| Background and purposeTropomyosin(TM) is a regulatory protein of the actomyosin system in muscle contraction. The present study was gene cloning of human smooth muscle TM isoform-β (hsmTM-β) to investigate the effect of smooth muscle TM (smTM) on vascular smooth muscle cell migration. It is helpful to explore the role of TM further in smooth muscle.Methods and resultsl.Gene cloning of hsmTM-pFrom human aorta cDNA library the cDNA fragment of hsmTM-p was amplified by nested primers-PCR. One of the ends of the PCR product was trimmed using dGTP and T4 DNA polymerase. The vector named 'pLIB-AS' was constructed on the base of the retrovirus expressing vector pLEB inserted by AS-RI-Nru fragment in sense orientation. pLIB-AS was digested by restriction endonuclease Nrul and EcoRI. The insert DNA and the vector DNA were purified by phenol/chloroform and ethanol, followed by DNA purification from 0.7% agarose gel using GENECLEARN Turbo Nucleic Acid Purification kit. The insert DNA was cloned into pLIB-AS in antisense orientation using DNA ligase, generating pLEB-AS-hsmTM-β. The recombinant plasmid was transformed into the competent XL10-Gold E.coli cells with the heat shock method, followed by extraction and purification of plasmid DNA in minimal scale with alkali lysis method. To check for desired constructions, restriction endonuclease EcoRI was used to examine the total lengths and the inserts were confirmed by PCR. The results showed that desired recombinant plasmid was successfully obtained in antisense inserting way, realizing hsmTM-p gene knockout. In addition, DNA SIS software was utilized for alignment of nucleotide sequences of TM-P cDNA from human smooth muscle and gizzard smooth muscle. It was found the homology was 84.7% in 772bp overlap.2.The transfection and expression of hsmTM-p recombinant plasmid According to retrovirus gene transfer and expression user manual, AmphoPack -293 cells and human coronary artery smooth muscle cell (CASMC) were cultured in DMEM and SmBM to the third passage, respectively. Using CalPhos?mammalian transfection kit, hsmTM-p recombinant plasmid 'pLIB-AS-hsmTM-β' containing antisense cDNA of hsmTM-p was transfected into AmphoPack-293 cells to produce retrovirus, which was then to infect CASMC for gene expression. On the same time, pLIB-EGFP containing cDNA of enchanced green fluorescent protein (EGFP) was transfected and expressed in the same way as above. Small and large amount protein from gizzard smooth muscle TM (gTM) were transfected into cells in hsmTM-β/AS expressing groups using Chariot?protein transfection kit. Finally the expression of protein was analyzed by Western blot. 3. Western Blot analysisAfter 12.5% SDS-PAGE electrophoresis of proteins derived from CASMC, the gels were transferred onto polyvinylidene fluoride (PVDF) membrane. For Western blot, the membrane was blocked with 5% non-fat dried milk power in PBS-0.5% Tween 20 (PBS-T). The first antibody is rabbit anti-tropomyosin polyclonal antibody, diluted in PBS-T (1:1000). A horseradish peroxidase (HRP) labeled goat anti-rabbit IgG was used as the secondary antibody (1:5000). The X-ray film was developed to visualize the protein bands, compared with Coomassie Brilliant Blue-stained PVDF membrane. The results of Western blot revealed that the expression of hsmTM-p protein was knockout, compared with the control group, EGFP expressing group and purified gTM as a standard. The expression of smooth muscle TM (smTM) appeared when cells were supplemented with gTM in hsmTM-p/AS expressing groups. The more amount of gTM was transfected, the more expression of smTM appears. 4.VSMC migration assayMigration of VSMC was assayed by a modification of the Boyden's Chamber method using microchemotaxis chambers and polycarbonate filter coated with 0.01% collagen previously in use. Cultured CASMCs were trypsinized and suspended at a concentration of 2 x105 in migration cell culture medium-DMEM containing 0.04% BSA. CASMC suspension was placed in the upper chamber, and migration cell culture medium w... |
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