| Objective: The objective of this study was to investigate the normal constituents of mRNA for connexins and, in cerebral vasospasm cell model, the alteration of mRNA for connexins from the smooth muscle cells of basilar arteries. It is expected to provide basis for further investigation on the role of gap junctions in cerebral vasospasm.Methods: 1) Cell culture and identification of basilar artery's smooth muscle cells. The media of rabbits' basilar arteries were taken and cultivated with the explant attachment method. The cultured cells were identified with light phase contrast microscopy and immunocytochemical staining. 2) Detection of mRNA for connexins in smooth muscle cells of rabbits' basilar arteries. The RT-PCR was performed after the total RNA was extracted from the smooth muscle cells with acid guanidium thiocyanate-phenol-chioroform extraction. The primers for Cx45, Cx43, Cx40, Cx37, and β-actin were used in the PCR. The RT-PCR products electrophoresed on the agarose gels containing ethidium bromide (EB) were observed and photographed. 3) The establishment of cell model of cerebral vasospasm. After the cultured cells were incubated in the medium containing Oxyhemoglobin, the length and ultrastructural changes of the cells were examined with electron microscopy. 4) The semiquantitation of mRNA for connexins in those contracted smooth muscle cells: The total RNA in the contractile smooth muscle cells was extracted. Semiquantitative RT-PCR was performed to measure the relative contain of mRNA for connexins in those cells. The average relative contain of mRNA for connexins in normal group was regarded as normal control. β-actin primers were used as internal control for semiquantitative RT-PCR. The products of PCR were electrophoresed in agarose gel stained with EB and then photographed and semiquantitated with densitometer scanning of the ethidium bromide-stained agarose gels. The density ratio of connexins RT-PCR band to theβ-actin RT-PCR band was used as the relative contain of connexins' mRNA in the cells. The results of RT-PCR were taken down as means ±SE and analyzed with t-test.Results: 1)Identification of cultured artery smooth muscle cells: (1) Under the light phase contrast microscopy, a few of cells with spindle-shape or long-spindle-shape had grown beside the explant for approximately 1 week. After 2 or 3 weeks, the cells reached confluence in flakes and were subcultured .The subcultured cells grew into monolayer or overlapping multilayers like typical "hill and valley" pattern . These observation conforms the characteristic of the shape of the artery smooth muscle cells. (2)Identification: With anti-α-actin immunocytochemical staining, 95% cultivated cells were positively stained. The result indicated that the cultivated cells was smooth muscle cells and the purity of the cells was high. 2) In normal group, the mRNA for, at least, four different connexins, namely Cx43, Cx40, Cx45and Cx37, were expressed in the basilar artery smooth muscle cells by RT-PCR. 3)Cerebral vasospasm cell model was successfully established. The cell average length of normal basilar artery smooth muscle cells was 52.04 um. The cell average length of smooth muscle cells cultivated with the medium containing Oxyhemoglobin is 37.64 um for 24 hours(24-hour group), shortened by 27.67 percent compared with that of normal cells, and 18.08 um for 72 hours(72- hour group),. shortened by 65.26 percent compared with that of normal cells. 4)The results of semiquantitative RT-PCR showed that the average relative contain of mRNA for Cx43 and Cx40 in the 24- hour group and the 72-hour group was distinctly higher than that in the normal group (P<0.05).The expression of mRNA for connexin43 and connexin40 in basilar artery smooth muscle cells was upregulated in the cell model of cerebral vasospasm. Conclusions: 1 Oxyhemoglobin can contract basilar artery smooth muscle cells. 2 Multiple mRNA for connexins were expressed in basilar artery smooth muscle cells. 3 The expression of mRNA for connexi... |
PDF Full Text Request