The Effects Of Connexin43on Cerebral Vasospasm In Vitro Experiment

Objective：Gap junction is the fastest and most direct communication mode betweenadjacent cells, which plays an important role to maintain the synergistic responsebetween vascular cells. Four kinds of connexin were found in the cardiovascularsystem, including Cx37, Cx40, Cx43and Cx45, and Cx43is the most important. Thedisorder of Cx43expression in vascular cells will lead to a series of pathologicalreactions and diseases. Previous studies have found that the Cx43gap junction andcerebral vasospasm (CVS) are strongly coupled. But the mechanism is still unknown.On the basis of previous studies, this issue makes an in-depth analysis of Cx43involved in CVS。 Through lots of in vitro experiments the issue wants topreliminary prove the molecular mechanism of Cx43participate in the CVS.Materials and methods：1.Primary culture and identification of Rats basilar artery smooth muscle cells(SMCs): Isolate SD-rat basilar arteries and cultivate buy explants attachment method,use the immunofluorescence techniques to identify the cultured vascular smoothmuscle cells.2.Construction of shRNA-Cx43adenovirus vector and detection of infectionefficiency of virus vector: Construct the plasmid, package and detect the titer by the293A cells; Infect the target cells by virus with different dilution, find out the besteffect dilution resulting from cells growth states and fluorescence, and use the besteffect dilution as the basis of subsequent test；Distinguish the changes of Cx43proteinexpression between before and after viral infection by the Western blotting analysis.3.Construction of the cerebral vasospasm cell model and observation of thechange of contraction signal communications: Stimulate smooth muscle cells by theOxyHb to construct the cerebral vasospasm cell model, use the Western blotting analysisto conduct the changes of Cx43protein expression in smooth muscle cells, conduct thechanges of the length of smooth muscle cells by the HE staining, and use the confocalmicroscopy to observe the signal exchange between the gap junction of SMC and SMC, SMC and EC.4.Impact of the reduction of the Cx43expression on signal communication on invitro model of cerebral vasospasm: Infect the smooth muscle cells by theshRNA-Cx43adenovirus, and construct the cerebral vasospasm double-culture model,use the Western blotting analysis to conduct the changes of Cx43protein expression in smoothmuscle cells, conduct the changes of the length of smooth muscle cells by the HEstaining, and use the confocal microscopy to observe the signal exchange between thegap junction of SMC and SMC, SMC and EC.Results:1. Cultured the basilar artery smooth muscle cells successfully. The cellphenotype and anti-α-actin antibody immunofluorescence staining indicated that thecells grown in good condition and their morphology were correct, the purity washigh.2. The PAV.KdSi.U/eGFP-U6-shCx43plasmid was constructed successfully,CPE could be seen after viral packaging formation, and the viral titer was1.07×1011PFU/mL.The Western blotting analysis found that Cx43expression level was lowest at24h, andthe experiment achieved the expected effect.3. Detected the Cx43expression and the length of smooth muscle cells ofdifferent kinds of cells, including the normal smooth muscle cells, the smooth musclecells after OxyHb stimulate24h, the smooth muscle cells after OxyHb stimulate48h,the smooth muscle cells after OxyHb stimulate72h. The result found out that theCx43expression gradually increased after the stimulation of OxyHb，and reached thepeak at48h, then the Cx43expression gradually declined. The length of smoothmuscle cell changed consistent with Cx43expression，the length of smooth musclecell gradually increased after the stimulation of OxyHb，and reached the peak at48h, then gradually declined. The length of infected smooth muscle cells longer thanthe uninfected cells.4. Used the confocal laser microscopy to detect the fluorescence intensity of the"receptor cells" in the double-cell co-culture model，and found out that muscle whichinfected by the shRNA-Cx43adenovirus could weaken the transportation of dyeCalcein AM and calcium ion between the smooth muscle cells，and also could weaken the transmittability of IP3from smooth muscle cells to endothelial cells. Nofluorescence appeared in "control cells" of each group.Conclusion：1.The Cx43expression in smooth muscle cells increased after the stimulation ofOxyHb，and the length of smooth muscle cells shorten at the same time. Reducing theexpression of Cx43in the smooth muscle cells can effectively alleviate thecontraction of smooth muscle cells.2.Changes of the expression of Cx43can affect the exchange of non-specificsubstances Calcein AM and shrinkage factor Ca2+between smooth muscle cells， thechanges also have effect on the transmission rates of IP3from smooth muscle cells toendothelial cells3.The expression of Cx43will increase in the vitro model of cerebral vasospasm,and the Cx43expression of smooth muscle cell is positively with the length of thesmooth muscle cells. The result demonstrates that Cx43is associated with smoothmuscle contraction, but the detailed mechanism needs further research; increasedexpression of Cx43can also affect the communication of factors associated withsmooth muscle cell among cells. The more information about Cx43expression, thestronger transmission of contract factor; the mechanism of Cx43participated in CVSmay provide new ideas and targets for prevention and treatment of cerebralvasospasm.