OBJECTIVE: To investigate the biological characteristics of human muscle satellite cell cultured in vitro. METHODS: Human muscle satellite cells were obtained from skeletal muscle biopsies of six patients during corrective orthopedic surgery, cultivated in growth medium for ten days, then in differentiation medium for additional five days. Human satellite cells were identified with monoclonal antibody against desmin. Cells were observed under phase contrast microscopy. RESULTS: Human muscle satellite cells proliferated in growth medium, and fused to form myotubes in differentiation medium. After 24 hours in differentiation medium, the confluent satellite cells began to fuse actively and achieved the top level at 72 hours. CONCLUSION: Human muscle satellite cell can proliferate and differentiate in appropriate culture condition. Immunocytochemical detection of desmin is the effective early method to determine satellite cell.
OBJECTIVE: To observe the changes of heme oxygenase-1 (HO-1) expression in the skeletal muscle after ischemia-reperfusion of hind limb in rats. METHODS: A model of hind limb ischemia was made by clamping femoral artery with a microvascular clip. Soleus muscle was obtained from the animals received sham operation, 4 h ischemia without reperfusion and 2 h, 4 h, 8 h, 16 h, 24 h reperfusion after 4 h ischemia. Soleus histology and malondialdehyde (MDA) content were measured. The levels of HO-1 mRNA and protein were measured in different time by Northern blotting, Western blotting and immunohistochemistry technique. RESULTS: After ischemia-reperfusion of limb, HO-1 mRNA increased at the 2nd hour, reached a peak at the 8th hour, and returned toward baseline at the 24th hour. The change of protein level was essentially in agreement with that of mRNA. Immunohistochemical results showed that HO-1 expressed primarily in skeletal muscle cytoplasma. There were no positive signals of mRNA and protein in sham group and in ischemia group. After limb reperfusion, MDA contents in the soleus muscle increased significantly when compared with that in the sham group (P lt; 0.05). MDA content of the 8th after reperfusion decreased significantly when compared with that of the 4 h after reperfusion (P lt; 0.05). CONCLUSION: Ischemia-reperfusion can induce HO-1 expression in skeletal muscle in rats, which may provide protection for injured tissue.
Objective To investigate the early change of brain-derived neurotrophic factor (BDNF) in denervated red and white muscles and the regeneration of nerves innervating the muscles and to discuss the effect of the target organs on regeneration of the injured nerves.Methods Forty Wistar rats were divided into 5 groups. The sciatic nerves in 4 groups were sheared to make the models of the denervated muscles and the other one as control group. The amount of BDNF in muscles was measured with immunohistochemistry 1 day, 3 days, 7 days and 14 days after injury. The models of the regeneration of the nerves were made in another 15 rats whose sciatic nerves were disconnected with forceps. The nerve conduction velocity and electromyogram were tested with neuroelectrophysiology7 days and 14 days after injury. Results The expression of BDNF in soleus increased significantly on the 1st day, the 3rd day and the 7th day (P＜0.01); theexpression ingastrocnemius was lower, but there was no significant difference(P＞0.05) on the 1st day, the 3rd day,the 7th day and the 14th day when compared with control group. After 14 days of injury in the nerves innervating GAS and SOL, the nerve conduction velocities and the amplitudes of wave M recovered to (36.60±7.40)% and (19.9±6.4)% of normal value, and (42.50±3.50)% and (13.7±4.0)% of normal value respectively; there were no significant differences between the two muscles(P＞0.05).Conclusion There is- difference in BDNF amount between the denervated red and white muscles, but the recovery of the two kinds of the motornerves is similar,and the neurotrophism of denervated muscles was determined by all kinds of neurotrophic factors.
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.
Objective To investigate the influence of clenbuterol on the expression of nerve growth factor (NGF) in denervated red and white muscles and the neurotrophism of the denervated muscles.Methods Sixty-four Wister rats, weighed 200-250 g, were divided into 8 groups(8 rats per group), including 4 experimental groups and 4 control groups. The denervated model was made in rats by dissection of sciatic nerves. Clenbuterol was given at a dose of 200 μg/kg per day in the experimental group, saline in the control group. The expression of NGF was measured with immunohistochemistry after 1, 3, 7 and 14 days of injury. The culture methods of dorsal root ganglions of the chick embryos were used to measure the neurotrophism of extracts of the muscles. Results Compared with the control groups, the NGF content of gastrocnemious(GAS) increased on the 1st day (Plt;0.05) and the NGF content of soleus(SOL) increased greatly on the 1st, 3rd and 7th dayafter injury in the experimental groups (Plt;0.01). In the experimental groups, the NGF amount of GAS reached the highest value on the 1st day after injury(Plt;0.01) and then decreased gradually. And the NGF amount of SOL had slight difference between different time. The NGF content of the SOL was higher than that of GASon the 7th day (Plt;0.05). The sensory neurotrophism of the extracts was similar between SOL and GAS.Conclusion Clenbuterol can change the expression of NGF in denervated muscles, but the change was different in SOL and GAS. The sensory neurotrophism of the denervated muscles were determined by all of the neurotrophic factors in them.
OBJECTIVE: To investigate the effects of bone morphogenetic protein (BMP) on the proliferation and collagen synthesis of skeletal muscle satellite cells. METHODS: Skeletal muscle satellite cells were harvested and cultured in vitro. The 0 ng/ml, 50 ng/ml, 100 ng/ml, 500 ng/ml, and 1000 ng/ml BMP were used to induce skeletal muscle satellite cells for 48 hours. Cell proliferation, rate of myotube formation and collagen-1 synthesis were measured. RESULTS: BMP promoted cell proliferation and reduced the rate of myotube formation. Collagen synthesis increased when skeletal muscle satellite cells were induced with more than 500 ng/ml BMP. And the higher the concentration of BMP was, the ber this effect became. CONCLUSION: BMP can enhance the proliferation of skeletal muscle satellite cells and change their differentiation from myoblasts to osteoblasts.
OBJECTIVE: To study the influence of the electric stimulation of denervated muscle atrophy. METHODS: Sixteen SD rats were made the model of denervated skeletal muscle in two lower limbs by cutting off the sciatic nerve and femoral nerve. The right gastrocnemius muscle was stimulated with JNR-II nerve amp; muscle recovery instrument by skin as the experimental side and the left was not treated as the control side. The muscle histology, ultrastructure, fibrillation potential amplitude, Na(+)-K(+)-ATPase and Ca(2+)-ATPase activities were observed 2 weeks and 4 weeks after operation. RESULTS: Electric stimulation could protect mitochondria and sarcoplasmic reticulum from the degeneration. The reduction rates of muscle cell diameter and cross section in the experimental side were slower significantly than those in control side. There was no influence on fibrillation potential amplitude in the both sides after electric stimulation. The reduction rates of Na(+)-K(+)-ATPase activity in the experimental side were slower 15.59% and 27.38% respectively than those in the control side. The reduction rates of Ca(2+)-ATPase activity in the experimental side were slower 4.83% and 21.64% respectively than those in the control side. CONCLUSION: The electric stimulation can protect muscle histology, electrophysiology and enzymic histochemistry of denervated skeletal muscle from the degeneration. The electric stimulation is an effective method to prevent and treat muscle atrophy.
Objective To explore the effect of basic fibroblast growth factor（bFGF）and epidermal growth factor（EGF）on the growth of muscle derived stem cells(MDSCs). Methods MDSCs were isolated from hindlimb muscle of 15 new born Kunming mice through serial preplates. 2% fetal bovine serum-containing DMEM was used to induce MDSCs to differentiate into skeletal muscle lineage. The expressions of stem cell marker Sca-1 and skeletal musclecell marker αSarcomeric actin were examined by immunocytochemistry. The effect of bFGF and EGF on the proliferation of MDSCs was determined by MTT colorimetric microassay. The solo effect of bFGF or EGF at different concentrations (6.25,12.50, 25.00, 50.00, and 100.00 ng/ml) was examined at 96 h and the combined effect (100.00 ng/ml) was examined at 24,48,72 and 96 h.Results MDSCs were successfully isolated from the hindlimb of neonatal mice. Over 90% of MDSCs showed Sca-1 positive immunoreactivity. MDSCs could give rise to α Sarcomeric actin positive myotubes in differentiation cultures. The proliferative effect of bFGF and EGF on MDSCs increased with the elevated concentration.bFGF began to show significant proliferative effect at 12.50 ng/ml (P＜0.05). The effect increased significantly when the concentration reached 25.00 ng/ml from 12.50 ng/ml (P＜0.01) and reached a saturation point. The effect at 50.00 ng/ml or 100.00 ng/ml showed no significant increase when compared with thatat 25.00 ng/ml. EGF had a similar effect to bFGF except that the saturation concentration was 50.00 ng/ml. EGF showed significant effect at 72 h and bFGF at 96 h (Plt;0.01). When they were applied together, significant effect was shownat 24 h (Plt;0.01) and much higher effect was observed at 48, 72 and 96 h (Plt;0.05). Conclusion Both bFGF and EGF can promote the proliferation of MDSCs. The combined application reacts faster and ber.