中华血液学杂志
中華血液學雜誌
중화혈액학잡지
Chinese Journal of Hematology
2008年
4期
247-251
,共5页
李炳宗%庄文卓%陈萍%傅晋翔
李炳宗%莊文卓%陳萍%傅晉翔
리병종%장문탁%진평%부진상
RNA干扰%缺氧诱导因子-1α%RPMI8226细胞%成瘤性
RNA榦擾%缺氧誘導因子-1α%RPMI8226細胞%成瘤性
RNA간우%결양유도인자-1α%RPMI8226세포%성류성
RNA interference%Hypoxia-inducible factor-1α%RPMI8226 cells%Tumorigenesis
目的 利用RNA干扰(RNAi)技术抑制多发性骨髓瘤(MM)细胞系RPMI8226细胞中缺氧诱导因子-1α(hypoxia-inducible factor,HIF-1α)表达,探讨对RPMI8226细胞在裸鼠体内成瘤性的影响.方法 利用短发夹RNA(shRNA)表达载体pSilencer 2.1-U6 hygro构建RNAi载体,在RPMI8226细胞中抑制HIF-1α的表达.应用RT-PCR技术和Western blot方法 ,鉴定RNAi后HIF-1α的表达.ELISA法测定细胞上清中血管内皮生长因子(VEGF)表达的改变.流式细胞术检测RNAi前后细胞周期的改变.研究缺氧(PO2为1%)条件下,抑制HIF-1α的表达对其靶基因VEGF和葡萄糖转运蛋白(Glut-1)表达的影响.利用异种移植瘤模型观察HIF-1α干扰后对肿瘤生长的影响.结果 RNAi后,HIF-1α的表达在mRNA和蛋白水平均明显降低.常氧条件下,RNAi组(RPMI8226-i1和RPMI8226-i2)和未干扰组(RPMI8226-c和RPMI8226)细胞上清中VEGF浓度无明显差异;缺氧条件下四组细胞上清中VEGF浓度分别为(506.0±53.2)、(494.7±63.1)、(984.4±61.9)和(938.2±62.2)pg/ml.RPMI8226-i1和RPMI8226-i2组较RPMI8226-c和RPMI8226组明显降低(P＜0.05).G0/G1期累积受到抑制,S期细胞比例增加.缺氧条件下VEGF和Glut-1等靶基因的表达明显下调(P＜0.05).裸鼠皮下成瘤性实验表明,抑制HIF-1α的表达,能够明显抑制肿瘤的生长.结论 RNAi抑制RPMI8226细胞HIF-1α表达,可能通过抑制肿瘤的血管新生和糖代谢过程来发挥肿瘤抑制作用.
目的 利用RNA榦擾(RNAi)技術抑製多髮性骨髓瘤(MM)細胞繫RPMI8226細胞中缺氧誘導因子-1α(hypoxia-inducible factor,HIF-1α)錶達,探討對RPMI8226細胞在裸鼠體內成瘤性的影響.方法 利用短髮夾RNA(shRNA)錶達載體pSilencer 2.1-U6 hygro構建RNAi載體,在RPMI8226細胞中抑製HIF-1α的錶達.應用RT-PCR技術和Western blot方法 ,鑒定RNAi後HIF-1α的錶達.ELISA法測定細胞上清中血管內皮生長因子(VEGF)錶達的改變.流式細胞術檢測RNAi前後細胞週期的改變.研究缺氧(PO2為1%)條件下,抑製HIF-1α的錶達對其靶基因VEGF和葡萄糖轉運蛋白(Glut-1)錶達的影響.利用異種移植瘤模型觀察HIF-1α榦擾後對腫瘤生長的影響.結果 RNAi後,HIF-1α的錶達在mRNA和蛋白水平均明顯降低.常氧條件下,RNAi組(RPMI8226-i1和RPMI8226-i2)和未榦擾組(RPMI8226-c和RPMI8226)細胞上清中VEGF濃度無明顯差異;缺氧條件下四組細胞上清中VEGF濃度分彆為(506.0±53.2)、(494.7±63.1)、(984.4±61.9)和(938.2±62.2)pg/ml.RPMI8226-i1和RPMI8226-i2組較RPMI8226-c和RPMI8226組明顯降低(P＜0.05).G0/G1期纍積受到抑製,S期細胞比例增加.缺氧條件下VEGF和Glut-1等靶基因的錶達明顯下調(P＜0.05).裸鼠皮下成瘤性實驗錶明,抑製HIF-1α的錶達,能夠明顯抑製腫瘤的生長.結論 RNAi抑製RPMI8226細胞HIF-1α錶達,可能通過抑製腫瘤的血管新生和糖代謝過程來髮揮腫瘤抑製作用.
목적 이용RNA간우(RNAi)기술억제다발성골수류(MM)세포계RPMI8226세포중결양유도인자-1α(hypoxia-inducible factor,HIF-1α)표체,탐토대RPMI8226세포재라서체내성류성적영향.방법 이용단발협RNA(shRNA)표체재체pSilencer 2.1-U6 hygro구건RNAi재체,재RPMI8226세포중억제HIF-1α적표체.응용RT-PCR기술화Western blot방법 ,감정RNAi후HIF-1α적표체.ELISA법측정세포상청중혈관내피생장인자(VEGF)표체적개변.류식세포술검측RNAi전후세포주기적개변.연구결양(PO2위1%)조건하,억제HIF-1α적표체대기파기인VEGF화포도당전운단백(Glut-1)표체적영향.이용이충이식류모형관찰HIF-1α간우후대종류생장적영향.결과 RNAi후,HIF-1α적표체재mRNA화단백수평균명현강저.상양조건하,RNAi조(RPMI8226-i1화RPMI8226-i2)화미간우조(RPMI8226-c화RPMI8226)세포상청중VEGF농도무명현차이;결양조건하사조세포상청중VEGF농도분별위(506.0±53.2)、(494.7±63.1)、(984.4±61.9)화(938.2±62.2)pg/ml.RPMI8226-i1화RPMI8226-i2조교RPMI8226-c화RPMI8226조명현강저(P＜0.05).G0/G1기루적수도억제,S기세포비례증가.결양조건하VEGF화Glut-1등파기인적표체명현하조(P＜0.05).라서피하성류성실험표명,억제HIF-1α적표체,능구명현억제종류적생장.결론 RNAi억제RPMI8226세포HIF-1α표체,가능통과억제종류적혈관신생화당대사과정래발휘종류억제작용.
Objective To explore the influence of inhibition of hypoxia-inducible factor-1α(HIF-1α)by RNA interference(RNAi)on tumorigenesis of human myeloma cell line(HMCL)RPMI8226 cells in nude mice.Method RNAi vector of HIF-1α was constructed with commercial shRNA expression vector pSilencer 2.1-U6 hygro.RT-PCR and western blot were used to detect HIF-1αmRNA and protein expression respectively.Vascular endothelial growth factor(VEGF)secretion and cell cycle changes were detected by ELISA and flow cytometry respectively.Expression of target gene of HIF-1α,VEGF and Glut-1 were testedunder hypoxia condition.Tumorigenesis was observed after transfected cells were injected subcutaneously innude mice.Results After interference,expression of HIF-1α decreased significantly at both mRNA and protein level.Under normoxia condition,VEGF concentrations in HIF-1α inhibited cells (RPMI8226-i1 and RPMI8226-i2)and non-inhibited cells(RPMI8226-c and RPMI8226)showed no differences.While under hypoxia condition,VEGF concentration in the above four cells was(506.0±53.2),(494.7±63.1).(984.4±61.9)and(938.2±62.2)pg/ml,respectively,being significantly lower in RPMI8226-i1 and RPMI8226-i2 cells than in RPMI8226-c and RPMI8226 cells(P＜0.05).HIF-1α interference was found to suppress the cells shift from S-phase to G1 induced by hypoxia.VEGF and Glut-1 expressions were markedly attenuated(P＜0.05).The growth rate of HIF-1α inhibition tumors in subcutaneous xenograft model decreased drastically.Conclusions RNAi inhibits HIF-1α expression.Reduced tumor growth by HIF-1α inhibition may partly through inhibiton of angiogenesis and glycolysis metabolism.
