Objective： To evaluate the inhibitory effects of PIN1 antiseuse gene on the proliferation of htnnan osteosarcoma cells. Methods： Different doses of antisense PIN1 gene （0,20,50,100,200,250μl） were transfected int...Objective： To evaluate the inhibitory effects of PIN1 antiseuse gene on the proliferation of htnnan osteosarcoma cells. Methods： Different doses of antisense PIN1 gene （0,20,50,100,200,250μl） were transfected into osteosarcoma MG-63 cells. The cells and the culture supernatants before and after transfection were collected. The cell growth curve was made using MTT method. The cell growth cycle and apoptosis were detected by FCM. The expression of PIN1 was detected by Western blot. The expression of PIN1 mRNA was detected by reverse transcription polymerase chain reaction （RT-PCR）. Results： MTT and FCM assays indicated that the transfection of antisense PIN1 gene could inhibit profiferation of MG-63 cells and lead to cell apoptosis. Western-blot assays revealed the MG-63 cells transfected with antisense PIN1 gene had weaker expression than those without transfection with antisense PIN1 gene, and the band intensity was negatively related with doses. The cells transfected with different doses of gene （0,20,50,100,200,250μl） had different absobance rate（0.854±0.136,0.866±0.138,0.732±0.154,0.611 ± 0.121, 0. 547 ± 0. 109, 0. 398 ± 0.113, 0. 320 ± 0.151）, with significant difference assessed by F and q test （P〈0.05）. The absorbance rate of PINI mRNA was 0.983±0.125,0.988±0.127, 0.915±0.157, 0.786 ± 0.125,0.608 ± 0.124,0.433 ± 0.130,0.410 ± 0. 158 respectively （ P 〈 0.05）. Conclusion. The expression of PIN1 mRNA in MG-63 cells could be inhibited by antiseuse PIN1 gene, and then the expression of PIN1 was reduced and depressed, and so the proliferation of hmnan osteosarcoma cells MG-63 was inhibited.展开更多
目的:应用RNA干扰技术沉默肺癌A549细胞中PIN1(protein interacting with N1MA1)基因的表达,探讨其对A549细胞增殖、细胞周期和裸鼠成瘤能力的影响。方法:构建靶向PIN1基因的shRNA真核表达质粒pGPU6-GFP-Neo-PIN1和无义对照质粒pGPU6-GF...目的:应用RNA干扰技术沉默肺癌A549细胞中PIN1(protein interacting with N1MA1)基因的表达,探讨其对A549细胞增殖、细胞周期和裸鼠成瘤能力的影响。方法:构建靶向PIN1基因的shRNA真核表达质粒pGPU6-GFP-Neo-PIN1和无义对照质粒pGPU6-GFP-Neo,以脂质体法转染A549细胞,G418筛选稳定沉默PIN1基因的细胞株。Real-time PCR和Western blotting验证PIN1基因在mRNA和蛋白水平的表达,MTT法和流式细胞术检测A549细胞增殖和细胞周期分布。将稳定沉默PIN1的A549细胞与对照细胞皮下接种裸鼠,观察接种后肿瘤生长情况。结果:成功构建了pGPU6-GFP-Neo-PIN1载体,转染A549细胞并筛选获得稳定克隆。稳定转染pGPU6-GFP-Neo-PIN1的A549细胞中PIN1mRNA表达量较pGPU6-GFP-Neo转染组下降了89.3%;蛋白表达同时也显著抑制。PIN1基因沉默组的A549细胞增殖速率明显下降(P<0.01),细胞出现G1期阻滞。小鼠体内实验显示,PIN1沉默的A549细胞在裸鼠体内成瘤能力降低(P<0.01)。结论:pGPU6-GFP-Neo-PIN1质粒稳定转染肺癌A549细胞能有效沉默PIN1基因的表达,从而抑制A549细胞的增殖、影响细胞周期和抑制成瘤能力。展开更多
Objective: To clone and construct eukaryotic expressing vectors of sense and antisense human Pin1 (hPin1) genes. Methods: Total RNA was extracted from MG-63 cells, then the hPin1 cDNA was amplified by RT-PCR. The same...Objective: To clone and construct eukaryotic expressing vectors of sense and antisense human Pin1 (hPin1) genes. Methods: Total RNA was extracted from MG-63 cells, then the hPin1 cDNA was amplified by RT-PCR. The same time the sense and antisense hPin1 genes were formed by binding BamH I and Hind III in cis and trans-directions. At the end they were cloned into the eukaryotic expressing vector pIRES2-EGFP in cis and trans directions using DNA recombinant technology. The recombinant vectors were further identified by digestion of BamH I and Hind III. Results: The results of sequencing showed that the orientation of the ligations and the reading frame were correct. After digested by BamH I and Hind III, two fragments exhibiting 5.3 kb and 0.99 kb were formed in sense and antisense eukaryotic expressing vectors. Electrophoretic results were completely coincident with theoretical calculation. Conclusion: Human Pin1 sense and antisense genes were successfully cloned and eukaryotic expressing vectors were successfully constructed.展开更多
文摘Objective： To evaluate the inhibitory effects of PIN1 antiseuse gene on the proliferation of htnnan osteosarcoma cells. Methods： Different doses of antisense PIN1 gene （0,20,50,100,200,250μl） were transfected into osteosarcoma MG-63 cells. The cells and the culture supernatants before and after transfection were collected. The cell growth curve was made using MTT method. The cell growth cycle and apoptosis were detected by FCM. The expression of PIN1 was detected by Western blot. The expression of PIN1 mRNA was detected by reverse transcription polymerase chain reaction （RT-PCR）. Results： MTT and FCM assays indicated that the transfection of antisense PIN1 gene could inhibit profiferation of MG-63 cells and lead to cell apoptosis. Western-blot assays revealed the MG-63 cells transfected with antisense PIN1 gene had weaker expression than those without transfection with antisense PIN1 gene, and the band intensity was negatively related with doses. The cells transfected with different doses of gene （0,20,50,100,200,250μl） had different absobance rate（0.854±0.136,0.866±0.138,0.732±0.154,0.611 ± 0.121, 0. 547 ± 0. 109, 0. 398 ± 0.113, 0. 320 ± 0.151）, with significant difference assessed by F and q test （P〈0.05）. The absorbance rate of PINI mRNA was 0.983±0.125,0.988±0.127, 0.915±0.157, 0.786 ± 0.125,0.608 ± 0.124,0.433 ± 0.130,0.410 ± 0. 158 respectively （ P 〈 0.05）. Conclusion. The expression of PIN1 mRNA in MG-63 cells could be inhibited by antiseuse PIN1 gene, and then the expression of PIN1 was reduced and depressed, and so the proliferation of hmnan osteosarcoma cells MG-63 was inhibited.
文摘Objective: To clone and construct eukaryotic expressing vectors of sense and antisense human Pin1 (hPin1) genes. Methods: Total RNA was extracted from MG-63 cells, then the hPin1 cDNA was amplified by RT-PCR. The same time the sense and antisense hPin1 genes were formed by binding BamH I and Hind III in cis and trans-directions. At the end they were cloned into the eukaryotic expressing vector pIRES2-EGFP in cis and trans directions using DNA recombinant technology. The recombinant vectors were further identified by digestion of BamH I and Hind III. Results: The results of sequencing showed that the orientation of the ligations and the reading frame were correct. After digested by BamH I and Hind III, two fragments exhibiting 5.3 kb and 0.99 kb were formed in sense and antisense eukaryotic expressing vectors. Electrophoretic results were completely coincident with theoretical calculation. Conclusion: Human Pin1 sense and antisense genes were successfully cloned and eukaryotic expressing vectors were successfully constructed.
