CAJ | 학술논문

目的：研究S期激酶相关蛋白2反义寡核苷酸（Skp2 antisense oligodeoxynucleotide ，Skp2 ASODN ）对大肠癌细胞生长和增殖的影响及可能机制。方法采用脂质体介导不同浓度的 Skp2 ASODN 转染 SW480细胞，终浓度分别设置0．050、0．125、0．250、0．500、1．000、2．000、4．000μmol/L ，以Skp2无义寡核苷酸（NSODN ）及空白组作对照。采用倒置显微镜观察、噻唑蓝比色法（MTT）和流式细胞术（FCM ），观察不同浓度的Skp2 ASODN 对SW480细胞生长、增殖和细胞周期的影响。运用RT-PCR和免疫细胞化学检测SW480细胞中Skp2、P27kip1 mRNA和蛋白表达情况。结果倒置显微镜下观察见SW480细胞形态未见改变，生长速度减慢。Skp2 ASODN浓度达到0．125μmol/L时，抑制率为14．48％（P＜0．05）。随着Skp2 ASODN浓度增加，其对SW480细胞的生长和增殖的抑制作用增强（P＜0．01），并存在浓度依赖效应。SW480细胞G0/G1期增多（P＜0．01），S期减少（P＜0．05），细胞周期进程受到阻滞。RT-PCR及免疫细胞化学显示：Skp2 mRNA及Skp2蛋白表达降低（P＜0．05），P27kip1 mRNA未见改变（P＞0．05），但其蛋白表达明显升高（P＜0．01）。结论 Skp2 ASODN对SW480细胞的生长和增殖具有抑制作用。其可能的机制为通过Skp2 ASODN的基因封闭作用，降低了Skp2对P27kip1泛素化降解作用，从而阻滞细胞周期进行。
목적：연구S기격매상관단백2반의과핵감산（Skp2 antisense oligodeoxynucleotide ，Skp2 ASODN ）대대장암세포생장화증식적영향급가능궤제。방법채용지질체개도불동농도적 Skp2 ASODN 전염 SW480세포，종농도분별설치0．050、0．125、0．250、0．500、1．000、2．000、4．000μmol/L ，이Skp2무의과핵감산（NSODN ）급공백조작대조。채용도치현미경관찰、새서람비색법（MTT）화류식세포술（FCM ），관찰불동농도적Skp2 ASODN 대SW480세포생장、증식화세포주기적영향。운용RT-PCR화면역세포화학검측SW480세포중Skp2、P27kip1 mRNA화단백표체정황。결과도치현미경하관찰견SW480세포형태미견개변，생장속도감만。Skp2 ASODN농도체도0．125μmol/L시，억제솔위14．48％（P＜0．05）。수착Skp2 ASODN농도증가，기대SW480세포적생장화증식적억제작용증강（P＜0．01），병존재농도의뢰효응。SW480세포G0/G1기증다（P＜0．01），S기감소（P＜0．05），세포주기진정수도조체。RT-PCR급면역세포화학현시：Skp2 mRNA급Skp2단백표체강저（P＜0．05），P27kip1 mRNA미견개변（P＞0．05），단기단백표체명현승고（P＜0．01）。결론 Skp2 ASODN대SW480세포적생장화증식구유억제작용。기가능적궤제위통과Skp2 ASODN적기인봉폐작용，강저료Skp2대P27kip1범소화강해작용，종이조체세포주기진행。
Objective To investigate the effect of S-phase kinase-associated protein 2 antisense oligodeoxynucleotide (Skp2 ASODN) on the growth and proliferation of colorectal cancer cells SW480 and its mechanism .Methods The liposome-mediated dif-ferent concentrations of Skp2 ASODN was adopted to transfect SW480 cells ,the final concentrations of 0 .050 ,0 .125 ,0 .250 ,0 .500 , 1 .000 ,2 .000 ,4 .000 μmol/L were respectively set up with Skp2 nonsense oligodeoxynucleotides(NSODN) and blank group as con-trol .Then the inhibited effect of growth and proliferation of colorectal cancer cells were measured by the inverted microscope obser-vationandmethylthiazolyltetrazolium(MTT),thecellcyclewassurveyedbytheflowcytometry(FCM).TheexpressionofmRNA and protein of Skp2 and P27kip1 were inspected by reverse transcription-polymerase chain reaction(RT-PCR) and immunocytochem-istry methods .Results The inverted microscope observed that the SW480 cells had no change in form and grew at a slower speed . When the Skp2 ASODN concentration reached 0 .125 μmol/L ,the inhibition rate was 14 .48% (P<0 .05) .As the Skp2 ASODN concentration increase ,its inhibition action on the growth and proliferation of SW480 cells was enhanced in a dose-dependent man-ner(P<0 .01) .The SW480 cells were increased in G0/G1 phase(P<0 .01) and decreased in S phase(P<0 .05) ,the progression of cell cycle was blocked .RT-PCR and the immunocytochemistry showed that both Skp2 mRNA and protein levels were down-regula-ted(P<0 .05) .Although the P27kip1 mRNA level had no change(P>0 .05) ,but its protein expression was elevates obviously (P<0 .01) .Conclusion Skp2 ASODN can inhibit the growth and proliferation of SW480 cells .Its possible mechanism is that the ubiq-uitin degradation of the Skp2 to P27kip1 is decreased by the gene occlusion effect of Skp2 ASODN ,thus the cell cycle progression is arrested .