中华妇产科杂志
中華婦產科雜誌
중화부산과잡지
Chinese Journal of Obstetrics and Gynecology
2015年
10期
770-776
,共7页
子宫内膜异位症%RNA,小分子干扰%环氧化酶2%血管内皮生长因子A%基质金属蛋白酶9%6-酮前列腺素F1α%细胞凋亡%间质细胞
子宮內膜異位癥%RNA,小分子榦擾%環氧化酶2%血管內皮生長因子A%基質金屬蛋白酶9%6-酮前列腺素F1α%細胞凋亡%間質細胞
자궁내막이위증%RNA,소분자간우%배양화매2%혈관내피생장인자A%기질금속단백매9%6-동전렬선소F1α%세포조망%간질세포
Endometriosis%RNA%small interfering%Cyclooxygenase 2%Vascular endothelial growth factor A%Matrix metalloproteinase 9%6-Ketoprostaglandin F1 alpha%Apoptosis%Stromal cells
目的 应用小分子干扰RNA(siRNA)靶向沉默环氧合酶2(COX-2)基因,观察对子宫内膜异位症(内异症)在位、异位子宫内膜间质细胞(ESC)COX-2、血管内皮生长因子(VEGF)、基质金属蛋白酶9(MMP-9)表达、6-酮前列腺素F1α(6-keto-PGF1α)含量及细胞凋亡的影响.方法 分离、原代培养内异症患者在位、异位ESC各30份,将在位、异位ESC分别分为3组(n=10):干扰组、阴性对照组、空白对照组,前两组分别转染COX-2 siRNA转染复合物和阴性对照转染复合物,空白对照组不进行任何转染;并以正常ESC 10份作为正常对照组.应用逆转录实时定量PCR技术和蛋白印迹法分别检测转染前、后各组在位、异位ESC中COX-2、VEGF、MMP-9 mRNA和蛋白的表达;ELISA法检测各组ESC中6-keto-PGF1α的含量;流式细胞仪检测各组ESC的细胞凋亡率.结果 转染siRNA沉默COX-2基因后,干扰组异位ESC COX-2、VEGF、MMP-9 mRNA相对表达量分别为0.75±0.12、1.62±0.47、0.88±0.25,干扰组在位ESC分别为0.87±0.06、1.76±0.59、1.04±0.32,干扰组异位ESC COX-2、VEGF、MMP-9蛋白表达水平分别为0.323±0.018、0.474±0.016、0.339±0.009,干扰组在位ESC分别为0.457±0.019、0.500± 0.012、0.361±0.008;与阴性对照组和空白对照组相比,干扰组在位、异位ESC中COX-2、VEGF、MMP-9 mRNA的相对表达量和蛋白的表达水平均降低,分别比较,差异均有统计学意义(P<0.05);与干扰组在位ESC相比,其异位ESC中COX-2、VEGF、MMP-9 mRNA的相对表达量和蛋白的表达水平下降更为明显(P<0.05);阴性对照组与空白对照组相比,其在位、异位ESC中COX-2、VEGF、MMP-9 mRNA的相对表达量和蛋白的表达水平分别比较,差异均无统计学意义(P>0.05).ELISA法检测结果显示,空白对照组在位、异位ESC的6-keto-PGF1α含量分别为(32.4±2.6)、(38.2±3.7)pg/ml,干扰组在位、异位ESC 6-keto-PGF1α含量分别为(17.1±2.4)、(20.9±2.7)pg/ml;与正常对照组[(17.7±1.9)pg/ml]相比,空白对照组在位、异位ESC的6-keto-PGF1α含量均升高,分别比较,差异均有统计学意义(P<0.05),且异位ESC的6-keto-PGF1α含量高于在位ESC(P<0.05);与阴性对照组和空白对照组相比,干扰组在位、异位ESC的6-keto-PGF1α含量均降低,分别比较,差异均有统计学意义(P<0.05),与干扰组异位ESC相比,其在位ESC的6-keto-PGF1α含量下降不明显(P>0.05).细胞凋亡检测结果显示,干扰组在位、异位ESC细胞凋亡率分别为(33.76±0.06)%、(47.18±0.12)%;与阴性对照组和空白对照组相比,干扰组在位、异位ESC细胞凋亡率增加,分别比较,差异均有统计学意义(P<0.01);与干扰组在位ESC相比,其异位ESC细胞凋亡率更高(P<0.05);与空白对照组相比,阴性对照组在位、异位ESC细胞凋亡率变化不显著(P>0.05).结论 siRNA靶向沉默COX-2基因能明显抑制内异症在位、异位ESC中COX-2、VEGF、MMP-9 mRNA和蛋白的表达,显著增加细胞凋亡率,并通过抑制COX-2活性而降低6-keto-PGF1α含量,且异位ESC均较在位ESC变化更明显.
目的 應用小分子榦擾RNA(siRNA)靶嚮沉默環氧閤酶2(COX-2)基因,觀察對子宮內膜異位癥(內異癥)在位、異位子宮內膜間質細胞(ESC)COX-2、血管內皮生長因子(VEGF)、基質金屬蛋白酶9(MMP-9)錶達、6-酮前列腺素F1α(6-keto-PGF1α)含量及細胞凋亡的影響.方法 分離、原代培養內異癥患者在位、異位ESC各30份,將在位、異位ESC分彆分為3組(n=10):榦擾組、陰性對照組、空白對照組,前兩組分彆轉染COX-2 siRNA轉染複閤物和陰性對照轉染複閤物,空白對照組不進行任何轉染;併以正常ESC 10份作為正常對照組.應用逆轉錄實時定量PCR技術和蛋白印跡法分彆檢測轉染前、後各組在位、異位ESC中COX-2、VEGF、MMP-9 mRNA和蛋白的錶達;ELISA法檢測各組ESC中6-keto-PGF1α的含量;流式細胞儀檢測各組ESC的細胞凋亡率.結果 轉染siRNA沉默COX-2基因後,榦擾組異位ESC COX-2、VEGF、MMP-9 mRNA相對錶達量分彆為0.75±0.12、1.62±0.47、0.88±0.25,榦擾組在位ESC分彆為0.87±0.06、1.76±0.59、1.04±0.32,榦擾組異位ESC COX-2、VEGF、MMP-9蛋白錶達水平分彆為0.323±0.018、0.474±0.016、0.339±0.009,榦擾組在位ESC分彆為0.457±0.019、0.500± 0.012、0.361±0.008;與陰性對照組和空白對照組相比,榦擾組在位、異位ESC中COX-2、VEGF、MMP-9 mRNA的相對錶達量和蛋白的錶達水平均降低,分彆比較,差異均有統計學意義(P<0.05);與榦擾組在位ESC相比,其異位ESC中COX-2、VEGF、MMP-9 mRNA的相對錶達量和蛋白的錶達水平下降更為明顯(P<0.05);陰性對照組與空白對照組相比,其在位、異位ESC中COX-2、VEGF、MMP-9 mRNA的相對錶達量和蛋白的錶達水平分彆比較,差異均無統計學意義(P>0.05).ELISA法檢測結果顯示,空白對照組在位、異位ESC的6-keto-PGF1α含量分彆為(32.4±2.6)、(38.2±3.7)pg/ml,榦擾組在位、異位ESC 6-keto-PGF1α含量分彆為(17.1±2.4)、(20.9±2.7)pg/ml;與正常對照組[(17.7±1.9)pg/ml]相比,空白對照組在位、異位ESC的6-keto-PGF1α含量均升高,分彆比較,差異均有統計學意義(P<0.05),且異位ESC的6-keto-PGF1α含量高于在位ESC(P<0.05);與陰性對照組和空白對照組相比,榦擾組在位、異位ESC的6-keto-PGF1α含量均降低,分彆比較,差異均有統計學意義(P<0.05),與榦擾組異位ESC相比,其在位ESC的6-keto-PGF1α含量下降不明顯(P>0.05).細胞凋亡檢測結果顯示,榦擾組在位、異位ESC細胞凋亡率分彆為(33.76±0.06)%、(47.18±0.12)%;與陰性對照組和空白對照組相比,榦擾組在位、異位ESC細胞凋亡率增加,分彆比較,差異均有統計學意義(P<0.01);與榦擾組在位ESC相比,其異位ESC細胞凋亡率更高(P<0.05);與空白對照組相比,陰性對照組在位、異位ESC細胞凋亡率變化不顯著(P>0.05).結論 siRNA靶嚮沉默COX-2基因能明顯抑製內異癥在位、異位ESC中COX-2、VEGF、MMP-9 mRNA和蛋白的錶達,顯著增加細胞凋亡率,併通過抑製COX-2活性而降低6-keto-PGF1α含量,且異位ESC均較在位ESC變化更明顯.
목적 응용소분자간우RNA(siRNA)파향침묵배양합매2(COX-2)기인,관찰대자궁내막이위증(내이증)재위、이위자궁내막간질세포(ESC)COX-2、혈관내피생장인자(VEGF)、기질금속단백매9(MMP-9)표체、6-동전렬선소F1α(6-keto-PGF1α)함량급세포조망적영향.방법 분리、원대배양내이증환자재위、이위ESC각30빈,장재위、이위ESC분별분위3조(n=10):간우조、음성대조조、공백대조조,전량조분별전염COX-2 siRNA전염복합물화음성대조전염복합물,공백대조조불진행임하전염;병이정상ESC 10빈작위정상대조조.응용역전록실시정량PCR기술화단백인적법분별검측전염전、후각조재위、이위ESC중COX-2、VEGF、MMP-9 mRNA화단백적표체;ELISA법검측각조ESC중6-keto-PGF1α적함량;류식세포의검측각조ESC적세포조망솔.결과 전염siRNA침묵COX-2기인후,간우조이위ESC COX-2、VEGF、MMP-9 mRNA상대표체량분별위0.75±0.12、1.62±0.47、0.88±0.25,간우조재위ESC분별위0.87±0.06、1.76±0.59、1.04±0.32,간우조이위ESC COX-2、VEGF、MMP-9단백표체수평분별위0.323±0.018、0.474±0.016、0.339±0.009,간우조재위ESC분별위0.457±0.019、0.500± 0.012、0.361±0.008;여음성대조조화공백대조조상비,간우조재위、이위ESC중COX-2、VEGF、MMP-9 mRNA적상대표체량화단백적표체수평균강저,분별비교,차이균유통계학의의(P<0.05);여간우조재위ESC상비,기이위ESC중COX-2、VEGF、MMP-9 mRNA적상대표체량화단백적표체수평하강경위명현(P<0.05);음성대조조여공백대조조상비,기재위、이위ESC중COX-2、VEGF、MMP-9 mRNA적상대표체량화단백적표체수평분별비교,차이균무통계학의의(P>0.05).ELISA법검측결과현시,공백대조조재위、이위ESC적6-keto-PGF1α함량분별위(32.4±2.6)、(38.2±3.7)pg/ml,간우조재위、이위ESC 6-keto-PGF1α함량분별위(17.1±2.4)、(20.9±2.7)pg/ml;여정상대조조[(17.7±1.9)pg/ml]상비,공백대조조재위、이위ESC적6-keto-PGF1α함량균승고,분별비교,차이균유통계학의의(P<0.05),차이위ESC적6-keto-PGF1α함량고우재위ESC(P<0.05);여음성대조조화공백대조조상비,간우조재위、이위ESC적6-keto-PGF1α함량균강저,분별비교,차이균유통계학의의(P<0.05),여간우조이위ESC상비,기재위ESC적6-keto-PGF1α함량하강불명현(P>0.05).세포조망검측결과현시,간우조재위、이위ESC세포조망솔분별위(33.76±0.06)%、(47.18±0.12)%;여음성대조조화공백대조조상비,간우조재위、이위ESC세포조망솔증가,분별비교,차이균유통계학의의(P<0.01);여간우조재위ESC상비,기이위ESC세포조망솔경고(P<0.05);여공백대조조상비,음성대조조재위、이위ESC세포조망솔변화불현저(P>0.05).결론 siRNA파향침묵COX-2기인능명현억제내이증재위、이위ESC중COX-2、VEGF、MMP-9 mRNA화단백적표체,현저증가세포조망솔,병통과억제COX-2활성이강저6-keto-PGF1α함량,차이위ESC균교재위ESC변화경명현.
Objective To investigate the effect of targeted interruption of cyclooxygenase-2 (COX-2) gene by small interference RNA (siRNA) on the expression of COX-2, vascular endothelial growth factor (VEGF) and matrix metalloproteinase-9 (MMP-9) in eutopic and ectopic endometrial stromal cells (ESC) with endometriosis, and the effect on the content of 6-keto-prostaglandin-F1α (6-keto-PGF1α, metabolites of COX) and the apoptosis of eutopic and ectopic ESC with endometriosis. Methods Ectopic and eutopic ESC from 30 women with endometriosis were isolated and cultured respectively. Then, ESC were classified into three groups: interference group, negative control group and blank control group. ESC in interference group were injected into siRNA transfection complex while ESC in negative control group were injected into negative control transfection complex. ESC from 10 participants without endometriosis were the normal control group. The mRNA and protein expression of COX-2, VEGF, MMP-9 in pre-transfected and post-transfected eutopic and ectopic ESC were detected through real time reverse transcription PCR and western blot. The content of 6-keto-PGF1α was determined by ELISA, the apoptotic cells were detected by flow cytometry. Results After interruption of COX-2 gene, there were no significant difference in the mRNA and protein expression of COX-2, VEGF and MMP-9 between the negative control group and blank control group (P>0.05); the mRNA and protein expression of the three genes in interference group were significantly lower than those in negative control group and blank control group (P<0.05); the mRNA expression of the three genes in interference group of eutopic ESC were 0.87±0.06, 1.76±0.59, 1.04±0.32, in interference group of ectopic ESC were 0.75±0.12, 1.62±0.47, 0.88±0.25, the protein expression of the three genes in interference group of eutopic ESC were 0.457 ± 0.019, 0.500 ± 0.012, 0.361 ± 0.008, in interference group of ectopic ESC were 0.323 ± 0.018, 0.474 ± 0.016, 0.339 ± 0.009;the mRNA and protein expression of the three genes in ectopic ESC had a more reduction than those in eutopic ESC (P<0.05). The results from ELISA revealed that the content of 6-keto-PGF1α in the normal control group [(17.7 ± 1.9) pg/ml] were significantly lower than those in the blank control group (P<0.05), the content of 6-keto-PGF1α in ectopic ESC were significantly higher than that in eutopic ESC (P<0.05), the content of 6-keto-PGF1α in the blank control group of eutopic and ectopic ESC were (32.4±2.6) pg/ml, (38.2±3.7) pg/ml;there was no significant difference in the content of 6-keto-PGF1α between the negative control group and blank control group (P>0.05);compared with those of negative control group and blank control group, the content of 6-keto-PGF1αin interference group decreased significantly (P<0.05), the content of 6-keto-PGF1α in interference group of eutopic and ectopic ESC were (17.1 ± 2.4) pg/ml, (20.9 ± 2.7) pg/ml; the content of 6-keto-PGF1α in eutopic ESC had a slightly more reduction than that in ectopic ESC (P>0.05). The results from flow cytometry displayed that, there was no significant difference in apoptotic cells between the negative control group and blank control group (P>0.05);compared with those of negative control group and blank control group, more apoptotic cells were detected in interference group and the difference was significant (P<0.01);the apoptotic cells in ectopic ESC were significantly more than that in eutopic ESC (P<0.05); the apoptosis rate in interference group of eutopic and ectopic ESC were (33.76 ± 0.06)%, (47.18 ± 0.12)%. Conclusions Our results suggested the targeted interruption of COX-2 gene by siRNA effectively inhibited the mRNA and protein expression of COX-2, VEGF and MMP-9 in both eutopic ESC and ectopic ESC with endometriosis, greatly increased the apoptotic rate of cells and obviously reduced the content of 6-keto-PGF1αby inhibiting the activity of COX-2. And the changes in ectopic endometrium were more evident than those in eutopic endometrium.
