摘要
力生长因子(Mechano growth factor, MGF)是一种新发现的生长因子,由Igf-1基因剪接变异产生。该生长因子在拉伸刺激的骨骼肌或损伤的肌肉、神经等组织中表达被提高。进一步的研究表明:MGF及其E肽(MGF-Ct24E)具有促进肌肉肥大、修复损伤的功能,因而对相关疾病可能具有治疗作用。但迄今为止,该蛋白还没有进行理化性质和生物学功能的直接研究,原因在于尚未获得MGF蛋白样品。我们实验室在以往的研究中发现,MGF mRNA在拉伸刺激的成骨细胞中高表达,提示该因子也可能是骨组织中的力响应分子,并对成骨细胞的功能产生影响。这方面的研究目前还未见其他研究人员的报道。所以本课题首先开展MGF以及MGF-Ct24E的制备,并进一步研究它们对成骨细胞功能的影响。
研究目的:构建适合中试化生产的MGF和MGF-Ct24E大肠杆菌表达系统,建立一套有效的蛋白纯化工艺,制备满足实验需要的蛋白样品。制备MGF特异性多克隆抗体,用于分析拉伸刺激后成骨细胞内MGF蛋白质的表达。构建携带Mgf和Igf-1基因的重组腺病毒。通过生长因子直接作用和基因转染,研究MGF及其E肽对成骨细胞增殖、迁移、分化的影响,并分析其相关机制。
研究方法:以MGF DNA为模版,PCR扩增携带限制性内切酶位点和标签序列的截短型MGF(des(1-3)MGF)和MGF-Ct24E的重组序列,分别插入质粒pMAL-c2x并转化大肠杆菌BL21,发酵表达融合蛋白MBP/des(1-3)MGF和MBP/MGF-Ct24E,通过离子交换层析和金属亲和层析等技术纯化融合蛋白,经肠激酶酶切,rpHPLC去除MBP后,获纯净的des(1-3)MGF和MGF-Ct24E样品。质谱测定样品分子量,等电聚焦测定等电点进行鉴定。MGF-Ct24E作抗原免疫动物,制备MGF特异性抗体,并利用抗体进行western blot分析拉伸刺激下MGF在成骨细胞中的表达变化,免疫荧光细胞技术分析MGF在细胞内的分布特点。构建携带Mgf和Igf-1基因的腺病毒转染系统,感染成骨细胞株MC3T3-E1。通过多肽直接作用和基因转染研究MGF、MGF-Ct24E以及IGF-1对成骨细胞增殖、迁移、分化的影响,结合抑制剂PD98059、LY294002的使用,分析信号通路MAPK-Erk1/2和PI3K-Akt与MGF功能的关联。细胞增殖通过MTT法检测,流式细胞仪分析细胞周期;Millicell-PCF细胞培养小室分析多肽作用下细胞的迁移;碱性磷酸酶活性、胞外基质蛋白表达、钙沉积分析评价细胞分化。
研究结果:
1、将des(1-3)MGF、MGF-Ct24E的重组序列克隆入PMAL-c2x质粒,构成重组质粒pMGF和pMGF24E,重组质粒转化BL21,在IPTG的诱导下成功表达融合蛋白MBP/ des(1-3) MGF和MBP/MGF-Ct24E。
2、补料分批发酵制备重组蛋白,工艺优化后确定采用M9-YE培养基,含天然有机氮源的补料液,37°C,30%溶氧发酵,0.2mM IPTG诱导蛋白表达。MBP/ des(1-3)MGF包含体占细菌总蛋白30%,MBP/MGF-Ct24E可溶蛋白占细菌总蛋白的35%。柱层析法可以获得纯度95%以上的融合蛋白,包含体复性率80%以上。融合蛋白经EK酶切,rpHPLC分离获得纯度达98%以上的目的蛋白des(1-3)MGF和MGF-Ct24E,质谱鉴定二者的分子量与理论值相符。
3、MGF-Ct24E免疫4次获得含特异性抗体的兔血清,经纯化后的抗体,western blot鉴定具有抗MGF特异性。Western blot分析显示拉伸刺激成骨细胞6、12、24h,MGF的表达量分别是对照组的2.7、5.2、1.1倍。免疫荧光检测发现MGF具有核分布特性。
4、构建了携带Mgf和Igf-1基因的重组腺病毒,成功感染成骨细胞MC3T3-E1,实现两种生长因子的高水平表达。
5、MGF (des(1-3)MGF)、MGF-Ct24E、IGF-1三种多肽作用成骨细胞MC3T3-E1。分析细胞增殖和迁移发现,MGF-Ct24E和MGF具有比IGF-1更显著的促进细胞增殖的作用。三种生长因子都对成骨细胞有趋化作用,其中MGF最强,而MGF-Ct24E最弱。MGF-Ct24E和MGF的促增殖作用与其显著激活MAPK-Erk1/2途径有关,促迁移能力部分的与MAPK-Erk1/2和PI3K-Akt活化有关,其中PI3K-Akt处于主要地位。
6、细胞分化检测发现,MGF-Ct24E和MGF具有延迟成骨细胞分化的作用。1nM的多肽处理,MGF-Ct24E和MGF在细胞分化早期降低了ALP活性,而IGF-1表现出促进作用,在分化晚期MGF-Ct24E对细胞的矿化也表现出抑制作用,而MGF没有这种效应。另外MGF-Ct24E和MGF抑制了Col1的表达,但提高了OPN的表达。MGF-Ct24E和MGF对成骨细胞分化的延迟效应与Erk1/2的激活有关,抑制Erk1/2活化,ALP的表达被逆转,而MGF和IGF-1激活Akt促进了细胞分化。进一步研究发现MGF-Ct24E和MGF对转录因子Cbfα-1的核转运有抑制作用,这可能是细胞分化受到抑制的原因之一。
结论:本研究成功构建了MGF和MGF-Ct24E的大肠杆菌表达系统,并实现了发酵表达,建立了一套完整的重组蛋白纯化和复性工艺,证明MGF可以通过原核表达的方式获得。获得的MGF-Ct24E可以免疫动物获得抗MGF特异性抗体,利用该抗体通过western blot分析,在蛋白质水平证实拉伸刺激能够刺激成骨细胞表达MGF。通过细胞实验发现,MGF及其E肽具有显著的促进成骨细胞增殖的作用,但对分化表现出一定的延迟,这些功能与E肽显著激活MAPK-Erk1/2有关,而MGF还具有显著激活Akt的能力,Akt活化促进了细胞迁移并且是成骨细胞分化所必须的。
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.
引文
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