Objective To investigate the myogenic differentiation of mesenchymal stem cells (MSCs) after being transplanted into the local muscle tissues. Methods The serious muscleinjured model was established by the way of radiation injury, incising, and freezing injury in 36 mouses. Purified MSCs derived from bone marrow of male mouse and MSCs induced by5-azacytidine(5-Aza-CR) were transplanted into the local of normal muscle tissues and injured muscle tissues of femal mouse. The quantity of MSCs and the myogenic differentiation of implanted MSCs were detected by the method of double labeling, which included fluorescence in situ DNA hybridization (FISH) and immuno-histochemistry on the 1st, 3rd, 6th, 9th, 12th, and 15th day after transplantation. Results The quantity of implanted MSCs decreased as timepassed. MSCs’ differentiation into myoblasts and positive expression of desmin were observed on the 15th day in purified MSCs group and on the 6th day in induced MSCs groups. Conclusion MSCs could differentiate into myoblasts after being implanted into the local of muscle tissues. The differentiationoccurs earlier in the induced MSCs group than that in purified MSCs group.
Objective To study the effect of allogenic different cells injected into denervated muscles on nerve regeneration. Methods Thirty-six adult female SD rats, weighed 120-150 g, were divided into four groups randomly (n=9, each group). Left sciatic nerves were cut down on germfree conditions and given primary suture of epineurium. Different cells were injected into the muscles of calf at once after operation every seven days and in all four times (group A: 1 ml Schwann cells at concentration of 1×106/ml; group B: 1 ml mixed cells of Schwann cells and myoblast cells at concentration of 1×106/ml; group C: 1 ml extract from the culture medium of kidney endothelial cells; and group D: 1 ml culture medium without FCS as control ). After 3 months, the specimen was observed on macrobody and histology, and the densities of neurilemma cell and myoceptor were counted. Results The means of proximate neurilemma cells were 0.187 7±0.054 2 in group A, 0.155 1±0.032 1 in group B, 0.072 4±0.023 7 in group C, and 0.187 7±0.054 2 in group D. The densities of myoceptor were 6.000±0.866 in group A，9.000±2.291 in group B,12.780±1.394 in group C, 3.110±0.782 in group D. Conclusion Schwann cells, mixed cells of Schwann cells with myoblast cells, and the extract from kidney endothelial cells canall accelerate the nerve regeneration. And the effect of extract from the kidney endothelial cell is superior to that of Schwann cell and mixed cell.
Objective To explore the possibilityof constructing tissue engineering muscles by combining allogeneic myoblasts with small instestinal submucosa(SIS) in rabbits.Methods A large number of purified myoblasts were obtained with multiprocedure digestion and repeated attachment method from skeletal muscles taken from extremities of immature rabbits which were born 7 days ago. The myoblasts were labeled with BrdU, and then combined with SIS to construct tissue engineering muscles. This kind of tissue engineering muscles were grafted into the gastrocnemius muscle defect (1.5 cm in length, 1.0 cmin width) of fifteen rabbits as the experimental group. The SIS was grafted into the same position in the control group. The rabbits were sacrificed 4, 6, 8 weeks after operation. The tissue engineering muscles were evaluated by macroscopic、histological and immunohistochemical observations, and by quantitative analysis of local immunocyte in the grafting site. Results Allogeneic myoblasts with SIS were combined perfectly in vitro. The SIS was connected tightly to surrounding skeletal muscles and inflammation response was obvious 4 weeks after grafting.The SIS began to break down and inflammation response became slight 6 and 8 weeks after operation. Compared with that of 8th week, the quantitative analysis oflocal immunocyte in 4th and 6th week in both experimental and control group hassignificance(Plt;0.05). Newly formed muscle tissues were found around SIS in the experimental group in 4th, 6th, and 8th week. Expression of BrdU and myosin immunohistochemical staining were positive in the experimental group and negative inthe control group.Conclusion Tissue engineering muscles of rabbits which are constructed by combining allogeneic myoblasts with SIS can survive and proliferate.
Objective To investigate a change in the differentiation and biological function of the cultured rat fibroblast (FB) transfected by the myoblast determining gene (MyoD) and the connexin 43 (Cx43) gene and to explore the possible mechanism of the MyoD and Cx43 genes on treatment of ischemic heart disease (IHD). Methods The gene cloning technology was used to construct the eukaryotic expressed plasmid vector pLenti6/V5-DEST-MyoD and pLenti6/V5DEST-Cx43 in which MyoD cDNA or Cx43 cDNA was inserted. The RFL-6 FB cells were transfected with exogenetic MyoD cDNA or Cx43 cDNA via lipofectamine, followed by the Blasticidin (50 μg/ml) selection, according to the lentiviral expression system (ViraPower) protocol. The expression and the biological functions of MyoD and Cx43 in the transfectants were testified by RT-PCR, Western blot, and molecular and immunocytochemical methods. The mophological structure changes of the cells were observed under microscope before and after the transfection. Results The expression of MyoD and Cx43 was detected in the MyoD and Cx43 genes transfected FB with RT-PCR and Western blot. The immunocytochemical methods indicated the expressionsof the MyoD and Cx43 genes, while desmin and αactin were found in these cells. The myotubes were found from the cultures incubated a week in the differentiation medium, in which the transfected cells had a characteristic of the filamentsin their cytoplasm and showed a myoblast morphology. Conclusion MyoD cDNA can induce the cultured FB to differentiate into the myoblasts and Cx43 cDNA can enhance the gap junctional intercellular communication between the cell and the cell. Thus, a further experimental foundation for the therapy of IHD can be provided.
Objective To investigate the expression of micro-dystrophin gene in myoblast cultured in vitro, to explore the possibil ity of combining myoblast transplantation with gene transfer for Duchenne muscular dystrophy therapy. Methods Competent Escherichia coli JM109 was prepared, which transformed with plasmid pSL139, and positive clones were picked to cultivate. Plasmid was extracted with Alkal ine lysis method and cutted with both Pvu I and Cla I enzyme. Agarose gel electrophoresis was employed to take pictures. Ten healthy 5-7 days old male C57/BL10 mice were selected, weighing4-5 g, the primary and subcultured myoblasts were cultured with multi-step enzymatic digestion and differential adhesionmethod, and Desmin immunofluorescent method was used to identfy. The 3rd generation myoblasts that were transfected with plasmid pSL139 mediated by l iposome served as the experimental group, untransfected cells served as the control group. After 48 hours of transfection, the expressions of micro-dystrophin mRNA and protein in myoblasts were detected with RTPCR and cell immunofluorescent methods, and the transfection efficiency was caculated. Results After pSL139 plasmids being digested and for 40 minutes agarose gel of electrophoresis, 3.75 kb fragment of target gene and vector were observed. The cells were almost uniform, and triangular or diamond shape after 24-48 hours of culture; the cells turned to fusion manner and could be passaged after 4-6 days. Desmin immunofluorescent result showed that green fluorescence was seen in cytoplasm of most 2nd myoblasts, and the purity of the myoblasts was above 90%. At 48 hours after transfection of myoblasts with plasmid pSL139, RT- PCR results showed that about 300 bp fragment was seen in the experimental group and the control group, and the brightness was higher in experimental group. Immunofluorescent staining displayed that green fluorescence was seen in the cytoplasm of the myoblasts in the experimental group and no green fluorescence in the control group; the expression efficiency of positive cells for micro-dystrophin was 45%-55% in experimental group. Conclusion Micro-dystrophin gene can highly express at the levels of mRNA and protein respectively in myoblasts transfected with plasmid pSL139 mediated by l iposome.
Objective To research the protective effects of different allogeneic cells injected into denervated muscles on ventricornual motor neuron. Methods Thirty-six adult female SD rats, weighting 120-150 g, were individed into four groups randomly and each group had nine. Left ischiadic nerves of all the SD rats, which were cut down on germfree conditions,were operated by primary suture of epineurium. Different cells were injected into the triceps muscles of calf in each group after operation with once a week for 4 weeks:1 ml Schwann cells (1×106/ml) in group A, 1 ml mixed cells ofSchwann cells and myoblast cells (1∶1,1×106/ml) in group B, 1 ml extract from the mixed cells of Schwann cells, myoblast cells and endotheliocytes (1∶1∶1,1×106/ml)in group C,and 1 ml culture medium without FCS as control group(group D). The observation of enzymohistochemistry and C-Jun expression in the ventricornual motor neuron was made after three months of operation. Results After 3 months of operation, the expressions of C-Jun in groups A, B and C were superiorto that in group D; the number of neuron was more than that of group D. The expressions of C-Jun in the ventricornual motor neuron were as follows: 128.591±0.766 in group A, 116.729±0.778 in group B, 100.071±2.017 in group C and 144.648±2.083 in group D; showing statistically significant difference between groupsA, B, C and D(P＜0.01). Enzymohistochemistry showed the well outlined and wellstacked cell body of neuron in groups A, B and C, and illdefined boundary of cytoplasm and nucleus. There was statistically significant defference in enzyme activity of the ventricornual motor neuron between groups(P＜0.01). Conclusion All of the Schwann cells,mixed cells of Schwann cells with myoblast cells,and the extract from Schwann cells, myoblast cells and endotheliocytes can protect the ventricornual motor neuron. And the protectiveeffect of the extract from Schwann cells, myoblast cells and endotheliocytes is superior to that of Schwann cells and mixed cells.
【Abstract】 Objective To investigate the effect of IGF-1 on the growth of primary human embryonicmyoblasts. Methods The method of incorporation of 3H-TdR was used to evaluate the abil ity of prol iferation of myoblasts.The count per minute (CPM) values of myoblasts at different concentrations(1, 2, 4, 8, 16 and 32 ng/mL) of IGF-1 were measured,and dose-effect curves were drawn to choose the optional concentration of IGF-1 to promote the prol iferation. Then theexperimental group of myoblasts received the addition of the optional concentration of IGF-1 in the growth medium, the controlgroup just received the growth medium. The flow cytometry was used to detect the cell cycle . The method of incorporation of3H-TdR was used to measure the peak-CPM. The myotube fusion rate was measured in myoblasts with different concentrations(0, 5,10, 15, 20, 25 and 30 ng/ mL) of IGF-1 in fusion medium, the dose-effect curves were also drawn, so as to decided the optional concentrationof IGF-1 in stimulating differentiation. Fusion medium with optional concentration of IGF-1 was used in experimentalgroup, and the control group just with fusion medium. The fusion rate of myotube and the synthesis of creatine kinase(CK) weredetected in both groups. Results The optional concentration of 5 ng/mL IGF-1 was chosen for stimulating prol iferation . It was shown that the time of cell cycle of control was 96 hours, but that of the experimental group was reduced to 60 hours. The results of flow cytometry showed that the time of G1 phase, S phase and G2M phase was 70.03, 25.01 and 0.96 hoursrespectively in control group, and were 22.66, 16.47 and 20.87 hours respectively in experimental group. The time-CPM value curves showed that the peak-CPM emerged at 96 hours in control group and 48 hours in experimental group, which was in agreementwith the results of the flow cytometry. The optional concentration stimulating prol iferation was 20 ng/mL IGF-1. Compared with control, the quantity of CK was increased by 2 000 mU/mL and the fusion rate was elevated by 30% in experimental group. Conclusion The concentrations of 20 ng/mL IGF-1 can elevat obviously the fusion rate and the quantity of CK. IGF-1 can enhance the prol iferation and differentiation of myoblasts via inducing the number of myoblasts at G1 phase and increasing the number of myoblasts at S and G2M phases.
Objective To investigate the effects of sodium hyaluronate solution on the proliferation and differentiation of myoblasts. Methods The 3rd subculture myoblasts from muscle of infant SD rat were cultured in four growth media, in which the concentrations of sodium hyaluronate were 0.05% (group A) , 0.1%( group B), 0.2% (group C)and 0 (group D, control group), respectively. The proliferation rate of myoblasts in each medium was observed through growth curves by means of count and MTT. At the same time, the subculture myoblasts were cultured in differentiated media in which the concentrations of sodium hyaluronate were 0 and 0.1%. The capacity of fusion of myoblasts was compared between two kinds of differentiated media. Results There were the nearly same proliferation curse in Groups A, B and C: increasing by logarithm at 2 days and reaching peak value at 4 days. The myoblasts in Group D increased slowly: increasing by logarithm at 3 days, doubling at 5 days and reaching peak value at 6 days. MTT has the similar curse to counting. The myoblast proliferation of Group B was more than that of the other groups. The peak value of myoblast fusion was 35% at 6 days in common differentiated media; slowly reached 11.7% at 7 days in the differentiated media in which the concentrations of sodiumhyaluronate was 0.1%.Conclusion Sodium hyaluronate at certain concentration can be a decent media for myoblasts, it can accelerate proliferation and differentiation of myoblasts.
OBJECTIVE: To investigate the biological characteristics of continuously subcultured human embryonic skeletal myoblasts, and choose the optimal seeding cells for muscle tissue engineering. METHODS: Human embryonic skeletal myoblasts were subcultured in vitro. The growth curve, rate of myotube formation(RMF) were used to evaluate the proliferative and differentiation ability of myoblasts, and to investigate the influence of fibroblasts contamination on myoblasts. RESULTS: The beginning 6 passages of myoblasts showed b proliferative and differentiation ability. From the 8th to 20th passage, the rate of fibroblasts contamination was increased, it mainly showed the growth characteristics of fibroblasts with increased proliferation and low differentiation. After subcultured to the 20th passage, the degeneration of myoblasts was obvious. CONCLUSION: The myoblasts within 6 passages should be used as the seeding cells of muscle tissue engineering because of b proliferative ability and high rate of myotube formation.
Objective To explore heterotopic chondrogenesis of canine myoblasts induced by cartilage-derived morphogenetic protein 2 (CDMP-2) and transforming growth factor β1 (TGF-β1) which were seeded on poly (lactide-co-glycolide) (PLGA) scaffolds after implantation in a subcutaneous pocket of nude mice. Methods Myoblasts from rectus femoris of 1-year-old Beagle were seeded on PLGA scaffolds and cultured in medium containing CDMP-2 and TGF-β1 for 2 weeks in vitro. Then induced myoblasts-PLGA scaffold, uninduced myoblasts-PLGA scaffold, CDMP-2 and TGF-β1-PLGA scaffold, and simple PLGA scaffold were implanted into 4 zygomorphic back subcutaneous pockets of 24 nude mice in groups A, B, C, and D, respectively. At 8 and 12 weeks, the samples were harvested for general observation, HE staining and toluidine blue staining, immunohistochemical staining for collagen type I and collagen type II; the mRNA expressions of collagen type I, collagen type II, Aggrecan, and Sox9 were determined by RT-PCR, the glycosaminoglycans (GAG) content by Alician blue staining, and the compressive elastic modulus by biomechanics. Results In group A, cartilaginoid tissue was milky white with smooth surface and slight elasticity at 8 weeks, and had similar appearance and elasticity to normal cartilage tissue at 12 weeks. In group B, few residual tissue remained at 8 weeks, and was completely degraded at 12 weeks. In groups C and D, the implants disappeared at 8 weeks. HE staining showed that mature cartilage lacuna formed of group A at 8 and 12 weeks; no cartilage lacuna formed in group B at 8 weeks. Toluidine blue staining confirmed that new cartilage cells were oval and arranged in line, with lacuna and blue-staining positive cytoplasm and extracellular matrix in group A at 8 and 12 weeks; no blue metachromatic extracellular matrix was seen in group B at 8 weeks. Collagen type I and collagen type II expressed positively in group A, did not expressed in group B by immunohistochemical staining. At 8 weeks, the mRNA expressions of collagen type I, collagen type II, Aggrecan, and Sox9 were detected by RT-PCR in group A at 8 and 12 weeks, but negative results were shown in group B. The compressive elastic modulus and GAG content of group A were (90.79 ± 1.78) MPa and (10.20 ± 1.07) μg/mL respectively at 12 weeks, showing significant differences when compared with normal meniscus (P lt; 0.05). Conclusion Induced myoblasts-PLGA scaffolds can stably express chondrogenic phenotype in a heterotopic model of cartilage transplantation and represent a suitable tool for tissue engineering of menisci.