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 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.
ObjectiveTo research the effect of chondroitin sulfate (CS) on the proliferation of myoblasts and the formation of myotube. MethodsThe myoblasts at passage 5 were used to prepare the cells suspension (1×108 cells/mL), and the experiment was divided into 4 groups based on CS concentration in the medium:group A (0 μg/mL), group B (50 μg/mL), group C (100 μg/mL), and group D (200 μg/mL). The cell morphology and myotube formation were observed by inverted microscope at 4, 5, and 8 days after treatment; MTT was used to detect the cell proliferation at 6 days, and the number of myotube was calculated by HE staining at 8 days. ResultsCells showed spindle shape after adherent, with ovoid nuclei and dense cytoplasm under inverted microscope. When the cell adherent rate was 90%, cells arranged in whorls swirled and showed long fusiform adherent growth; and then nuclei fusion resulted in formation of multincleated myotubes. At 8 days, most myoblasts fused to form myotube in group A, but less myotube was observed in groups B and C, and the least myotube in group D. The absorbance (A) values of groups A, B, C, and D were 0.045 2±0.004 4, 0.540 4±0.096 7, 0.660 9±0.143 4, and 1.069 0±0.039 0 respectively, showing significant difference between other groups (P<0.05) except between groups B and C P>0.05). HE staining observation showed that most myoblasts fused to form myotube in group A, but less myotube in groups B and C, and the least myotube in group D. The number of myotube of groups A, B, C, and D were 222.01±30.02, 193.13±42.46, 170.26±11.96, and 136.88±16.78 respectively, showing no significant difference among groups (F=1.658, P=0.252). ConclusionCS can significantly promote the proliferation of myoblast, the promotion is the biggest when CS concentration is 200 μg/mL.
【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 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 investigate the effect of myoblast transplantation on duchenne muscular dystrophy (DMD) and to explore the method and feasibil ity of applying gene therapy to DMD. Methods Myoblast of C57/BL10 mice were cultured using multiple-step enzyme digestion method and differential velocity adherent technique. The morphology of the cells was observed with inverted phase contrast microscope. The cells at passage 4 were labeled with 5-BrdU. Twenty-four DMDmodel mice (mdx mice: aged 4-6 weeks, male, 13.8-24.6 g) were randomly divided into two groups (n=12 per group): group A, 1 × 106/mL labeled myoblast were injected via ven caudal is twice at an interval of 2 weeks; group B: 1 mL DMEM/F12 was injected in the same manner serving as a control group. The mice were killed 4 weeks after operation and the motor abil ity of the mice was detected by one-time exhaustive swimming before their death. HE staining and immunohistochemistry staining observation for 5-BrdU, desmin, and dystrophin (Dys) were preformed, and the imaging analysis was conducted. Results The primary myoblast could be sub-cultured 5-7 days after culture, providing stable passage and sufficient cells. The time of onetime exhaustive swimming was (60.72 ± 5.76) minutes in group A and (47.77 ± 5.40) minutes in group B, there was significant significance between two groups (P lt; 0.01). At 4 weeks after injection, HE staining showed that in group A, there were round and transparent-stained myocytes and the percentage of centrally nucleated fibers (CNF) was 67%; while in group B, there were uneven muscle fiber with such pathological changes as hypertrophia, atrophia, degeneration, and necrosis, and the percentage of CNF was above 80%. Immunohistochemistry staining revealed that the expression of 5-BrdU, desmin, and Dys was positive in group A; while in group B, those expressions were l ittle or negative. Image analysis result displayed that integral absorbency (IA) value of desmin was 489.70 ± 451.83 in group A and 71.15 ± 61.14 in group B (P lt; 0.05) and the ratio of positive area to thetotal vision area was 0.314 3 ± 0.197 3 in group A and 0.102 8 ± 0.062 8 in group B (P lt; 0.05); the Dys IA value was 5 424.64 ± 2 658.01 in group A and 902.12 ± 593.51 in group B (P gt; 0.05) and the ratio of positive area to the total vision area was 0.323 7 ± 0.117 7 in group A and 0.035 2 ± 0.032 9 in group B (P lt; 0.05). Conclusion Myoblast transplantation has certain therapeutic effect on DMD of mice.
【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.
Objective To introduce the current situation and futureof myoblast transfer therapy (MTT) in clinical application Methods The latest fifteenyear literatures were extensively reviewed, concerninggene therapy for Duchenne’s muscular dystrophy, Parkinson’s disease, myelopathy, permanent facial paralysis, angiocardiopathy, injuries of bone, joint and muscle, hematopathy, and pituitary dwarf. Results In medical field, MTT is an ideal method to treat some common diseases. The problems were immunologic rejection and better carriers for myoblasts implantation. Conclusion It is the focus on the use of myoblast as a vector to carry exogenous gene in some disease therapy. The major problems of MTT include transplantation immunity, cell fusion and target protein expression. It is easy to gain,culture and transfuse to the host for myoblasts, these merits are beneficial to clinical application.
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.