家蝇幼虫凝集素的分离纯化及其抑制MCF-7细胞活性的研究
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摘要
家蝇幼虫血淋巴粗提液经过亲和层析,分离出3种与D-半乳糖特异性结合的蛋白,经高效液相色谱和聚丙烯酰胺凝胶电泳,分离出分子质量为38 ku的家蝇幼虫凝集素(MLL-2),该组分具有明显的凝血活性,并且MLL-2是一种热敏感型凝集素。圆二CD色谱结果表明,MLL-2的二级结构主要是以β折叠和无规卷曲的构象存在。MTT实验结果显示,MLL-2对人乳腺癌MCF-7细胞的生长具有明显的抑制作用,其抑制效果具有时间和浓度依赖性。通过HE染色、Hochest33258染色和吖啶橙染色从形态学上表明,MLL-2处理过的人乳腺癌MCF-7细胞出现凋亡小体、染色质浓缩等典型的凋亡现象。MLL-2处理过的人乳腺癌MCF-7细胞经TNUEL检测出DNA链断裂,证明发生凋亡现象。经流式细胞仪分析人乳腺癌MCF-7细胞被阻遏在G_2/M期,并且凋亡细胞随作用时间的延长而增加。在人乳腺癌MCF-7细胞凋亡过程中,作为线粒体途径信使的Ca~(2+)明显增加,并且线粒体途径上游的p53蛋白、中游的Bcl-2/Bad也发生改变,抑癌基因Bad升高,原癌基因Bcl-2略有降低。而且与对照组比较,线粒体途径下游的Caspase-3酶活性表达率明显增加(P＜0.01)。因此,MLL-2通过诱导人乳腺癌MCF-7细胞发生凋亡,从而起到抑制肿瘤细胞生长的作用,并且线粒体途径是诱导其发生凋亡的主要途径之一。
A new lectin was purified from Musca domestica larvae after affinity chromatography and HPLC. Approximately three D-galactose specific lectins were isolated from the crude extract of Musca domestica larvae haemolymph, By HPLC procedure and SDS-PAGE, a new 38 ku Musca domestica larvae lectin (MLL-2) was purified ultimately. Circular dichroism detected the main backbone conformations of MLL-2 areβ-sheet and Random coil. The haemagglutinating activity of the lectin was instability lectin for temperature. Anti-tumor activity of MLL-2 was demonstrated by its inhibition on the proliferation of human breast cancer MCF-7 cells in a time-and dose-dependent manner. The morphology of MCF-7 cells showed changes such as nuclear chromatin condensation and fragmentation. DNA fragment in human breast cancer human breast cancer MCF-7 cells has been detected by TUNEL. Flow cytometric analysis also demonstrated that MLL-2 induced apoptosis of MCF-7 cells through cell arrest at G_2/M phase. Western blotting revealed that MLL-2 induced apoptosis in MCF-7 cells was associated with p53, Bcl-2 and Bad protein. The expression of p53, Bad protein was enhanced. The expression of Bcl-2 protein was decreased; In addition, the caspase-3 relative activity in MCF-7 cells treated with MLL-2 for 48h group was significantly increased compared with control group. MLL-2 induces apoptosis in MCF-7cells and its antitumor activity may be applicable for the treatment of cancers.
A new lectin was purified from Musca domestica larvae after affinity chromatography and HPLC. Approximately three D-galactose specific lectins were isolated from the crude extract of Musca domestica larvae haemolymph, By HPLC procedure and SDS-PAGE, a new 38 ku Musca domestica larvae lectin (MLL-2) was purified ultimately. Circular dichroism detected the main backbone conformations of MLL-2 areβ-sheet and Random coil. The haemagglutinating activity of the lectin was instability lectin for temperature. Anti-tumor activity of MLL-2 was demonstrated by its inhibition on the proliferation of human breast cancer MCF-7 cells in a time-and dose-dependent manner. The morphology of MCF-7 cells showed changes such as nuclear chromatin condensation and fragmentation. DNA fragment in human breast cancer human breast cancer MCF-7 cells has been detected by TUNEL. Flow cytometric analysis also demonstrated that MLL-2 induced apoptosis of MCF-7 cells through cell arrest at G_2/M phase. Western blotting revealed that MLL-2 induced apoptosis in MCF-7 cells was associated with p53, Bcl-2 and Bad protein. The expression of p53, Bad protein was enhanced. The expression of Bcl-2 protein was decreased; In addition, the caspase-3 relative activity in MCF-7 cells treated with MLL-2 for 48h group was significantly increased compared with control group. MLL-2 induces apoptosis in MCF-7cells and its antitumor activity may be applicable for the treatment of cancers.
引文
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