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 effect of surface propertyof different polyether-ester block copolymers［poly(ethylene glycol-terephthalate)/poly(butylene terephthalate), PEGT/PBT］ on the growth of smooth muscle cells (SMCs) and endothelial cells(ECs). Methods Three kinds of copolymers were synthesized, which were 1000-T20 (group A), 1000PEGT70/PBT30 (group B) and 600PEGT70/PBT30 (group C). The water-uptake and contact angle of three polyether-ester membranes were determined. The canine aorta smooth muscle cells and external jugular vein endothelial cells were primarily harvested, subcultured, and then identified. The proliferation of SMCs and ECs on the different polyether-ester membranes were investigated. Results The water-uptake of three copolymers arranged as the sequence of group C＜group A＜group B, and contact angle as the sequence of group C＞group A＞group B, indicating group B being more hydrophilic. However, smooth musclecells andendothelial cells grew poorly on the membrane of group B after low density seeding, but proliferated well on the membranes of group A and group C. Conclusion In contrast with more hydrophilic 1000PEGT70/PBT30, moderately hydrophilic 1000-T20 and 600PEGT70/PBT30 has better compatibility with vascular cells. The above results indicate that the vascular cells can grow well on moderately hydrophilic PEGT/PBT and that PEGT/PBT can be used in vascular tissue engineering.
OBJECTIVE: To investigate the characteristic and phenotype of ectomesenchymal stem cells of human fetal facial processes and the procedure of spontaneous differentiation to smooth muscle cells. METHODS: The primary ectomesenchymal cells of E 50 human fetal facial processes were isolated by 2.5 g/L trypsin and cultured with DMEM/F 12 with 10(-6) U/L leukemia inhibitor factor(LIF). The morphology and growth rate were observed by inverted microscop. After being withdrawn LIF, the characteristic of cells were identified by immunohistochemistry and RT-PCR. Ultrastructure was observed by transmission electron microscope. RESULTS: The cultured cells displayed monolayer growth and were fibroblast-like with 2-4 processes. The cells were stainely positived for anti-human natural killer cell marker-1, Vimentin, S-100, neuron specific enolase, myoglobin and VIII factor, but negatively for glial fibrillary acidic protein, neural fiblament, alpha-SMA and cytokeratin in immunohistochemistry. Two days after being withdrawn the LIF, cells expressed alpha-SMA in protein and mRNA levels. The cells were rich in muscular filament-like structure and dense bodies under transmission electron microscope. CONCLUSION: Cultured cells are undifferentiated ectomesenchymal stem cells. The cells have the potential for differentiating spontaneously to smooth muscle cell.
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.
ObjectiveTo observe whether multipoint target muscle injection of botulinum toxin type A (BTX-A) in the treatment of spastic cerebral palsy in children is better than non-multipoint target muscle injection.
MethodsFrom February to October 2013, 42 children with spastic cerebral palsy were treated in our hospital. According to the treatment sequence, the children were numbered. Those with an odd number were designated into multipoint target muscle injection group (group A), and those with an even number were put into non-multipoint target muscle ordinary injection group (group B). Each group had 21 children, and all of them were treated with the injection of BTX-A. Modified Ashworth Scoring (MAS) was performed for all the children before treatment, and 2 weeks, one month, and three months after treatment. The change of dorsiflexion range of motion with knee flexion and extension was recorded and compared. The analysis was done by using multilevel statistical method.
ResultsBoth groups of children had significantly improved their ankle range and modified Ashworth score (P<0.05). No interaction between measurement time and group was detected, and the differences between the two groups had no statistical significance (P>0.05).
ConclusionLower muscle tone, greater ankle mobility and better motor function can be achieved after Botulinum toxin A treatment. For now, we cannot draw the conclusion that the effect of multipoint target muscle injection is better than that of non-multipoint target muscle injection in the treatment of spastic cerebral palsy in children.
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.
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.
【Abstract】 Objective To construct tissue engineered skeletal muscle in vivo using glial cell derived neurotrophic factor (GDNF) genetically modified myoblast (Mb) on acellular collagen sponge with hypoglossal nerve implantation, and to observe whether structural or functional connection could be established between engineered tissue and motor nerve or not. Methods Mbs were isolated from 7 male Lewis rats at age of 2 days, cultured and genetically modified by recombinant adenovirus carrying GDNF cDNA (MbGDNF). Calf skin-derived acellular collagen sponge was used as scaffold; cell adhesion was detected by scanning electron microscope after 24 hours. Hypoglossal nerve was implanted into Mb-scaffold complex (Mb group, n=27) or MbGDNF-scaffold complex (MbGDNF group, n=27) in 54 female Lewis rats at age of 8 weeks. HE staining was performed at 1, 6, and 12 weeks postoperatively, and immunohistochemistry staining and fluorescence in situ hybridization were used. Results MbGDNF could highly expressed GDNF gene. Mb and MbGDNF could adhere to the scaffold and grew well. HE staining showed tight junctions between implant and peripheral tissue with new muscle fiber and no distinguished line at 12 weeks in 2 groups. Immunohistochemistry staining showed that positive cells of myogenin and slow skeletal myosin were detected, as well as positive cells of actylcholine receptor α1 at 1, 6, and 12 weeks. The positive cells of Y chromosome decreased with time. At 1, 6, and 12 weeks, the positive neurons were 261.0 ± 6.6, 227.3 ± 8.5, and 173.3 ± 9.1, respectively in MbGDNF group, and were 234.7 ± 5.5, 196.0 ± 13.5, and 166.7 ± 11.7, respectively in Mb group; significant differences were found between 2 groups at 1 and 6 weeks (P lt; 0.05), no significant difference at 12 weeks (P gt; 0.05). Conclusion Connection can be established between engineered tissue and implanted hypoglossal nerve. Recombinant GDNF produced by MbGDNF might play a critical role in protecting central motor neurons from apoptosis by means of retrograde transportation.
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 define how to preserve the severed limbs to prolong the period of replantation. METHODS: The original articles about preservation of severed limbs in recent years were reviewed, it was suggested that the period of replantation was determined by the injury of skeletal muscle. RESULTS: When the environment of severed limbs was changed, the injures of skeletal muscle could be decreased. CONCLUSION: After the severed limbs are reasonably preserved, the period of replantation may be prolonged.
