凋亡、坏死合体滋养细胞微粒对内皮细胞增殖与凋亡的影响

目的:探讨凋亡、坏死合体滋养细胞微粒对人脐静脉内皮细胞(HUVECs)增殖、凋亡的影响。方法:培养原代HUVECs,物理方法诱导滋养细胞凋亡、坏死。采用三步超速离心法制备凋亡合体滋养细胞微粒(a STBM)、坏死合体滋养细胞微粒(n STBM)(实验组),同时制备RBC膜(对照组),并检测胎盘碱性磷酸酶(PLAP)含量。将不同浓度(20、80、320μg/ml)a STBM、n STBM和RBC膜分别与HUVECs共同孵育4、16、32h。采用MTT法检测细胞增殖抑制率,采用流式细胞仪AV-PI双标记观察细胞凋亡情况。结果:(1)80μg/ml a STBM组作用4、16、32h时的细胞增殖抑制率分别为29.19±0.47、44.73±0.69、54.01±1.61;80μg/ml n STBM组作用4、16、32h的细胞增殖抑制率分别为57.15±0.70、64.35±0.89、71.67±0.63。a STBM、n STBM均能明显抑制HUVEC增殖,抑制作用呈浓度时间依赖性;n STBM对HUVECs的增殖抑制性明显高于a STBM,差异有统计学意义(P0.001)。RBC膜处理组对HUVECs增殖无明显影响,与空白组比较差异无统计学意义(P0.05)。(2)80μg/ml a STBM组的细胞早期凋亡率、晚期凋亡-坏死率分别为29.05%和7.46%。80μg/ml n STBM组分别为15.93%和30.31%。RBC膜处理组对HUVECs的凋亡无明显影响,与空白对照组相比,差异无统计学意义(P0.05)。a STBM、n STBM组均能引起HUVECs凋亡,n STBM作用HUVECs的晚期凋亡率高于a STBM组,两者有统计学意义(P0.001)。a STBM主要诱导HUVECs早期凋亡。结论:a STBM、n STBM对内皮细胞有抑制细胞增殖作用,a STBM主要以促进内皮细胞早期凋亡为主,而n STBM以内皮细胞晚期凋亡为主,n STBM对内皮细胞造成的损害强于a STBM。

Apoptotic or necrotic syncytiotrophoblast micro-particles induce human umbilical vein endothelial cells proliferation inhibition and apoptosis

Objective: To study the effects of a STBM and n STBM on the proliferation and apoptosis of human umbilical vein endothelial cells. Methods: Primary culture human umbilical vein endothelial cells,trophoblasts were induced necrosis and apoptosis,which were used to prepare a STBM and n STBM by modification of the method of Smith et al. RBC ghost particles was prepared also. The enrichment of PALP protein in STBM was used to assess the purity of STBM by ELISA( Enzyme linked immunosorbent assay). MTT( Methyl thiazolyl tetrazolium assay) was used to detect the inhibition rate of cell proliferation of human umbilical vein endothelial cells which was pulsed with a STBM and n STBM at different concentrations( 20,80,320 ug /ml),in groups after( 4,16,32) hours. The apoptosis was analyzed by flow cytometry. Results:( 1) Cell proliferation inhibition: Incubation with 80 ug /ml n STBM in 4,16,32 hours,produced higher inhibitory effect on cell proliferation [( 29. 19 ± 0. 47),( 44. 73 ± 0. 69),( 54. 01 ±1. 61) ],vs 80μg / ml a STBM[( 57. 15 ± 0. 70),( 64. 35 ± 0. 89),( 71. 67 ± 0. 63) ]( P < 0.001). The rate of cell proliferation with a STBM and n STBM was significantly higher than the red cell membrance,however,the rate of cell proliferation with n STBM is higher than a STBM( P< 0. 001).( 2) The apoptosis rates: Incubation with 80 ug / ml a STBM,the early apoptosis rate was 29. 05% 、the late apoptosis rate was 7. 46%,vs 80μg / ml n STBM( 15. 93%,30. 31%).The red cell membrance produce no change between the normal group( P > 0. 05). The n STBM produce late apoptosis significantly higer than the a STBM( P < 0. 001). Conclusion: Apoptotic and necrotic syncytiotrophoblast micro-particles both induce human umbilical vein endothelial cells proliferation inhibition and apoptosis,however,a STBM induce cells early apoptosis,n STBM late apoptosis. So,n STBM but not a STBM injure cells more.