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.

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.

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.

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.

Objective To study the effective sterilization method of laparoscopic instruments. Methods Using 2 different methods to sterilize the same laparoscopic instruments,and then compared the effects of sterilization. Results The bactericial rate of the 2 methods were both 100%. Conclusion The Huiri sterilizing method only need 40 minutes,which fits the requirement of one by one operation.

Objective:To investigate the normal range of exhaled nitric oxide (FeNO) in 6-18-year-old children in China, so as to provide a data base for the establishment of FeNO standards for Chinese children.Methods:A multi-center study was conducted on 5 949 children aged 6-18 (3 101 males and 2 848 females) in 16 pro-vinces of 7 administrative districts in China.According to the technical standard recommended by American Thoracic Society/European Respiratory Association, FeNO was measured, and the relationship of FeNO with the sex, age, height, weight, body mass index and region was discussed.Results:The geometric mean FeNO value of Chinese children aged 6-18 was 14.1 ppb, and its 95% confidence interval (skewness distribution) was 1.0-38.2 ppb.The geometric mean FeNO values of children aged 6-11 and 12-18 were 13.1 ppb and 15.7 ppb, respectively, and their 95% confidence intervals (skewness distribution) were 1.0-38.1 ppb and 2.0-38.2 ppb.For children at and under 11 years old, FeNO decreased with age, with a mean decline of 1 ppb per year.The multiple linear regression results suggested that there was a significant correlation between FeNO and age for children aged 6-11, and FeNO of children aged 12-18 was significantly correlated with the gender, height, and region(all P<0.01). Conclusions:FeNO values of Chinese children and adolescents in this study are higher than those obtained by the previous study conducted from 2010 to 2012.For children aged 12-18, 16 ppb is recommended as the clinical cut-off point.For children at or under 11 years old, the influence of age on FeNO should be considered, and the cut-off point of FeNO decreases by 1 ppb as the age is reduced by one year.

Inflammation-driven endothelial dysfunction is the major initiating factor in atherosclerosis, while the underlying mechanism remains elusive. Here, we report that the non-canonical stimulator of interferon genes (STING)-PKR-like ER kinase (PERK) pathway was significantly activated in both human and mice atherosclerotic arteries. Typically, STING activation leads to the activation of interferon regulatory factor 3 (IRF3) and nuclear factor-kappa B (NF-κB)/p65, thereby facilitating IFN signals and inflammation. In contrast, our study reveals the activated non-canonical STING-PERK pathway increases scaffold protein bromodomain protein 4 (BRD4) expression, which encourages the formation of super-enhancers on the proximal promoter regions of the proinflammatory cytokines, thereby enabling the transactivation of these cytokines by integrating activated IRF3 and NF-κB via a condensation process. Endothelium-specific STING and BRD4 deficiency significantly decreased the plaque area and inflammation. Mechanistically, this pathway is triggered by leaked mitochondrial DNA (mtDNA) via mitochondrial permeability transition pore (mPTP), formed by voltage-dependent anion channel 1 (VDAC1) oligomer interaction with oxidized mtDNA upon cholesterol oxidation stimulation. Especially, compared to macrophages, endothelial STING activation plays a more pronounced role in atherosclerosis. We propose a non-canonical STING-PERK pathway-dependent epigenetic paradigm in atherosclerosis that integrates IRF3, NF-κB and BRD4 in inflammatory responses, which provides emerging therapeutic modalities for vascular endothelial dysfunction.