Objective To investigate the effect of exogenous erythropoietin (EPO) on the denervated muscle atrophy. Methods Twenty-four SD male rats, weighting 200-220 g were made the models of denervated gastrocnemius muscle after sciatic nerves were transected under the piriform muscle at the right lower leg, and were randomly divided into two groups (n=12). rhEPO (2 500 U/kg) was injected daily into the denervated gastrocnemius muscle in EPO group, and normal sal ine was injected into the denervated gastrocnemius muscle in control group. To observe the general state of health of the experimental animal, the muscle wet weight, the muscle cell diameter, the cross section area, the protein amount, thepercentage of the apoptotic muscle cells, and the Na+-K+-ATPase and Ca2+-ATPase activities were measured 2 and 4 weeks after operation. Results All experimental animals were survived during experiment without cut infection, and all animals could walk with pull ing the right knee. At 4 weeks after operation, 7 cases showed ulcer in the right heel, inculding 5 in the control group and 2 in the EPO group. At 2 and 4 weeks after operation, the muscle wet weight in EPO group was (885.59 ± 112.35) and (697.62 ± 94.74) g, respectively; in control group, it was (760.63 ± 109.05) and (458.71 ± 58.76) g, respectively; indicating significant differences between two groups (P lt; 0.01). The protein amount in EPO group was (77.37 ± 5.24) and (66.37 ± 4.87) mg/mL, respectivly；in control group, it was (65.39 ± 4.97) and (54.62 ± 6.32) mg/mL；indicating significant differences between two groups (P lt; 0.01). At 2 and 4 weeks after operation, the myofibrillar shapes were nearly normal in EPO group while there were muscle fiber atrophy, some collapse and obviously hyperblastosis between muscle bundle. There were significant differences in the muscle cell diameter and the cross section between two groups (P lt; 0.01). However, the percentage of the apoptotic muscle cells was 11.80% ± 1.74% and 28.47% ± 1.81% in control group, respectively, which was significantly smaller than that in EPO group (21.48% ± 2.21% and 55.89% ± 2.88%, P lt; 0.01). At 2 and 4 weeks after operation, Na+-K+-ATPaseand Ca2+-ATPase activities in EPO group were higher than those in control group (P lt; 0.01). Conclusion EPO can delay the denervated muscle atrophy.
Objective To investgate the effects of neurotrophic factor 3 (NT-3) genes modified SC on facil itating nerve regeneration and protecting neuronal survival after the sciatic nerve transection in rats. Methods The double sciatic nerves were harvested from 3-day-old Wistar rats and the SCs were separated, cultured and purified with double enzyem digestion and adherent culture. The third generation purified SCs were used. The NT-3 cDNA gene was transfected into culturedSCs by using cationic l iposome. The NT-3 expression were identified by ELISA after 1, 2, 4 and 8 weeks. The plasmids expressing NT-3 genes were transfected into SCs with l ipofectamine. The purity of SCs were detecting before and after modified with NT-3. The nerve-grafting complexes were constructed by SCs (3 × 107/mL) modified NT-3, third generation SCs (3 × 107/mL), NT-3 gene, respectively. And the nerve-grafting complexes were combined with ECM gel and PLGA conduit. Forty-eight adult SD rats were made the models of the right sciatic nerve defect (10 mm). According to the nerve-grafting complexes which were repaired the sciatic nerve defects, the models were divided into 4 groups randomly (n=12): group A (ECM gel and PLGA conduits), group B (SC, ECM gel and PLGA conduits), group C (NT-3 gene, ECM gel and PLGA conduits) and group D (NT-3 modified SC, ECM gel and PLGA conduits). At 2, 4, 6, 8 and 12 weeks after operation, the nerve gross were observed. Electrophysiological examination, histological observation and transmission electron microscope observation were performed at 12 weeks after operation. Results The concentrations of NT-3 protein were 0.39 ± 0.25, 0.76 ± 0.22, 1.06 ± 0.38 and 1.61 ± 0.35 at 1, 2, 4 and 8 weeks after operation; showing statistically significant differences (P lt; 0.05). The purity of SCs was 94.7% ± 2.1% and 95.6% ± 2.5% before and after modified with NT-3, respectively; showing a statistically significant difference (P lt; 0.05). The feet of injury rats began inflammation and ulcer, which healed at 12 weeks in group D, followed by groups C and B, but which was serious in group A gradually. The observations of gross, sections under microscope and transmission electron microscope at 12 weeks showed the regeneration of defect nerve was best in group D, followed by groups C and B, and group A was worst. There were statistically significant differences (P lt; 0.05) in latent period, ampl itude, motor nerve conduction velocity, the number and thickness of axon, the diameter of nerve fiber, the percentage of the nerve tissue area between group A and groupsB, C, D, between groups B, C and group D at 12 weeks. At 12 weeks after operation, the transmission electron microscope showed observation the maturation of medullary sheath was best in group D, followed by groups C and B, and group A was worst. Conclusion The nerve-grafting complex of NT-3 genes modified SCs could repair injured nerve. The competence is superior to SCs and neurotrophic factors.
Objective To observe the influence of the transforming growth factor β1(TGF-β1) on the denervated mouse musclederived stem cells（MDSCs） producing the connective tissue growth factor（CTGF）at different time points in vitro. Methods MDSCs from the primarycultureof the denervated mouse skeletal muscle were isolated and purified by the preplate technique, and they were identified before the culture and after the culturein vitro with TGF-β1 (10 ng/ml) for 24 hours. Then, MDSCs were randomlydivided into 6 groups (Groups A, B, C, D, E and F) according to the different time points, and were cultured in vitro with TGF-β1 (10 ng/ml) for 0, 3, 6, 12, 24 and 48 hours, respectively. The levels of CTGF mRNA in MDSCs were measured by the real time RT-PCR and the expression of CTGF protein was detected by the CTGF Western blot. Results The immunohistochemistry revealed that before the adding of TGF-β1, MDSCs highly expressed Sca-1, with a positivityrate of 96%; however, after the adding of TGF-β1, the positive expression of Sca-1 decreased greatly, with a negativity rate gt;99%. The Western blot test showed that the ratios of CTGF to the average absorbance of βactin in Groups A-F were 0.788±0.123, 1.063±0.143, 2.154±0.153, 2.997±0.136, 3.796±0.153 and 3.802±0.175, respectively. In Groups AD，the absorbance increased gradually, with a significant difference between the abovementioned groups (Plt;0.05). However, in Groups D-F, there was no significant difference between the groups as the promotive tendency became less significant (P＞0.05). The RT-PCR test showed that the △Ct values in GroupsA-F were 1.659±0.215, 1.897±0.134, 2.188±0.259, 2.814±0.263,2.903±0.125 and 3.101±0.186, respectively. In Groups A-D, the increase in the △Ct value was gradual, but the differences were significant between the groups (Plt;0.05). But in Groups E and F, the promotive tendency became less significant（Pgt;0.05）. Conclusion TGF-β1 can promote the production of CTGF inthe mouse MDSCs cultured in vitro and the time-dependent relation exists for 3-12 hours.