ABSTRACT
Objective To explore the effect of Cyclin A on origin recognition complex-1 (ORC1) expression of rat vascular smooth cells (VSMCs).Methods Primary VSMCs of thoracic aorta in rats was obtained by the adherence method of tissue culture.The synchrony of cell was obtained by the method of double-thymidine block.In different cell cycles of VSMCs,the expression of ORC1 mRNA was determined by RT-PCR and the protein expression of ORC1 was observed by flow cytometry.Results Synchronized VSMCs were obtained and identified by the methods of double-thymidine block,colchicine treatment and serum starvation.Synchronized growth was monitored by flow cytometry.All the synchronized VSMC's distribution ration was (89.22±3.54) ％ at G0/G1 phase,(66.74 ±7.16)％ at G1/S phase,(63.24 ±4.06)％ at S phase and (51.64 ± 11.18)％ at G2/M phase and there was statistically significant difference compared with other phase(P ＜0.01).There was no significant effect of Cyclin A on ORC1 mRNA expression at a quiescent stage of VSMC.At G2/M phase peaked ORC1 was (52.133 3 ± 2.122 1)％,at G1/S phase was(10.916 7 ± 0.531 1)％,at S phase was (7.656 7 ± 0.412 4)％,and there was statistically significant difference (P ＜0.01).At G2/M phase ORC1 downed to the lowest point was (1.276 7 ± 0.161 7) ％,at G1/S phase was (13.371 0 ± 1.057 3)％,at S phase was (3.043 3 ± 0.538 0)％,and there was statistically significant difference (P ＜ 0.01),suggesting that Cyclin A might prevent ORC1 binding the chromatin of VSMCs.Conclusion Cyclin A may be an important regulative factor at the initiation of ORC1 in VSMCs.
ABSTRACT
Objective To investigate the influence of RNA interference targeting ORC1 gene on the proliferation of rat vascular smooth muscle cells(VSMCss).Methods VSMCss were transfected with siRNA targeting ORC1 gene by liposome.The expression of ORC1 protein was detected by Western blotting.MTT test and ~(3)H thymidine~()(~(3)H-TdR) incorporation were used to detect VSMCss proliferation.The expression of proliferating cell nuclear antigen(PCNA) in VSMCss was detected by immunocytochemistry.Results After transfected of the three pairs of siRNA targeting ORC1 genes respectively,the expression of ORC1 was all lower than that in the control group(non-transfection and negative siRNA).Especially,that in the group transfected of the second pair positive siRNA decreased most significantly.The optical density of MTT,~(3)H thymidine incorporation and the expression of PCNA decreased significantly in VSMCss transfected with siRNA targeting ORC1 gene as compared with that in the control group.Conclusion ORC1 gene silenced by RNA interference can inhibit VSMCss proliferation.
ABSTRACT
Objective To explore origin recognition complex 1(ORC1) expression in the rat vascular muscle cells at different phases of proliferation.Methods Vascular smooth muscle cells(VSMCs) of thoracic aorta of rats in primary culture were obtained by the adherence method of tissue culture.Total RNA of VSMCs was extracted.The expression of ORC1 mRNA of VSMCs at different phases of proliferation was determined by reverse transcription polymerase chain reaction(RT-PCR) and the expression of ORC1 protein by immunocytochemistry and laser confocal microscopy.Results Cultured VSMCs were confirmed by light microscope and immunocytochemistry.The expression of ORC1 mRNA in the quiescence stage of VSMCs was not found.After VSMCs were stimulated with serum,the level of ORC1mRNA had an obvious increase at 6 h,peaked at 12 to 24 h and decreased in the following 24 h.The expression of ORC1 protein was also not found in the quiescence stage of VSMCs,but the level of ORC1 protein during proliferation of VSMCs was significantly increased.Conclusion ORC1 may have an important role during the process of VSMCs proliferation in rats.
ABSTRACT
Objective To explore the expression of origin recognition complex1(ORC1) during DNA replication progress of rat vascular muscle cells(VSMCs).Methods VSMCs of rat thoracic aorta were obtained by the adherence method of tissue culture.The growth curve was drawn by MTT.The association between DNA replication and the expression of ORC1 mRNA and protein in different growth phases of VSMCs was analyzed.Results The expression of ORC1 mRNA and protein in quiescence stage of VSMCs was not found.After stimulated with serum,the expression of ORC1 mRNA in rat VSMCs increased significantly,peaked at 12-24 h.The expression of ORC1 protein was similar to ORC1 mRNA in VSMCs.Meanwhile,the higher DNA replication of stimulated VSMCs was observed,peaked at 12-24 h after serum addition. Conclusion ORC1 may be involved in the DNA replication of rat VSMCs during the progress of proliferation.