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Pancreatic cancer (pancreatic carcinoma) serious harm to human life and health of malignant tumor, its high degree of malignancy and the incidence of occult difficult early diagnosis and treatment, clinical diagnosis are mostly of advanced cancer. Very poor prognosis of pancreatic cancer, the overall 5-year survival rate of less than 5%. In addition to early surgery, there is no other effective treatment measures, but also because of the difficulty of early diagnosis, only about 14% of patients can choose surgery. In recent years, the incidence of pancreatic cancer at home and abroad showed a rising trend year by year, it was reported nearly a decade of increased incidence of 3-7 times. 1991, Hrudan statistics, pancreatic cancer the U.S. District tumor cause of death, after lung cancer, colon cancer and breast cancer. In China, with the improvement of people's living standards, changes in diet, the incidence of pancreatic cancer increased from 1980, the twenty-fifth. Shanghai Center for Disease Control statistics in 2000, the incidence rate of pancreatic cancer per 100,000 population, with 6 cases, of which 5.5 people were killed and almost close to mortality, morbidity, this figure is shocking. Is generally believed that the tumor is a disorder caused due to the regulation of cell growth mechanism. Normal cell growth and proliferation gene regulation by the two categories, one is positively regulated signal to promote cell growth and proliferation, and prevent the occurrence of terminal differentiation, known in most of the oncogene (Oncogene) is such a case. The other for the negative regulation of signal to promote cell maturation, terminal differentiation, anti-cancer gene (anti-Oncogene) may play this role. These two signals to maintain homeostasis, precise regulation of normal cell growth, proliferation and death. Once the normal relationship between the two is destroyed, then make the regulation of cell disorders resulting in malignant cells. The Ras gene located on 1lq13 H-ras, K-ras and N-ras gene family. K-ras gene encodes a protein located on the cell membrane guanylate binding protein is responsible for the external signal transduction to the cell interior. The encoding of the K-Ras p21 ras protein having GTP activity, mutant ras protein can lead to lost GTP activity, GTP in a continuous activated state in sustained activation of the signal transduction stimulation, the cells continue to proliferate, and become cancerous. Pancreatic cancer K-ras gene exon 12 point mutation at codon has proven to be an early event in pancreatic tumors, is an important indicator of early diagnosis of pancreatic cancer. K-ras gene mutation in pancreatic average rate of around 80%, which is the most common mutation in codon 12. Codon 12 point mutations the Youyi GAT (46%), GTT (32%), and CGT (13%), three forms of the most common. Studies have shown that inhibition of the expression of mutant K-ras, can effectively inhibit the growth of pancreatic cancer cells. RNA interference (RNA interference) technology is a new technology of a suppressor genes discovered in recent years, because of its specificity and high efficiency in cancer research became a hot spot. The experiment was based on mutations in the K-ras gene sequence to build two pancreatic cancer cell lines specific shRNA plasmid vector pGenesil-1-P1 pGenesil-1-M2 and negative control pGenesil-1-HK. Observed RNA interference for the therapeutic effect of the K-ras gene mutations in pancreatic cancer, and verify that the two non-specific shRNA sequences cross-inhibition on pancreatic cancer cells, finding new ways for gene therapy of pancreatic cancer. Objective: To study the short hairpin RNA (shRNA) on human pancreatic cancer cells PANC-1 and Miapaca-2 mutant K-ras gene expression inhibition, as well as the two shRNA fragments of non-specific pancreatic cancer cells whether cross-inhibition. Methods: Construction of mutant K-ras genes in PANC-1 cells recombinant plasmid the carrier pGenesil-1-P1, and the recombinant plasmid for Miapaca-2 cells of mutant K-ras gene the carrier pGenesil-1-M2-negative control the plasmid pGenesil-1-HK. 2 cell cultures transfected with Lipofectamine 2000 and applied recombinant the plasmid pGenesil-1-P1 to pancreatic cancer cells PANC-1, the recombinant plasmid vector pGenesil-1-M2 transfection in Miapaca-2 pancreatic cancer cells. And cross two recombinant plasmid vector transfected two pancreatic cancer cells. 3 cell RNA extracted 48 hours after transfection, total protein, application of RT-PCR, immunoblotting (Western blotting) was detected by RNA interference of pancreatic cancer cells mutant K-ras gene expression. 4 Application CCK-8 Measurement of cell growth curve. Results: sequencing the plasmid vector was constructed successfully observed by fluorescence microscopy recombinant plasmid successfully transfected into pancreatic cancer cells, RT-PCR product by agarose electrophoresis and Western results showed that the mRNA and protein expression of specific transfection group were reduced (p lt; 0.05), rather than the the specific transfection group of K-ras gene mRNA and protein expression showed no significant change. Application of CCK-8 was used to detect cell growth curve showed specificity transfected cells growth was inhibited (p lt; 0.05), rather than the specific growth of cells transfected group was significantly inhibited. Conclusion: After the specific shRNA recombinant plasmid pancreatic cancer cells, pancreatic cancer cells mutant K-ras gene mRNA and protein expression and cell growth was inhibited, rather than specific and negative control plasmid transfected pancreatic cancer cells mRNA and protein expression, and cell growth is not inhibited.
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