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.
OBJECTIVE Because of its special biological characteristics, myoblast might play a role in gene delivery and cell-to-biomaterial interactions. In this paper, the biological features of myoblast and its application on gene therapy and tissue engineering was discussed. METHODS Documents about proliferation and differentiation of myoblast were reviewed in details. The prospects of its application on gene therapy and tissue engineering were also presented. RESULTS Myoblast was important in muscle regeneration. The activation of myoblast to proliferate and differentiate was the very beginning of regeneration after injury. The cultured myoblast had high potential to proliferate, it was ready to fuse with each other and to form myotube (the special behavior of myoblast differentiation). Myoblast transplantation had been studied as a possible treatment for inherited myopathies, such as Duchenne muscular dystrophy. The transplanted myoblast could fuse with host myofibers, so the delivered target gene integrated into host. Several myoblast-mediated gene delivery system had been established, including the gene delivery of human factor IX (hFIX), erythropoietin (EPO) and clony stimulating factor-1 (CSF-1). Results from animal experiments demonstrated that myoblast-mediated gene delivery could be used as gene therapy for some inherited diseases. And recently, some authors have shown great interest in the interaction between myoblast and type I collagen gels. It was found that myoblast could keep on proliferating and differentiating in collagen gels and could form discoid, tubular materials. CONCLUSION Myoblast has great importance in gene therapy and tissue engineering. It is suggested that more efforts should be made in this field.
Objective To study the construction feasibility of a biodegradable artificial esophagus by the squamous epithelial cells and the myoblast cells seeded on the small intestinal submucosa(SIS) and to investigate the growth patternand angiogenesis of the co-cultured human embryonic squamous epithelial cells and the skeletal myoblasts in vivo. Methods The squamous epithelial cells and the myoblast cells were obtained from the 20-week aborted fetus. Both of their cellswere marked by 5-BrdU in vitro.The isolated cells were then seeded on the SIS and co-cultured in vitro for 24 hours, and then the compound of the cells and the SIS was transplanted into the subcutaneous tissue of the athymismus mice. The observation on the morphology and the cytokeratin AE3 and α-actin specified immunohistochemistry of the squamous epithelial cells and the myoblastcells was performed at each of the following time points: 3 days, 1 week, 2 weeks, and 3 weeks after transplantation. Results The morphological observation indicated that the cultured cells could penetrate into the small intestinal submucosa and form several-layered cell structures, and that the compound of the cells and the SIS could have angiogenesis within 2-3 weeks. The 5-BrdU specified immunohistochemical observation suggested that the cells growing in the small intestinal submucosa scaffold might be the cells transplanted.The cytokeratin AE3 specified and α-actin specified immunohistochemical studies demonstrated that the transplanted cells could differentiate in vivo. Conclusion It is possible to fabricate the framework of a biodegradable artificial esophagus with the epithelial cells and the myoblast cells seeded on the small intestinal submucosa.
Objective To explore the human stromal cell-derived factor 1α (hSDF-1α) and human vascular endothel ial growth factor 165 (hVEGF165) mRNA expressions of the transfected cells after hSDF-1α gene and hVEGF165 gene were transfected into rat myoblasts in vitro so as to lay a foundation for further study on the synergistic effects of 2 genes on tissue engineered skeletal muscle vascularization. Methods The myoblasts of 1-day-old Sprague Dawley rats were cultured and purified by trypsin digestion assay in vitro and were identified by immunohistochemistry staining of Desmin. pproximately 70%-80% of confluent myoblasts were transfected with enhanced green fluorescent protein (EGFP)-hSDF-1α and EGFP-hVEGF165 genes in vitro (transfected group) and were not transfected (control group). The expressions of hSDF-1αand hVEGF165 mRNA and protein in the transfected cells were detected by RT-PCR, ELISA, and Western blot espectively.Results The cultured cells were identified as myoblasts by immunohistochemistry staining of Desmin. The expression ofgreen fluorescent protein was observed in transfected cells, indicating that hSDF-1α and hVEGF165 genes were transfected into myoblasts successfully. The mRNA and protein expressions of the 2 genes were positive in the transfected group by RT-PCR and Western bolt assay at 2, 4, 6, and 8 days after transfection, and were negative in the control group. The expressions of hSDF- 1α and hVEGF165 showed a stable low level in the control group, but the expressions of the proteins increased at 2 days and then showed gradual downtrend with time in the transfected group by ELISA assay. There were significant differences in the expressions of hSDF-1α and hVEGF165 proteins between different time points in the transfected group, and between 2 groups (P lt; 0.05). Conclusion hSDF-1α and hVEGF165 genes are successfully transfected into myoblasts in vitro, and mRNA and proteins of hSDF-1α and hVEGF165 can be expressed in the transfected myoblasts, which may provide the experimental evidence for the expressions of hSDF-1α and hVEGF165 mRNA and proteins in vivo successfully.
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 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 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.
【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 explore the facilitative effects of different allogenic cells injected into the denervated muscles on the nerve regeneration, the protection of the myoceptor degeneration, and the promotion for rehabilitation of the muscular function. Methods Schwann cells, myoblast cells, and renal endothelial cells were prepared from 400 SD rats aged 7 days and weighing 20.0±2.3 g. Thirty-six adult female SD rats weighing 120-150 g were randomly divided into 4 groups(n=9). Under the asepsis condition, the left ischiadic nerves of all the SD rats were cut off, and the primary suture of the epineurium was performed. After operation, the different corresponding cells were injected into the triceps muscles of the rat calf in each group once per week for 4 times in all. One ml of Schwann cells (1×106/ml) was injected into the rats in Group A; 1 ml of the mixed cells of Schwann cells and myoblast cells (1×106/ml) was injected into the rats in Group B; 1 ml of the extract from the mixed cells of Schwann cells, myoblast cells, and renal endothelial cells (1×106/ml) was injected into the rats in Group C; 1 ml of the culture medium without any serum was injected into the rats in Group D as a control. After operation, observation was made for the general condition of the rats; 3 months after operation, enzymohistochemistry and the CJun expression were performedin the ventricornual motor neuron. At the proximal and the distal ends of the nerve suture, the density of neurilemma cells in the unit area and the area size of the regenerated nerve fibers were observed and measured. Results The affected limbs of the rats in Groups A, B and C improved 13 months after operation. The ulcers and swelling at the ankles gradually relieved and the rats could move normally 3 months after operation. However, the affected limbsof the rats in Group D still had ulcers and swelling, with an obvious contracture of the toes and a difficult movement. Three months after operation, the number of the target muscle myoceptor, the number of the Actin positive cells, the activity of the various enzymes in the denervated muscles, and the histological changes of the regenerated nerves were better in Group C than in Groups A and B (P＜0.01); and they were all better in Groups A, B and C than in Group D（Plt;0.01）. Conclusion Schwann cells, the mixture of Schwann cells and myoblast cells, and the extract from the mixture of Schwann cells, myoblast cells and renal endothelial cells can all promote neurotization and rehabilitation of the muscular function, and protect against the myoceptor degeneration. However, the effect of the extract is superior to that of Schwann cells or the mixed cells.
【Abstract】 Objective To investigate the role of myosin l ight chain (Myl) in myogenesis in vitro. Methods The extraocular muscle, diaphragm and gastrocnemius muscle myoblasts (eMb, dMb and gMb) were isolated and purified from 12 3-week-old C57BL/6 mice by using the enzyme digestion and Preplate technique, and then were subcultivated. The Myl expression in Mb was detected by RT-PCR and Western blot analysis; the Mb prol iferation activity was tested by methylene blue assay, and the myotube formation was observed. After anti-Myl antibody (1, 2, 3, 8, 16 ng/mL) was induced in the Mb culture (experimental group), the abil ity of prol iferation of myoblasts and the myotube formation were identified. Meanwhile, the Mb which was cultured without anti-Myl antibody was indentified as the control group. Results The results of RT-PCR and Western blot analysis showed that Myl1 and Myl4 mRNA and Myl protein were expressed in eMb, dMb and gMb at 24 hours after seeding, and their expression level were lower in eMb than in dMb and gMb (P lt; 0.01), and the latter two did not show any significant difference (P gt; 0.05). Myl2 and Myl3 mRNA was not detected in these three myoblasts. The prol iferation assay showed that the eMb prol iferated faster as compared with dMb and gMb (P lt; 0.01). eMb began to yield myotubes at 40 hours after seeding and dMb and gMb at 16 hours after seeding. At 6 days, the number of myotubes derived from eMb was (137.2 ± 24.5)/ field, which was significantly larger than that of myotubes from dMb [(47.6 ± 15.5) / field ] and gMb [(39.8 ± 5.1) field ] (P lt; 0.01). There was not statistically significant difference between the latter two groups (P gt; 0.05). After the antibody treatment, the absorbency values of the eMb, dMb and gMb in the experimental groups at each antibody concentration point were significantly higher than those in the corresponding control groups (P lt; 0.05), and the dose-dependent way was performed.The numbers of myotubes from dMb at 16 hours were (48.2 ± 7.1)/ well in the experimental group and (23.4 ± 4.9)/ well in the control group, and at 6 days were (40.6 ± 10.2)/ field in the experimental group and (63.1 ± 6.1)/ field in the control group.There was statistically significant difference between the experimental and control groups (P lt; 0.01). Conclusion Myl may play a role in myogenesis through the negative effect on the myoblast prol iferation.