穿膜肽-凋亡蛋白融合蛋白导入人脐静脉内皮细胞和人肺腺癌Anip973细胞的生物学特性

Biological features of cell penetrate peptide-apoptin fusion protein introduced into human umbilical vein endotheliocytes and human adenocarcinoma of lung Anip973 cells

目的:在大肠杆菌内表达穿膜肽与凋亡蛋白的融合蛋白,观察其诱导肺腺癌Anip973细胞凋亡的活性,并了解其是否有剂量依赖性。方法:实验于2005-08/2006-01在哈尔滨医科大学生物化学与分子生物学教研室完成。①在大肠杆菌中表达穿膜肽-凋亡蛋白并用IDA-Ni2+亲和层析纯化。②细胞增殖抑制实验:取对数生长期人脐静脉内皮细胞和Anip973细胞接种于96孔培养板中,培养12h后加入终浓度分别为0,10,20,30,40,50mg/L融合蛋白培养48h,弃上清,每孔中加入四甲基偶氮唑盐溶液穴5g/L雪20μL,继续孵育4h,检测吸光度,计算肿瘤生长抑制率。③取对数生长期人脐静脉内皮细胞、Anip973细胞,每种细胞分成两组:给药组加入终浓度为30mg/L穿膜肽-凋亡蛋白,对照组加入相同体积的磷酸盐缓冲液,继续培养48h。苏木精-伊红染色,光镜下观察细胞形态;溴乙啶/丫啶橙荧光染色观察细胞凋亡率。结果:①细胞增殖抑制实验结果表明浓度为10～50mg/L穿膜肽-凋亡蛋白对人脐静脉内皮细胞没有明显抑制作用,对Anip973细胞呈剂量依赖性抑制作用,半数抑制浓度为27mg/L。②30mg/L穿膜肽-凋亡蛋白作用48h后,人脐静脉内皮细胞形态没有显著改变,对照组和给药组的凋亡率分别为穴2.9±0.4雪%和穴3.1±0.6雪%,没有显著差异(P>0.05);Anip973细胞可见到细胞皱缩,染色质浓缩和出现凋亡小体等凋亡特征性形态变化,给药组和对照组凋亡率分别为穴36.8±1.7雪%和穴6.7±0.5雪%,差异显著(P<0.01)。结论:穿膜肽-凋亡蛋白能诱导肿瘤细胞凋亡,这种作用呈剂量依赖性,而对正常细胞没有毒性作用。

AIM： To express cell penetrate peptide （CPP）-apeptin fusion protein in E. Coil and to investigate its activity of inducing apeptosis of human lung adenocarcinoma cell line Anip973, and to understand whether it has dose dependent or not. METHODS： The experiment was carried out in the Department of Biochemistry and Molecular Biology, Harbin Medical University from August 2005 to January 2006. ①CPP-apeptin fusion protein was expressed in E. Coil and purified via IDA-Ni^2＋ affinity chromatography. ②Cytostasis trial： Endotheliocytes and Anip973 cells were gained from human umbilical vein in logarithmic growth phase and inoculated in 96-well culture plate. 12 hours after culture, the fusion protein of the concentration of 0,10,20,30, 40,50 mg/L was added to culture for 48 hours. Throwing away supernatant, and then added with （5 g/L） 20 p,L MTF solution in each well incubating for 4 hours to detect absorbance （A） and calculate inhibition rate of tumor growth. ③Endotheliocytes and Anip973 cells were gained from human umbilical vein in logarithmic growth phase, and each cell were assigned to two groups： Administration group was added with 30 mg/L CPP-apoptin; Phosphate buffer of the same volume was added in the control group cultured for 48 hours. Cell formation was observed under light microscope after hematoxylin and eosin （HE） staining. Apoptosis rate was observed with ethidium bromide/acridine orange （EB/AO） staining. RESULTS： ① MTT assay showed 10-50 mg/L CPP-apeptin had no obvi ous inhibitory effect on endotheliocytes of human umbilical vein, but a dose-dependent inhibitory effect on Anip973 cells, with an 50% inhibiting concentration of 27 mg/L. ② Endotheliocytes of human umbilical vein showed no morphological changes when treated with 30 mg/L CPP-apoptin for 48 hours. There were no significant differences between the apoptosis rate of control group and adsninistration group [ （2.9±0.4）% and （3.1±0.6）%, respectively, P 〈 0.05]. On the same condition, a series of typical morphological changes occurred to Anip973 cells,including cellular shrinkage, chromatin condensation,appearance of apoptosis body, and so on. The apeptosis rates in the administration group and the eontrol group were （36.8±1.7）% and （6.7±0.5）%, respectively, which had significantly differ±ence （P 〈 0.01 ）. CONCLUSION：. CPP-apeptin can induee tumor cells apeptosis, which shows dose-dependent effect, but has no toxic effect on normal cells.