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1.
AIM:To elucidate the effect of antisense oligo-peptide nucleic acid (PNA) against PDGF-B mRNA on in vivo proliferation of VSMCs and vascular restenosis. METHODS:A rabbit vascular restenotic model was constructed and a synthesized PNA was transfected into the vascular cells using a therapeutic ultrasound for the gene drug delivery. The proliferation of intimal VSMCs was observed by monitoring the expression of proliferating cell nuclear antigen (PCNA) and platelet-derived growth factor B chain mRNA (PDGF-B mRNA) with the LSAB and in situ hybridization, respectively. The intimal thickness and area were measured. RESULTS:PNA delivered by therapeutic ultrasound significantly inhibited the expression of PCNA and PDGF-B mRNA by intimal VSMCs 1 week after denudation with the inhibitory rates of 84.19% and 64.95%, respectively and reduced the intimal thickness and area. CONCLUSION:The PNA against PDGF-B mRNA delivered by therapeatic ultrasound inhibits the proliferation of rabbit VSMCs and the formation of intima.  相似文献   

2.
AIM: To elucidate the in vivo mechanisms of the proliferation of vascular smooth muscle cells (VSMCS) in injuried arteries. METHODS: A VSMCS proliferative model was constructed by injury of rabbit iliac arteries with balloon catheters and a probe designed for rabbit platelet-derived growth factor B chain (PDGF-B ) mRNA was used to detect the expression of it by intimal VSMCS on the vascular cross sections using an in situ hybridization technique at the indicated times. The relation of this expression to the proliferation of VSMCS by their expression of proliferating cell nuclear antigen (PCNA) and vascular intimal areas were estimated. RESULTS: The expression of PDGF-B mRNA of intimal VSMCS was increased when calculating the intimal PDGF-B mRNA positive cells per millimetre area at ×400 magnification with average numbers of 31.93±14.64 in 1 week group, 26.50±9.25 in 2 weeks group and 24.85±13.65 in 4 weeks group. This was in accordance with the expression of PCNA by VSMCS and the increase of intimal areas. CONCLUSION: The local production of PDGF-B by VSMCS via an autocrine mechanism is responsible for the continuous proliferation of these cells and formation of neointima after the injury. The probe designed is very useful for detecting rabbit PDGF-B mRNA.  相似文献   

3.
AIM: To investigate the effects of c-fos antisense oligoneuleotide and p21 genetic transfection on the intimal proliferation of venous autografts. METHODS: The external jugule veins were autografted into common carotid arteries in the same side in 20 New Zealand rabbits, which were divided evenly into experimental and control group randomly. The transplanted veins of experimental group were immersed in the adenovirus-mediated p21 gene solution for 15 minutes just before anastomosis and coated with c-fos antisense oligoneucleotide glue gel just after anastomosis, while the control was only treated with empty vector. The transplanted vascular sample were taken at 2 weeks after operation. The intimal thickness (IT), degree of restenosis (DR), expression of proliferating cell nuclear antigen (PCNA), quantity of VSMC were determined by immunohischemistry. RESULTS: The IT, DR and expression of PCNA, VSMC were decreased, compared to control group. CONCLUSION: Transfection of c-fos antisense oligoneuleotide and p21 gene inhibits the intimal proliferation of venous antografs.  相似文献   

4.
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5.
AIM:To investigate the effects of lentivirus-mediated transfection of shRNA targeting α1D-adrenergic receptor (Adra1d) gene on calcium ion (Ca2+) and calmodulin (CaM) in vascular smooth muscle cells (VSMCs) of rat aorta. METHODS:Single oligonucleotide sequences of shRNA targeting rat Adra1d gene were design and synthesized, and then the shRNA was constructed and cloned into GV248 vector. The U6-shRNA carrier and expression vector were transfected into 293T cells together and packed with lentivirus, and the supernatant was collected and concentrated by overspeed centrifugation. The VSMCs of rat aorta were transfected with recombinant lentivirus vector. The interference effects were identified by RT-qPCR and Western blot. The concentration of Ca2+ in VSMCs was detected by laser confocal inspection, and the expression of CaM at mRNA and protein levels in the VSMCs was determined by RT-qPCR and Western blot. RESULTS:The lentiviral shRNA expression vector was successfully constructed. The titer of the concentrated virus was 3×1011 TU/L. The mRNA and protein expression levels of Adra1d in the rat aortic VSMCs were significantly reduced after transfection. The interference efficiency of Lv-shRNA4-Adr to Adrald gene was greater than 85%. After target silencing of Adra1d gene, compared with scrambled group, the Ca2+ fluorescence intensity of rat aortic VSMCs was significantly increased. Moreover, the mRNA and protein expression levels of CaM were also increased significantly. CONCLUSION:A lentiviral shRNA expression vector targeting rat Adra1d gene was successfully constructed, which significantly increased Ca2+ concentration and CaM expression in rat aortic VSMCs.  相似文献   

6.
AIM: To explore a new gene therapeutic strategy for vein graft restenosis by investigating the effects of adenovirus-mediated human growth arrest-specific homeobox (Ad5-hGax) gene delivery on the proliferation, migration and cell cycle distribution of serum-induced rabbit vascular smooth muscle cells (VSMCs). METHODS: The recombinant adenovirus vector containing hGax gene was constructed and transfected into rabbit VSMCs. The expression of hGax in VSMCs was detected by RT-PCR and immunofluorescent staining. Methyl thiazolyl tetrazolium (MTT) assay was used to assess the effect of hGax over-expression on serum-induced proliferation of VSMCs. Wound healing method was applied to examine the distance of serum-induce VSMCs migration. Flow cytometry was employed to analyze the cell cycle distribution. RESULTS: The recombinant adenovirus vector Ad5-hGax was successfully constructed. The results of RT-PCR and immunofluorescent staining revealed that the hGax -transfected cells contained a 174 bp specific fragment of hGax gene and target protein 48 h after transfection. The proliferation of serum-induced VSMCs was significantly inhibited by overexpression of hGax gene as compared with control group. The migration of serum-induced VSMCs was inhibited after hGax gene delivery. Flow cytometry showed that 72 h after serum induction, the cells in G0/G1 phase in Ad5-hGax group were significantly increased, whereas the cells in G2/M+S phase were significantly decreased. CONCLUSION: Overexpression of hGax gene inhibits the proliferation and migration of serum-induced rabbit VSMCs, and arrests the cells in G0/G1 phase. It is likely that hGax gene is a potential target for the gene therapy of vein graft restenosis.  相似文献   

7.
AIM: To evaluate the role of human angiotensin II (AngII) type 1 receptor (AT1R) antisense cDNA (ahAT1) on migration of cultured artery smooth muscle cells (VSMCs). METHODS: Two recombinant adenoviral vectors, Ad/CMV.ahAT1 containing full length antisense cDNA targeting to human AT1R mRNA, and Ad/CMV.LacZ containing LacZ called report gene, were constructed by orientation clone technology and homologous recombination, and then were used to transfect VSMCs in vitro. AT1R expression detected by RT-PCR and immunohistochemistry, and migration of VSMCs measured by Boyden's Chamer methods, were compared between transfected and nontransfected VSMCs. RESULTS: Forty-eight hours after Ad/CMV. ahAT1 transfection, the level of AT1R mRNA decreased markedly (50% of control group), and AT1R protein expression was significantly less (P＜0.01 vs control-group and Ad/CMV.LacZ-group, respectively) in VSMCs. So it was migration distance of VSMCs among the three groups. CONCLUSION: These results indicate that antisense cDNA targeting to human AT1R transfer in vitro mediated by adenoviral vector has a powerful inhibitory effect on migration of VSMCs by attenuating AT1R expression.  相似文献   

8.
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9.
AIM: To investigate the influence of Sini decoction (SND) on the proliferation and apoptosis of rabbit abdominal aorta smooth muscle cells after ballon injury and discuss the effect of vascular smooth muscle cell's (VSMCs) proliferation and apoptosis in post-percutaneous coronary intervention (PCI) restenosis (RS) and the feasibility of SND preventing post-PCI RS. METHODS: The animal model of rabbit abdominal aorta ballon injury was set up and the therapertic group was treated with SND. The shape of proliferative and apoptotic cell were investigated by electron microscope. Immunohistochemistry staining was performed using α-actin,PCNA and Cyclin E monoclonal antibodies. In situ Cell Death Detection Kit was used to identify apoptotic cells. Abdomial aorta angiography was operated in the 84th day subgroup and the stenosis degree was evalued by quantitative angiographic analysis. RESULTS: As compared with the control group, the therapeutic group displayed a lower proliferative percentage and a higher apoptosic percentage (P<0.05). Moreover, the apoptosic peek time was on the 14th day after operation,which was longer than the control group. CONCLUSION: SND effectly inhibited the proliferation of VSMCs and iuduced apoptosis in VSMCs.  相似文献   

10.
AIM: To observe the effects of local transfection of vascular endothelial growth factor 165 (VEGF165) gene on inhibiting intimal hyperplasia and restenosis of artery in rabbits after operation injury, and its possible mechanisms. METHODS: Microsurgery injury was used to establish the intimal injury model of right external iliac artery in rabbits. 105 male New Zealand rabbits were randomly divided into 3 groups (35 rabbits in each group). Group A was physiological saline control group, group B was pBudCE4.1-transfected group, group C was pBudCE4.1/VEGF165-transfected group. The physiological saline, pBudCE4.1 and pBudCE4.1/VEGF165 transfection solutions were injected into injured vessel walls of above-mentioned groups. The injured vascular specimen was harvested for pathologic examination, electric microscope observation, RT-PCR examining and immunohistochemical staining. RESULTS: Rabbit intimal thickness and area of vessel walls in group C at every time point after operation were significantly less than those in group A and group B (P<0.01). The stenosis ratio of vessels in group C at 28 days after operation decreased by 51.6% and 49.8%, respectively, as compared with groups A and B. The expression of VEGF165 mRNA and VEGF165 positive cells in Group C were increased significantly than those in group A and B at every time point after operation (P<0.01). CONCLUSION: Local transfection of VEGF165 gene restrains intimal hyperplasia and restenosis of vessels, which lays a foundation for future gene therapy of vascular intimal hyperplasia.  相似文献   

11.
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12.
AIM: To study the effects of angiotensin Ⅱ type 2 receptor (AT2R) gene transfer on neointimal hyperplasia in rat carotid arteries after balloon angioplasty. METHODS:AT2R gene was transfected into rat carotid arteries with pAdCMV/AT2R after the establishment of rat carotid balloon injury restenosis model. The arteries were harvested at 7 days, 14 days and 21 days after gene transfer. The expression of AT2R, AT1R, PCNA in arteries and morphology analysis were evaluated by RT-PCR, immunohistochemistry and HE staining. The expressions of AT2R and PCNA were measured by double immunofluorescence staining and confocal microscope. RESULTS:pAdCMV/AT2R delivered into injured rat carotid arteries significantly up-regulated the levels of AT2R mRNA and protein in neointima from day 7 to day 21, but the levels of AT1R was not significantly different (P>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<0.01］. At day 21, compared with no transfection group and pAd-GFP transfection group, pAdCMV/AT2R transfection reduced I/M (intimal/medial area) ratio significantly (0.78±0.06 vs 1.44±0.22, 1.36±0.21， respectively, P<0.01). No significant difference between pAd-GFP group and no transfection group was observed. CONCLUSION: Gene transfer of AT2R from lumen may effectively inhibit VSMC proliferation and neointimal hyperplasia in the rat carotid arteries after balloon angioplasty. The cross-talk between AT1R and AT2R may operate via signaling pathway, but not via counteraction of receptor expression.  相似文献   

13.
AIM: To investigate the effect of chloride channel CLCN2 antisense oligonucleotide on the cell injury of malignant U251 glioma cells induced by cisplatin (DDP). METHODS: The experiment was divided into 4 groups: control group (nonsense oligonucleotide), CLCN2 antisense oligonucleotide group, DDP group (DDP＋nonsense oligonucleotide), DDP＋CLCN2 antisense oligonucleotide group. The viability of U251 cells was measured by MTT assay, CLCN2 mRNA level was determined by RT-PCR, cell apoptosis was measured by TUNEL assay. RESULTS: Compared to the control group, the cell viability, CLCN2 and cyclinD1 mRNA decreased in CLCN2 antisense oligonucleotide group, DDP treated group and CLCN2 antisense oligonucleotide with DDP treated group, cells apoptosis increased. Compared to DDP group, the cell viability (P<0.05) and CLCN2 mRNA decreased in CLCN2 antisense oligonucleotide with DDP treated group, and cells apoptosis increased (P<0.01). Compared to CLCN2 antisense oligonucleotide group, CLCN2 mRNA significantly decreased (P<0.01) in CLCN2 antisense oligonucleotide with DDP treated group. CONCLUSION: CLCN2 antisense oligonucleotide inhibits the expression of CLCN2 mRNA in U251 cells. Inhibition of CLCN2 mRNA facilitates the cell injury of U251 cells induced by DDP. The decrease in CLCN2 mRNA is involved in the mechanism of cell injury by DDP.  相似文献   

14.
AIM: To investigate the different expressions of cell cycle related genes in hyperplastic and hypertrophic vascular smooth muscle cells caused by platelet-derived growth factor (PDGF-BB) and angiotensinⅡ(AngⅡ). METHODS: Rat aorta media smooth muscle cells were cultured. PDGF-BB and AngⅡ were added into serum-free medium at a concentration of 20 μg/L and 10-6 mol/L , respectively. Vascular smooth muscle cells (VSMCs) were harvested after stimulated for 24 hours. The expression of cell cycle related genes was measured by DNA chips(Atlas cDNA Expression Arrays, Clontech Laboratories, Inc.). RESULTS: The expression of cyclin D3 mRNA ,cyclin G1 mRNA,p57 mRNA,p16 mRNA,E2F-3 mRNA and DP2 mRNA were higher in PDGF-BB than those in AngⅡstimulated VSMCs. p15 mRNA,p19 mRNA,E2F-1 mRNA, E2F-5mRNA,and N-myc mRNA were only detected in PDGF-BB stimulated group. But the expression of p53-associated protein mRNA were higher in AngⅡstimulation group. The expression of PCNA mRNA, c-myc binding protein mRNA,p53-dependent cell growth regulater mRNA,cyclinC mRNA, cyclinB1 mRNA, E2F-3mRNA were similar in the two groups. CONCLUSION: The procession of cell cycle relys on the coordination of many regulater molecules expressed in different phases. Our study preliminarily definite the genes that express during PDGF-stimulated VSMC's hyperplasia and Ang II-stimulated VSMC's hypertrophy.  相似文献   

15.
LIAN Bing  WANG Ji-qun  JIN Lin 《园艺学报》2009,25(8):1533-1537
AIM: To explore the effects of small interfering RNA (siRNA) targeting PCNA gene on nasopharyngeal carcinoma CNE2 cells growth and cycle.METHODS: Three synthesized siRNA targeting PCNA gene was transfected into CNE2 cells by using LipofectamineTM reagent. The PCNA mRNA and PCNA protein were detected by real-time polymerase chain reaction (RT-PCR) and immunohistochemical method. Inverted phase contrast microscope was used to determine the CEN2 cells growth before and after PCNA-siRNA transfected. Flow cytometry was used to observe the cell cycle.RESULTS: In CNE2 cells after PCNA-siRNA transfection, the expressions of PCNA mRNA and protein were down-regulated at different degree. Inhibition ratio of PCNA mRNA was 98.5%. Meanwhile, the cell cycle was suffocated at G0/G1 stage.CONCLUSION: The synthesized PCNA-siRNA effectively interferes nasopharyngeal carcinoma cells by down-regulating the expressions of the PCNA mRNA and its protein, therefore inhibits the growth of CNE2 cells. Future application of PCNA-siRNA in the gene therapy of nasopharyngeal carcinoma might be expected．  相似文献   

16.
AIM：To evaluate the effects of tetramethylpyrazine (TMP) on calcineurin (CaN) and proliferating cell nuclear antigen (PCNA) gene expression in the proliferation of vascular smooth muscle cells (VSMCs) treated by angiotensin Ⅱ (AngⅡ).METHODS：A cell proliferating model of VSMCs induced by AngⅡ was established.PCNA gene exprersion was observed by immunocytochemical staining and image analysis technique;Calcineurin (CaN) activity was detected by enzyme reaction phosphorus measurement.RESULTS：AngⅡ significantly stimulated the proliferation of VSMCs,cell proliferation activity,CaN activity and the expression levels of PCAN were higher than those in control (P<0.01).While treated with TMP,the CaN activity and PCNA expression were obviously lower than those in AngⅡ group (P<0.01).CONCLUSION：The VSMCs proliferation induced by AngII can be inhibited by tetramethylpyrazine significantly,and the inhibiting mechanism of TMP may be related to inhibiting CaN activity and restraining the expression of PCNA in a dose and time-dependent manner.  相似文献   

17.
AIM: To assess the effect of estrogen on the gene expression of caveolin-1 in rat vascular smooth muscle cells (VSMCs). METHODS: Wistar rats were ovariectomized and subjected to subcutaneous implantation of placebo pellets (OVX+V group) or estradiol pellets (OVX+E group). 2 weeks after implantation, the expression of caveolin-1 gene in endothelium-denuded aortic tissue was examined by RT-PCR. Furthermore, Northern blotting was used to analyze the mRNA expression of caveolin-1 in cultured rat VSMCs. RESULTS: RT-PCR showed that expression of caveolin-1 gene was significantly higher in OVX+E group than that in OVX+V group. Northern blot analysis showed that the mRNA expression of caveolin-1 was higher in VSMCs pretreated with 17β-estradiol (17β-E2) than that in VSMCs without 17β-E2 pretreatment. CONCLUSION: Estrogen up-regulates the gene expression of caveolin-1 in the vascular wall, partially indicating the cardiovascular effect of estrogen.  相似文献   

18.
AIM: To investigate the expression of tissue factor (TF) induced by homocysteine (Hcy) and the effect of Hcy on activation of nuclear factor-κB (NF-κB) in human vascular smooth muscle cells (VSMCs).METHODS: Human umbilical artery VSMCs were cultured by tissue explanting method,and were incubated with different dosage of Hcy/ PDTC (NF-κB inhibitor) in different time.RT-PCR was used to measure the expression of TF mRNA,and flow cytometry was used to detect the expression of TF on the surface of VSMCs.Western blotting was performed to measure the NF-κB protein level in the nuclear,flow cytometry was used to examine the expression of iNOS and the expression of TF on the surface of VSMCs.RESULTS: Hcy induced VSMCs TF mRNA expression significantly after the VSMCs were incubated with Hcy at concentrations of 10,100,500 μmol/L,respectively.There was low expression level of TF protein on the surface of the control VSMCs and Hcy also induced VSMCs TF protein expressionn on the cell surface at different concentrations.Additionally,Hcy rapidly induced the activation of NF-κB and inhibited this effect significantly by PDTC.Hcy alone did not induce the expression of iNOS in VSMCs.CONCLUSION: Hcy induces human VSMCs expression of TF in mRNA and protein.These effects were partly mediated by NF-κB.These results suggest that Hcy may play an important role in atherosclerosis and thrombosis.  相似文献   

19.
AIM: To observe the antiproliferative effect of c-myc antisense oligonucleotide in rat thymus lymphocytes. METHODS: Rat thymus lymphocytes were separated by Ficoll-Urografin density gradient centrifugation. Lipofectin was used to introduce antisense, sense and mismatched oligonucleotides for c-myc to rat thymus lymphocytes. The antiproliferative effect was assayed by incorporation of [3H]-TdR and MTS cell proliferation assay. TR-PCR was used to detect the expression of c-myc mRNA. RESULTS: c-myc antisense oligonucleotide inhibited ratthymus lymphocytes proliferation[(0.14±0.03)A vs(0.32±0.16)A,P<0.05],but this ef ect had no relationship with the concentration of c-myc antisense oligonucleot ide.c-myc antisense oligonucleotide decreased the expression of c-myc mRNA in rat thymus lymphocytes. CONCLUSION: c-myc antisense oligonucleotide inhibited rat thymus lymphocyte proliferation.  相似文献   

20.
AIM: To study the expression of Pim-1 in vascular smooth muscle cells (VSMCs) induced by platelet-derived growth factor BB (PDGF-BB). METHODS: VSMCs isolated from rats were treated with different concentrations of PDGF-BB for different time. The proliferation of VSMCs was detected by cell counting. The mRNA expression of Pim-1 was measured by real-time RT-PCR. The STAT3 activity was determined by Western blotting. Actinomycin D, AG490, and small interfering RNA (siRNA) for Pim-1 or STAT3 were used to investigate the underlying mechanisms. RESULTS: Pim-1 gene silencing attenuated the proliferation of VSMCs in response to PDGF-BB. The mRNA expression of Pim-1 was up-regulated by PDGF-BB at concentrations of 10 μg/L~50 μg/L for 1 h, and was maximally induced at the concentration of 20 μg/L. The time of Pim-1 mRNA expression maximally occurred 30 min after PDGF-BB exposure. Incubation of VSMCs with PDGF-BB resulted in a significant activation of STAT3. VSMCs pretreated with actinomycin D showed a significant decrease in the mRNA expression of Pim-1. Treatment with AG490 or knockdown of STAT3 in VSMCs resulted in inactivation of STAT3, and significantly suppressed the mRNA expression of Pim-1. CONCLUSION: PDGF-BB-induced VSMC proliferation is partly attributed to Pim-1. VSMCs strongly increase Pim-1 mRNA upon stimulation with PDGF-BB, and STAT3 signaling pathway appears to be efficient for regulation of Pim-1 expression. This process may play a critical role in development of vascular remodeling.  相似文献   

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