Objective: To construct recombinant adenovirus vector of Antisense BTEB2 cDNA and study the effect of antisense BTEB2 on proliferation of vascular smooth muscle cells and neointimal hyperplasia.Methods:BTEB2 cDNA was prepared by RT PCR technique and was subcloned reversedly into shuttle plasmid pDC315 to constructed recombinant shuttle plasmid pDC315AS BTEB2.Then the recombinant shuttle plasmid and adenovirus genomic plasmid pBHGlox?E1,3Cre were cotransfected into 293 cell to obtain recombinant adenovirus. The PCR technique was used to detect target gene fragment and adenovirus genomic characteristic fragment. After the recombinant adenovirus infected vascular smooth muscle cells, antisense RNA expression of BTEB2 was detected by RT PCR. Results:There was recombinant adenovirus containing BTEB2 cDNA in the lysate of cotransfected 293 cells.The recombinant adenovirus infected 293 cells and replicated in 293 cells. The expression of BTEB2 antisense RNA was very obvious in vascular smooth muscle cells after infected by recombinant adenovirus. Conclusion:The construction of recombinant adenovirus vector of Antisense BTEB2 has been achieved, and Antisense RNA of BTEB2 can express in vascular smooth muscle cells. This study has paved the way for furthur research.

Objective To study the effect of rat angiotensin Ⅱ receptor (AT2R) gene transfer mediated by adenoviral vector on neointimal proliferation after rat carotid artery injury. Methods AT2R gene was transducted into the carotid artery by adenoviral vector with GFP after carotid balloon injury. Restenosis models were established in Wistar rats. Immunostaining was performed to examin the expression of AT2R in local arteries. Neointima/media area ratio at day 21 after gene transfer was measured by morphometric analysis. Results The transduction rate of AT2R gene in rat carotid arteries was 40% and the gene expressed constantly in the vessel walls. Neointima/media area ratio of the gene transduction group was significantly reduced (48%) compared with the control group at day 21 after gene transfer. Conclusion The results suggest the possibility that AT2R gene transfer can inhibit proliferation of arterial intima after balloon injury and it is a potential therapeutic approach to prevent neointimal hyperplasia.

Objective To construct the recombinant adenovirus of Angiotensin Ⅱ type 2 receptor (AT2R), and study the effect of AT2R on prevention of neointimal hyperplasia in rat carotid arteries after balloon angioplasty. Methods The recombinant adenovirus of AT2R with green fluorescent protein was constructed by using the method of homogenous recombination in bacteria. AT2R gene was transduced into rat carotid arteries with pAdCMV/AT2R after the reproduction of rat carotid balloon injury restenosis model. The expression and the transfection efficiency of AT2R gene were evaluated by RT-PCR, immunofluorescence staining, and confocal microscope. The intimal/medial area ratio was measured by digital analysis system. Results The titre of purified recombinant adenovirus was 1.5?10 12pfu/ml, and it was proved by PCR. pAdCMV/AT2R transfection efficiency in carotid artery was about 40% at day14, and localized to the neointima, as well as to the media and the adventitia. pAdCMV/AT2R delivered into injured rat carotid arteries significantly up-regulated AT2RmRNA and protein expression in neointima from day 7 to 21 after injury. Compared with pAd-GFP transfection group, pAdCMV/AT2R transfection reduced I/M ratio significantly on day 21(0.78?0.06 vs. 1.36?0.21 respectively, P

Objective To study the change of the expression of cellular growth factor receptor and its effect on the proliferation and migration of vascular smooth muscle cells (VSMCs) under electrical field. Methods 1) A electrophoresis apparatus was designed for applying the direct current electric fields to the cultured VSMCs, which were separated from the aorta of rats. Interval photographs were used to analyse the direction and distance of VSMCs migration. Cellular growth factors such as platelet derived growth factor receptor (PDGFR), angiotensin II type 1 receptor (AT1R) and its type 2 receptor(AT2R) were studied by immunohistochemistry or immunofluorescence to show the mechanism relation to the migration of VSMCs. Results Under the direct current electric field, the expression of PDGFR was increased obviously and distributed asymmetricly on the membrane of VSMCs. The receptor mainly focused on the cathode side of cellular membrane in electric field. The AT1R were also enhanced in the cells after exposure to the electric field, but the expression of AT2R was uncharged. The longer exposure time to the 150 mV/mm electric field, the more electrotaxis the cells showed. The distance of cellular migration increased obviously with the time to exposure the electric field comparing with the control group. There was no significant difference in the expression of proliferating cell nuclear antigen (PCNA) between two groups. Conclusion External electric fields applying to the rat VSMCs can change the growth characteristic and enhance directional migration of VSMCs. These effects maybe related to the up-regulated expression and re-distribution of some cellular growth factors such as PDGFR, AT1R and AT2R.

AIM: To study the effects of BTEB2 antisense RNA on the proliferation of vascular smooth muscle cells(VSMC) and its mechanism. METHODS: After the recombinant adenovirus Ad.ASBTEB2 infected VSMC, antisense RNA and protein expression of BTEB2 were evaluated respectively by RT-PCR and Western blotting. Proliferation and cell cycle progress of VSMC were analyzed respectively by MTT test and flow cytometry. The expression of PCNA, AT1R and PDGF-BB were detected by immunocytochemistry. RESULTS: The expression of BTEB2 antisense RNA was demonstrated in VSMC after infected by recombinant adenovirus. Ad.ASBTEB2 infection significantly inhibited BTEB2 protein expression and proliferation of VSMC induced by serum, and resulted in G_0/G_1 blocking. The inhibitory effects of BTEB2 antisense RNA on the expression of PCNA, AT1R and PDGF-BB were demonstrated by immunohistochemistry. CONCLUSION: BTEB2 antisense RNA significantly inhibits the proliferation of VSMC, probably by suppressing the expression of VSMC proliferation related genes, such as PCNA, AT1R and PDGF-BB.

Objective To construct recombinant adenoviral vector expressing basic transcription element binding protein 2 (BTEB2) antisense RNA and study the effects of BTEB2 antisense RNA on the proliferation of vascular smooth muscle cells (VSMCS). Methods BTEB2 cDNA was prepared by RT-PCR technique and was subcloned reversedly into the shuttle plasmid to construct recombinant shuttle plasmid, and then the recombinant shuttle plasmid and adenovirus genomic plasmid pBHG were cotransfected into 293 cells to obtain the recombinant adenovirus. The PCR technique was used to detect the target gene fragment and adenovirus genomic characteristic fragment. After transfection of the recombinant adenovirus into VSMCs, BTEB2 antisense RNA was detected by RT-PCR. The effects of BTEB2 antisense RNA on BTEB2 protein expression, proliferation, and cell cycles of VSMCs were analyzed by Western blot, MTT test, and flow cytometry, respectively. Results The recombinant adenovirus was proved correct by PCR. The expression of BTEB2 antisense RNA was very obvious in VSMCs after infection by recombinant adenovirus. Recombinant adenovirus infection significantly inhibited BTEB2 protein expression and proliferation of VSMCs and resulted in G 0/G 1 block. Conclusion The recombinant adenoviral vector expressing BTEB2 antisense RNA has been constructed. BTEB2 antisense RNA can significantly inhibit the proliferation of VSMCs.

AIM To evaluate the beneficial effects of isoliensinine on paraquat(PQ)-induced acute lung injury and pulmonary fibrosis and explore the mechanism of its action. METHODS PQ (45 mg·kg-1, ip)-induced acute lung injury and PQ (100 mg·kg-1, ig)-induced pulmonary fibrosis were prepared. At 8, 24 and 48 h after PQ administration, the effects of isoliensinine (20 mg·kg-1, ig, 3 times a day, from 24 h before PQ administration to the end of experiment) on activity of superoxide dismutase (SOD), alkaline phosphatase (ALP) and the content of malondialdehyde (MDA) in plasma and bronchoalveolar lavage fluid (BALF) of acute lung injury groups were evaluated respectively. On the 14 d following PQ ingestion, the effects of isoliensinine (10, 20 and 40 mg·kg-1, ig, twice a day, from 24 h before PQ administration to the end of experiment) on hydroxyproline content, transforming growth factor β1 (TGF-β1) and matrix metalloproteinase-2 (MMP-2) expressions and the histopathological changes in lung tissues of pulmonary fibrosis groups were observed. RESULTS In the acute lung injury model, isoliensinine (20 mg·kg-1) significantly increased SOD activity, and decreased MDA content and ALP activity, as well as ameliorated the histopathological damage of lung tissue compared with PQ group. However, the indexes mentioned above in isoliensinine alone group did not change obviously compared with normal saline group. In the pulmonary fibrosis model, isoliensinine (10, 20 and 40 mg·kg-1) resulted in a dose-dependent decrease of hydroxyproline content compared with PQ group [(2.11±0.21), (1.94±0.24) and (1.89±0.26), respectively, vs (2.44±0.33) mg·g-1 wet tissue]. The expressions of TGF-β1 and MMP-2 in the lung tissue of the isoliensinine 40 mg·kg-1+PQ group were significantly less than those of the PQ group. Furthermore, isoliensinine could improve the histopathological changes of fibrosis as comparison with PQ group. CONCLUSION Isoliensinine has protective effects on PQ-induced acute lung injury and pulmonary fibrosis.

0.05).pAdCMV/AT2R transfection significantly decreased the expression of PCNA in neointima at day 14 [(27.29?5.81)% vs(72.25?4.47)%,(68.43?9.12)%,P

Objective:To construct the recombinant adenovirus of AT2R by using the method of homogenous recombination in bacteria. Methods:AT2R gene was get from the vector of PUHD-AT2R by PCR ,and subcloned into shuttle vector of pAdTrack-CMV, forming transfer vector of pAdTrack-CMV/AT2R.Then it was linearized with PmeⅠ and transformed into Adeasier-1 cell. The DNA of identified recombinant plasmid was digested with PacⅠ and transfected to 293cells to package adenovirus. The PCR technique was used to detect target gene . The titre of the Ad-AT2R was measured with the aid of GFP expression. Results:PCR test indicated each the recombinant adenovirus contained the insert of AT2R. The titre of purified recombinant adenovirus was 1.5?10 12pfu/ml. Conclusion:The method of homologous recombination in bacteria is more convenient and efficient compared with that in cell .The prepared Ad-AT2R paves a sound foundation for further study.

Objective:To identify the sensitivity,reliability in diagnosis and prediction the outcome for neonatal sepsis and septic shock.Methods:Plasma PCT levels of 108 neonates with sepsis(40 with septic shock) were measured by semi-quantitative solid phase immunoassay.At the same time,the plasma CRP and IL-6 levels were observed.Results:The positive rate of PCT was significantly higher in patients with neonatal sepsis and septic shock compared with control group and plasma PCT levels of septic shock group was the highest.The prevalence and fatality rate of septic shock were associated to the plasma levels of PCT,the higher was the PCT,the much higher was the prevalence and fatality rate of septic shock.The plasma PCT levels were significantly correlated with the plasma levels of CRP and IL-6.The sensitivity(87.5%),specificity(82.4%),positive predictive values(74.5%),accuracy(84.3%) of PCT all higher than that of CRP and IL-6.Conclusion:Increased plasma PCT levels in the early stage of neonatal sepsis and septic shock were associated with the severity of illness.PCT is superior than CRP and IL-6 as a marker of severe bacterial infection.