上海交通大学学报（医学版）
上海交通大學學報（醫學版）
상해교통대학학보（의학판）
JOURNAL OF SHANGHAI JIAOTONG UNIVERSITY(MEDICAL SCIENCE)
2009年
10期
1163-1168
,共6页
胰岛素样生长因子-1受体%RNA干扰%真核表达载体%肝癌%FAK
胰島素樣生長因子-1受體%RNA榦擾%真覈錶達載體%肝癌%FAK
이도소양생장인자-1수체%RNA간우%진핵표체재체%간암%FAK
insulin like growth factor-1 receptor%RNA interference%eukaryotic expression vector%hepatocellular carcinoma%FAK
目的 构建胰岛素样生长因子-1受体(IGF1R)RNA干扰真核表达载体,探讨人肝癌细胞株MHCC-97H在IGF1R基因抑制前后黏附和侵袭能力的变化及相关信号分子的变化.方法 以pGCsi-U6-Neo-GFP为空白质粒载体,以IGF1R为靶基因,按GeneBank IGF1R基因核苷酸序列和Tusch1设计原则,选择5对2条互补的带发夹结构的核苷酸序列,连接到pGCsi-U6-Neo-GFP空载体中.转化大肠杆菌Stable3,扩增后提取质粒,进行酶切鉴定和测序分析.转染模型细胞(293T),进行RT-PCR及Western blotting检测,筛选出沉默效果最好的质粒载体.用脂质体转染MHCC-97H肝癌细胞,G418筛选并建立IGF1R基因沉默的MHCC-97H肝癌细胞株.检测MHCC-97H细胞株黏附和侵袭能力及FAK蛋白的表达变化.结果 构建的IGF1R pGCsi-U6-Neo-GFP shRNA,经酶切和DNA测序证实与设计完全一致;RT-PCR及Western blotting检测发现IGF1R沉默效率达88%.MHCC-97H细胞IGF1R被沉默后黏附和侵袭能力下降,FAK蛋白表达下降.结论 IGF1R pGCsi-U6-Neo-GFP shRNA能够降低MHCC-97H细胞黏附和侵袭能力并抑制FAK蛋白的表达.
目的 構建胰島素樣生長因子-1受體(IGF1R)RNA榦擾真覈錶達載體,探討人肝癌細胞株MHCC-97H在IGF1R基因抑製前後黏附和侵襲能力的變化及相關信號分子的變化.方法 以pGCsi-U6-Neo-GFP為空白質粒載體,以IGF1R為靶基因,按GeneBank IGF1R基因覈苷痠序列和Tusch1設計原則,選擇5對2條互補的帶髮夾結構的覈苷痠序列,連接到pGCsi-U6-Neo-GFP空載體中.轉化大腸桿菌Stable3,擴增後提取質粒,進行酶切鑒定和測序分析.轉染模型細胞(293T),進行RT-PCR及Western blotting檢測,篩選齣沉默效果最好的質粒載體.用脂質體轉染MHCC-97H肝癌細胞,G418篩選併建立IGF1R基因沉默的MHCC-97H肝癌細胞株.檢測MHCC-97H細胞株黏附和侵襲能力及FAK蛋白的錶達變化.結果 構建的IGF1R pGCsi-U6-Neo-GFP shRNA,經酶切和DNA測序證實與設計完全一緻;RT-PCR及Western blotting檢測髮現IGF1R沉默效率達88%.MHCC-97H細胞IGF1R被沉默後黏附和侵襲能力下降,FAK蛋白錶達下降.結論 IGF1R pGCsi-U6-Neo-GFP shRNA能夠降低MHCC-97H細胞黏附和侵襲能力併抑製FAK蛋白的錶達.
목적 구건이도소양생장인자-1수체(IGF1R)RNA간우진핵표체재체,탐토인간암세포주MHCC-97H재IGF1R기인억제전후점부화침습능력적변화급상관신호분자적변화.방법 이pGCsi-U6-Neo-GFP위공백질립재체,이IGF1R위파기인,안GeneBank IGF1R기인핵감산서렬화Tusch1설계원칙,선택5대2조호보적대발협결구적핵감산서렬,련접도pGCsi-U6-Neo-GFP공재체중.전화대장간균Stable3,확증후제취질립,진행매절감정화측서분석.전염모형세포(293T),진행RT-PCR급Western blotting검측,사선출침묵효과최호적질립재체.용지질체전염MHCC-97H간암세포,G418사선병건립IGF1R기인침묵적MHCC-97H간암세포주.검측MHCC-97H세포주점부화침습능력급FAK단백적표체변화.결과 구건적IGF1R pGCsi-U6-Neo-GFP shRNA,경매절화DNA측서증실여설계완전일치;RT-PCR급Western blotting검측발현IGF1R침묵효솔체88%.MHCC-97H세포IGF1R피침묵후점부화침습능력하강,FAK단백표체하강.결론 IGF1R pGCsi-U6-Neo-GFP shRNA능구강저MHCC-97H세포점부화침습능력병억제FAK단백적표체.
Objective To construct short-hairpin RNA (shRNA) eukaryotic express vectors targeting of insulin like growth factor-1 receptor (IGF1R) gene, and to explore the changes of adhesion, invasion and FAK protein expression of MHCC-97 H hepatocellular carcinoma cells with RNA interference. Methods The shRNA oligonucleotide fragments were designed and synthesized based on the sequence of IGF1R mRNA. Double strands were formed after annealing and inserted into pGCsi-U6-Neo-GFP vector. The recombinant was transformed into Stable 3, then plasmids were extracted and identified by restriction enzyme and sequencing analysis. The most effective vectors were selected by RT-PCR and Western blotting after transfecting 293T cells. The best one was used to transform MHCC-97H cells which were selected with G418 to obtain positive colons. The changes of adhesion, invasion and FAK protein expression in MHCC-97H cells were studied. Results The restriction enzyme analysis and sequencing analysis demonstrated that shRNA had been inserted into vectors, and their sequences were the same as the design. It was indicated by RT-PCR and Western blotting that the silencing efficacy of IGF1R was 88%. The ability of adhesion and invasion significantly decreased after IGF1R silencing in MHCC-97H cells, and so was the expression of FAK protein. Conclusion IGF1R pGCsi-U6-Neo-GFP shRNA can significantly decrease the abilities of adhesion and invasion in MHCC-97H cells, and inhibit the expression of FAK protein.
