人类白细胞抗原G基因表达水平下降对滋养细胞增殖和侵袭能力的影响

Effects of human leukocyte antigen-G on proliferation and invasion of JEG-3 cell line

目的探讨人类白细胞抗原G（HLA-G）基因表达水平下降对具有滋养细胞特点的绒毛膜癌（绒癌）细胞株JEG-3细胞增殖和侵袭能力的影响，了解HLA—G基因在子痫前期发生、发展中的作用。方法采用高表达HLA—G的JEG-3细胞进行体外培养，将培养好的细胞分为实验组（转染HLA—G小分子干扰RNA）、阴性对照组（转染阴性对照小分子干扰RNA）和空白对照组（不进行细胞转染，仅转染脂质体）3组进行细胞转染。应用RT—PCR技术和蛋白印迹法检测转染后各组JEG-3细胞中HIJA—G mRNA和蛋白的表达水平；四甲基偶氮唑蓝（MTT）比色法、流式细胞技术、穿膜小室侵袭实验分别检测转染后各组JEG-3细胞增殖能力、细胞周期、凋亡抑制率和侵袭能力的变化。结果（1）转染后JEG-3细胞中HLA-G mRNA和蛋白的表达水平，实验组分别为0．0013±0．0014、0．0163±0．0007，分别与空白对照组（分别为0．1923±0．0384、0．2184±0．0153）比较，差异均有统计学意义（P〈0．05）；阴性对照组分别为（0．1606±0．0133、0．2020±0．0132），分别与空白对照组比较，差异均无统计学意义（P〉0．05）。（2）转染48h后JEG-3细胞的增殖能力，实验组的积分吸光度（IA）值为0．44±0．04，与空白对照组（0．75±0．13）比较，差异有统计学意义（P〈0．01）；阴性对照组为0．69±0．10，与空白对照组比较，差异无统计学意义（P〉0．05）。（3）转染72h后的细胞周期比例，实验组G2／M期细胞比例为（10．9±2．2）％，低于空白对照组[（15．4±1．9）％]；S期细胞比例为（58．6±0．8）％，高于空白对照组[（52．9±2．3）％]。两组分别比较，差异均有统计学意义（P〈0．05）。（4）转染72h后的细胞凋亡率，实验组为（14．5±2．7）％，高于阴性对照组[（5．3±1．1）％]和空白对照组[（4．7±0．6）％]，差异均有统计学意义（P〈0．01）。（5）转染72h后穿膜小室中的穿膜细胞数，实验组为（121±12）个，低于空白对照组[（452±17）个]，差异有统计学意义（P〈0．01）。结论HLA-G基因表达水平的下降可以影响滋养细胞的增殖、凋亡、侵袭和细胞周期。HLA-G基因可能通过调节滋养细胞增殖、侵袭等过程参与子痫前期的发生。

Objective 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. Methods 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. Results （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 differenee eompared with blank eontrol group（P 〉 0. 05 ）, （ 2 ） The integral absorbanee （ IA ） values of the HLA-G transfeetion group and blank eontrol group were 0. 44 ± 0. 04 and 0. 75 ± 0. 13 respeetively, whieh was signifieantly different（P 〈0. 01 ） ;the IA value of negative control group was 0. 69 ±0. 10, whieh was not signifieantly different eompared with blank group（ P 〉 0. 05 ）. （ 3 ） The ratios of G2/M and S phase eells in transfeetion group were（ 10.9 ± 2.2 ） % and （ 58. 6 ± 0. 8 ） % respeetively, signifieantly different eompared 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 ）. Conclusion HLA-G is probably involved in the onset of preeclampsia by regulating proliferation and invasion of trophoblast.