及乳腺癌等患者体内，其血浆水平却低于正常，最近，流行病学研究发现，脂联素与某些肥胖相关的肿瘤发生呈直接负相关性，亦有文献报道，体外实验中，脂联素在生理浓度就能明显抑制肿瘤细胞的生长增殖活性。

应用要点：实验采用BrdU掺入实验、TUNEL、流式细胞仪检测方法，发现gapM1对人ER阴性乳腺癌细胞MDA-MB-231有明显的抑制增殖和诱导凋亡作用。提示，肥胖患者体内血脂联素和gapM1水平的降低是导致其肥胖相关肿瘤(乳腺癌)发病率升高的一个重要原因；提高肥胖者体内gapM1水平，可能降低其肥胖相关肿瘤(乳腺癌)的发生率。

同行评价：实验结果显示：采用低于脂联素生理浓度的gapM1作用于乳腺癌细胞MDA-MB-231，可使细胞增殖明显受到抑制；其抑制作用有明显的浓度-效应依赖关系；并可改变细胞周期时相分布，随gapM1剂量升高，诱导细胞发生凋亡的作用增强。文章对肥胖患者乳腺癌的预防及治疗具有指导意义。

摘要
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 （0，0.5，2，8 mg/L）处理细胞48 h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48 h，0.5，2，8 mg/L组细胞的凋亡率高于0 mg/L组( P < 0.05)。④流式细胞仪分析结果：gapM1作用MDA-MB-231细胞 48 h，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。
关键词：脂联素；乳腺肿瘤；细胞周期；细胞增殖；细胞凋亡；组织构建

秦蕾，桂律.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[J].中国组织工程研究与临床康复，2008，12(2):266-270 [www.zglckf.com/zglckf/ejournal/upfiles/08-2/2k-266(ps).pdf]

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

Abstract

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 gapM1 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. ①MDA-MB-231 cells were in vitro incubated with increasing concentration of gapM1 (0, 0.5, 2, and 8 mg/L, R&D company) for 48 hours. The morphous was observed by microscopy. ②The 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: ①Inverted microscopic observation showed there was no typical morphological change in MDA-MB-231 cell incubated with increasing concentration of gapM1, but more detached cells appeared. Moreover, cell number was decreased with the increase in gapM1 concentration. ②BrdU 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). ③TUNEL method indicated that after 48 hours of treatment with gapM1, 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). ④Flow cytometry analysis showed that gapM1 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: gapM1 has the capabilities of inhibiting proliferation and inducing apoptosis in MDA-MB-231 cells in vitro.　　

Qin L, Gui L.Globular domain of adiponectin mediates antiproliferative and apoptotic responses in human breast cancer cell MDA-MB-231.Zhongguo Zuzhi Gongcheng Yanjiu yu Linchuang Kangfu 2008;12(2):266-270(China)
[www.zglckf.com/zglckf/ejournal/upfiles/08-2/2k-266(ps).pdf]