| Background and objective:Excessive proliferation of VSMC is the common pathologic basis for artherosclerosis and restenosis after revascularization. DNA replication is an essential character of VSMC proliferation. ORC is a complex of DNA replication origin protein in eukaryotic cells, and it cooperates with other origin factors on chromatin involves in the initiation of DNA replication. ORC is composed of six subunits, which are called ORC1~6 respectively, and ORC1 is the largest subunit of ORC. In early G1 phase, ORC binds to DNA replication origin site on chromatin, which provides a platform for the other initiation factors (such as Cdc6, Cdt1, Mcm2~7) to combine together and form the pre-RC. Activated by various kinase, pre-RC promotes cell from G1 phase to S phase and commences DNA replication. In mammalian cells, the ORC2~6 subunits bind to the DNA, ORC1 is removed from the chromatin as cells progress into S phase and rebound to chromatin only as cells progress into G1 phase. It was defined as"ORC cycle". So, It is a widely accepted opinion that ORC1 is the key factor to regulate cell proliferation.Rapamycin is one of the most usually used drugs of drug-eluting stent, and its inhibitive effect on VSMC proliferation is complex, it maybe through PI3K-AKT-mTOR pathway to inhibit mTOR, then inhibit composition of proteins and keep cells mainly in G0/G1 phase. Our earlier studies found that ORC1 gene silenced by RNA interference can inhibit VSMC proliferation and keep cell mainly in G0/G1 phase. So, both rapamycin and ORC1 gene scilence can inhibit the proliferation of VSMC. However, in cell cycle, rapamycin and ORC1, which site of action is more prior ? How does the expression level of ORC1 change in cell cycle during rapamycin inhibit the proliferation of VSMC ? it is not clear. In this study, cultured VSMC was obtained from SD rat thoracic aorta to explore the effect and mechanism of rapamycin on the proliferation of VSMC in rats and the action site of rapamycin and ORC1 in cell cycle, in order to provide theory evidences for precaution and treatment of vessel diseases such as As and restenosis after revascularization.Methods:(1) VSMC was obtained by the adherence method of tissue culture, VSMC growth curve and the optimization concentration of rapamycin were determined by MTT.(2) VSMC was divided randomly into two groups, three bottles/wells per group. control groups (CG) were cultured in DMEM with 10% FBS, and experimental groups (EG) were cultured in DMEM with 10μmol/L rapamycin and 10% FBS. Synchronized cell was obtained by serum starvation method. Expression of PCNA, Bcl-2, Bax protein were observed by immunocytochemistry, VSMC ultramicrostructure was analyzed by electron microscope, changes of cell cycle, such as the percentage of cells at G0/G1 phase, at S phase and at G2 phase, were monitored by FCM, cell cycle related factors (cyclin D1 and cyclin A) and apoptosis factor P53 protein were displayed by Western Blot, expression of ORC1 at transcription and protein level were displayed by RT-PCR and Western Blot when VSMC was cultured for 48 h.Results:(1) VSMC was isolated successfully and its good proliferative activity was obtained. Compared with CG, VSMC proliferation in EG was inhibited significantly by 10μmol/L rapamycin cultured for 48 h (P<0.05).(2) Compared with that in CG after 10% FBS cultured for 48 h, positive expression ratio of PCNA protein in EG decreased significantly (20±2.1% vs 80±3.0%, P<0.05), expression level of P53 protein increased gradually (38.2±2.4 vs 5.0±2.6 at 12 h, 70.0±2.7 vs 23.0±2.8 at 48 h, P<0.05), Bcl-2 expression level significantly falled (10±5.0% vs 94±3.0%, P<0.05) but Bax expression significantly increased (95±2.0% vs 2±0.5%, P<0.05), which caused the ratio of Bcl-2/Bax decreased significantly.(3) Microscopic structure in CG showed that nucleus margin was irregular, chromatospherite was clear, ecphyma of cell surface increased, organelle abundant and crista mitochondriales increased without chromatin assemble observed. However, VSMC in EG showed that nucleus margin was regular, ecphyma of cell surface decreased, some chromatin assembled to membrane of nucleus, forming massive or crescent.(4) Both in CG and in EG at 0 h, VSMC was mainly in G0/G1 phase (80.0±0.5% vs 79.9±0.5%), accompany with less cells in S phase (10.4±0.2% vs 10.0±0.3%). After 10% FBS stimulus in CG, percentage of G0/G1 phase gradually decreased (60.0±0.2% at 12 h, 55.0±0.3% at 24 h and 53.0±0.3% at 48 h), and the ratio of S phase significantly changed (10.4±0.2% at 12 h, 31.3±0.3% at 24 h and 23.3±0.1% at 48 h). However, VSMC of post-FBS stimulus in EG was lasted mainly in G0/G1 phase all the time (81.3±0.6% at 12 h, 84.7±0.6% at 24 h and 85.7±0.1% at 48 h), with less ratio of cells at S phase (8.7±0.2% at 12 h, 4.8±0.3% at 24 h and 5.5±0.3% at 48 h). Compared with CG at the same time, percentage of G0/G1 phase increased gradually but S phase and G2 phase decreased gradually from 12 h to 48 h in EG.(5) Compared with that in CG at the same time, expression level of cyclinD1 slightly increased (12 h :37.4±0.1 vs 29.3±0.5, 48 h :67.2±0.9 vs 52.1±0.8, P>0.05), but expression level of cyclinA significantly decreased in EG (12 h :80.6±0.7 vs 170.0±0.8, 48 h :63.2±0.6 vs 160.2±0.1, P<0.05).(6) The expression level of ORC1mRNA in CG significantly increased from 12 h~ 48 h, and its peak was at 12 h (0 h:1.2±0.2, 12 h:1.1±0.3, 24 h:0.6±0.1, 48 h:0.3±0.2). But ORC1mRNA expression in EG were kept at high level (0 h:1.2±0.1, 12 h:1.2±0.2, 24 h:1.2±0.2, 48 h:1.1±0.2). Compared with CG at the same time, expression of ORC1 mRNA were significantly high at 24 h and 48 h in EG (24 h:1.2±0.2 vs 0.6±0.1, 48 h:1.1±0.2 vs 0.3±0.2, P<0.05). The expression change of ORC1 protein in both EG and CG were similar to that of ORC1mRNA.Conclusions:(1) Rapamycin inhibits the proliferation of VSMC.(2) Rapamycin not only inhibits the proliferation of VSMC, but also promotes its apoptosis.(3) Rapamycin's inhibitive effect on VSMC proliferation probable is through preventing transformation of G0/G1 phase to S phase and inducing cell keep quiescence phase to come true.(4) Rapamycin induces high expression of ORC1 indicating that the effect of ORC1 in cell cycle on the upstem of rapamycin, and further supported the view that ORC1 involves in the initiation process of DNA replication.
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