PP-1在成骨细胞力学信号转导NF-κB通路中对IKK磷酸化水平的影响

发布时间：2018-03-15 12:56

本文选题：蛋白质磷酸酶-1(PP-1)　切入点：成骨细胞　出处：《第四军医大学》2011年硕士论文　论文类型：学位论文

【摘要】：蛋白质磷酸化或去磷酸化调节是力学信号从细胞外传向细胞内并导致特定生物学效应的关键调节机制。成骨细胞作为维持骨组织应力改建平衡的主体细胞,既是应力刺激的效应细胞,又是机械生化信号传递的传感器。研究发现,机械应力能够激活成骨细胞NF-κB信号转导通路,进而完成对成骨细胞基因表达的调控。目前,有关蛋白质激酶在NF-κB力学信号通路中对IKK复合体的磷酸化作用的已经得到一定程度的阐述,但与此相比,有关蛋白质磷酸酶在细胞NF-κB力学信号转导通路中,对IKK复合体去磷酸化调控机制并不清楚。目的: 本研究通过构建成骨细胞牵张应力模型,利用基因转染技术,建立稳定高表达蛋白质磷酸酶-1(protein phasphatase-1, PP-1)的MC3T3-E1成骨样细胞系,观察过表达的PP-1在小鼠成骨样细胞MC3T3-E1力学信号转导NF-κB通路中对IKK磷酸化水平的影响。方法: 实验一:首先根据GenBank中已知的小鼠PP-1基因序列(NM_031868),设计并合成引物,采用RT-PCR技术从MC3T3-E1细胞中扩增蛋白质磷酸酶-1(PP-1)基因,并将获得的基因定向插入pCI-neo真核表达载体中,利用PCR、双酶切以及测序鉴定。在转染前24h,取对数生长期细胞接种于6孔板中。当细胞生长成70%汇合时,用脂质体将表达载体转染入MC3T3-E1细胞,,以未转染细胞为对照组,转染后48h,向培养基中加入G418加压有限稀释筛选以建立稳定高表达PP-1的MC3T3-E1成骨样细胞系,采用Western blot法检测PP-1的蛋白表达情况。实验二:通过多通道细胞牵张应力加载系统,对稳定高表达PP-1蛋白的MC3T3-E1小鼠成骨样细胞株施以8%形变率的周期性牵张力,作用5、15、30和60分钟后,分别将细胞裂解,利用Western blot分析,以转染pCI-neo空载体组为对照,分别用抗pIKKα-Ser180及pIKKβ-Ser181的特异化磷酸化抗体检测IKK磷酸化水平。结果: 一、将获得的重组载体质粒进行PCR、双酶切及测序鉴定,结果显示,阳性克隆扩增出特异性目的基因大小的条带;将提取的质粒双酶切,电泳结果表明,可切出符合载体大小的条带及符合插入片段大小的特异条带,另外,经宝生物工程有限公司测序鉴定与目的序列完全一致,表明表达载体pCI-neo-PP-1构建正确。 Western blot检测结果显示,在转染组出现了明显的特异性条带,目的蛋白表达水平显著高于未转染成骨细胞对照组,PP-1蛋白能在转染pCI-neo-PP-1的MC3T3-E1细胞中稳定高表达。二、运用多通道细胞牵张应力加载系统对MC3T3-E1成骨样细胞株施以8%形变率的周期性牵张力后,发现无论哪个时间点,与转染空载体pCI-neo对照相比,过表达的PP-1均能明显减少IKK的磷酸化水平。结论: 本课题成功构建了pCI-neo-PP-1真核表达载体,并进一步建立了稳定高表达PP-1蛋白的MC3T3-E1成骨样细胞系;另外,在周期性牵张力作用下诱导的成骨细胞NF-κB通路中,过表达的PP-1基因能够抑制牵张应力诱导的IKK的磷酸化水平,为进一步深入研究PP-1在细胞力学信号转导通路中的功能及作用机制奠定了实验基础。
[Abstract]:Protein phosphorylation or dephosphorylation regulation is the mechanical signal from the cell to the outside cells and result in specific key regulatory mechanism. The biological effect of osteoblasts as maintaining bone tissue stress remodeling balance subject cells and effector cells is stress stimulation, and students of mechanical sensor signal transmission. The study found that mechanical stress can activate osteoblasts NF- B signal transduction pathway, and then complete the regulation of osteoblast gene expression. At present, the protein kinase in NF- kappa B signaling pathway on mechanical IKK complex phosphorylation has been a certain degree of elaboration, but compared with this, the protein phosphatase in cells NF- kappa B mechanical signal transduction pathway, the IKK complex dephosphorylation regulation mechanism is not clear.
Objective:
This study builds into stretch model of bone cells, using gene transfection technique, establish the stable expression of protein phosphatase -1 (protein phasphatase-1 PP-1) MC3T3-E1 osteoblast like cells, observed the expression of PP-1 on phosphorylation of IKK osteoblast like cells MC3T3-E1 signal transduction pathway in NF- kappa B mice.
Method:
Experiment one: first according to the mouse PP-1 gene sequence in GenBank (NM_031868), the primers were designed from MC3T3-E1 cells by RT-PCR -1 amplification of protein phosphatase (PP-1) gene, PCR gene and the use of directional will be inserted into the eukaryotic expression vector pCI-neo, and double enzyme digestion and DNA sequencing.
Before transfection of 24h cells in the logarithmic growth phase were seeded in 6 well plates. When the cells grow into 70% confluence, the expression vector was transfected into MC3T3-E1 cells by liposome, untransfected cells as the control group, 48h after transfection, G418 added to the medium pressure limited dilution to establish stable high expression screening PP-1 MC3T3-E1 osteoblast like cells by Western blot assay, expression of PP-1 protein.
Experiment two: stretch stress loading system by multi channel cell, on the stability of the high expression of PP-1 protein in MC3T3-E1 mouse osteoblast like cell lines with 8% deformation rate of cyclic tension force, the role of 5,15,30 and 60 minutes respectively, cell lysis, Western analysis using blot, pCI-neo transfected by empty vector group, respectively. For the detection of phosphorylated IKK phosphate specific antibody against pIKK alpha -Ser180 and pIKK beta -Ser181 level.
Result:
First, the recombinant plasmids obtained by PCR, double enzyme digestion and sequencing, the results showed that the positive clone amplified specific band gene size; the extracted plasmid was digested by electrophoresis results showed that can cut out in line with the size of carrier tape and meet specific insertion segment size. In addition, the zone, Bao biological Engineering Co. Ltd. sequencing and the target sequence is completely consistent, showed that the expression vector pCI-neo-PP-1 was constructed correctly.
Western blot detection showed that there was a significant specific band in the transfection group, and the target protein expression level was significantly higher than that in the control group without transfection. PP-1 protein could be highly expressed in pCI-neo-PP-1 transfected MC3T3-E1 cells.
Two, using a multi-channel cell tensile stress loading system, a 8% strain rate cyclic tension force was applied to MC3T3-E1 osteoblast like cell line. It was found that at any time point, the over expression of PP-1 could significantly reduce the phosphorylation level of IKK compared with the empty vector pCI-neo.
Conclusion:
This study successfully constructed pCI-neo-PP-1 eukaryotic expression vector, and further establish a stable and high expression of PP-1 protein in MC3T3-E1 osteoblast like cells; in addition, in under the action of cyclic stretch induced osteoblast NF- kappa B pathway, overexpression of PP-1 can suppress the stretch stress induced phosphorylation of IKK the level, which lays the foundation for further Research on the function and mechanism of PP-1 cells in the mechanical signal transduction pathway.

【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R346

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