中华皮肤科杂志
中華皮膚科雜誌
중화피부과잡지
Chinese Journal of Dermatology
2011年
7期
497-500
,共4页
周永静%龚丹丹%邱志远%彭辉勇%范钰
週永靜%龔丹丹%邱誌遠%彭輝勇%範鈺
주영정%공단단%구지원%팽휘용%범옥
黑色素瘤,实验性%细胞系,肿瘤%中期因子%RNA,小分子干扰
黑色素瘤,實驗性%細胞繫,腫瘤%中期因子%RNA,小分子榦擾
흑색소류,실험성%세포계,종류%중기인자%RNA,소분자간우
Melanoma,experimental%Cell line,tumor%Midkine%RNA,small interfering
目的 探讨中期因子(midkine,MK)基因小干扰RNA(siRNA)对黑素瘤细胞侵袭的影响.方法 根据MK基因mRNA序列特点,设计并用化学方法合成3个siRNA,转染人黑素瘤A375细胞后,以荧光实时定量PCR方法筛选出效果最好的siRNA,以此siRNA转染人黑素瘤A375细胞.同时设空白对照组(不予任何处理)和空载对照组(仅用脂质体转染).分别以荧光实时定量PCR和Western印迹方法观察MK基因mRNA和蛋白水平,然后以噻唑蓝法检测细胞黏附率,以Boyden小室模型方法检测细胞侵袭力.结果 所设计的siRNA均能有效下调MK基因mRNA表达水平,以S3号作用最强.以该siRNA转染A375细胞后,荧光实时定量PCR结果显示,转染组A375细胞MK基因mRNA水平明显下降,且呈浓度(24 h时r=-0.906;48 h时r=-0.922;72 h时r=-0.939,P均<0.01)和时间(3.125 nmol/L r=-0.889;6.25 nmol/L r=-0.935;12.5 nmol/L r=-0.928,P均<0.01)依赖性.细胞黏附试验显示,3.125、6.25和12.5 nmol/L siRNA组黏附率分别为73.66%±2.25%、49.36%±2.16%和28.35%±1.68%,呈浓度依赖性下降(r=-0.936,P<0.01).Boyden小室试验结果显示,3.125、6.25和12.5 nmol/L siRNA组穿过滤膜的细胞数分别为23.9±1.6、12.1±1.5、5.6±1.2,而空白对照组为36.8±1.5,穿膜细胞数呈浓度依赖性下降(r=-0.915,P<0.05).结论 MK基因在黑素瘤细胞黏附、侵袭中发挥重要作用;以siRNA转染黑素瘤细胞抑制该基因表达可抑制黑素瘤细胞黏附、侵袭能力.
目的 探討中期因子(midkine,MK)基因小榦擾RNA(siRNA)對黑素瘤細胞侵襲的影響.方法 根據MK基因mRNA序列特點,設計併用化學方法閤成3箇siRNA,轉染人黑素瘤A375細胞後,以熒光實時定量PCR方法篩選齣效果最好的siRNA,以此siRNA轉染人黑素瘤A375細胞.同時設空白對照組(不予任何處理)和空載對照組(僅用脂質體轉染).分彆以熒光實時定量PCR和Western印跡方法觀察MK基因mRNA和蛋白水平,然後以噻唑藍法檢測細胞黏附率,以Boyden小室模型方法檢測細胞侵襲力.結果 所設計的siRNA均能有效下調MK基因mRNA錶達水平,以S3號作用最彊.以該siRNA轉染A375細胞後,熒光實時定量PCR結果顯示,轉染組A375細胞MK基因mRNA水平明顯下降,且呈濃度(24 h時r=-0.906;48 h時r=-0.922;72 h時r=-0.939,P均<0.01)和時間(3.125 nmol/L r=-0.889;6.25 nmol/L r=-0.935;12.5 nmol/L r=-0.928,P均<0.01)依賴性.細胞黏附試驗顯示,3.125、6.25和12.5 nmol/L siRNA組黏附率分彆為73.66%±2.25%、49.36%±2.16%和28.35%±1.68%,呈濃度依賴性下降(r=-0.936,P<0.01).Boyden小室試驗結果顯示,3.125、6.25和12.5 nmol/L siRNA組穿過濾膜的細胞數分彆為23.9±1.6、12.1±1.5、5.6±1.2,而空白對照組為36.8±1.5,穿膜細胞數呈濃度依賴性下降(r=-0.915,P<0.05).結論 MK基因在黑素瘤細胞黏附、侵襲中髮揮重要作用;以siRNA轉染黑素瘤細胞抑製該基因錶達可抑製黑素瘤細胞黏附、侵襲能力.
목적 탐토중기인자(midkine,MK)기인소간우RNA(siRNA)대흑소류세포침습적영향.방법 근거MK기인mRNA서렬특점,설계병용화학방법합성3개siRNA,전염인흑소류A375세포후,이형광실시정량PCR방법사선출효과최호적siRNA,이차siRNA전염인흑소류A375세포.동시설공백대조조(불여임하처리)화공재대조조(부용지질체전염).분별이형광실시정량PCR화Western인적방법관찰MK기인mRNA화단백수평,연후이새서람법검측세포점부솔,이Boyden소실모형방법검측세포침습력.결과 소설계적siRNA균능유효하조MK기인mRNA표체수평,이S3호작용최강.이해siRNA전염A375세포후,형광실시정량PCR결과현시,전염조A375세포MK기인mRNA수평명현하강,차정농도(24 h시r=-0.906;48 h시r=-0.922;72 h시r=-0.939,P균<0.01)화시간(3.125 nmol/L r=-0.889;6.25 nmol/L r=-0.935;12.5 nmol/L r=-0.928,P균<0.01)의뢰성.세포점부시험현시,3.125、6.25화12.5 nmol/L siRNA조점부솔분별위73.66%±2.25%、49.36%±2.16%화28.35%±1.68%,정농도의뢰성하강(r=-0.936,P<0.01).Boyden소실시험결과현시,3.125、6.25화12.5 nmol/L siRNA조천과려막적세포수분별위23.9±1.6、12.1±1.5、5.6±1.2,이공백대조조위36.8±1.5,천막세포수정농도의뢰성하강(r=-0.915,P<0.05).결론 MK기인재흑소류세포점부、침습중발휘중요작용;이siRNA전염흑소류세포억제해기인표체가억제흑소류세포점부、침습능력.
Objective To study the effects of midkine (MK) gene-targeting small interfering RNA (siRNA)on the invasion of melanoma cells.Methods Three MK gene-targeting siRNAs (S1,S2 and S3)were designed,constructed,and transfected into human A375 melanoma cells.Real-time PCR was performed to measure the expression of MK gene and to screen the siRNA with best efficacy.Then,A375 cells were transfected with the optimal siRNA of various doses (3.125,6.25 and 12.5 nmol/L)followed by additional culture of various durations(24,48,72 hours).Some A375 cells remaining untreated served as the blank control group,and some transfected only with liposomes served as the vector control group.Reverse transcription (RT) -PCR and Western blot were conducted to detect the mRNA and protein expression of MK,respectively,MTT assay to observe the adhesion of A375 cells,and Boyden chamber was used to evaluate cell invasion.Results The expression of MK mRNA was downregulated by all the three siRNAs,especially by the siRNA S3,which was used in the following transfection experiment.Real-time quantitative PCR revealed that the MK mRNA expression was reduced by the siRNA in a dose- (r24hours=-0.906,r4Bhours=-0.922,r72hours=-0.939,all P<0.01)and time-dependent(r3.125nmol/L=-0.889,r625nmol/L=-0.935,r125nmol/L=-0.928,all P<0.01)manner.MTT assay showed that the percentage of adhesing cells was 73.66%±2.25%,49.36%±2.16%and 28.35%±1.68%in A375 cells transfected with the siRNA of 3.125,6.25 and 12.5 nmol/L,respectively.The number of cells migrating across the chamber filter was 23.9±1.6,12.1±1.5,5.6±1.2 among A375 cells transfected with the siRNA of 3.125,6.25 and 12.5 nmol/L,respectively,significantly lower than that in the blank control group(36.8±1.5).The percentage of adhesing cells and number of migrating cells decreased with the dose of siRNA(r=-0.936,-0.915,P<0.01,0.05,respectively).Conclusions MK gene might play an important role in the adhesion and invasion of melanonla cells.To down-regulate the expression of MK gene by siRNA may suppress the adhesion and invasion of melanoma cells.
