PURPOSE: To observe dystrophin formation and histological improvement in dystrophic muscle of mdx mouse after normal myoblast injection. MATERIALS AND METHODS: Cultured myoblasts from genetically normal rats were injected into the right quadriceps femoris of a 6-week-old mdx mouse (n=9). dPBS was injected into the left quadriceps femoris as a control. One, 2, and 3 months after injection, The control and experimental group were compared histologically and by dystrophin immunostaining. RESULTS: When compared with controls 3 months postoperatively, quadriceps femoris in the experimental group exhibited greater cross-sectional area and total fiber number, and the experimental animals contained more normal-appearing and less abnormalappearing fibers than the control group. Most of the fibers in the experimental group showed positive results in dystrophin immunostaining, whereas immunostaining of mdx muscle fibers in the control group was completely negative. CONCLUSION: This study shows that normal myoblast injection improved the muscle architecture histologically and produced dystrophin protein in dystrophic muscle.
Animals ; Dystrophin ; Mice ; Mice, Inbred mdx ; Muscular Dystrophies ; Myoblasts* ; Quadriceps Muscle ; Rats

BACKGROUND: Duchenne muscular dystrophy is an X-linked recessive disorder leading to death in the late teens or early twenties. There is no effective pharmacological therapy for now. L-carnitine (LCAR), a naturally occurring compound facilitating the transport of fatty acid into mitochondria for -oxidation, has been getting an attention for its antiapoptotic and osmoprotective effect. The aim of this study is to evaluate if LCAR administration reduces dystrophic progression and enhances exercise tolerance in dystrophin deficient (mdx) mice. METHODS: Mdx mice (n=5) and wild type mice (n=5), aged 3 weeks were treated with oral LCAR (75mg/kg/day) for 6 weeks. Five each mdx and wild type mice were recruited for their counter-control. The animals underwent a 30-minute run on a horizontal treadmill for evaluating their exercise endurance. After 6-week training, baseline and post exercise serum CK of each group were analyzed. We examined sarcolemma integrity and muscle histology after exercise. Immunofluorescent stain and Western blot analysis for dystrophin-dystroglycan complex were also performed. RESULTS: LCAR-treated mdx mice showed higher exercise tolerance and lower serum CK value compared with those of control mice. The area of Evans blue dye uptake in LCAR-treated mdx mice was much smaller than that of control mdx mice. There was no remarkable difference in dystrophin-dystroglycan complex expression between treated and control mdx mice. CONCLUSIONS: LCAR seems to enhance exercise tolerance and decrease the breakdown of sarcolemma during strenuous exercise. Our study suggests the possibility of adjunctive therapeutic use of L-carnitine to the patients with Duchenne muscular dystrophy.
Adolescent ; Animals ; Blotting, Western ; Carnitine* ; Dystrophin ; Evans Blue ; Exercise Tolerance* ; Humans ; Mice* ; Mice, Inbred mdx ; Mitochondria ; Muscular Dystrophies* ; Muscular Dystrophy, Duchenne ; Sarcolemma

OBJECTIVE: To investigate the effect of steroid administration on the apoptosis and heat shock protein 70 (HSP70) expression after exercise in the animal model of Duchenne muscular dystrophy. METHOD: We measured Bcl-2, BAX and HSP70 expression by western blotting. 20 control and 20 mdx mice were divided into free-living (n=10) and exercise (n=10) groups. Free-living and exercise groups were further divided into steroid-treated and sham-treated groups to evaluate the effect of steroid administration. RESULTS: Apoptosis was most prominent in the sham-treated exercise group, while apoptosis was significantly reduced in the steroid-treated exercise group. HSP70 expression was maximized in sham-treated exercise group, whereas steroid administration inhibited HSP70 expression after exercise in muscular dystrophy animal model. Exercise loading was found to cause severe apoptosis but steroid administration alleviated apoptotic damage in mdx mice. CONCLUSION: HSP70 expression was suppressed in the steroid-treated exercise group, which suggests steroid might have major preventive effect in exercise-induced apoptosis of muscular dystrophy animal model.
Animals ; Apoptosis ; Blotting, Western ; Heat-Shock Proteins ; Hot Temperature ; HSP70 Heat-Shock Proteins ; Mice ; Mice, Inbred mdx ; Models, Animal ; Muscular Dystrophies

OBJECTIVE: To investigate the effect of exercise and steroid to the muscle of animal model of Duchenne muscular dystrophy. METHOD: We used 15 mdx and 15 control mice. To grade exercise loading, control and mdx mice were divided into free-living, exercise and immobilization groups. Free-living and exercise groups were further divided into steroid-treated and sham-treated groups to evaluate the effect of steroid administration. We measured the apoptotic changes using in situ DNA nick-end labling (TUNEL), DNA fragmentation assay and western blots for Bcl-2 and BAX. RESULT: With TUNEL method, the largest number of myonuclei became positive in sham-treated exercise group while apoptosis was significantly reduced in steroid-treated exercise group in mdx mice. Steroid-treated free-living group showed higher rate of apoptotic change than sham-treated free-living group. With western blots for Bcl-2 and BAX, the value of BAX/Bcl-2 ratio was highest in sham-treated exercise group and among free living mdx mice, it was higher in steroid-treated group than sham-treated one. CONCLUSION: Apoptosis can be minimized in free living condition while exercise loading or immobilization can cause apoptotic change in muscular dystrophy animal model. Steroid administration induces apoptosis in free living muscle and it alleviates apoptotic damage caused by exercise loading in mdx mice.
Animals ; Apoptosis* ; Blotting, Western ; DNA ; DNA Fragmentation ; Immobilization ; In Situ Nick-End Labeling ; Mice ; Mice, Inbred mdx* ; Models, Animal ; Muscle, Skeletal* ; Muscular Dystrophies ; Muscular Dystrophy, Duchenne ; Social Conditions

Duchenne muscular dystrophy (DMD) is an X-linked, recessive and lethal genetic disease, which usually caused by gene mutations and the underlying mechanisms are complicated and diverse. The causal gene of DMD is the largest one in human that locates in the region of Xp21.2, encoding dystrophin. Currently there is no effective treatment for DMD patients. The treatment of DMD depends on gene mutation and molecular mechanism study of the disease, which requires reliable disease models such as mdx mouse model. Recently, researchers have increasingly discovered gene therapy strategies for DMD, and the efficacy has been demonstrated in DMD animal models. In addition, induced pluripotent stem cell technology can provide patient-specific cell source, offering a new platform for mechanism and therapy study of DMD.
Animals ; Disease Models, Animal ; Dystrophin ; genetics ; Genetic Therapy ; trends ; Humans ; Induced Pluripotent Stem Cells ; Mice ; Mice, Inbred mdx ; genetics ; Muscular Dystrophy, Duchenne ; genetics ; therapy

OBJECTIVETo construct the retroviral vector containing human micro-dystrophin gene and detect the expression of human micro-dystrophin in mdx mice bone marrow-derived mesenchymal stem cells (MSCs) after retrovirus infection.

METHODSRetroviral vector for micro-dystrophin gene was constructed and transferred into the packing cell PA317 mediated by Lipofectamine 2000. The retroviral supernatant containing the target genes were subsequently used to infect mdx mice MSCs. Micro-dystrophin expression was examined by methods of immunofluorescence staining and reverse transcriptase-polymerase chain reaction.

RESULTSMicro-dystrophin retroviral vector was successfully constructed and transferred into PA317 cells, and 48 h after infection with the recombinant retrovirus in mdx mice MSCs, 319 bp fragment could be detected by electrophoresis in the RT-PCR products. The red particles could be detected in some infected mdx mice MSCs with immunofluorescence staining. CONCLUSION mdx mice MSCs infected with retrovirus containing micro-dystrophin gene can express micro-dystrophin protein.

Animals ; Bone Marrow Cells ; cytology ; metabolism ; Dystrophin ; biosynthesis ; genetics ; Humans ; Mesenchymal Stromal Cells ; cytology ; metabolism ; Mice ; Mice, Inbred mdx ; Muscular Dystrophy, Animal ; metabolism ; Retroviridae Infections ; Transfection

Duchenne muscular dystrophy (DMD) is a common X-linked disease characterized by widespread muscle damage that invariably leads to paralysis and death. There is currently no therapy for this disease. This study was purposed to investigate the feasibility to use adult adipose-derived mesenchymal stem cells (AD-MSCs) in the therapy of DMD. The Flk-1(+) MSCs were isolated from adipose tissue of adult GFP mice; the phenotype and cell cycle of MSCs were analyzed by flow cytometry; the AD-MSCs were directionally differentiated by myoblast and endotheliablast induction system in vitro and were identified by immumofluorecence staining and RT-PCR; the AD-MSCs were transplanted into CTX-injured mice model or mdx mice (DMD animal model) through tail vein; the distribution and differentiation of AD-MSCs were detected by immunofluorescence staining and RT-PCR respectively, and statistic analysis was performed. The results showed that the Flk-1(+) AD-MSCs could be induced to differentiate into myoblasts and endothelial cells in vitro. After transplanted into CTX-injured mice model or mdx mice, GFP-positive cells could be detected in damaged muscle, and these donor-derived cells were also positive for MHC, vWF, or Pax7. Flk-1(+) AD-MSC transplantation also partly reconstituted the expression of dystrophin, and reduced the percentage of centronucleated myofibers in mdx mice. It is concluded that Flk-1(+) AD-MSCs represent a possible tool for future cell therapy applications in DMD disease, as they can be delivered through the circulation for their potential of muscle homing. And Flk-1(+) AD-MSCs also show the ability to contribute to muscle repair, improvement of blood supply and long term reconstitution of dystrophy muscle.
Adipose Tissue ; cytology ; Animals ; Cell Differentiation ; Cells, Cultured ; Dystrophin ; biosynthesis ; Mesenchymal Stem Cell Transplantation ; Mesenchymal Stromal Cells ; cytology ; Mice ; Mice, Inbred C57BL ; Mice, Inbred mdx ; Mice, Transgenic ; Muscle Cells ; cytology ; Muscular Dystrophy, Duchenne ; pathology ; therapy ; Myoblasts ; cytology

OBJECTIVETo investigate the effect of bone marrow stem cell transplantation (BMT) on the diaphragm muscles of mdx mice, a mouse model of Duchenne muscular dystrophy (DMD).

METHODSThe bone marrow-derived stem cells form male SD rats was transplanted through the tail vein into 18 female 8-week-old mdx mice, which were sacrificed at 4, 8 and 12 weeks after BMT (6 at each time point), respectively. The diaphragm muscles of the mice were subjected to HE staining, immunofluorescence detection of dystrophin, reverse transcription (RT)-PCR analysis of dystrophin mRNA transcripts and PCR analysis of Sry (sex-determining region on the Y chromosome) gene, with age-matched female C57 mice and untreated mdx mice as the controls.

RESULTSThe proportion of centrally nucleated fibers (CNF) in the diaphragm muscle of the recipient mdx mice was (15.58+/-0.91) %, (12.50+/-1.87) % and (10.17+/-1.17) % at 4, 8 and 12 weeks after BMT, respectively, significantly smaller than that of untreated mdx mice [(19.5+/-1.87) %], and the fibers after BMT showed less inflammatory infiltration. Compared with the untreated mice, the recipient mdx mice showed green fluorescence on significantly more diaphragm muscle cell membranes [with the proportion of dystrophin-positive fibers of (1.00+/-0.32) %, (6.00+/-1.05) % and (11.92+/-1.11) % at 4, 8, and 12 weeks after BMT]. RT-PCR of dystrophin mRNA also demonstrated significantly higher relative levels of dystrophin in the recipient mdx mice (0.19+/-0.05, 0.26+/-0.06 and 0.36+/-0.04 at 4, 8 and 12 weeks after BMT) than in untreated mdx mice, and Sry gene was present in the recipient mice.

CONCLUSIONBMT can partially restore dystrophin expression and ameliorate the pathology in the diaphragm muscles of mdx mice, and has great potential to produce general therapeutic effect in patients with DMD.

Animals ; Bone Marrow Transplantation ; methods ; Diaphragm ; metabolism ; pathology ; Dystrophin ; biosynthesis ; genetics ; Female ; Male ; Mice ; Mice, Inbred C57BL ; Mice, Inbred mdx ; Muscular Dystrophy, Duchenne ; metabolism ; pathology ; surgery ; Rats ; Rats, Sprague-Dawley ; Transplantation, Heterologous

OBJECTIVETo investigate the dynamic changes of dystrophin expression in mdx mice after bone marrow stem cells transplantation.

METHODSThe bone marrow stem cells of C57 BL/6 mice (aged 6 to 8 weeks) were injected intravenously into the mdx mice (aged 7 to 9 weeks), which were preconditioned with 7Gy gamma ray. The amount of dystrophin;expression in gastrocnemius was detected by immunofluorescence, reverse transcription-polymerase chain reaction and Western blot at week 5, 8, 12 and 16 after transplantation.

RESULTSAt week 5 after bone marrow stem cells transplantation, the dystrophin expression detected in mdx mice were very low; however, its expression increased along with time. At week 16 week, about 12% muscle cells of all transplanted mice expressed dystrophin. There were less centrally placed myonuclei than the control mdx mice, whereas peripheral myonuclei increased.

CONCLUSIONSAfter having been injected into mdx mice, the allogenic bone marrow stem cells have a trend to reach the injured muscle tissues and differentiate to fibers that can express dystrophin and the expression increased with time. The bone marrow stem cells participates in the repair and regeneration of the injured tissues permanently and constantly.

Animals ; Bone Marrow Cells ; cytology ; metabolism ; Cell Differentiation ; Disease Models, Animal ; Dystrophin ; biosynthesis ; Hematopoietic Stem Cell Transplantation ; Male ; Mice ; Mice, Inbred C57BL ; Mice, Inbred mdx ; Muscular Dystrophy, Duchenne ; metabolism ; surgery ; Transplantation, Homologous

OBJECTIVETo investigate the differentiation of rat bone marrow mesenchymal stem cells (MSCs) into myocytes and their expression of dystrophin/utrophin after transplantation in mdx mice.

METHODSBrdU-labeled fifth-passage rat MSCs were transplanted in mdx mice with previous total body gamma irradiation (7 Gy). At 4, 8, 12 and 16 weeks after the transplantation, the mice were sacrificed to detect dystrophin/BrdU and utrophin expressions in the gastrocnemius muscle using immunofluorescence assay, RT-PCR and Western blotting. Five normal C57 BL/6 mice and 5 mdx mice served as the positive and negative controls, respectively.

RESULTSFour weeks after MSC transplantation, less than 1% of the muscle fibers of the mdx mice expressed dystrophin, which increased to 15% at 16 weeks. Donor-derived nuclei were detected in both single and clusters of dystrophin-positive fibers. Some BrdU-positive nuclei were centrally located, and some peripherally within myofibers. Utrophin expression decreased over time after transplantation.

CONCLUSIONThe myofibers of mdx mice with MSC transplantation express dystrophin, which is derived partially from the transplanted MSCs. Dystrophin expression from the transplanted MSCs partially inhibits the upregulation of utrophin in mdx mouse muscle, showing a complementary relation between them.

Animals ; Bone Marrow Cells ; cytology ; Cell Differentiation ; physiology ; Dystrophin ; genetics ; metabolism ; Mesenchymal Stem Cell Transplantation ; Mesenchymal Stromal Cells ; cytology ; Mice ; Mice, Inbred C57BL ; Mice, Inbred mdx ; metabolism ; Muscle Fibers, Skeletal ; cytology ; metabolism ; Muscular Dystrophy, Animal ; metabolism ; therapy ; Rats ; Utrophin ; metabolism