Objective To observe the eotaxin expression of rat airway smooth muscle cells ( ASMCs) induced by serum from asthmatic rats, and explore the possible mechanism. Methods ASMCs isolated fromrat tracheas were cultured in vivo. Then they were treated with serum from asthmatic rats, or treated with serum and dexamethasone simultaneously. The level of eotaxin protein in supernatant and eotaxin mRNA in ASMCs were measured by ELISA and reverse transcription-polymerase chain reaction. The expression of cAMP in ASMCs was examined by radioimmunoassay. Results After the treatment with sensitized serum, the eotaxin level in supernatant and mRNA expression in ASMCs were significantly higher [ ( 107. 09 ±7. 12) ng/L vs. ( 0. 63 ±0. 56) ng/L, P lt; 0. 05; 1. 39 ±0. 04 vs. 0. 05 ±0. 01, P lt;0. 05] , and the level of cAMP in ASMCs was significantly lower compared with the control group [ ( 17. 58 ±3. 62) ng/L vs. ( 32. 39 ±3. 36) ng/L, P lt; 0. 05] . After intervened by the sensitized serum and dexamethasone simultaneously, the protein and mRNA expressions of eotaxin were lower compared with those intervened by sensitized serumalone [ ( 64. 18 ±4. 04) ng/L and 0. 77 ±0. 19] . The level of eotaxin in supernatant was negatively correlated with cAMP level in ASMCs ( r = - 0. 788, P lt; 0. 01) . Conclusions There is anautocrine function in ASMCs as inflammatory cells after stimulation with sensitized serum. Eotaxin may play an important roll in the pathogenesis of asthma via a cAMP-dependent pathway.
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.
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.
【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.
Previous studies have shown that growth arrest, dedifferentiation, and loss of original function occur in cells after multiple generations of culture, which are attributed to the lack of stress stimulation. To investigate the effects of multi-modal biomimetic stress (MMBS) on the biological function of human bladder smooth muscle cells (HBSMCs), a MMBS culture system was established to simulate the stress environment suffered by the bladder, and HBSMCs were loaded with different biomimetic stress for 24 h. Then, cell growth, proliferation and functional differentiation were detected. The results showed that MMBS promoted the growth and proliferation of HBSMCs, and 80 cm H2O pressure with 4% stretch stress were the most effective in promoting the growth and proliferation of HBSMCs and the expression level of α-smooth muscle actin and smooth muscle protein 22-α. These results suggest that the MMBS culture system will be beneficial in regulating the growth and functional differentiation of HBSMCs in the construction of tissue engineered bladder.
Objective
To observe whether umbilical cord mesenchymal stem cells (UCMSCs) can differentiate into the smooth muscle cells (SMCs) induced by bladder SMCs (BSMCs) conditioned medium so as to seek an alternative seed cells for the repair and reconstruction of the urology system.
Methods
UCMSCs and BSMCs were harvested from umbilical cord of full-term births and bladder tissues which were obtained from patients who underwent a radical cystectomy. BSMCs conditioned medium was prepared by mixing supernatant of BSMCs at passages 1-5 with complete medium at ratio of 1
∶
1. UCMSCs at passage 3 were cultured with BSMCs conditioned medium (induced group, group A) and complete medium (control group, group B), respectively; simple BSMCs served as positive control group (group C). The morphological changes of co-cultured UCMSCs were observed by inverted phase microscope, the expressions of α-smooth muscle actin (α-SMA), Calponin, and smooth muscle myosin heavy chain (SM-MHC) of UCMSCs were tested by immunofluorescence staining and Western blot at 7 and 14 days.
Results
The morphology of UCMSCs in group A started to change from a polygonal and short spindle shape to a large and spindle shape after co-culture, which was similar to BSMCs morphology; but the morphology of UCMSCs did not change obviously in group B. Immunofluorescence staining showed that the expressions of α-SMA, Calponin, and SM-MHC were positive in group C. At 7 days, the expression of α-SMA could be observed in groups A and B; at 14 days, the positive expression of α-SMA increased gradually in group A, but it did not increase in group B. At 7 days, a positive expression of Calponin could be observed in group A, and positive expression increased obviously at 14 days; the expression of Calponin could not be observed at 7 and 14 days in group B. However, the expression of SM-MHC could not be observed in groups A and B. The results of Western blot showed the expressions of α-SMA, Calponin, and SM-MHC protein were consistent with the results of immunofluorescence staining.
Conclusion
UCMSCs have the potential of differentiation into SMCs and may be a potential seed cells for bladder tissue engineering.
ObjectiveTo explore the relationship between the pressure level within the scope of promoting proliferation and cell injury of human bladder smooth muscle cells (HBSMCs).
MethodHBSMCs in vitro were divided into the experimental group and control group. The cells in the experimental group were exposed to 40 cm H2O (1 cm H2O=0.098 kPa) pressure and those in the control group were cultured in normal condition for 24 hours. We investigated the cell morphology and cytoskeleton with indirect immunofluorescence staining for α-actin. Propidium iodide (PI) staining was applied to evaluate the level of cell apoptosis.
ResultsThere was no significant difference in the cell morphology between the two groups. However, the expression of α-actin in the experimental group[(50.93±1.99)%] was significantly reduced comparing with that in the control group[(24.70±1.61)%] (t=32.404, P<0.001). The results of PI staining showed that compared with the control group[(3.50±2.12)%], the number of PI staining positive cells in the experimental group [(9.00±1.41)%] was significantly higher (t=6.110, P<0.001).
ConclusionsPressure condition can promotes cell proliferation, but at the same time, it can also lead to cell injury of HBSMCs.
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.
Objective To investigate the effects of simvastatin on the collagen synthesis of rat pulmonary arterial smooth muscle cells ( PASMCs ) induced by hypoxia. Methods Under hypoxic condition, rat PASMCs were cultured with different concentrations of simvastatin. Collagen synthesis of PASMCs with or without simvastatin were measured by 3H-proline incorporation assay. The mRNA expression of TGF-β1 and the contents of super oxide dismrtase ( SOD) ,malondialdehyde ( MDA) in mediumwere also measured. Results The incorporation data of 3H-TdR in the hypoxia group was significantly increased as compared with that in the control group ( P lt;0. 01) , and simvastatin significantly reduced the incorporation data of 3H-TdR induced by hypoxia. The expression of TGF-β1 mRNA in the hypoxia group was significantly increased as compared with that in the control group ( P lt; 0. 01 ) , and simvastatin could significantly inhibited hypoxia-induced expression of TGF-β1 mRNA in a dose-dependent manner. Compared with the hypoxia group, the expression of TGF-β1 mRNA decreased by 55% in simvastatin( 10 - 6mol /L) group ( P lt; 0. 01) , and by 70% ( P lt; 0. 01) in simvastatin ( 10 - 5mol /L) group. Compared with the control group, the activity of SOD was reduced and the contents of MDA were increased significantly in the hypoxia group. Simvastatin can increase the activity of SOD and reduced the content of MDA in a dose-dependent manner. Conclusions Simvastatin can decreases collagen synthesis of PASMCs. This effect might be explained that simvastatin can reduce lipid peroxide and expression of TGF-β1 mRNA.
Objective To investigate the effect of adiponectin on proliferation of airway smooth muscle cells( ASMCs) , and explore its possible mechanism. Methods ASMCs were derived fromrat airway tissue and were cultured in vitro. RT-PCR was used to verify the expression of adiponectin receptors on ASMCs. Then ASMCs were treated with adiponectin at different concentrations( 5, 10, 20, 40, 80 μg/mL) for different periods of time( 1, 12, 24, 48, 72 hours) , respectively. The absorbsence ratios of adiponectin at different concentrations were determined by MTT assay. The adenosine monophosphate-activated protein kinase( AMPK) and phosphorylated AMPK( pho-AMPK) in ASMCs were quantified by Western blot after being treated with adiponectin at different concentrations ( 5, 10, 20, 40 μg/mL) for 48 hours. ResultsThe inhibition of adiponectin on ASMCs was showed in dose-dependent manner( r = 0. 324, P lt; 0. 01) and time-dependent manner( r = 0. 607, P lt; 0. 05) . Western blot indicated that the expression of pho-AMPK increased with the increased concentrations of adiponectin( r =0. 607, P lt; 0. 01) . The ratio of pho-AMPK/AMPK were ( 27. 66 ±1. 03) % , ( 31. 91 ±0. 86 ) %, ( 75. 52 ±2. 67) % , and ( 84. 50 ±1. 05) % ,respectively, with significant differences between each concentrations of adiponectin( P lt; 0. 05) . There was no expression of pho-AMPK in the control group. Conclusion Adiponectin can significantly inhibit ASMCs’proliferation by activating AMPK.
