Construction Of Eukaryotic Expression Vector Of Notch1 Gene And Its Effects On Eca109 Cells Apoptosis

Objective:Esophageal carcinoma(EC) is one of the most common malignant tumors in the world.It is the sixth most prevalent malignancy worldwide,the forth in the China. Esophageal carcinoma exists in 2 main histopathological types-esophageal squamous cell carcinoma(ESCC) and esophageal adenocarcinoma(EADC).In China,about 90%of esophageal cancers are ESCC.China is a high-incidence country of esophageal carcinoma,more than 70%of all esophageal cancer patients in the world are Chinese. Those methods of tumor therapy used currently are not perfect,such as surgical operation, chemotherapy,radiotherapy.The basic research of therapeutic measure still need breakthrough.Now,there are many gene therapy methods for esophageal carcinoma. Among them,gene correction and induced apoptosis are two important ways,especially the research on the process of cell apoptosis has been paid more and more attention to. Notch proteins are transmembrane receptor family,which widely expressed and highly conserved throughout evolution.As a signal transduction pathway,the Notch gene turned out to be not only plays a critical role in normal tissue and cells differentiation and growth but also correlate to the occurrence and growth of tumorous.Some scholars found that Notch1 expressed abnormally in tumorous,for instance,in the uterine cervix cancer, endometrial cancer,renal carcinoma,lung cancer,breast cancer and neuroblastoma. Therefore Notch1 was highly valued more and more as a new method to prevent and treat the tumor.The relationship between Notch1 protein and ESCC has not been detected yet. Our experiments were to construct a eukaryotic expression vector of Notch1 and investigate the effects on cell apoptosis of Notch1 gene in Eca109.We hoped it would provide a theory base of research on ESCC and feasibility of treating esophageal cancer with Notch1 gene in future.Methods:1.Construction a eukaryotic expression vector of Notch1 gene:A eukaryotic expression vector pcDNA3.1(+)-Notch1 was constructed by inserting Notch1 into pcDNA3.1(+) between the enzyme cutting site BamHⅠand XhoⅠ.Total RNA was isolated from human placenta using Trizol reagent.Then RNA was reversely transcribed to cDNA.After the target fragment linked to T vector,PCR,double digests and sequencing were used to detect the correct series of pcDNA3.1(+).PCR and double digests were used to confirm the success on construction of recombinant eukaryotic expression vector pcDNA3.1(+)-Notch1.2.Effects on cell apoptosis of Notch1 gene in Eca109:Notch1 gene was transfected into Eca109.Then observe them with inverted microscope.Cells apoptosis was measured by flow cytometry.Results:1.The results of endonuclease digestion and DNA sequencing indicated target fragment was inserted into the vector correctly and eukaryotic expression vector of pcDNA3.1 (+)-Notch1 was constructed successfully.2.The results of morphology showed that there were no significant differences in 24 hours among Notch1 transfected Eca109 cells after 24h,48h and 72h.In 48h and 72h,the number of apoptosis cells was increased significantly.3.The apoptosis rate of pcDNA3.1(+)-Notch1 transfected Eca109 cells in 24h,48h and 72h were 20.57%,21.50%,38.03%based on Annexin V-FITC,the untransfected group were 4.40%,5.57%,8.80%and the control group were 3.88%,6.26%,8.55%.The apoptosis rate was significantly higher in PcDNA3.1(+)-Notch1 transfected Eca109 cells than the untransfected group and the control group.(p＜0.05) Conclusions:1.Eukaryotic expression vector-pcDNA3.1(+)-Notch1 was constructed successfully.2.Notch1 gene had pro-apoptosis bioactivity in ESCC Eca109 cells.