HLA抗原%组织相容性抗原Ⅰ类%滋养层%p38丝裂原活化蛋白激酶类%RNA,小分子干扰%先兆子痫
HLA抗原%組織相容性抗原Ⅰ類%滋養層%p38絲裂原活化蛋白激酶類%RNA,小分子榦擾%先兆子癇
HLA항원%조직상용성항원Ⅰ류%자양층%p38사렬원활화단백격매류%RNA,소분자간우%선조자간
HLA antigens%Histocompatibility antigens class Ⅰ%Trophoblasts%p38 Mitogenactivated protein kinases%RNA,small interfering%Pre-eclampsia
目的 探讨人类白细胞抗原G(HLA-G)基因表达变化对滋养细胞侵袭力、滋养细胞中p38丝裂原活化蛋白激酶(p38MAPK)及磷酸化p38MAPK(p-p38MAPK)蛋白表达的影响.方法 对滋养细胞株HTR-8/SVneo细胞进行体外培养并分组,将细胞分为实验组[转染HLA-G小分子干扰RNA(siRNA)]、阴性对照组(转染阴性对照siRNA)和空白对照组(仅转染脂质体2000).分别用逆转录(RT)PCR技术测定转染后细胞中HLA-G mRNA的表达;蛋白印迹法测定HLA-G蛋白的表达来验证核糖核酸干扰(RNAi)敲减效率;穿膜小室侵袭实验检测细胞的侵袭力;蛋白印迹法检测p-p38MAPK积分吸光度(IA)与p38MAPK IA的比值;检测加入抑制剂SB203580后穿膜小室的侵袭细 胞数.结果 (1)HLA-G mRNA表达:实验组为0.26±0.08,阴性对照组为0.71±0.11,空白对照组为0.79±0.07.实验组与阴性对照组比较,差异有统计学意义(P＜0.01);阴性对照组与空白对照组比较,差异无统计学意义(P＞0.05);实验组HLA-G mRNA表达抑制率为(69.8±6.3)%,阴性对照组HLA-G mRNA表达抑制率为(14.9±2.2)%,空白对照组为0.(2)HLA-G蛋白表达:实验组为0.20±0.15,阴性对照组为0.75±0.12,空白对照组为0.76±0.21.实验组与阴性对照组比较,差异有统计学意义(P＜0.01);阴性对照组与空白对照组比较,差异无统计学意义(P＞0.05);实验组HLA-G蛋白表达抑制率为(81.1±14.4)%,阴性对照组HLA-G蛋白表达抑制率为(18.0±7.7)%,空白对照组为0,实验组与阴性对照组比较,差异有统计学意义(P＜0.01).(3)穿膜小室下室的侵袭细胞数:实验组为(57±38)个,阴性对照组为(364±79)个,空白对照组为(260±84)个.实验组与阴性对照组比较,差异有统计学意义(P＜0.01);阴性对照组与空白对照组比较,差异无统计学意义(P＞0.05).(4)p-p38MAPK与p38MAPK蛋白比值:实验组为0.74±0.04,阴性对照组为0.47±0.09,空白对照组为0.36±0.21.实验组比值明显高于其他两组,差异有统计学意义(P＜0.01).(5)加入抑制剂前、后p-p38MAPK与p38MAPK蛋白比值:实验组分别为0.89±0.09、0.16±0.04,阴性对照组分别为0.76±0.08、0.14±0.03,空白对照组分别为0.51±0.05、0.03±0.01,各组加入抑制剂SB203580前、后p-p38MAPK与p38MAPK比值分别比较,差异均有统计学意义(P＜0.01).(6)加入抑制剂前、后穿膜小室下室的滋养细胞侵袭力:实验组分别为(51±13)和(90±21)个,前后比较,差异有统计学意义(P＜0.01);阴性对照组分别为(290±52)和(298±33)个,前后比较,差异无统计学意义(P＞0.05);空白对照组分别为(290±73)和(264±64)个,前后比较,差异无统计学意义(P＞0.05).结论 HLA-G基因可能通过p38MAPK信号通路调节滋养细胞的侵袭力.提示滋养细胞HLA-G低表达可导致病理妊娠的发生.
目的 探討人類白細胞抗原G(HLA-G)基因錶達變化對滋養細胞侵襲力、滋養細胞中p38絲裂原活化蛋白激酶(p38MAPK)及燐痠化p38MAPK(p-p38MAPK)蛋白錶達的影響.方法 對滋養細胞株HTR-8/SVneo細胞進行體外培養併分組,將細胞分為實驗組[轉染HLA-G小分子榦擾RNA(siRNA)]、陰性對照組(轉染陰性對照siRNA)和空白對照組(僅轉染脂質體2000).分彆用逆轉錄(RT)PCR技術測定轉染後細胞中HLA-G mRNA的錶達;蛋白印跡法測定HLA-G蛋白的錶達來驗證覈糖覈痠榦擾(RNAi)敲減效率;穿膜小室侵襲實驗檢測細胞的侵襲力;蛋白印跡法檢測p-p38MAPK積分吸光度(IA)與p38MAPK IA的比值;檢測加入抑製劑SB203580後穿膜小室的侵襲細 胞數.結果 (1)HLA-G mRNA錶達:實驗組為0.26±0.08,陰性對照組為0.71±0.11,空白對照組為0.79±0.07.實驗組與陰性對照組比較,差異有統計學意義(P＜0.01);陰性對照組與空白對照組比較,差異無統計學意義(P＞0.05);實驗組HLA-G mRNA錶達抑製率為(69.8±6.3)%,陰性對照組HLA-G mRNA錶達抑製率為(14.9±2.2)%,空白對照組為0.(2)HLA-G蛋白錶達:實驗組為0.20±0.15,陰性對照組為0.75±0.12,空白對照組為0.76±0.21.實驗組與陰性對照組比較,差異有統計學意義(P＜0.01);陰性對照組與空白對照組比較,差異無統計學意義(P＞0.05);實驗組HLA-G蛋白錶達抑製率為(81.1±14.4)%,陰性對照組HLA-G蛋白錶達抑製率為(18.0±7.7)%,空白對照組為0,實驗組與陰性對照組比較,差異有統計學意義(P＜0.01).(3)穿膜小室下室的侵襲細胞數:實驗組為(57±38)箇,陰性對照組為(364±79)箇,空白對照組為(260±84)箇.實驗組與陰性對照組比較,差異有統計學意義(P＜0.01);陰性對照組與空白對照組比較,差異無統計學意義(P＞0.05).(4)p-p38MAPK與p38MAPK蛋白比值:實驗組為0.74±0.04,陰性對照組為0.47±0.09,空白對照組為0.36±0.21.實驗組比值明顯高于其他兩組,差異有統計學意義(P＜0.01).(5)加入抑製劑前、後p-p38MAPK與p38MAPK蛋白比值:實驗組分彆為0.89±0.09、0.16±0.04,陰性對照組分彆為0.76±0.08、0.14±0.03,空白對照組分彆為0.51±0.05、0.03±0.01,各組加入抑製劑SB203580前、後p-p38MAPK與p38MAPK比值分彆比較,差異均有統計學意義(P＜0.01).(6)加入抑製劑前、後穿膜小室下室的滋養細胞侵襲力:實驗組分彆為(51±13)和(90±21)箇,前後比較,差異有統計學意義(P＜0.01);陰性對照組分彆為(290±52)和(298±33)箇,前後比較,差異無統計學意義(P＞0.05);空白對照組分彆為(290±73)和(264±64)箇,前後比較,差異無統計學意義(P＞0.05).結論 HLA-G基因可能通過p38MAPK信號通路調節滋養細胞的侵襲力.提示滋養細胞HLA-G低錶達可導緻病理妊娠的髮生.
목적 탐토인류백세포항원G(HLA-G)기인표체변화대자양세포침습력、자양세포중p38사렬원활화단백격매(p38MAPK)급린산화p38MAPK(p-p38MAPK)단백표체적영향.방법 대자양세포주HTR-8/SVneo세포진행체외배양병분조,장세포분위실험조[전염HLA-G소분자간우RNA(siRNA)]、음성대조조(전염음성대조siRNA)화공백대조조(부전염지질체2000).분별용역전록(RT)PCR기술측정전염후세포중HLA-G mRNA적표체;단백인적법측정HLA-G단백적표체래험증핵당핵산간우(RNAi)고감효솔;천막소실침습실험검측세포적침습력;단백인적법검측p-p38MAPK적분흡광도(IA)여p38MAPK IA적비치;검측가입억제제SB203580후천막소실적침습세 포수.결과 (1)HLA-G mRNA표체:실험조위0.26±0.08,음성대조조위0.71±0.11,공백대조조위0.79±0.07.실험조여음성대조조비교,차이유통계학의의(P＜0.01);음성대조조여공백대조조비교,차이무통계학의의(P＞0.05);실험조HLA-G mRNA표체억제솔위(69.8±6.3)%,음성대조조HLA-G mRNA표체억제솔위(14.9±2.2)%,공백대조조위0.(2)HLA-G단백표체:실험조위0.20±0.15,음성대조조위0.75±0.12,공백대조조위0.76±0.21.실험조여음성대조조비교,차이유통계학의의(P＜0.01);음성대조조여공백대조조비교,차이무통계학의의(P＞0.05);실험조HLA-G단백표체억제솔위(81.1±14.4)%,음성대조조HLA-G단백표체억제솔위(18.0±7.7)%,공백대조조위0,실험조여음성대조조비교,차이유통계학의의(P＜0.01).(3)천막소실하실적침습세포수:실험조위(57±38)개,음성대조조위(364±79)개,공백대조조위(260±84)개.실험조여음성대조조비교,차이유통계학의의(P＜0.01);음성대조조여공백대조조비교,차이무통계학의의(P＞0.05).(4)p-p38MAPK여p38MAPK단백비치:실험조위0.74±0.04,음성대조조위0.47±0.09,공백대조조위0.36±0.21.실험조비치명현고우기타량조,차이유통계학의의(P＜0.01).(5)가입억제제전、후p-p38MAPK여p38MAPK단백비치:실험조분별위0.89±0.09、0.16±0.04,음성대조조분별위0.76±0.08、0.14±0.03,공백대조조분별위0.51±0.05、0.03±0.01,각조가입억제제SB203580전、후p-p38MAPK여p38MAPK비치분별비교,차이균유통계학의의(P＜0.01).(6)가입억제제전、후천막소실하실적자양세포침습력:실험조분별위(51±13)화(90±21)개,전후비교,차이유통계학의의(P＜0.01);음성대조조분별위(290±52)화(298±33)개,전후비교,차이무통계학의의(P＞0.05);공백대조조분별위(290±73)화(264±64)개,전후비교,차이무통계학의의(P＞0.05).결론 HLA-G기인가능통과p38MAPK신호통로조절자양세포적침습력.제시자양세포HLA-G저표체가도치병리임신적발생.
Objective To investigate the role of human leukocyte antigen-G ( HLA-G ) on the invasion and the molecular mechanism involved in this cellular progress in HTR-8/SVneo cell line. Methods There were three groups: groups of transfection, negative control and blank control, which corresponding to treatment by HLA-G specific siRNA, negative siRNA and only lipofectamine 2000 using lipofection technology in HTR-8/SVneo cell line. The efficiency of down-regulated of HLA-G was detected by reverse transcription-polymerase chain reaction and western blot analysis in mRNA and protein level,respectively. Changes of p38 mitogen-activated protein kinases (p-p38MAPK)/p38MAPK protein levels and the cell invasion were respectively detected by western blot analysis and transwell test. Results ( 1 ) The mRNA levels of HLA-G transfection group, negative control group and blank control group were 0. 26 ±0. 08, 0. 71 ±0. 11, 0. 79 ±0. 07, respectively. There was significant difference between transfection group and negative control group ( P ＜ 0. 01 ), while there was no significant difference between negative control group and blank control group ( P ＞ 0. 05 ). The efficiencies of down-regulated of HLA-G were ( 69. 8 ±6. 3)%, ( 14. 9 ± 2. 2 )%, 0 in transfection group, negative control group and blank control group respectively in mRNA level. (2)In protein levels, HLA-G were 0. 20 ±0. 15, 0. 75 ±0. 12, 0. 76 ±0. 21 in transfection group, negative control group and blank control group, respectively. There was significant difference between transfection group and negative control group ( P ＜ 0. 01 ), whereas there was no significant difference between negative control group and blank control group ( P ＞ 0. 05 ). The efficiencies of down-regulated of HLA-G were (81. 1 ± 14.4)%, ( 18.0 ± 7.7)%, 0 in transfection group, negative control group and blank control group respectively. ( 3 ) The invasive number of transfection group, negative control group and blank control group were 57 ± 38,364 ± 79 and 260 ± 84, respectively, with a significant difference between transfection group and negative control group (P ＜ 0. 01 ). There was no significant difference between negative control group and blank control group ( P ＞ 0. 05 ). ( 4 ) The p-p38MAPK/p38MAPK values of the HLA-G transfection group, negative control group and blank control group were 0. 74 ±0.04, 0. 47 ± 0. 09 and 0. 36 ± 0. 21, respectively. HLA-G transfection group was significantly different compared with the other two groups( P ＜0. 01 ). (5)Without or with SB203580, the p-p38MAPK/ p38MAPK values of the HLA-G transfection group were 0. 89 ± 0. 09 and 0. 16 ± 0. 04, the values of negative control group and blank control group were 0.76 ±0.08, 0. 14 ±0.03 and 0.51 ±0.05, 0.03 ±0.01, respectively. There was significant difference between without SB203580 and with SB203580 ( P ＜ 0. 01 ). (6)Without or with SB203580, the invasive number of transfection group were 51 ± 13 and 90 ± 21 ,respectively,which was significantly different ( P ＜ 0. 01 ). The invasive number of negative control group and blank control group were 290 ± 52, 298 ± 33 and 290 ± 73, 264 ± 64, respeczively, which was no significant difference between without SB203580 and with SB203580 (P ＞ 0. 05 ). Conclusions HLA-G gene may regulate invasion of trophoblast-derived cell line HTR-8/SVneo via p38MAPK signaling pathway. The lower expression of HLA-G in trophoblast cells may lead to the occurrence of pathologic pregnancy.
