中华妇产科杂志
中華婦產科雜誌
중화부산과잡지
CHINESE JOUNAL OF OBSTETRICS AND GYNECOLOGY
2013年
7期
532-537
,共6页
吕琼莹%张蔚%程静%张文婷%钟亚娟
呂瓊瑩%張蔚%程靜%張文婷%鐘亞娟
려경형%장위%정정%장문정%종아연
宫颈肿瘤%HeLa细胞%葡糖醛酸糖苷酶%RNA干扰%肿瘤侵润
宮頸腫瘤%HeLa細胞%葡糖醛痠糖苷酶%RNA榦擾%腫瘤侵潤
궁경종류%HeLa세포%포당철산당감매%RNA간우%종류침윤
Uterine cervical neoplasms%Hela cells%Glucuronidase%RNA interference%Neoplasm invasiveness
目的 利用RNA干扰(RNAi)技术沉默宫颈癌细胞系HeLa细胞中乙酰肝素酶(HPA)基因的表达,并探讨其对宫颈癌细胞侵袭力的影响.方法 根据GenBank数据库提供的HPA基因核苷酸序列,设计4对针对HPA基因的短发夹状RNA(shRNA)特异性寡核苷酸序列(即shRNA-HPA-592、shRNA-HPA-995、shRNA-HPA-1351、shRNA-HPA-1658),并设计1条随机无关序列作阴性对照,分 别克隆到pYr-1.1质粒中.用脂质体法将上述质粒转染入HeLa细胞,以未转染质粒者为空白对照,采用逆转录(RT)-PCR技术、免疫荧光法分别检测转染后HeLa细胞中HPA mRNA和蛋白的表达,筛选出其中对HPA基因沉默效果最佳的质粒.将该质粒转染入HeLa细胞,采用穿膜(transwell)小室体外侵袭实验检测转染后HeLa细胞侵袭力的变化.结果 RT-PCR技术检测显示,转染shRNA-HPA-592、shRNA-HPA-995、shRNA-HPA-1351、shRNA-HPA-1658质粒后,HeLa细胞中HPA mRNA的表达水平分别为0.54±0.05、0.89 ±0.18、0.82±0.22、0.91 ±0.47,阴性对照和空白对照细胞中HPAmRNA的表达水平分别为1.31 ±0.72和1.09±0.16；免疫荧光法检测显示,转染上述不同质粒后HeLa细胞的胞质中均可见绿色荧光,HPA蛋白均呈阳性表达,其中转染shRNA-HPA-592质粒后HeLa细胞的胞质中绿色荧光强度最弱.故筛选出的对HPA基因沉默效果最佳的质粒即为shRNA-HPA-592质粒.体外侵袭实验检测显示,转染shRNA-HPA-592质粒后HeLa细胞的穿膜细胞数为(44±4)个,明显低于空白对照、阴性对照[分别为(182±6)、(258±17)个],差异有统计学意义(P＜0.01)；而阴性对照与空白对照比较,差异无统计学意义(P＞0.05).结论 本研究成功筛选出了靶向HPA基因的shRNA表达质粒,转染宫颈癌HeLa细胞后可高效抑制其HPA基因的表达,明显降低宫颈癌细胞的侵袭力,为进一步研究HPA基因的表达与宫颈癌侵袭的机制奠定了基础.
目的 利用RNA榦擾(RNAi)技術沉默宮頸癌細胞繫HeLa細胞中乙酰肝素酶(HPA)基因的錶達,併探討其對宮頸癌細胞侵襲力的影響.方法 根據GenBank數據庫提供的HPA基因覈苷痠序列,設計4對針對HPA基因的短髮夾狀RNA(shRNA)特異性寡覈苷痠序列(即shRNA-HPA-592、shRNA-HPA-995、shRNA-HPA-1351、shRNA-HPA-1658),併設計1條隨機無關序列作陰性對照,分 彆剋隆到pYr-1.1質粒中.用脂質體法將上述質粒轉染入HeLa細胞,以未轉染質粒者為空白對照,採用逆轉錄(RT)-PCR技術、免疫熒光法分彆檢測轉染後HeLa細胞中HPA mRNA和蛋白的錶達,篩選齣其中對HPA基因沉默效果最佳的質粒.將該質粒轉染入HeLa細胞,採用穿膜(transwell)小室體外侵襲實驗檢測轉染後HeLa細胞侵襲力的變化.結果 RT-PCR技術檢測顯示,轉染shRNA-HPA-592、shRNA-HPA-995、shRNA-HPA-1351、shRNA-HPA-1658質粒後,HeLa細胞中HPA mRNA的錶達水平分彆為0.54±0.05、0.89 ±0.18、0.82±0.22、0.91 ±0.47,陰性對照和空白對照細胞中HPAmRNA的錶達水平分彆為1.31 ±0.72和1.09±0.16；免疫熒光法檢測顯示,轉染上述不同質粒後HeLa細胞的胞質中均可見綠色熒光,HPA蛋白均呈暘性錶達,其中轉染shRNA-HPA-592質粒後HeLa細胞的胞質中綠色熒光彊度最弱.故篩選齣的對HPA基因沉默效果最佳的質粒即為shRNA-HPA-592質粒.體外侵襲實驗檢測顯示,轉染shRNA-HPA-592質粒後HeLa細胞的穿膜細胞數為(44±4)箇,明顯低于空白對照、陰性對照[分彆為(182±6)、(258±17)箇],差異有統計學意義(P＜0.01)；而陰性對照與空白對照比較,差異無統計學意義(P＞0.05).結論 本研究成功篩選齣瞭靶嚮HPA基因的shRNA錶達質粒,轉染宮頸癌HeLa細胞後可高效抑製其HPA基因的錶達,明顯降低宮頸癌細胞的侵襲力,為進一步研究HPA基因的錶達與宮頸癌侵襲的機製奠定瞭基礎.
목적 이용RNA간우(RNAi)기술침묵궁경암세포계HeLa세포중을선간소매(HPA)기인적표체,병탐토기대궁경암세포침습력적영향.방법 근거GenBank수거고제공적HPA기인핵감산서렬,설계4대침대HPA기인적단발협상RNA(shRNA)특이성과핵감산서렬(즉shRNA-HPA-592、shRNA-HPA-995、shRNA-HPA-1351、shRNA-HPA-1658),병설계1조수궤무관서렬작음성대조,분 별극륭도pYr-1.1질립중.용지질체법장상술질립전염입HeLa세포,이미전염질립자위공백대조,채용역전록(RT)-PCR기술、면역형광법분별검측전염후HeLa세포중HPA mRNA화단백적표체,사선출기중대HPA기인침묵효과최가적질립.장해질립전염입HeLa세포,채용천막(transwell)소실체외침습실험검측전염후HeLa세포침습력적변화.결과 RT-PCR기술검측현시,전염shRNA-HPA-592、shRNA-HPA-995、shRNA-HPA-1351、shRNA-HPA-1658질립후,HeLa세포중HPA mRNA적표체수평분별위0.54±0.05、0.89 ±0.18、0.82±0.22、0.91 ±0.47,음성대조화공백대조세포중HPAmRNA적표체수평분별위1.31 ±0.72화1.09±0.16；면역형광법검측현시,전염상술불동질립후HeLa세포적포질중균가견록색형광,HPA단백균정양성표체,기중전염shRNA-HPA-592질립후HeLa세포적포질중록색형광강도최약.고사선출적대HPA기인침묵효과최가적질립즉위shRNA-HPA-592질립.체외침습실험검측현시,전염shRNA-HPA-592질립후HeLa세포적천막세포수위(44±4)개,명현저우공백대조、음성대조[분별위(182±6)、(258±17)개],차이유통계학의의(P＜0.01)；이음성대조여공백대조비교,차이무통계학의의(P＞0.05).결론 본연구성공사선출료파향HPA기인적shRNA표체질립,전염궁경암HeLa세포후가고효억제기HPA기인적표체,명현강저궁경암세포적침습력,위진일보연구HPA기인적표체여궁경암침습적궤제전정료기출.
Objective Design and synthesize short hairpin RNA (shRNA) expression vector of RNA for specific silencing of heparanase (HPA) gene,screened plasmid which silence effects is the best.Observe the function of ceil invasion after inhibiting the expression of HPA in cervical carcinoma cell lines (HeLa).Methods The genomic sequence of HPA gene was retrieved from GenBank database.Designed four pairs of specific oligonucleotide sequences and a negative control according to the shRNA design principles.They were inserted into the vector pYr-1.1,vectors,and transfected into HeLa cells via lipofectamine.Reverse transcription (RT)-PCR and immunofluorescence were employed to detect the expression of HPA gene in the transfected cells at the mRNA and protein levels,respectively.The plasmid were screened and transfected into HeLa cells,then transwell small room stromal invasion experiment were employed to observe the cervical carcinoma cell invasion.Results RT-PCR results of transfected HeLa cells shown that the mRNA amplification multiples were 0.54 ±0.05 in the HPA-592 group,0.89 ±0.18 in HPA-995 group,0.82 ±0.22 in the HPA-1351 group,0.91 ±0.47 in HPA-1658 group.While,they were 1.31 ±0.72 and 1.09 ±0.16 in negative control and blank control group,respectively.Green fluorescence was visible in the cytoplasm,which indicated that the HPA protein was expressed in the cytoplasm,of them the weakest green fluorescence in the HPA-592 group.The relative numbers of invasive cells among the HeLa cells were as follows:182 ±6 in the blank control group,258 ± 17 in the negative control group,and 44 ± 4 in the HPA-592-specific interference group(P ＜ 0.01).Conclusion Successfully screened shRNA vector targeting human HPA,efficiently inhibit expression of HPA gene when transfected into HeLa cells,and significantly reduced the invasion capacity of cervical carcinoma cells.
