| Objective:AMF gene was transfected into primary cultured rat myoblasts withconstructed lentivirus vector, and the myoblasts were transplanted into ratgastrocnemius denervation and reinnervation model, with the intention ofassessing if AMF, as a malignant tumor-related cytokine, could improvemotor plate regeneration and neuromuscular function restoration byautologous myoblast-mediated gene transfer; and also provide experimentalevidence for AMF application research as well as neuromuscular functionalrecovery after direct nerve implantation.Methods:1. Primary cultured rat myoblasts were amplified after verification andpurification.2. Constructed FIV lentivirus vector transduct AMF and eGFP geneinto primary cultured rat myoblast, and verify if the myoblasts can express bioactive AMF.3. Establish rat gastrocnemius denervation and reinnervation model,and devided into three groups: Group A (primary cultured myoblast),Group B (myoblasts with AMF and eGFP gene), Group C (myoblastswith eGFP gene). After direct nerve implantation, at day0,7, autologousmyoblasts was transplanted into gastrocnemius of rat in each group.4. After periods of8,16, and24weeks from the last myoblaststransplantation, gastrocnemius frozen sections were made for observationof transplanted myoblasts survival. In the same time, The nerve conductionvelocity (NCV) of the tibial nerve, peak-to-peak value (PPV), area underthe curve (AUC) of the compound muscle action potentials (CMAP) andthe Tibial Functional Index (TFI) were measured. The regeneratedendplates in gastrocnemius were examined by histochemical staining andthe morphological changes of gastrocnemius were detected by HE staining.Results:1. After periods of21days' primary culture and purification,5.6×106myoblast with98.0%purity can be obtained from1cm3muscle;2. Myoblast was successfully transfected with constructed FIVlentivirus vector and the best multiplicyties of infection(MOI) was100.NIH-3T3fibroblast cell motility was significantly enhanced by addingAMF to the culture medium (AMF ONLY,159.0±23.5/field, P<0.05whencompared to CONTROL,94.6±36.1/field), indicates that transfected myoblast can express bioactive AMF.3. Rat gastrocnemius denervation and reinnervation model wassuccessfully established and transplanted myoblast survival can beobserved in gastrocnemius at8,16, and24weeks afer transplantation.4. After periods of8,16, and24weeks from the last myoblaststransplantation, TFI(group A:-69.3±11.8,-46.5±8.0,-33.6±9.1；groupB:-57.5±9.1,-36.8±7.1,-25.2±6.4；group C:-67.7±8.2,-49.7±9.2,-34.2±8.7),PPV(group A:8.5±2.3mV,29.5±7.4%,12.4±2.4mV,40.4±7.1%,18.6±2.9mV,62.5±9.3%；group B:11.8±1.9mV,39.0±5.2%,15.0±1.6mV,39.0±5.2%,22.1±1.5mV,39.0±5.2%；group C:8.7±1.5mV,30.0±5.4%,11.5±1.8mV,39.2±6.2%,18.1±1.7mV,60.9±5.4%),AUC(group A:10.5±2.1mV*ms,33.0±5.9%,13.7±2.5mV*ms,41.2±6.8%,19.0±3.4mV*ms,60.5±8.6%；group B:13.6±2.1mV*ms,40.9±7.3%,16..8±3.1mV*ms,50.7±9.3%,23.0±1.7mV*ms,72.3±6.7%；group C:9.6±2.6mV*ms,31.3±8.1%,13.7±2.9mV*ms,42.4±9.9%,18.2±2.1mV*ms,59.4±6.7%)values and endplates examination(groupA:90.1±15.1/section；group B:120.7±18.6/section；group C:86±16.4/section) demonstrated that group B is significantly higher than groupA and group C(P<0.01), indicated that autologous myoblast-mediatedAMF gene transfer can significantly improve the neuromuscular functionalrecovery; and eGFP showed no influence towards myoblast nor theexperiment results (P>0.05). NCV values may not be reliable in this study(P<0.05and P>0.05). After comparing the histochemical-stainedtissues, no morphological changes and indications of tumorigenesis weredetected in gastrocnemius.Conclusions:Autologous myoblast-mediated AMF gene transfer can significantlyimprove the neuromuscular functional recovery and endplate regenerationin rat gastrocnemius denervation and reinnervation model, with no sideeffect observed. AMF had positive effects on neuromuscular reparation,may serve as an important factor in peripheral nerve restorationmicroenvironment and is likely to be used in clinical therapeutics in thefuture.
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