卵巢肿瘤%RNA,小分子干扰%血蛋白质类%糖蛋白类%蛋白酶抑制蛋白质类,分泌
卵巢腫瘤%RNA,小分子榦擾%血蛋白質類%糖蛋白類%蛋白酶抑製蛋白質類,分泌
란소종류%RNA,소분자간우%혈단백질류%당단백류%단백매억제단백질류,분비
Ovarian neoplasms%RNA,small interfering%Blood proteins%Glycoproteins%Proteinase inhibitory proteins,secretory
目的 探讨ITIH4基因表达下调对卵巢上皮性癌(卵巢癌)细胞生物学功能的影响.方法 设计4对ITIH4基因小分子干扰RNA(siRNA)片段,分别为ITIH4-546、ITIH4-795、ITIH4-917和ITIH4-1568,脂质体法瞬时转染ITIH4 mRNA高表达的卵巢癌细胞系HO8910pm细胞,荧光定量逆转录(RT) PCR技术检测转染后HO8910pm细胞中ITIH4 mRNA的表达,选择最佳沉默效应的siRNA干扰片段(即ITIH4-917),构建重组表达载体pGPU6/GFP/Neo-shRNA-ITIH4-917质粒,转染H08910pm细胞,氨基糖苷类抗生素(G418)筛选,获得稳定转染细胞系pGPU6/GFP/Neo-shRNA-ITIH4-917-HO8910pm细胞,用于以下实验.实验分为3组,即重组表达载体pGPU6/GFP/Neo-shRNA-ITIH4-917质粒转染HO8910pm细胞组(ITIH4-917转染组)、空载体pGPU6/GFP/Neo-shRNA质粒转染HO8910pm细胞组(空载体组)、阴性对照载体pGPU6/GFP/Neo-shRNA-ITIH4-NC质粒转染HO8910pm细胞组(阴性对照组),采用荧光定量RT-PCR技术和蛋白印迹法分别检测3组细胞中ITIH4 mRNA和蛋白的表达,四甲基偶氮唑蓝(MTT)比色法检测3组细胞的增殖情况[以吸光度(A)值表示],流式细胞仪检测3组细胞的细胞周期比例,体外集落形成实验检测3组细胞的集落形成情况,穿膜(transwell)小室体外侵袭实验检测3组细胞的体外迁移、侵袭能力(以A值表示).结果 荧光定量PCR技术检测显示,ITIH4-546、ITIH4-795、ITI H4-917和ITIH4-1568瞬时转染后,HO8910pm细胞中ITIH4 mRNA的表达水平分别为1.24±0.74、0.30±0.09、0.26±0.15和0.71±0.35,以ITIH4-917瞬时转染后HO8910pm细胞中ITIH4 mRNA表达水平最低,故选择ITIH4-917用于以下实验.荧光定量RT-PCR技术检测显示,ITIH4-917转染组细胞中ITIH4 mRNA的相对拷贝数为0.34±0.10,明显低于空载体组(1.87±0.12,P=0.008)和阴性对照组(1.58 ±0.21,P=0.032)；蛋白印迹法检测显示,ITIH4-917转染组细胞中ITIH4蛋白的相对表达水平为0.51,明显低于空载体组(1.64,P=0.012)和阴性对照组(1.74,P=0.014).MTT比色法检测显示,ITIH4-917转染组细胞的增殖速度明显快于空载体组和阴性对照组,分别比较,差异均有统计学意义(P =0.001).流式细胞仪检测显示,ITIH4-917转染组细胞S+G2/M期细胞的比例为54.2％,明显高于空载体组和阴性对照组(分别为26.3％和31.3％,P＜0.05).体外集落形成实验检测显示,ITIH4-917转染组细胞的集落形成率为(55.7±0.7)％,明显高于空载体组[(29.7±0.9)％,P=0.037]和阴性对照组[(31.4±0.3)％,P=0.043].体外迁移和侵袭能力实验显示,ITIH4-917转染组细胞的迁移能力为0.40±0.18,明显高于阴性对照组(0.30±0.03,P=0.031)和空载体组(0.25±0.03,P=0.028)；ITIH4-917转染组细胞的侵袭能力为1.31±0.34,虽高于阴性对照组的1.05±0.68和空载体组的1.14±0.08,但差异无统计学意义(P＞0.05).结论 采用siRNA干扰片段对卵巢癌细胞中ITIH4基因进行干扰,有效地抑制其ITIH4基因的表达,并使卵巢癌细胞的增殖速度加快、迁移能力增强.
目的 探討ITIH4基因錶達下調對卵巢上皮性癌(卵巢癌)細胞生物學功能的影響.方法 設計4對ITIH4基因小分子榦擾RNA(siRNA)片段,分彆為ITIH4-546、ITIH4-795、ITIH4-917和ITIH4-1568,脂質體法瞬時轉染ITIH4 mRNA高錶達的卵巢癌細胞繫HO8910pm細胞,熒光定量逆轉錄(RT) PCR技術檢測轉染後HO8910pm細胞中ITIH4 mRNA的錶達,選擇最佳沉默效應的siRNA榦擾片段(即ITIH4-917),構建重組錶達載體pGPU6/GFP/Neo-shRNA-ITIH4-917質粒,轉染H08910pm細胞,氨基糖苷類抗生素(G418)篩選,穫得穩定轉染細胞繫pGPU6/GFP/Neo-shRNA-ITIH4-917-HO8910pm細胞,用于以下實驗.實驗分為3組,即重組錶達載體pGPU6/GFP/Neo-shRNA-ITIH4-917質粒轉染HO8910pm細胞組(ITIH4-917轉染組)、空載體pGPU6/GFP/Neo-shRNA質粒轉染HO8910pm細胞組(空載體組)、陰性對照載體pGPU6/GFP/Neo-shRNA-ITIH4-NC質粒轉染HO8910pm細胞組(陰性對照組),採用熒光定量RT-PCR技術和蛋白印跡法分彆檢測3組細胞中ITIH4 mRNA和蛋白的錶達,四甲基偶氮唑藍(MTT)比色法檢測3組細胞的增殖情況[以吸光度(A)值錶示],流式細胞儀檢測3組細胞的細胞週期比例,體外集落形成實驗檢測3組細胞的集落形成情況,穿膜(transwell)小室體外侵襲實驗檢測3組細胞的體外遷移、侵襲能力(以A值錶示).結果 熒光定量PCR技術檢測顯示,ITIH4-546、ITIH4-795、ITI H4-917和ITIH4-1568瞬時轉染後,HO8910pm細胞中ITIH4 mRNA的錶達水平分彆為1.24±0.74、0.30±0.09、0.26±0.15和0.71±0.35,以ITIH4-917瞬時轉染後HO8910pm細胞中ITIH4 mRNA錶達水平最低,故選擇ITIH4-917用于以下實驗.熒光定量RT-PCR技術檢測顯示,ITIH4-917轉染組細胞中ITIH4 mRNA的相對拷貝數為0.34±0.10,明顯低于空載體組(1.87±0.12,P=0.008)和陰性對照組(1.58 ±0.21,P=0.032)；蛋白印跡法檢測顯示,ITIH4-917轉染組細胞中ITIH4蛋白的相對錶達水平為0.51,明顯低于空載體組(1.64,P=0.012)和陰性對照組(1.74,P=0.014).MTT比色法檢測顯示,ITIH4-917轉染組細胞的增殖速度明顯快于空載體組和陰性對照組,分彆比較,差異均有統計學意義(P =0.001).流式細胞儀檢測顯示,ITIH4-917轉染組細胞S+G2/M期細胞的比例為54.2％,明顯高于空載體組和陰性對照組(分彆為26.3％和31.3％,P＜0.05).體外集落形成實驗檢測顯示,ITIH4-917轉染組細胞的集落形成率為(55.7±0.7)％,明顯高于空載體組[(29.7±0.9)％,P=0.037]和陰性對照組[(31.4±0.3)％,P=0.043].體外遷移和侵襲能力實驗顯示,ITIH4-917轉染組細胞的遷移能力為0.40±0.18,明顯高于陰性對照組(0.30±0.03,P=0.031)和空載體組(0.25±0.03,P=0.028)；ITIH4-917轉染組細胞的侵襲能力為1.31±0.34,雖高于陰性對照組的1.05±0.68和空載體組的1.14±0.08,但差異無統計學意義(P＞0.05).結論 採用siRNA榦擾片段對卵巢癌細胞中ITIH4基因進行榦擾,有效地抑製其ITIH4基因的錶達,併使卵巢癌細胞的增殖速度加快、遷移能力增彊.
목적 탐토ITIH4기인표체하조대란소상피성암(란소암)세포생물학공능적영향.방법 설계4대ITIH4기인소분자간우RNA(siRNA)편단,분별위ITIH4-546、ITIH4-795、ITIH4-917화ITIH4-1568,지질체법순시전염ITIH4 mRNA고표체적란소암세포계HO8910pm세포,형광정량역전록(RT) PCR기술검측전염후HO8910pm세포중ITIH4 mRNA적표체,선택최가침묵효응적siRNA간우편단(즉ITIH4-917),구건중조표체재체pGPU6/GFP/Neo-shRNA-ITIH4-917질립,전염H08910pm세포,안기당감류항생소(G418)사선,획득은정전염세포계pGPU6/GFP/Neo-shRNA-ITIH4-917-HO8910pm세포,용우이하실험.실험분위3조,즉중조표체재체pGPU6/GFP/Neo-shRNA-ITIH4-917질립전염HO8910pm세포조(ITIH4-917전염조)、공재체pGPU6/GFP/Neo-shRNA질립전염HO8910pm세포조(공재체조)、음성대조재체pGPU6/GFP/Neo-shRNA-ITIH4-NC질립전염HO8910pm세포조(음성대조조),채용형광정량RT-PCR기술화단백인적법분별검측3조세포중ITIH4 mRNA화단백적표체,사갑기우담서람(MTT)비색법검측3조세포적증식정황[이흡광도(A)치표시],류식세포의검측3조세포적세포주기비례,체외집락형성실험검측3조세포적집락형성정황,천막(transwell)소실체외침습실험검측3조세포적체외천이、침습능력(이A치표시).결과 형광정량PCR기술검측현시,ITIH4-546、ITIH4-795、ITI H4-917화ITIH4-1568순시전염후,HO8910pm세포중ITIH4 mRNA적표체수평분별위1.24±0.74、0.30±0.09、0.26±0.15화0.71±0.35,이ITIH4-917순시전염후HO8910pm세포중ITIH4 mRNA표체수평최저,고선택ITIH4-917용우이하실험.형광정량RT-PCR기술검측현시,ITIH4-917전염조세포중ITIH4 mRNA적상대고패수위0.34±0.10,명현저우공재체조(1.87±0.12,P=0.008)화음성대조조(1.58 ±0.21,P=0.032)；단백인적법검측현시,ITIH4-917전염조세포중ITIH4단백적상대표체수평위0.51,명현저우공재체조(1.64,P=0.012)화음성대조조(1.74,P=0.014).MTT비색법검측현시,ITIH4-917전염조세포적증식속도명현쾌우공재체조화음성대조조,분별비교,차이균유통계학의의(P =0.001).류식세포의검측현시,ITIH4-917전염조세포S+G2/M기세포적비례위54.2％,명현고우공재체조화음성대조조(분별위26.3％화31.3％,P＜0.05).체외집락형성실험검측현시,ITIH4-917전염조세포적집락형성솔위(55.7±0.7)％,명현고우공재체조[(29.7±0.9)％,P=0.037]화음성대조조[(31.4±0.3)％,P=0.043].체외천이화침습능력실험현시,ITIH4-917전염조세포적천이능력위0.40±0.18,명현고우음성대조조(0.30±0.03,P=0.031)화공재체조(0.25±0.03,P=0.028)；ITIH4-917전염조세포적침습능력위1.31±0.34,수고우음성대조조적1.05±0.68화공재체조적1.14±0.08,단차이무통계학의의(P＞0.05).결론 채용siRNA간우편단대란소암세포중ITIH4기인진행간우,유효지억제기ITIH4기인적표체,병사란소암세포적증식속도가쾌、천이능력증강.
Objective To investigate the efficiency of biology function of ITIH4 gene silenced by small interfering RNA (siRNA) on ovarian cancer.Methods The four pairs ITIH4 gene siRNA interference fragments(ITIH4-546,ITIH4-795,ITIH4-917 and ITIH4-1568) were designed respectively,and transfected into HO8910pm cells with ITIH4 mRNA high expression by liposomal method transiently.Quantitative PCR method was used to detect the ITIH4 mRNA expression in HO8910pm cells transfected with interference fragment.The ITIH4 917 was selected as the best silencing effect of siRNA interference fragment and then the recombinant plasmid expression vector pGPU6/GFP/Neo-shRNA-ITIH4-917 was constructed and transfected into HO8910pm cells.The stably transfected cells-pGPU6/GFP/Neo-shRNA-ITIH4-917-HO8910pm cells was obtained by screening of aminoglycoside antibiotics (G418).The experiment was divided into three groups,namely ITIH4-917 transfection group,the HO8910pm cell group transfected with pGPU6/GFP/Neo-shRNA plasmid (empty vector group),and the HO8910pm cell group transfected with pGPU6/GFP/Neo-shRNA-ITIH4-NC the plasmid (negative control group).Fluorescence quantitative reverse transcription(RT)PCR and western blot were used to detect the ITIH4 mRNA and protein expression.The cell proliferation,the cell cycle,colony formation of cells,cells migration and invasion in vitro were determined by using methyl thiazolyl tetrazolium (MTT),flow cytometry,colony formation assay and transmembrane (transwell) small chamber method [value represented by absorbance (A)],respectively.Results The fluorescent quantitative PCR results showed that the ITIH4 mRNA expression levels in ITIH4-917 HO8910pm cells was significantly lower than that in the control cells,the relative copy number was only 0.26 ± 0.15.Also the relative copy number of ITIH4 mRNA in ITIH4-917 transfection group cells was 0.34 ±0.10,it significantly lower than that in empty vector group (1.87 ±0.12,P =0.008) and negative control group (1.58 ±0.21,P =0.032) ; Western blot results showed that the ITIH4 relative expression levels of the protein in ITIH4-917 HO8910pm group cells,empty vector group and negative control group were 0.51,1.64 and 1.74,respectively,there were statistically significant differences (0.51 vs.1.64,P =0.012; 0.51 vs.1.74,P =0.014).MTT colorimetric assay showed that the proliferation of ITIH4-917 HO8910pm group cells was significantly faster than that in the empty vector group and negative control group,and there were statistically significant differences among them (P =0.001).The S ± G2/M phase cell ratio in ITIH4-917 HO8910pm group cells was 54.2％,which was significantly higher than that in the empty vector group or negative control group (26.3％ and 31.3％,respectively,all P ＜ 0.05).The colony formation rate (55.7 ± O.7) ％ in ITIH4-917 HO8910pm group cells was also significantly higher than that in empty vector group (29.7 ±0.9) ％ (P =0.037) and negative control group (31.4 ± 0.3) ％ (P =0.043).Migration and invasion experiments showed that cell migration in ITIH4-917 HO8910pm group cells was 0.40 ± 0.18,whicht was significantly higher than that in the negative control group or empty vector (0.30 ±0.03,P =0.031 ;0.25 ±0.03,P =0.028,respectively).Although the invasive ability of ITIH4-917 HO8910pm group cells (1.31 ±0.34) was higher than that in the control cells (1.05 ±0.68) and empty vector group (1.14 ±0.08),while there were not significant difference (P ＞ 0.05).Conclusion It would be to promote the cell doubling time and increase the migration capability in HO8910pm cells that ITIH4 expression was down-regulating by ITIH4 mRNA interference.
