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.
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 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 molecular signal mechanism of transform growth factor (TGF)-β induced arterial endothelial-mesenchymal transition. Methods Rat arterial endothelial cells were primarily cultured by ex-transplant method. The endothelial cells were incubated by combinant TGF-β (10 ng/mL) for 48 hours and then were detected by immunofluorescence staining and western blotting to observe the cell surface marker expression profile and Akt/mTOR signal activation. On the other hand, the endothelial cells were preincubated by Ly294002 (20 μmol/L) and rapamycin (10 nmol/L) to inhibit the Akt/mTOR signal, and then the cells were further treated with TGF-β (10 ng/mL) for 48 hours to observe the cell surface marker expression profile without Akt/mTOR signal activation. Results Rat artery endothelial cells by TGF-β after incubation, the expressions of smooth muscle cell markers α-smooth muscle actin (α-SMA) and smooth muscle-22α (SM-22α) were up-regulated, and the endothelial cell markers CD31 and vW factor were significantly down-regulated, at the same time, the expressions of phosphorylated Akt and mTOR were also up-regulated. However, after preincubation of Ly294002 (20 μmol/L) and rapamycin (10 nmol/L) to inhibit the phosphorylation of Akt and mTOR signal, above TGF-β-induced expressions of α-SMA and SM-22α in arterial endothelial cells were significantly suppressed and the expressions of CD31 and vWF were preserved. Conclusion TGF-β-induced arterial endothelial-mesenchymal transition is dependent on activation of Akt/mTOR signal, suggesting that Akt/mTOR-dependent arterial endothelial-mesenchymal transition would be one of the mechanisms for intima hyperplasia in transplant arteriosclerosis.
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.
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 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 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.