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.
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 effect of monocyte chemoattractant protein 1 (MCP-1) on the migration of the induced and differentiated mouse bone marrow mesenchymal stem cells (BMSCs) for raising the efficacy of intravenous transplantation of BMSCs. Methods The BMSCs were cultured with the method of differential adhesion and density gradient centrifugation of C57/BL10 mice, and were identified by alkal ine phosphatase Gomori modified staining after osteogenic inducing. At the 3rd passage, the BMSCs were induced to the myoblasts with 5-azacytidine (5-Aza). The chemotaxis of MCP-1 in the induced and differentiated BMSCs in vitro at concentrations of 25, 50, 100, 200, and 400 ng/mL was observed through the migration test, by counting the number of the migrated cells. The expression of the chemokine receptor 2 (CKR-2) in the induced and differentiated BMSCs was detected with the flow cytometry. Results The cells could be cultured with the methods of differential adhesion and density gradient centrifugation and still had higher prol iferative and differentiative potency; the induced cells at the 3rd passage could differenciate to the osteoblasts, confirming that the cells were BMSCs; the myogenic induced BMSCs possesed the sarcotubule structure. The number of the migrating BMSCs at MCP-1 concentrations of 25-400 ng/ mL were respectively 35.066 7 ± 6.584 2, 43.200 0 ± 6.460 8, 44.466 7 ± 4.823 5, 45.600 0 ± 8.650 3, and 50.733 3 ± 7.582 5; showing significant difference when compared with control group (28.333 3 ± 8.917 6, P lt; 0.05), and presenting significant difference among 25, 50, 400 ng/mL groups compared with each other (P lt; 0.05). The expression of CKR-2 in the mouse BMSCs (48.0%) was significantly higher (P lt; 0.001) than those of blank control (0.6%) and negative control (17.0%). Conclusion The results indicate that the MCP-1 can induce the migration of mouse BMSCs by MCP-1/CKR-2 pathway.