Tan Jing, Sun Min, Mo Xiao-hua, Lu Qi-yu, Zhou Xiao-hu
Department of Hepatobiliary and Pancreatic Surgery, The 2nd Affiliated Hospital of Kunming Medical University, Kunming 650101, China.
Nan Fang Yi Ke Da Xue Xue Bao. 2010 Aug;30(8):1847-50.
To study the transfection of pancreatic cancer cells BxPC-3 with recombinant plasmid pSilencer4.1-CMV neo-hTERT-siRNA and its silencing effects.
Pancreatic cancer cells BxPC-3 transfected with recombinant plasmid pSilencer4.1-CMV neo-hTERT-siRNA were selected as target and divided into five groups: (1) T1 group (pSilencer4.1CMV neo-hTERT1-siRNA), (2) T2 group (pSilencer4.1CMV neo-hTERT2-siRNA), (3) Lipofectamine (Lipofectamine), (4) mismatch group(pSilence4.1CMV, as negative control), (5) cell control group(without transfection). The expression of hTERT mRNA was detected by RT-PCR. The viability of cells was measured by MTT method. The cell cycle and cell apoptosis was measured by flow cytometry. The expression of telomerase protein was measured by Western blot.
Compared with Lipofectamine group, negative control group and cell control group, the expression of hTERT-mRNA and telomerase protein in cells was downregulated significantly(P<0.05), the viability of BxPC-3 cells was decreased significantly (P<0.05), the ratio of cells in G0/G1 stage was increased, the ratio of cells in S stage and G2/M stage was decreased, and the ratio of apoptotic cells was increased significantly in T1 group and T2 group.
Recombinant plasmid T1 and T2 can downregulate the expression of hTERT mRNA and telomerase protein in BxPC-3 cells , and has good RNAi silencing effects. T1 and T2 can also inhibit the growth of BxPC-3 cells, block the cell cycle, promote the apoptosis of cells, and has anti-pancreatic cancer effects in vitro.
研究重组质粒pSilencer4.1-CMV neo-hTERT-siRNA对胰腺癌BxPC-3细胞的转染及其沉默效应。
将转染重组质粒pSilencer4.1-CMV neo-hTERT-siRNA的胰腺癌BxPC-3细胞作为研究对象，分为五组：（1）T1组（pSilencer4.1CMV neo-hTERT1-siRNA），（2）T2组（pSilencer4.1CMV neo-hTERT2-siRNA），（3）脂质体组（Lipofectamine），（4）错配组（pSilence4.1CMV，作为阴性对照），（5）细胞对照组（未转染）。采用RT-PCR检测hTERT mRNA的表达。采用MTT法检测细胞活力。采用流式细胞术检测细胞周期和细胞凋亡。采用Western blot检测端粒酶蛋白的表达。
与脂质体组、阴性对照组和细胞对照组相比，T1组和T2组细胞中hTERT-mRNA和端粒酶蛋白的表达明显下调（P<0.05），BxPC-3细胞活力明显降低（P<0.05），G0/G1期细胞比例增加，S期和G2/M期细胞比例降低，凋亡细胞比例明显增加。
重组质粒T1和T2可下调BxPC-3细胞中hTERT mRNA和端粒酶蛋白的表达，具有良好的RNA干扰沉默效应。T1和T2还可抑制BxPC-3细胞生长，阻断细胞周期，促进细胞凋亡，并在体外具有抗胰腺癌作用。
