Research On The Preparation And Function Of Mechano-growth Factor

Mechano growth factor (MGF), a novel growth factor, is originated from alternative splicing of Igf-1 gene, which is mainly expressed in stretched skeletal muscle, damaged muscle and neuron. Further studies found that MGF and its E peptide (MGF-Ct24E) could promote muscle hypertrophy, repair damage, so as to play a role in treating relevant diseases. However, MGF was not obtained for the studies of the physicochemical property and biological function of MGF so far. Our previous studies concluded that MGF mRNA expressed highly in the cyclic stretched osteoblasts, which suggested that MGF might be a amechano-response molecule in bone and have influence on the function of osteoblasts. And in this study, we developed the technique of MGF and MGF-Ct24E preparation, and studied their effects on the function of osteoblasts.Objective: To construct efficiently expression systems for MGF and MGF-Ct24E producing on pilot scale. To establish optimal techniques to purify protein samples meeting the experimental needs. To prepare the specific anti-MGF polyclonal antibody for the analysis of MGF expression in the stretched osteoblasts. To construct recombined adenovirus containing Mgf or Igf-1 gene. To study the effects of MGF and MGF-Ct24E on the proliferation, migration and differentiation of osteoblasts, and to analyze its relevant mechanisms.Methods: Using MGF DNA as a template, the recombinat sequences of truncated MGF(des(1-3)MGF) and MGF-Ct24E carrying restriction endonuclease sites and tag sequences were amplified by PCR. Then, the sequences were inserted into plasmid pMAL-c2x and transform to E.coli BL21, expressing the fusion proteins of MBP/des(1-3)MGF and MBP/MGF-Ct24E by fermentation. These fusion proteins were purified through ion exchange chromatography, metal affinity chromatography, after enterokinase digestion, the MBP was removed by rpHPLC and the purified des(1-3)MGF and MGF-Ct24E samples were obtained. Molecular mass of samples were assayed by mass spectrometry, and isoelectric point was identified by isoelectric focusing. MGF specific antibodies were prepared through immuning animals with MGF-Ct24E as antigen. Western blot was introduced to analyze the MGF expression in cyclic stretched osteoblasts, while immunofluorescence was used to observe the distribution of MGF in cells. Adenovirus transfection system carrying Mgf and Igf-1 genes were constructed to infect MC3T3E1 osteoblast lines. The effects of MGF, MGF-Ct24E and IGF-1 on the proliferation, migration, differentiation of osteoblasts were studied by peptide treatment and gene transfection. With the inhibitors PD98059 and LY294002, the roles of MAPK-Erk1/2 and PI3K-Akt signaling pathways were studied. Cell proliferation and cell cycle were detected by MTT, flow cytometry respectively; cell migration was analyzed by Millicell-PCF cell culture chamber; cell differentiation was evaluated by alkaline phosphates activity, expression of extracellular matrix proteins and calcium deposition.Results:1. A prokaryotic expression system of des(1-3)MGF and MGF-Ct24E was constructed by cloning the recombination sequences into plasmid PMAL-c2x, after transforming E.coli BL21, fusion proteins MBP/des(1-3)MGF and MBP/MGF-Ct24E were successfully expressed by IPTG induction.2. The recombinant proteins were prepared by batch fermentation, after process optimization, the M9-YE medium, feeding liquid containing the natural organic nitrogen, 30% dissolved oxygen, 37°C and 0.2 mM IPTG were determined to produce recombination protein by fermentation. MBP/des(1-3)MGF inclusion body and MBP/MGF-Ct24E soluble protein accounted for 30%, 35% of total bacterial proteins, respectively. The fusion protein with purity above 95% could be obtained by column chromatography, the refolding rate of inclusion body above 80%. Purity of target proteins, des(1-3)MGF and MGF-Ct24E, were above 98% after EK digestation and rpHPLC separation. The molecular mass of these two proteins assayed by mass spectrometry was consistent with the theoretical values.3. Purified antibody was acquired from serum of rabbits immunized with MGF-Ct24E for 4 times, and anti-MGF specificity of which was identified by western blot. At the three time points (6 h, 12 h, 24 h) examined, the MGF level in the stretched osteoblasts groups was respectively 2.7, 5.2 and 1.1 fold higher than that in the control group. The subcellular distribution of MGF protein was revealed by immunofluorescence analysis to be restricted to the nucleus.4. Adenovirus carrying Mgf and Igf-1 genes was constructed and was successfully transfected into osteoblasts MC3T3-E1. These two growth factors were expressed higly.5. The osteoblasts MC3T3-E1 were treated with MGF(des(1-3)MGF), or MGF-Ct24E, or IGF-1. Cell proliferation and migration analysis showed that MGF-Ct24E and MGF had a more significant effect in promoting cell proliferation than that of IGF-1, while all these factors had chemotaxis to osteoblasts, of which MGF was the strongest, MGF-Ct24E the weakest. Proliferation was involved in activation of MAPK-Erk1/2 pathway by MGF-Ct24E and MGF, while migration was partly involved in the activation of MAPK-Erk1/2 and PI3K-Akt, of which PI3K-Akt was dominant.6. MGF-Ct24E and MGF delayed osteoblast differentiation. With 1nM peptide treatment, MGF-Ct24E and MGF decreased ALP activity at the early stage of osteoblast differentiation, while IGF-1 promoted differentiation; at the late stage, only MGF-Ct24E showed inhibition in osteoblast differentiation. In addition, MGF-Ct24E and MGF inhibited the Col1 expression, but increased the OPN expression. The effects of MGF-Ct24E and MGF on delayed osteoblast differentiation was associated with the activation of the Erk1/2. ALP expression was reversed by inhibiting Erk1/2 activation, however, the activation of Akt induced by MGF and IGF-1 promoted cell differentiation. Further study indicated that MGF-Ct24E and MGF inhibited nuclear transport of transcription factor Cbfα-1, which might be one of the reasons why cell differentiation was inhibited.Conclusion: This study successfully constructed E.coli expression system of MGF and MGF-Ct24E, achieved the fermentation expression, and established technology of recombinant protein purification and renaturation, which indicated that MGF could be obtained from prokaryotic expression. The MGF-Ct24E can be used to immune animals so as to obtain MGF specific antibody, western blot confirms that the stretch can stimulate the osteoblasts to express MGF protein. MGF and its peptide E promote the proliferation but delay the differentiation of osteoblasts. This function might be associated with specific activation of Erk1/2 induced by E peptide; while MGF has the capability significantly to activate Akt too. The activated Akt can promot cell migration and act as an important role on differentiation of osteoblasts.