Liu Hai-yan, Gu Wei-rong, Li Xiao-tian
Department of Obstetrics, Obstetrics and Gynecology Hospital, Fudan University, Shanghai 200011, China.
Zhonghua Fu Chan Ke Za Zhi. 2008 Jun;43(6):445-50.
To investigate the effect of human leukocyte antigen-G (HLA-G) on the growth and invasion of JEG-3 cell line and the role of HLA-G in the onset and development of pre-eclampsia.
The experiment was composed of three groups: groups of transfection, negative control and blank control, which corresponded to groups of HLA-G siRNA transfection, negative siRNA transfection and no transfection. HLA-G overexpressed choriocarcinoma cell line JEG-3 was used. The role of HLA-G in JEG-3 cell monolayer was examined by RNA interference technology using HLA-G specific small interfering RNA (siRNA). Expression of HLA-G was detected by reverse transcriptase-polymerase chain reaction and western blot analysis. Changes of cell cycle, apoptosis, proliferation and invasion were respectively detected by methyl thiazolyl tetrazolium (MTT), flow cytometry assay and transwell test.
(1) The mRNA and protein levels of HLA-G control group and blank control group were 0.0013 +/- 0.0014, 0.0163 +/- 0.0007 and 0.1923 +/- 0.0384, 0.2184 +/- 0.0153, respectively, which were both significantly different (P<0.05); the number of negative transfection group was 0.1606 +/- 0.0133 and 0.2020 +/- 0.0132, which had no significant difference compared with blank control group (P>0.05). (2) The integral absorbance (IA) values of the HLA-G transfection group and blank control group were 0.44 +/- 0.04 and 0.75 +/- 0.13 respectively, which was significantly different (P<0.01); the IA value of negative control group was 0.69 +/- 0.10, which was not significantly different compared with blank group (P>0.05). (3) The ratios of G2/M and S phase cells in transfection group were (10.9 +/- 2.2)% and (58.6 +/- 0.8)% respectively, significantly different compared with the blank control group [(15.4 +/- 1.9)% and (52.9 +/- 2.3)% respectively; P<0.01]. (4) The ratio of early apoptosis cells in transfection group [(14.5 +/- 2.7)%] was significantly increased compared with negative [(5.3 +/- 1.1)%] and blank control group [(4.7 +/- 0.6)% ; P<0.01]. (5) The invasion number of transfection group and blank control group were 121+/- 12 and 452 +/- 17 respectively, with a significant difference between them (P<0.01).
HLA-G is probably involved in the onset of preeclampsia by regulating proliferation and invasion of trophoblast.
探讨人类白细胞抗原-G(HLA-G)对JEG-3细胞系生长和侵袭的影响以及HLA-G在子痫前期发病及发展中的作用。
实验分为三组:转染组、阴性对照组和空白对照组,分别对应HLA-G小干扰RNA(siRNA)转染组、阴性siRNA转染组和未转染组。采用HLA-G过表达的绒毛膜癌细胞系JEG-3。运用针对HLA-G的特异性小干扰RNA(siRNA),通过RNA干扰技术研究HLA-G在JEG-3细胞单层中的作用。采用逆转录聚合酶链反应和蛋白质免疫印迹分析检测HLA-G的表达。分别通过噻唑蓝(MTT)、流式细胞术检测及Transwell实验检测细胞周期、凋亡、增殖及侵袭的变化。
(1)HLA-G对照组和空白对照组的mRNA水平分别为0.0013±0.0014、0.0163±0.0007,蛋白质水平分别为0.1923±0.0384、0.2184±0.0153,差异均有统计学意义(P<0.05);阴性转染组的mRNA水平为0.1606±0.0133,蛋白质水平为0.2020±0.0132,与空白对照组相比差异无统计学意义(P>0.05)。(2)HLA-G转染组和空白对照组的积分吸光度(IA)值分别为0.44±0.04、0.75±0.13,差异有统计学意义(P<0.01);阴性对照组的IA值为0.69±0.10,与空白组相比差异无统计学意义(P>0.05)。(3)转染组G2/M期和S期细胞比例分别为(10.9±2.2)%和(58.6±0.8)%,与空白对照组[分别为(15.4±1.9)%和(52.9±2.3)%]相比差异有统计学意义(P<0.01)。(4)转染组早期凋亡细胞比例[(14.5±2.7)%]较阴性对照组[(5.3±1.1)%]和空白对照组[(4.7±0.6)%]明显升高(P<0.01)。(5)转染组和空白对照组的侵袭细胞数分别为121±12和452±17,差异有统计学意义(P<0.01)。
HLA-G可能通过调节滋养细胞的增殖和侵袭参与子痫前期的发病。