Effects Of AAV9 Mediated VEGF Gene Overexpression On Mdx Mice

Objective: Duchenne muscular dystrophin(DMD) is an X-linked recessive lethal muscle disease, in the survival of male incidence rate is about1 / 3500,characterized by progressive limb weakness and muscle atrophy。patients generally 6-12 year old lose the ability to walk, about 20 years old died of heart and lung failure. caused by mutations in the dystrophin gene.The onset of DMD is caused by mutations in the Dmd gene resulting in loss of dystrophin protein. Dystrophin has a structural role as a cytoskeletal stabilization protein and protects cells against contraction-induced damage.Dystrophin also serves a signaling role through mechanotransduction of forces and localization of signal conduct proteins involved in intracellular signal transduction. muscle fibers without dystrophin protein are more sensitive to mechanical and chemical damage, caused the intracellular signaling pathway disorder at the same time, and a variety of mechanisms, such as the intracellular calcium overload, oxidative stress, inflammatory reaction,autophagy, apoptosis, lead to muscle tissue in a large number of inflammatory cell infiltration, necrosis of muscle fibers, eventually by fatty and fibrous connective tissue instead of.There is no known cure for DMD, and although the culpable gene has been identified for more than twenty years, research on treatments has produced few clinically relevant results. Although gene therapy for DMD patients provides hope, but Applied in clinical and to achieve the purpose of healing there are many intractable obstacles.After long term research and exploration, for DMD pathophysiological mechanisms have good understanding. Therefore, We can slow down the disease progression and improve clinical symptoms in patients with DMD by interfering with the cellular and molecular mechanisms that promote the development of muscular dystrophy.Vascular endothelial growth factor(VEGF) are major regulators of physiological and pathological angiogenesis, is implicated in the regulation of angiogenesis, muscle regeneration and improve perfusion. Studies have confirmed that in muscle damage model, VEGF can reduce muscle injury,promote muscle regeneration. In recent years, vascular targeted treatment,such as angiotensin converting enzyme inhibitor(ACEI) for improving cardiac function and vasodilatory capacity in DMD patients, clinical trial has proved its effectiveness. So whether VEGF can slow the progression of the disease in DMD patients, improve the clinical symptoms, it is worthy of further research.The mdx mice is a classic animal model for DMD research, formed from spontaneous point mutation of DMD gene in C57BL/10 mouse. In this experiment, We have injected AAV9 carrying VEGF gene into mdx mice,which induced VEGF overexpression. To further investigate whether the VEGF can improve the muscle pathology and muscle function in mdx mice.Methods: Our previous experiment observed pathological changes of mdx mice muscle is the most serious at 8W.In this experiment we first further evaluate quantitatively muscle damage of 8W mdx mice, provide reference data and an objective and feasible evaluation program for subsequent experiments.1 Observe the serological, pathological and muscle force changes of 8week old mdx mice.8w male C57BL/10ScSn-Dmdmdx/ JNju mice(for short mdx)were used as the experimental animals and C57BL/10 ScSn mice,served as control group(separately named mdx group,wild-type group).Each group included ten mice,5 for the forelimb grip detection and serum specimen achievement; 5 for hindlimb muscle test and conventional histochemical staining.selecting 5 to administrate grip strength test first,then remove the eye to achieve blood after anesthesia with 10% chloral hydrate(350mg / kg body weight) via intraperitoneal injection, serum samples were collected for serum CK value determination;another 5 for tibialis anterior muscle in situ strength test after anesthesia via intraperitoneal injection of 10% chloral hydrate(350mg / kg body weight). Then,tibialis anterior muscle, gastrocnemiusmuscle and diaphragm was extracted immediately(Weight of the anterior tibial muscle)and frozen in liquid nitrogen or for the embedding frozen processing. Muscle were done for HE staining to observe skeletal muscle pathologic changes and quantitatively evaluate regeneration and necrosis of muscle fibers.2 Observe the effects of AAV9 mediated VEGF gene overexpression on muscle tissue pathology and muscle force in mdx mice2.1 Firstly we should verify that AAV9 carrying gene can express in mice under the conditions of the laboratory.Selected three pairs of 6W littermate male wild-type mice. 3 not given any treatment and served as blank control. 3given AAV9-GFP(150ul/ mice 1×1012 vp/mL) via tail vein injection, three weeks after a single injection, observed the expression of green fluorescent protein(GFP) in muscle tissue under the confocal laser scanning microscope.2.2 6w male wild-type mice served as control group, 6W male mdx mice were selected and divided into two groups: mdx group(not given any treatment); AAV9-VEGF group(given AAV9-VEGF 150ul/ mice, tail vein injection).each group included 5 mice. Animals were analyzed 4 weeks after Single administration. Evaluation methods was the same with mentioned above. SPSS13.0 system for data analysis.Results:1 Serological, pathological and muscle force changes in 8 week old mdx mice.1.1 Serum CK level:The serum CK level of mdx mice was significantly higher than that of the wild-type group.1.2 Muscle pathology:In HE staining, the percentage of muscle fibers with centralized nuclei was significantly increased compared with the wild-type group in anterior tibial muscle,gastrocnemius and diaphragm.The minimal ‘Feret’s diameter’ variance coefficients of the muscle fiber size determined in anterior tibial muscle and diaphragm muscles of mdx was significantly greater than that in wild-type group.The percentage of inflammatory necrosis area in the anterior tibial muscle, gastrocnemius muscleand diaphragm of mdx mice was significantly higher than that in wild-type group.1.3 Muscle force:In 5 repeated grip strength test,the result of each round in mdx mice was lower than the wild-type group; Compared to the first measurements of grip strength,the 5th measurements decreased by about 56%,significantly higher than that of the wild-type group.In situ analysis of tibialis anterior muscle contractile function, At different stimulation frequencies maximum isometric force of mdx mice were all less than wild-type group;compared to initial maximum isometric contraction, the maximum isometric force after 9 consecutive eccentric contraction decreased by about80%,significantly higher than the wild-type group.2 AAV9 mediated VEGF gene overexpression improves muscle pathology and muscle function in mdx mice.2.1 The expression of green fluorescent protein in muscle tissue of C57/10 mice injecting AAV9-GFP was observed. this verifies that AAV9 could be successfully transfected into muscle cells and express the gene.2.2 Western blot detection:compared with the wild-type group and mdx group, the VEGF content in the muscle tissue of AAV9-VEGF group is higher.2.3 Quantitative comparison of muscle damage in AAV9-VEGF group,mdx group and wt group.2.3.1 The level of serum CK:The serum CK level of AAV9-VEGF(46249.5U/L) group was lower than mdx(73418U/L) group, higher than wt(2746.5U/L) group, there is significant difference statistically.2.3.2 Muscle pathology:The percentage of inflammatory necrotic area in AAV9-VEGF group is 0.992±0.22%, lower than the mdx group, higher than the wt group. There is significant difference statistically; The percentage of regenerated muscle fibers is 27.97±3.3% in AAV9-VEGF group, higher than mdx group and wt group, there is no significant difference statistically between the AAV9-VEGF group and mdx group; there is significant difference statistically between the AAV9-VEGF group and wt group.2.3.3 Muscle force:In 5 repeated grip strength tests,the result of eachround in AAV9-VEGF group was lower than the wt group,higher than the mdx group.There is significant difference statistically.Compared to the first measurements of grip strength,the 5th measurements in AAV9-VEGF group decrease by about 57%, significantly higher than that of the wt group,lower than that of the mdx group.There is significant difference statistically.Conclusion:VEGF can improve the muscle tissue damage of mdx mice to a certain extent, and provide a new direction for the treatment of DMD.How to make the best effect of VEGF still need to be further explored.