Effects Of AAV9 Mediated HIGF-1 Gene Overexpression On Mdx

Objective: Duchenne muscular dystrophin?DMD?is one of the most severe myodystrophy diseases.It is an X-linked recessive lethal muscle disease,in the survival of male incidence rate is about 1 / 3500,characterized by progressive limb weakness and muscle atrophy.The gene encoding dystrophin protein which is a very important part of the cytomembrane has a mutation,which leads to the cytomembrane incompleteness.The myocytes are fragile to any mechanical stimulation,followed by necrosis and inflammation.For the muscle tissues,the ability of regeneration can't cover the shortage of losing,so the fibrous connective tissues and adipose tissues substitute the original ones.The DMD patients always die of breathing and heart failure when they are at their twenties.Date to now,there is no known effective methods of treating.Like humans with DMD,mdx mice lack dystrophin due to an X-linked mutation providing an accepted model to study the human disease.The mice were first discovered for the high CK levels,and presenting similar muscular pathology with human patients.At present,utilizing vectors to deliver the normal dystrophin gene into the organism or to normalize the mutation has become hot,but what the obstacle in front of us is that the dystrophin gene is too large.In recent years,many people show their interests on the various symptoms of muscular dystrophy.They take various growth factors,through different signal pathways to promote the regeneration of muscle cells,thereby improving myopathy status.Insulin like growth factor-1?IGF-1?has become an important alternative direction in the treatment of DMD.IGF-1 is also called the �growth factor�?somatomedin C?,which is a polypeptide protein similar to insulin in the structure.It plays an important role in the growth of infants and the adults ' metabolism.It's the product of many autocrine and paracrine cells such as liver cells,renal cells,spleen cells.With the hypoglycemic,hypolipidemic and vasodilator effects and so on,it's also important in human cell mitogen,and it's essential to maintain cell differentiation relat ed protein level.When combined with some growth factors,it can promote cell differentiation and maturation.At present the research on mdx mice in IGF-1,mainly for two aspects: one is the observation of mdx/m IGF-1 transgenic mice,the other is a study on the effect of IGF-1 protein on mdx mice.Although the two methods are both effective,the first is not suitable for the clinical treatment,the second has more side effects and will take much more time and make work hard.In this experiment,we use AAV9 as the vector to deliver hIGF-1 gene.First,we injected into the tibialis anterior muscle locally to observe the effect of this method.Second we intravenously injected the mice to observe whether it can play a role in the whole body's skeletal muscle.Methods: our experiment is divided into two stages: the first is the method of local injection in which we injected the virus directly into the right tibialis anterior muscles of the mdx mice.Then we observed after a certain period of time.Followed by intravenous injection,and we observed after a certain period of time.1 the method of local injectionEight male mdx mice with similar weight were divided into two groups when they were at their age of 4 weeks.One group was injected into two points of the right tibialis anterior muscle with AAV9-GFP(20?l/ 1�10¹² vg/ml).The volume injected into each point was 10 ?l.So were the other group injected with AAV9-hIGF-1(20?l/ 1�10¹² vg/ml).In addition,four 4 weeks old male wild type mice with the C57BL/10 background were chosen as control.8 weeks later?age 12 weeks?,mice were tested strength in the right tibialis anterior muscle after anesthesia.Then they were picked eyeballs for their blood to test the CK levels.Finally we separated the right tibialis anterior muscles completely.We tested the muscles' weight and then embedded them to make frozen sections,so various indicators would be detected.2 the method of intravenous injectionSix male mdx mice with similar weight were divided into two groups when they were at their age of 6 weeks.One group was injected into the tail venous with AAV9-GFP(200?l/ 1x10¹² vg/ml).And the other group was injected with AAV9-hIGF-1(200?l/ 1x10¹² vg/ml).Three 4 weeks old male wild type mice with the C57BL/10 background were chosen as control.6 weeks later?age 12 weeks?,nine mice were harvested with the same method and detected all kinds of indicators as the mice in stage one.Spss13.0 system was used to analyze the data.Results: 1 The local injection of AAV9-hIGF-1improved the muscle pathology and muscle function of mdx mice 1.1 8 weeks after the injection of AAV9-GFP,the expression of green fluorescence protein was observed in the tibialis anterior muscle.The results showed that AAV9 can successfully transfect the muscle cells and express the genes carried by our laboratory.1.2 Immunofluorescence results showed that compared with the normal control group,GFP group,only hIGF-1 group expressed hIGF-1protein in tibialis anterior muscles.1.3 Compare the tibialis anterior muscles weight,hIGF-1group was 80.70�8.00 mg,GFP group was 58.35 � 4.26 mg,C57BL/10 group was 39.93 �0.81 mg.The difference was statistically significant between groups.But we had a question that the weight of GFP group was larger than that of C57BL/10 group.To find out the muscle weight difference between normal mdx mice and C57BL/10 mice,we tested a new group by four mdx mice never given any interaction.And the result 60.20�2.75 mg showed no difference between GFP group.1.4 The comparison of the degree of muscle damage quantitative between each group 1.4.1 Serum CK level: The serum CK level of wild type mice was 5076.67 �3959.18U/L,and the CK value of AAV9-GFP mice was 26345.64�5762.39 U/L,and the CK value of AAV9-hIGF-1 mdx mice was 39446.28�15905.25 U/L.The CK value of mdx mice was higher than that of normal wild type mice,but there was no significant difference between GFP group and hIGF-1 group;1.4.2 Muscle pathology: In HE staining,the necrosis area proportion of tibialis anterior muscle in GFP group was 6.33�2.51%,larger than that in group hIGF-1,and the proportion of the tibialis anterior muscle necrosis area in group hIGF-1 was 2.14�0.43%,the difference was statistically significant;but there was no significant difference between the two groups in the number of regenerated fibers;1.4.3 myodynamia: The muscle strength of the tibialis anterior muscle in group hIGF-1 was the largest,followed by GFP group,and that of the wild-type mice was the lowest,while that endurance test results of the tibialis anterior muscle was the opposite.2 the intravenous injection of AAV9-hIGF-1improved the muscle pathology and muscle function of mdx mice 2.1 6 weeks after the injection of AAV9-GFP,the expression of green fluorescence protein was the best in the tibialis anterior muscle,while only a little in other muscles;2.2 Immunofluorescence results showed that compared with the normal control group,GFP group,only hIGF-1 group expressed hIGF-1protein in tibialis anterior muscles.2.3 Compare the tibialis anterior muscles weight,hIGF-1group was 75.03�4.54 mg,GFP group was 55.63�13.34 mg,C57BL/10 group was 40.07�0.93 mg.The difference was statistically significant between groups.2.4 The comparison of the degree of muscle damage quantitative between each group 2.4.1 Serum CK level: The CK value of AAV9-GFP mice was 33418.57�11695.54 U/L,and the CK value of AAV9-hIGF-1 mdx mice was 31855.8�16394.13 U/L.The CK value of mdx mice was higher than that of normal wild type mice,but there was no significant difference between GFP group and hIGF-1 group;2.4.2 Muscle pathology: In HE staining,the necrosis area proportion of tibialis anterior muscle in GFP group was 6.34�2.51%,larger than that in group hIGF-1,and the proportion of the tibialis anterior muscle necrosis area in group hIGF-1 was 2.46�0.54%,the difference was statistically significant;but there was no significant difference between the two groups in the number of regenerated fibers;2.4.3 myodynamia: The muscle strength of the tibialis anterior muscle in group hIGF-1 was the largest,followed by GFP group,and that of the wild-type mice was the lowest;while the endurance test results showed that the normal control mice had the best endurance,followed by the mice in hIGF-1group.The mice in GFP group showed the worst endurance.Conclusions:1 AAV9 carrying hIGF-1 gene could be successfully expressed in mouse skeletal muscle;2.In vivo,the expression of hIGF-1 could promote the proliferation of skeletal muscle cells and decrease the necrosis in mdx mice;3 The expression of hIGF-1 could promote the recovery of skeleta l muscle function in mdx mice;4 The role AAV9 carrying gene hIGF-1 in mdx mice plays,and the long-term expression of side effects,still need further exploration.