CAJ | 학술논문

目的观察KAI1对视网膜母细胞瘤(RB) Y79细胞肿瘤转移相关基因表达的影响.方法实验研究.分别用携带KAI1及增强型绿色荧光蛋白(EGFP)融合基因的慢病毒载体Lenti-MPKG及阴性对照载体转染RB细胞株Y79细胞.应用抗性药物嘌呤霉素结合荧光显微镜观察、筛选稳定且高表达KAI1的Y79细胞(Y79-KAI1)及其对照细胞(Y79-KAI1/zero).应用实时荧光定量PCR、免疫印迹法和细胞免疫荧光法检测Y79、Y79-KAI1和Y79-KAI1/zero细胞KAI1的表达水平.应用人肿瘤转移PCR芯片技术分析Y79-KAI1和Y79-KAI1/zero细胞转移相关基因的差异表达.3组细胞KAI1基因mRNA及蛋白表达的差异采用单因素方差分析进行比较.结果成功建立了稳定且高表达KAI1的Y79-KAI1及其对照细胞Y79-KAI1/zero.实时荧光定量PCR检测结果显示,3组细胞KAI1基因mRNA相对值分别为183.67±21.20、1.42 ±0.55、1.00 ±0.00,Y79-KAI1细胞KAI1基因mRNA表达水平较对照Y79-KAI1/zero及Y79细胞显著增高(F=108.74,P=0.000).免疫印迹法检测结果显示,Y79-KAI1细胞KAI1蛋白表达水平明显高于Y79和Y79-KAI1/zero细胞(F=34.36,P=0.001).细胞免疫荧光染色结果显示,Y79-KAI1细胞质中有KAI1蛋白表达,围绕细胞核分布；Y79和Y79-KAI1/zero细胞中未检测到KAI1蛋白表达.PCR芯片技术共筛选肿瘤转移相关基因84个,其中上调表达2倍以上的基因共有7个,下调表达2倍以上基因的共有6个.结论 KAI1可导致Y79细胞部分肿瘤转移相关基因的表达水平改变,这些改变可能与KAI1抑制RB转移有关.
목적 관찰KAI1대시망막모세포류(RB) Y79세포종류전이상관기인표체적영향.방법 실험연구.분별용휴대KAI1급증강형록색형광단백(EGFP)융합기인적만병독재체Lenti-MPKG급음성대조재체전염RB세포주Y79세포.응용항성약물표령매소결합형광현미경관찰、사선은정차고표체KAI1적Y79세포(Y79-KAI1)급기대조세포(Y79-KAI1/zero).응용실시형광정량PCR、면역인적법화세포면역형광법검측Y79、Y79-KAI1화Y79-KAI1/zero세포KAI1적표체수평.응용인종류전이PCR심편기술분석Y79-KAI1화Y79-KAI1/zero세포전이상관기인적차이표체.3조세포KAI1기인mRNA급단백표체적차이채용단인소방차분석진행비교.결과 성공건립료은정차고표체KAI1적Y79-KAI1급기대조세포Y79-KAI1/zero.실시형광정량PCR검측결과현시,3조세포KAI1기인mRNA상대치분별위183.67±21.20、1.42 ±0.55、1.00 ±0.00,Y79-KAI1세포KAI1기인mRNA표체수평교대조Y79-KAI1/zero급Y79세포현저증고(F=108.74,P=0.000).면역인적법검측결과현시,Y79-KAI1세포KAI1단백표체수평명현고우Y79화Y79-KAI1/zero세포(F=34.36,P=0.001).세포면역형광염색결과현시,Y79-KAI1세포질중유KAI1단백표체,위요세포핵분포；Y79화Y79-KAI1/zero세포중미검측도KAI1단백표체.PCR심편기술공사선종류전이상관기인84개,기중상조표체2배이상적기인공유7개,하조표체2배이상기인적공유6개.결론 KAI1가도치Y79세포부분종류전이상관기인적표체수평개변,저사개변가능여KAI1억제RB전이유관.
Objective To investigate the expression changes of tumor metastasis-related genes after overexpression of KAI1 in retinoblastoma Y79 cells.Methods Experimental study.Y79 cells were transfected with a lentivirus vector containing KAI1 and enhanced green fluorescent protein (EGFP) fusion gene,or a control lentivirus vector containing EGFP.Positive transfectants stably expressing high levels of KAI1 were named Y79-KAI1 and control transfectants were named Y79-KAI1/zero.These transfectants were selected by puromycin resistance and analysis with fluorescent microscopy.The expression of KAI1 mRNA and its protein among Y79,Y79-KAI1 and Y79-KAIl/zero were detected by fluorescent quantitative RT-PCR and Western blot.Differential expression of tumor metastasis-related genes in Y79-KAI1 and Y79-KAI1/zero was analyzed with human tumor metastasis PCR array.One-way ANOVA was used to analyze the differences of KAI1 mRNA and protein expression among the three groups.Results The stably transfected cell lines of Y79-KAI1 and Y79-KAI1/zero were established.The result of fluorescent quantitative real-time PCR showed that the relative quantification of mRNA level of KAI1 gene in the three kinds of cells above was 183.67 ± 21.20,1.42 ± 0.55,1.00 ± 0.00,respectively.And the expression level of KAI1 mRNA in Y79-KAI1 cells was significantly higher than those in Y79-KAI1/zero and Y79 cells (F=108.74,P =0.000).The results of Western blot showed that the expression level of the KAI1 protein in Y79-KAI1 cells was significantly higher than those in Y79-KAI1/zero and Y79 cells (F =34.36,P =0.001).Immunofluorescent staining showed that Y79 and Y79-KAI1/zero cells had no detectable KAI1 expression,while Y79-KAI1 cells expressed KAI1 in the cytoplasm surrounding the nuclei.Among the 84 tumor metastasis-related genes examined,7 genes were up-regulated more than 2 folds and 6 genes were down-regulated over 50％.Conclusion Over-expression of KAI1 may result in differential expression of tumor metastasis-related genes in Y79 cells,which may be related to the inhibitory effect on the tumor metastasis of retinoblastoma.