| Angiogenesis was an important pathological process in a number of conditions, especially in the malignant diseases. In the solid malignant tissue, the neovascularization was constantly induced by the angiogenic factors from the normal host cell and tumor cells itself. Tumor cells received nutrients and removed wastes via newly formed capillaries. Therefore, angiogenesis is essential for the development and translocation of tumor cells. Some studies had confirmed that several inhibitors specific to the different angiogenic factors could inhibit the tumor growth and metastasis. For this reason, to intervene the angiogenesis induced by tumor cells may inhibit tumor's development and translocation of tumor.Angiogenin(ANG) is a basic, single-chain protein of molecular weight approximately 14.1KD, it was first detected in culture medium of human colon cancer cell line HT-29. Like other angiogenic proteins such as basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF), ANG can induce neovascularization significantly. Binding to endothelial cells' cell-surface binding protein (α-actin), angiogenin is endocytosed by endothelial cells. By this means angiogenin can enhance the ability of endothelial cells to digest extracellular matrix components and degrade basement membrane, thereby facilitating cell invasion and migration. Now, some study indicate that angiogenin play a critical role in the processes of angiogenesis, including participating the origination and mature of the neovascularization. The level of ANG in tumor tissues are higher than that in normal tissues. Thus, ANG could be a potential target for developing the angiogenesis antagonists. In the past years, ANG antagonists, including monoclone antibody mAb26-2F,cAb26-2F, antiangiogenin peptid—chANG,chGNA,ANI-E peptide which was derived from the phage clone, actin and anti-actin antibody, novobiocin and IL-1 etc, were designed to arm at different poins of angiogenesis course and acted on the ANG protein. In this study, retroviral vector pBabe/neo was used as expression vector because of its high infection efficiency and stable expression of outside gene. Then, the cDNA of ANG amplified by RT-PCR from A549 was cloned in the antisense orientation to vector. The pseudovirion produced in PA317 packaging cell that was transfected by the recombinant retroviral vector was used to infect A549 cell in vitro. The result demonstrated that antisense RNA could specifically bind to mRNA of ANG and block out expression of ANG gene at level of shearing, transport and translation.Methods:1. The cDNA of ANG mature peptide amplification by RT-PCRHuman lung cancer cell line A549 was cultured in RPMI1640 till the number of cells reached to 107, then general RNA was extracted using Trizol and quantified by UV spectrophotometer. When proceed RT-PCR, 1μl general RNA which was used as templat and 10pmol P1,P2 primers were added in reaction system which total volume was 50μl. The RT-PCR condition is: 48℃ 45min, 94℃ 2min, then proceed circulation: 94℃ 30s, 62℃ 1min, 68℃ 1min, after 30 circulations, reaction system was kept at 68℃ for 7min,finally preserved product at 4℃, 5μl product was electrophoresis in 1% sepharose.2. Clone the product of RT-PCR into T-vectorRT-PCR product was purified by PCR products purification kit, and thereafter cloned into pMD18-T vector. Then the recombination plasmid was transformed into E.coli DH5α and the positive clones were selected, from which we extracted recombination plasmid and verified it by sequencing. 3. Construction of retroviral vectorT-ANG recombination plasmid and retroviral vector pBabe/neo were cut with restriction endonuclease BamH I and ANG cDNA fragment reclaimed from Gel was linked using T4 DNA ligase to linearitied pBabe/neo which was dephosphorylation by Calf Intestine Alkaline Phosphatase. The connection product was transformed into E.coli DH5α and the plasmid in positive clones was extracted and its sequence orientation was determined by restriction endonu...
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