中华实验外科杂志
中華實驗外科雜誌
중화실험외과잡지
CHINESE JOURNAL OF EXPERIMENTAL SURGERY
2015年
2期
302-305
,共4页
金少文%王开美%徐康%徐鋆耀%褚忠华%王捷
金少文%王開美%徐康%徐鋆耀%褚忠華%王捷
금소문%왕개미%서강%서윤요%저충화%왕첩
肝细胞癌%肝再生磷酸酶-1%反转录病毒载体%生物学功能
肝細胞癌%肝再生燐痠酶-1%反轉錄病毒載體%生物學功能
간세포암%간재생린산매-1%반전록병독재체%생물학공능
Hepatocellular carcinoma%Phosphatase of regenerating liver-1%Retrovirus vector%Biological functions
目的 构建肝再生磷酸酶-1(PRL-1)真核表达载体,建立稳定过表达PRL-1的肝细胞癌Huh7细胞株,研究PRL-1在Huh7细胞中的生物学功能.方法 应用反转录-聚合酶链反应(RT-PCR)技术从人肝癌组织总RNA中扩增PRL-1基因编码区域(CDS)全长序列,与双酶切的pMSCV-PIG质粒连接,经PCR及测序鉴定pMSCV-血细胞凝集素(HA)-PRL-1载体构建成功.用293T细胞包装病毒,并感染Huh7细胞,1 mg/L嘌呤霉素持续加压筛选2周后,荧光显微镜、流式细胞仪、实时荧光定量聚合酶链反应(FQ-PCR)及Western blot检测PRL-1在Huh7中的表达.PRL-1稳定转染细胞株构建成功后,用细胞计数试剂盒(CCK-8)法进行细胞增殖实验、平板克隆形成实验和Transwell侵袭实验研究PRL-1在Huh7细胞中的生物学功能.结果 测序结果显示克隆的PRL-1基因CDS序列完全正确,且正确插入到pMSCV-PIG载体中,荧光显微镜及流式细胞仪检测结果显示细胞稳定转染效率在90％以上,FQ-PCR结果显示PRL-1稳定转染组其mRNA表达是对照组的(4.4±1.5)倍(P＜0.05),Western blot结果显示PRL-1在Huh7细胞中过表达.CCK-8细胞增殖实验结果显示PRL-1促进Huh7细胞增殖(P＜0.01).PRL-1稳定转染细胞2周克隆形成率为(26.7±1.9)％,明显高于对照组[(7.3±0.8)％,P＜0.01].PRL-1稳定转染组较对照组侵袭细胞数量增多(P＜0.01).结论 PRL-1肝细胞癌Huh7稳定转染细胞株构建成功,PRL-1促进Huh7细胞增殖、克隆形成及侵袭.
目的 構建肝再生燐痠酶-1(PRL-1)真覈錶達載體,建立穩定過錶達PRL-1的肝細胞癌Huh7細胞株,研究PRL-1在Huh7細胞中的生物學功能.方法 應用反轉錄-聚閤酶鏈反應(RT-PCR)技術從人肝癌組織總RNA中擴增PRL-1基因編碼區域(CDS)全長序列,與雙酶切的pMSCV-PIG質粒連接,經PCR及測序鑒定pMSCV-血細胞凝集素(HA)-PRL-1載體構建成功.用293T細胞包裝病毒,併感染Huh7細胞,1 mg/L嘌呤黴素持續加壓篩選2週後,熒光顯微鏡、流式細胞儀、實時熒光定量聚閤酶鏈反應(FQ-PCR)及Western blot檢測PRL-1在Huh7中的錶達.PRL-1穩定轉染細胞株構建成功後,用細胞計數試劑盒(CCK-8)法進行細胞增殖實驗、平闆剋隆形成實驗和Transwell侵襲實驗研究PRL-1在Huh7細胞中的生物學功能.結果 測序結果顯示剋隆的PRL-1基因CDS序列完全正確,且正確插入到pMSCV-PIG載體中,熒光顯微鏡及流式細胞儀檢測結果顯示細胞穩定轉染效率在90％以上,FQ-PCR結果顯示PRL-1穩定轉染組其mRNA錶達是對照組的(4.4±1.5)倍(P＜0.05),Western blot結果顯示PRL-1在Huh7細胞中過錶達.CCK-8細胞增殖實驗結果顯示PRL-1促進Huh7細胞增殖(P＜0.01).PRL-1穩定轉染細胞2週剋隆形成率為(26.7±1.9)％,明顯高于對照組[(7.3±0.8)％,P＜0.01].PRL-1穩定轉染組較對照組侵襲細胞數量增多(P＜0.01).結論 PRL-1肝細胞癌Huh7穩定轉染細胞株構建成功,PRL-1促進Huh7細胞增殖、剋隆形成及侵襲.
목적 구건간재생린산매-1(PRL-1)진핵표체재체,건립은정과표체PRL-1적간세포암Huh7세포주,연구PRL-1재Huh7세포중적생물학공능.방법 응용반전록-취합매련반응(RT-PCR)기술종인간암조직총RNA중확증PRL-1기인편마구역(CDS)전장서렬,여쌍매절적pMSCV-PIG질립련접,경PCR급측서감정pMSCV-혈세포응집소(HA)-PRL-1재체구건성공.용293T세포포장병독,병감염Huh7세포,1 mg/L표령매소지속가압사선2주후,형광현미경、류식세포의、실시형광정량취합매련반응(FQ-PCR)급Western blot검측PRL-1재Huh7중적표체.PRL-1은정전염세포주구건성공후,용세포계수시제합(CCK-8)법진행세포증식실험、평판극륭형성실험화Transwell침습실험연구PRL-1재Huh7세포중적생물학공능.결과 측서결과현시극륭적PRL-1기인CDS서렬완전정학,차정학삽입도pMSCV-PIG재체중,형광현미경급류식세포의검측결과현시세포은정전염효솔재90％이상,FQ-PCR결과현시PRL-1은정전염조기mRNA표체시대조조적(4.4±1.5)배(P＜0.05),Western blot결과현시PRL-1재Huh7세포중과표체.CCK-8세포증식실험결과현시PRL-1촉진Huh7세포증식(P＜0.01).PRL-1은정전염세포2주극륭형성솔위(26.7±1.9)％,명현고우대조조[(7.3±0.8)％,P＜0.01].PRL-1은정전염조교대조조침습세포수량증다(P＜0.01).결론 PRL-1간세포암Huh7은정전염세포주구건성공,PRL-1촉진Huh7세포증식、극륭형성급침습.
Objective To construct phosphatase of regenerating liver-1 (PRL-1) eukaryotic expression vector,establish stably-transfected Huh7 cell lines and investigate biological functions of PRL-1 in Huh7 cells.Methods The coding sequences (CDS) fragment of PRL-1 was amplified by reverse transcriptase-polymerase chain reaction (RT-PCR).Bgi Ⅱ/Xho Ⅰ restriction sites were introduced into the flank of the target fragment.Then,pMSCV-Hemagglutinin (HA)-PRL-1 vector was constructed by cloning the target fragment into pMSCV-PIG plasmid.PCR and DNA sequencing verified recombinant plasmid was successfully constructed.Huh7 cells were infected by retrovirus supernatant which was packaged using 293T cells.Following infection,cells were selected with 1 mg/L puromycin for 2 weeks.Then the expression of PRL-1 in Huh7 cells was detected by fluorescence microscope,flow cytomety,real-time fluorescent quantitative polymerase chain reaction (FQ-PCR) and Western blotting.Biological functions of PRL-1 in Huh7 cells were investigated by cell counting kit-8 (CCK-8) assay,plate colony formation assay and Transwell invasion assay.Results DNA sequencing demonstrated target fragment inserted into pMSCV-PIG vector was exactly correct.Fluorescence microscope and flow cytometry showed stably-transfected efficiency was over 90％.FQ-PCR assay showed the mRNA level of PRL-1 in overexpression group was (4.4 ±-1.5) times as compared with that in control group (P ＜ 0.05).Western blotting assay indicated PRL-1 was exogenously overexpressed in Huh7 cells.Compared with control cells,the proliferation rate increased in the PRL-1 stable transfected cells (P ＜ 0.01).Colony formation efficiency was higher in PRL-1 stably tranfected cells [(26.7 ± 1.9) ％ vs.(7.3 ± 0.8) ％,P ＜ 0.01],and overexpression group had much stronger invasive capability (P ＜ 0.01).Conclusion PRL-1 stably-transfected Huh7 cell line is successfully established and PRL-1 enhances proliferation,colony formation and invasion in Huh7 cells.
