OBJECTIVE: To investigate the feasibility to seed vascular endothelial cell(VEC) and vascular smooth muscle cell (VSMC) into tissue engineered blood vessel scaffold material. METHODS: 1. A blood vessel scaffold with a combined polymer was designed, which mainly is composed of rabbit VSMC and collagen with reinforcement by a non-spinning fabric mesh made of polyglycolic acid (PGA). 2. VEC were isolated from rabbit thoracic aorta by enzyme digestion methods and subcultured and purified. Then the cells were seeded into scaffold material. The morphological characteristics of tissue engineered blood vessel was analyzed by scanning electron microscopy. RESULTS: VEC could adhere well to the inner surface of the tissue engineered tubular scaffold material with a tenacity and elasticity. VSMC could sustain bioactivity of cell. CONCLUSION: Non-spinning PGA porous biodegradable materials coated with collagen is benefit for cells to adhere and grow. It will lay a foundation of a laminated structure of tissue engineered blood vessel.
摘要:目的: 探讨在血管紧张素Ⅱ(AngⅡ)诱导血管平滑肌细胞(VSMCs)NOX1基因表达增加中线粒体所起的作用。 方法 :体外培养大鼠主动脉VSMCs,用线粒体呼吸链的抑制剂阻断线粒体的作用,用荧光实时定量PCR检测NOX1基因表达的量。 结果 :AngⅡ能够诱导 NOX1基因的表达增加,线粒体呼吸链的抑制剂能够抑制上述这一作用。 结论 :在大鼠的VSMCs中,AngⅡ诱导NOX1的增加通过线粒体呼吸的作用。Abstract: Objective: To detect the role of mitochondria involved in Angiotensin Ⅱ(Ang Ⅱ) induced NOX1 gene expression. Methods :Rat aortic vascellum smooth muscle cells(VSMCs) were cultured in vitro,and were treated with or without some inhibitors of complexs in mitochondrial respiratory chain. Realtime RTPCR was used to calculate the expression of NOX1 mRNA. Results :AngⅡ stimulated NOX1 gene expression,while some inhibitors of complexs in mitochondrial respiratory chain attenuated this progress.〖WTHZ〗Conclusion : Mitochondrial respiratory chain mediates expression of NOX1 gene in VSMCs by AngⅡ.
Objective To investigate the influence of RNA interference targeting c-Jun gene on the proliferation of rat vascular smooth muscle cells (VSMCs). Methods The experiment was performed with c-Jun siRNA （c-Jun siRNA group）, control reverse sequence siRNA (control siRNA group) or no siRNA (control group). VSMCs were transfected with siRNA targeting c-Jun gene by liposome. Effects of c-Jun siRNA on mRNA and protein expressions of c-Jun were examined by RT-PCR analysis and Western blot respectively. MTT test and 3H-TdR incorporation were used to detect VSMCs proliferation. Cell cycle analysis of VSMCs in vitro was determined by flow cytometer. Results The expression levels of mRNA and protein of c-Jun in c-Jun siRNA group were significantly lower than those in control group （P＜0.05， P＜0.01). There was no significant difference between control group and control siRNA group （Pgt;0.05）. Proliferation activity of VSMCs decreased significantly in c-Jun siRNA group compared with that in control group （P＜0.05） and VSMCs was blocked in the G0/G1 phase of cell cycle significantly （P＜0.05）. There was no significant difference between control group and control siRNA group （Pgt;0.05）. Conclusion c-Jun gene silenced by RNA interference can inhibit VSMCs proliferation effectively in vitro.
Objective To study the inhibitory effect of RNA interference (RNAi) on bcl-2 expression of vascular smooth muscle cells （VSMCs) in rabbit. Methods The expression vector of bcl-2 gene-targeting small interference RNA (pshRNA-bcl-2) was constructed and was transfected into VSMCs by lipofectamine, and the unloaded vector was used as control. The expression of bcl-2 mRNA was identified by RT-PCR and Western blot, respectively. The growth of the transfected VSMCs was examined by MTT. Results The pshRNA-bcl-2 may inhibit the expression of bcl-2 gene at the levels of transcription and translation. There were significant differences (P＜0.01) of the expressions of bcl-2 mRNA between the VSMCs that were transfected with pshRNA-bcl-2 and the ones in plasmid transfected group and control group, respectively. There was a significant difference (P＜0.01) in the growth of VSMCs between the plasmid transfected and the control groups. Conclusion The plasmid containing the small interference RNA of bcl-2 may have an inhibitory effect on the cell growth and endogenous expression of bcl-2 gene at the levels of transcription and translation in VSMCs.
ObjectiveTo explore the effects of PKD1 gene on mouse aortic smooth muscle (MOVAS) cells autophagy.MethodsThe shRNA and over-expression lentiviral vectors for the target gene of PKD1 were constructed. MOVAS cells were infected by a number of successful packaging shRNA (PKD1 knockdown) or ETS-1 (PKD1 over-expressing) lentiviral vectors, and qPCR was used to test interference and over-expressing effects. Then qPCR and Western blotting were used to detect the expression levels of autophagy markers including Atg5, Beclin1 and LC3 in control group, shPKD1 group and ETS-1 group.ResultsCompared with the control group, PKD1 mRNA level was decreased in the shPKD1 group (P<0.05); ETS-1 and PKD1 mRNA levels were increased in the ETS-1 group (P<0.05). In contrast with the control group, the mRNA levels of autophagy markers including Atg5 (P<0.05) and Beclin1 (P<0.01) were obviously decreased in the shPKD1 group, but they were obviously increased in the ETS-1 group (P<0.001). Protein levels of Atg5, Beclin1 and LC3 were significantly decreased in the shPKD1 group (P<0.05), but they were increased obviously in the ETS-1 group (P<0.05) in contrast with the control group.ConclusionPKD1 gene is involved in MOVAS cells autophagy, low expression of PKD1 gene can inhibit autophagy and high expression of PKD1 promotes autophagy in vascular smooth muscle cells.
ObjectiveTo investigate the effect of ADAM33 gene silencing in VSMCs on the proliferation and lumen formation of airway vascular endothelial cells (VECs) in a co-culture system and the possible regulatory mechanism. MethodsThe Human aortic smooth muscle cells (HASMCs) and human pulmonary microvascular endothelial cells (HPMECs) were used to construct a cell co-culture system. ADAM33 gene expression was silenced by lentivirus transfection technique, and the subjects were divided into endothelial cell blank group, co-culture group, co-culture +shRNA negative control group, and co-culture + ADAM33-SHRNA group. The expressions of sADAM33, VEGFA,VEGER2, ang-1 and ang-2 in co-culture system were detected by ELISA. The proliferation and lumen formation of HPMECs were observed by CCK-8 and Transwell experiments. The protein expression of Tie2, PI3K, Akt, and mTOR key molecules in Tie2/PI3K/Akt/mTOR signaling pathway and the phosphorylation levels of AKT and mTOR were detected by Western-blotting method. Results① Compared with the co-culture group (0.851±0.036) and the co-culture + shRNA negative control group (0.828±0.047), the OD value of the co-culture + ADAM33shRNA group (0.699±0.038) was significantly decreased (P<0.05). ② Compared with the co-culture group (159.169±15.740) and the co-culture +shRNA negative control group (157.357±21.612), the tube length of the co-culture +ADAM33shRNA group (120.812±2.791) was also significantly decreased (P<0.05). ③ After ADAM33 gene expression of HASMCs was silted in co-culture system, the expression levels of VEGFA, VEGFR2, ang-1 and ang-2 were significantly decreased (P<0.05), while the expression levels of Tie2, PI3K, P-Akt and P-mtor were decreased (P<0.05). ConclusionsSilencing the expression of the ADAM33 gene could reduce the release of sADAM33 from the membrane of the airway VSMCs, regulate the proliferation and lumen formation of airway VECs by reducing the expression of VEGF/VEGFR and inhibiting the activities of the Tie2/PI3K/Akt/mTOR signaling pathways,and then participate in airway vascular remodeling in asthma.
【Abstract】Objective To investigate the irradiating effect of low intensive microwave (LIM) on pathological process of blood vessel restenosis(RS) and assess the probability of LIM irradiation to prevent was used RS.Methods Fortyfour male healthy New Zealand rabbits were randomly divided into 2 groups. Fogarty catheter traumatize to the tunica intima of iliac artery so as to establish RS models. Two thousand four hundred and fifty MHz microwave with different power of 2 ,5 and 10 mW/cm2 was used, locally to irradiate EIA in irradiating group (1 h/d). Specimens were obtained at different time of 3,7,14 and 28 d after operation. Morphological changes of tissues were observed with HE and EF staining and the area of tunica intima, tunica media and the rate of cavity stenosis were analyzed with image analysis system; apoptosis was detected with TUNEL; phenotype and microstructure of VSMC were observed with TEM. Results After microwave irradiating, inflammatory reaction in early period was suppressed, mural thrombus decreased, the proliferation and migration of VSMC depressed, the area of tunica intima and the rate of cavity stenosis obviously reduced comparing with the control group (P＜0.01). The rate of apoptosis cells showed that there were no obvious differences among each group on 3 d after operation (Pgt;0.05). At other different time, however, the rate of apoptosis cells in irradiating groups obviously increased than that of the control group (P＜0.01), particularly in the one with power of 5 mW/cm2 .The number of synthesis form VSMC in the control group occupied (93.50±3.45)% of the total number of VSMC on 14 d after operation. Most of VSMC appear contractile in irradiating group in which a lot of morphological changes of apoptosis in fibroblast and VSMC existed.Conclusion LIM irradiation could obviously prevented from pathologic procedure of RS. After LIM irradiating, inflammatory reaction in early period is suppressed, the proliferation and migration of VSMC depressed. LIM irradiation promotes cell apoptosis, effectively prohibites the occurring and development of RS. LIM irradiation has had relationship between quantity and effect, power span to effectively prohibit RS, particularly in the one with power of 5 mW/cm2.
Abstract: Objective To determine the effects of oxidative stress reaction on intima hyperplasia after autologous vein grafting. Methods Seventy female SpragueDawley(SD) rats were randomly divided into a control group(n=10) and an experimental group (n=60). The experimental group was then divided into six time points of one day; one, two, four, and six weeks; and two months after surgery; with 10 rats for each time point. Autologous vein grafting models were established. At each time point the designated rats were anaesthetized, and the grafts were isolated and stained with HE. The same length of external jugular vein was cut from each rat in the control group. The neointima to tunica media area ratios (I/M) were measured with acomputerized digital image analysis system. Nuclear factorkappa B (NF-κB) and copper zinc superoxide dismutase (CuZnSOD) were detected byimmunohistochemistry. The concentration of malondialdehyde (MDA) in serum was analyzed by colorimetry. Results In the control group, expression levels of NF-κB and CuZnSOD were low. In the experimental group, expression of NF-κB increased after the operation and peaked two weeks later. The plateau was sustained for about one month, and then the level of expression declined gradually, reaching the baseline at the twomonth time point. The expression of CuZnSOD increased gradually after the operation and peaked one week later, then declined to the normal level after 2-3 weeks at the plateau. In the control group, the concentration of serum MDA was 4.966±1.346 nmol/ml. In the experimental -group, the-MDA concentration increased dramatically after the operation, then-declined from its highest level at the oneday time point (21.161±2.174 nmol/ml) to the normal level at two months (6.208±2.908 nmol/ml) after the operation (P＜0.05). In the control group, I/M was 0.2096±0.0253, while in the experimental group, it was higher one week after the operation (0.6806±0.0737) and peaked at four weeks (1.4527±0.0824), falling to 1.0353±00656 at six weeks and 0.9583±0.0516 attwo months (P＜0.05) for the experimental and control groups). Conclusion Endothelial cell injury initiates an oxidative stress reaction after autologous vein grafting and augments inflammation by activating NF-κB, thus playing an important role in inducing restenosis of the grafted vein.
Vascular smooth muscle cells (VSMCs) phenotype switching plays an essential role in the pathogenesis of various vascular diseases. The present study aims to investigate the role of receptor-interacting protein kinases 1(RIPK1) in VSMCs phenotypic switching induced by Angiotensin Ⅱ(Ang Ⅱ). Expression of mRNA and protein of RIPK1, markers of VSMCs phenotypic switching and secretion, phosphorylation of the P65 subunit of NF-κB were measured by real-time PCR and Western blot. Meanwhile, EdU incorporation assay and wound scratch assay were performed to determine the cell proliferation and migration respectively. At the same time, Necrostatin-1(Nec-1, an known RIPK1 inhibitor) and RIPK1-specific small interference RNA (siRNA) were used to inhibit the expression of RIPK1. The experimental data demonstrated that the mRNA and protein levels of RIPK1 and P65 phosphorylation were increased significantly in the process of VSMC phenotypic switching induced by Ang II. Moreover, the expression of RIPK1 and P65 phosphorylation were significantly down-regulated in VSMCs pretreated with Nec-1 or trans-fected with RIPK1-siRNA. Furthermore, the proliferation, secretion and migration of VSMCs were also markedly suppressed after inhibition of RIPK1 by Nec-1 or its specific siRNA. The results suggested that RIPK1 might be involved in VSMC phenotypic switching induced by Ang II, which was possibly via up-regulating the NF-κB signaling pathway.