| At present,lung cancer has become the most frenquent malignant tumor in the world which its mortality and morbidity is increasing fastest.It becomes NO.1 in leading malignant tumors,which is harmful to human health and life, also is the first killer in all cancers.Tumor metastasis is not only the malignant marker and characteristics of lung cancer,but also the key cause of failure to cure and high mortality in lung cancer.Studying and understanding the molecular mechanism of its invasion and metastasis provide a new targeting molecules and pathway . It has been proved that the regulation of biological functions including cell growth, viability, migration, and adhesion of small cell lung cancer (SCLC) cells depends largely on the autocrine or paracrine stimulation of growth factor receptors and chemokine receptors. CXCR4 is a seven-transmembrane G protein-coupled receptor and is also known as a coreceptor for HIV . SDF-1 , the natural ligand for CXCR4, is a member of the CXC chemokine family that has chemotactic activity for hematopoietic progenitor cells. In hematopoietic cells, it has been shown that CXCR4 interact to provide homing to the bone marrow. SDF-1 can enhance migration and proliferation potency in hematopoietic progenitor cells (CXCR4+ ). Recently, CXCR4, the chemokine receptor for stromal cell-derived factor-1 (SDF-1 ), was found to be the major chemokine receptor commonly expressed in SCLC ,has been shown to play an important role in proliferation and metastasis of SCLC. In this study,a recombinant plasmid generating short hairpin RNA was constructed,and to transfect into human small cell lung cancer ce11s - NCI-H446 by liposome.The down-expression of CXCR4 cell clones were chosen by G418.The change of cell proliferation, apoptosis, cell cycle and chemotactic activity, and the change of expression of VEGF,bFGF,and the change of [Ca2+] in cytosol, and the change of expression of bcl-2,PCNA were observed by MTT assay,flow cytometry,cloning test ,chemotaxis assay,RT-PCR,Western blot. The molecular mechanisms of pshRNA-CXCR4 transfection for inhibitting proliferation and metastasis were investigated. PART ONE Objective:To construct a recombinant plasmid generating short hairpin RNA in mammalian cells,and to investigate suppression of CXCR4 mRNA and protein in small cell lung cancer cells- NCI-H446. Methods : Two oligonucleotides CXCR4 cDNA were designed referring to that of GenBank then double-stranded RNA was derived through annealing,finally cloned into Psilencer2.1-U6 neo vector digestedby two restricted endoenzymes according to its special orientation.Human small cell lung cancer ce11s - NCI-H446 were transfected with recombinant plasmid.CXCR4 mRNA was detected by RT-PCR,and CXCR4 protein was detected by flow cytometry in vitro. Results: DNA sequencing showed that the sequence of recombinant vector pshRNA-CXCR4 was successfully constructed and suppressed the expression of CXCR4 mRNA and protein in small cell lung cancer cells- NCI-H446. Conclusion:The recombinant plasmid constructed can suppress the expression of CXCR4 mRNA and protein in transfected cells. PART TWO OBJECTIVE:This study was to investigate effect of recombinant vector pshRNA-CXCR4,on metastasis of transfected NCI-H446 cells. METHODS:There were 4 groups in our study, normal control group(SDF-1α-negative), pshRNA-CXCR4 experimental group(SDF-1α-negative), normal control group(SDF-1α- positive), pshRNA-CXCR4 experimental group(SDF-1α- positive). In the different time ,the cell proliferation, apoptosis, cell cycle and chemotactic activity were observed by MTT assay, flow cytometry, cloning test and chemotaxis assay.RESULTS:According to the results of normal control group(SDF-1α- positive)and pshRNA-CXCR4 experimental group(SDF-1α- positive), the OD-value were 2.457±0.168 and 1.365±0.025(p﹤0.005) 72h after culture, the cloning efficiency were 43.6±3.5% and 20.3±4.8%(p﹤0.05), the apoptosis rate were 5.23±1.01%and 17.54±5.61(p﹤0.05), the numbers of chemotaxis migration cells were 136.27±10.89 and 40.5... |
PDF Full Text Request