| Objective:Gap junction intercellular communication (GJIC) is the most direct pathwayfor electrochemical signals transmitting between adjacent cells，which play animportant role in regulating process of synergy contraction or relaxation in bloodvessels. Connexin43(Cx43) is the most widely distributed in vascular wall andmainly adjust the conformation and function through phosphorylation process.Preliminary studies in vivo have shown that the Cx43and its Ser368sitephosphorylation level were closely related to the pathological process of cerebralvasospasm (CVS) after experimental subarachnoid hemorrhage (SAH), based on this,this study is to detect the change of total Cx43(T-Cx43) and its Ser368sitephosphorylation(P-Cx43) level in vitro cell model of CVS, change and analysis thelevel of P-Cx43to investigate its potential roles in the pathogenesis of CVS.Materials and methods：1. Primary culture and identification of SD-Rats basilar artery smooth musclecells(SMCs), separated out basilar artery under the sterile condition quickly, Themedia of rabbits’ basilar arteries were taken and cultivated with the explantattachment method. The cultured vascular smooth muscle cells were identified withlight phase contrast microscopy and cell immunofluorescence.2. The establishment of OxyHb-induced cell model of cerebral vasospasm. Afterthe cultured cells were incubated in the medium containing10-6M Oxyhemoglobinfor24h,48h72h, and96h, the length of the cells were examined with opticalmicroscopy after HE staining in random and double blind way. Detected VSMCsphenotype molecular markers α-actin, calponin, OPN to reflect the change of cellcontraction state, determined the total protein expression of Cx43(T-Cx43) as well asthe change of its Ser368site phosphorylation (P-Cx43).3. Construction of recombinant lentiviral vector carrying mutational Cx43onS368. The Cx43cDNA was amplified from pCMV-Cx43cDNA plasmid by PCR and the mutational Cx43on S368was done by site directed mutagenesis. Gatewaytechnology was used to clone the mutational Cx43cDNA into the vector PLV. Des3d.p/neo labeled with enhanced green fluorescent protein (EGFP). The recombinantvector PLV.Ex3d.p/neo-EF1A<Cx43>IRES/EGFP was indentified by PCR and DNAsequencing and was used to cotransfection293T cells with packaging plasmids. Thetransfected293T cells was observed by fluorescence microscope.The titer of viruswas tested by crystal violet dye according to the cloned amount of293T cells.4. Cx43Ser368site mutation influence on cerebral vasospasm in vitroexperiment. Infection efficiency of virus vector in VSMCs detection. At firstcompared the infection efficiency of different MOI values, the second observedexpression of GFP at different time points24h,48h,72h,96h, then detected proteinlevel finally, choosed the optimal MOI to infected VSMCs used to downstreamexperiment.Experimental group,1)PLV.Ex3d.p/neo-EF1A<Cx43>IRES/EGFP infecte-d+OxyHb group,2)PLV.-EGFP infected+OxyHb group,3) uninfected+OxyHb, eachgroup contained normal control, then observe the changes of cell shrinkage degree atcorresponding time points, at the same time, detected the contraction molecularmarkers a-actin, calponin through western blot as the evidence of cell contractionstate, determined expression of T-Cx43as well as the P-Cx43in it.Results：1. The cell phenotype and anti-α-actin antibody immunofluorescence stainingindicated that the cultured cells were smooth muscle cells and the purity of the cellswas higher than95%.2. Established the OxyHb-induced cerebral vasospasm cell model successfully.The average length of cells were69.26±19.23μm(normal group),40.57±12.18μm(24h),29.98±20.19μm(48h),35.87±19.56μm(72h),42.25±17.94μm (96h) respective-ly. Contraction degree of VSMCs induced by OxyHb increased as the incubation timeincreased and reached a peak at48h, and then relaxation with increasing timegradually. The detection results of phenotype molecular markers shown that α-actin,calponin increased gradually and reached the maximum at48h, and then reducedgradually at72h and96h, even though higher than normal control at this two timepoint, the correlation coefficient with the contraction degree of VSMCs were respectively-0.879and-0.945, the expression trend of OPN was just opposite,indicated that the relative expression of these biomarker could be strong evidence ofcell contraction state. And the expression of T-Cx43shown the same trend as α-actinand calponin, but the P-Cx43was different, it had obvious change before48h, andthen increased to a peak (49.2%±10.8compared with normal control10.5%±16.3)significantly at72h, declined slightly at96h but much higher than normal. The rate ofP-Cx43accounted for T-Cx43fallen to minimum gradually at48h, and thenincreased..3. DNA sequencing demonstrated that the recombinant lentiviral vectorPLV.Ex3d.p/neo-EF1A<Cx43>IRES/EGFP content mutational Cx43on S368wasconstructed successfully. Under the fluorescence microscopy, abundant of greenfluorescent can be seized in293T cells. The recombinant lentiviral vector waspackaged in293T cells with a viral titer of9.7×107TU/ml.5. Cx43Ser368site mutation influence on cerebral vasospasm in vitroexperiment. The infected efficiency would be best, when MOI values taken100, andhad no obvious effect on the growth of cells, since the infection of the24h, greenfluorescence was observed, peaked at72h, infection rate were more than98%, andmaintained stability, Western blot detection of P-Cx43in mutations groups was verylow expression after72h, suggested that had achieved the desired effect in proteinexpression level. In mutation group the average length of cells at24h and48h pointwere respectively36.4±16.36μm,28.73±9.53μm, had no obvious differencecompared with that were38.64±16.32μm(24h)、30.96±19.98μm(48h) in uninfectedgroup, but28.12±14.66μm(72h),30.14±20.42μm(96h) were significantly shorter thanthat were36.46±17.53μm(72h) and45.34±17.94μm(96h) in uninfected group, theexpression of biomarkers also matched the results. The expression of T-Cx43had nostatistical difference, but expression tendency of P-Cx43was not obvious as that incontrol group, the proportion of P-Cx43stayed at lower level after48h that was notconsistent with the control. Conclusion：1. OxyHb incubation could induce T-Cx43and P-Cx43expression increased butdifferent time phase, and the change of P-Cx43was significantly related to the cellcontraction state.2. The lentiviral vector express mutational Cx43on S368has been constructedsuccessfully.3. After specific mutation at Cx43phosphorylation site (S368), had led tocontinuous contraction of VSMCs induced by OxyHb, relief was not obvious in late,that Cx43S368phosphorylation increased may play a protective role in thepathologic process of CVS could promote disease relief, Probably because ofphosphorylation rate increased lead to decreased of GJIC, further study of Cx43phosphorylation may provide a new train of thought and targets for the preventionand treatment of CVS. |
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