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.
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 explore an effective method of culturing the canine bladder smooth muscle cells, observe the morphological characteristics of the bladder smooth muscle cells growing on acellular small intestinal submucosa（SIS） and offer an experimental basis for reconstruction of the bladder smooth muscle structure by the tissue engineering techniques. Methods The enzymetreatment method and the explant method were respectively used to isolate and harvest the canine bladder smooth muscle cells, and then a primary culture of these cells was performed. The canine bladder smooth musclecells were seeded on the SIS scaffold, and the composite of the bladder smooth muscle cells and the SIS scaffold were co cultured for a further observation. At 5,7 and 9 days of the co culture, the specimens were taken; the bladder smooth muscle cells growing on the SIS scaffold were observed by the hematoxylin staining, the HE staining, and the scanning electron microscopy. The composite of the bladder smooth muscle cells on the SIS scaffold was used as the experimental group, and the bladder smooth muscle cells with no SIS were used as the control group. In each group, 9 holes were chosen for the seeded bladder smooth muscle cells, and then the cells were collected at 3, 5 and 7 days for the cell counting after the enzyme treatment. Morphological characteristics of the cells were observed under the phase contrast microscope and the transmission electron microscope. Expression of the cell specific marker protein was assessed by the immunohistochemical examinaiton. The proliferation of the cells was assessed by the cell counting after the seeding on the SIS scaffold. Results The primary bladder smooth muscle cells that had been harvested by the enzyme treatment method were rapidly proliferated, and the cells had good morphological characteristics. After the primary culture in vitrofor 5 days, the bladder smooth muscle cells grew in confluence. When the bladder smooth muscle cells were seeded by the explant method, a small amount of the spindleshaped bladder smooth muscle cells emigrated from the explant at 3 days. The cells were characterized by the welldeveloped actin filaments inthe cytoplasm and the dense patches in the cell membrane under the transmissionelectron microscope. The immunohistochemical staining showed the canine bladdersmooth muscle cells with positive reacting α actin antibodies. The bladder smooth muscle cells adhered to the surface of the SIS scaffold, growing and proliferating there. After the culture in vitro for 5 days, the smooth muscle cells covered all the surface of the scaffold, showing a singlelayer cellular structure. The cell counts at 3, 5 and 7 days in the experimental group were（16.85±0.79）×105，（39.74±2.16）×105 and （37.15±2.02）×105, respectively. Thecell counts in the control group were（19.43±0.54）×105，（34.50±1.85）×105 and （33.07±1.31）×105, respectively. There was a significant difference between the two groups at 5 days （P＜0.05）. ConclusionWith the enzyme treatment method, the primarily cultured canine bladder smooth muscle cells can produce a great amount of good and active cells in vitro. The acellular SIS can offer an excellent bio scaffold to support the bladder smooth muscle cells to adhere and grow, which has provided the technical foundation for a further experiment on the tissue engineered bladder reconstruction.
ObjectiveTo investigate the role and potential mechanisms of neuropilin-1 (NRP1) in the pathogenesis of vein graft failure.MethodsThe rat vascular smooth muscle cells (VSMCs) were transfected with NRP1-shRNA adenovirus and negative control adenovirus respectively. Cell counting kit-8, flow cytometry, Transwell and Western blot were used to investigate the effects of inhibition of NRP1 on VSMCs proliferation viability, apoptosis, migration capacity and its downstream signaling pathway protein expression.ResultsThe proliferation and migration of rat VSMCs could be inhibited after down-regulation of NRP1, and the increase of apoptosis was also observed. Moreover, inhibition of NRP1 significantly reduced Akt and NF-κB phosphorylation in rat VSMCs, but had little effect on activation of ERK1/2.ConclusionNRP1 may promote vein graft hyperplastic remodeling by regulating the proliferation and migration of VSMCs through PI3K/Akt and NF-κB pathways, but further animal study is required.
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.
Objective To extract and identify primary culture rat pulmonary arterial smooth cells ( PASMCs) , and investigate the effects of hypoxia on the proliferation of PASMCs. Methods Rat PASMCs were separated by the method of tissue block anchorage, and the cellular morphology was observed under light microscope. The cells were identified by projection electron microscopy, and α-smooth muscle actin ( α-SMactin)in the cells was identified by immunohistochemistry and immunofluorescence. The primary cultured PASMCs were exposed to normoxic and/ or hypoxia condition for 2, 6, 12, 24, 48 hours respectively, thenMTT assay and PCNA ( proliferating cell nuclear antigen) immunohistochemistry were used to detect the proliferation of PASMCs. Results The cells tended to be long spindle and grew in the “peak-valley”mode under light microscope. Immunology results showed that endochylema was stained in brownish yellow, and the positive rate was beyond 96% . There were dense patch, dense body and many filaments in endochylema under projection electron microscopy. MTT assay demonstrated that the A values of PASMCs expose to hypoxia were higher than that of nomoxia. Comparing with normoxia, the A values of PASMCs exposed to hypoxia increased after 12 hours ( P lt;0. 05) , significantly increased after 24 hours ( P lt;0. 01) . Compared with 2 hours’exposure to hypoxia, the A values increased after 12 hours( P lt; 0. 05) , markedly increased after 24 hours ( P lt; 0. 01 ) , which after 48 hours was similar with 24 hours. The result of PCNA immunohistochemistry was consistent with that of MTT. Conclusions The tissue explants adherent method is simple and convenient, and can easily obtain rat PASMCs with high purity and stability. Hypoxia canpromote the proliferation of PASMCs.
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 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 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.
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.