目的 探討絞股藍次級皂苷H （Gypensapogenin H，GH）對人乳腺癌MCF-7細胞的誘導凋亡作用。方法 采用5、10、20、40、60、80 μmol/L的GH處理人乳腺癌MCF-7細胞48 h后，MTT法測定GH對細胞增殖的影響；20 μmol/L的GH作用細胞24 h后，相差顯微鏡下觀察細胞的形態(tài)學變化，DAPI染色，熒光顯微鏡下觀察凋亡小體的形成；GH作用細胞6、12、24 h后，流式細胞儀測定細胞凋亡率；免疫印跡法檢測GH對Bcl-2、Bax、Cytochrome C、Caspase 3等蛋白表達的影響。結(jié)果 GH抑制細胞增殖作用呈明顯的濃度相關(guān)性，半數(shù)抑制濃度（IC₅₀）為（8.67±1.22）μmol/L；GH組細胞形態(tài)與對照組比較發(fā)生顯著變化，主要表現(xiàn)為細胞皺縮、染色質(zhì)濃縮、細胞核變形；細胞經(jīng)DAPI染色后在熒光顯微鏡下可觀察到有凋亡小體形成；隨GH作用時間延長，細胞凋亡率明顯上升，與對照組比較，GH作用12、24 h細胞早期凋亡率顯著升高（P<0.05、0.01）；蛋白免疫印跡顯示，與對照組比較，GH作用6、24 h Bax，6、12、24 h Cleaved Caspase 3和Cytochrome C蛋白水平顯著增加（P<0.05、0.01）；6、12、24 h Bcl-2和12、24 h Caspase 3蛋白表達量顯著減少（P<0.05、0.01）。結(jié)論 GH通過線粒體通路誘導乳腺癌MCF-7細胞凋亡。

Objective To investigate the cell apoptosis of human breast cancer MCF-7 cells induced by gypensapogenin H in vitro. Methods After human breast cancer MCF-7 cells were treatment with GH of 5, 10, 20, 40, 60 and 80 μmol/L for 48 h, the effect of GH on cell proliferation was measured by MTT assay. After cells were treated by GH for 24 h, the morphological changes were observed under Phase contrast microscope, and apoptotic bodys were observed under fluorescence microscope with DAPI staining. Additionally, flow cytometry was used to evaluate the apoptosis rate, Western blotting was used to detect the effect of GH treating for 6, 12, and 24 h on the expression of Bcl-2, Bax, Cytochrome c, Caspase 3 and other proteins. Results GH inhibited cell proliferation in a concentration-dependent manner, and the half inhibitory concentration (IC50) was (8.67±1.22) umol/L. Compared with control group, the cell morphology of GH group changed significantly, mainly in cell shrinkage, chromatin concentration and nuclear deformation. After cells were stained with DAPI, the formation of apoptotic cells could be observed under the fluorescence microscope. With the extension of GH, the apoptosis rate of cells increased significantly, compared with control group, the early apoptotic rate of GH group increased significantly at 12 and 24 hours (P<0.05 and 0.01). Western Blotting showed that compared with control group, the levels of Bax, Cleaved Caspase 3 and Cytochrome C protein increased significantly (P<0.05 and 0.01) and the levels of Bcl-2 and Caspase 3 protein decreased significantly (P<0.05, 0.01). Conclusion GH induces apoptosis of breast cancer MCF-7 cells through mitochondrial pathway

國家自然科學基金（81703389）