本文選題：蛋白質(zhì)磷酸酶-1(PP-1)　切入點：成骨細(xì)胞　出處：《第四軍醫(yī)大學(xué)》2011年碩士論文　論文類型：學(xué)位論文

【摘要】：蛋白質(zhì)磷酸化或去磷酸化調(diào)節(jié)是力學(xué)信號從細(xì)胞外傳向細(xì)胞內(nèi)并導(dǎo)致特定生物學(xué)效應(yīng)的關(guān)鍵調(diào)節(jié)機(jī)制。成骨細(xì)胞作為維持骨組織應(yīng)力改建平衡的主體細(xì)胞,既是應(yīng)力刺激的效應(yīng)細(xì)胞,又是機(jī)械生化信號傳遞的傳感器。研究發(fā)現(xiàn),機(jī)械應(yīng)力能夠激活成骨細(xì)胞NF-κB信號轉(zhuǎn)導(dǎo)通路,進(jìn)而完成對成骨細(xì)胞基因表達(dá)的調(diào)控。目前,有關(guān)蛋白質(zhì)激酶在NF-κB力學(xué)信號通路中對IKK復(fù)合體的磷酸化作用的已經(jīng)得到一定程度的闡述,但與此相比,有關(guān)蛋白質(zhì)磷酸酶在細(xì)胞NF-κB力學(xué)信號轉(zhuǎn)導(dǎo)通路中,對IKK復(fù)合體去磷酸化調(diào)控機(jī)制并不清楚。 目的: 本研究通過構(gòu)建成骨細(xì)胞牽張應(yīng)力模型,利用基因轉(zhuǎn)染技術(shù),建立穩(wěn)定高表達(dá)蛋白質(zhì)磷酸酶-1(protein phasphatase-1, PP-1)的MC3T3-E1成骨樣細(xì)胞系,觀察過表達(dá)的PP-1在小鼠成骨樣細(xì)胞MC3T3-E1力學(xué)信號轉(zhuǎn)導(dǎo)NF-κB通路中對IKK磷酸化水平的影響。 方法: 實驗一:首先根據(jù)GenBank中已知的小鼠PP-1基因序列(NM_031868),設(shè)計并合成引物,采用RT-PCR技術(shù)從MC3T3-E1細(xì)胞中擴(kuò)增蛋白質(zhì)磷酸酶-1(PP-1)基因,并將獲得的基因定向插入pCI-neo真核表達(dá)載體中,利用PCR、雙酶切以及測序鑒定。 在轉(zhuǎn)染前24h,取對數(shù)生長期細(xì)胞接種于6孔板中。當(dāng)細(xì)胞生長成70%匯合時,用脂質(zhì)體將表達(dá)載體轉(zhuǎn)染入MC3T3-E1細(xì)胞,,以未轉(zhuǎn)染細(xì)胞為對照組,轉(zhuǎn)染后48h,向培養(yǎng)基中加入G418加壓有限稀釋篩選以建立穩(wěn)定高表達(dá)PP-1的MC3T3-E1成骨樣細(xì)胞系,采用Western blot法檢測PP-1的蛋白表達(dá)情況。 實驗二:通過多通道細(xì)胞牽張應(yīng)力加載系統(tǒng),對穩(wěn)定高表達(dá)PP-1蛋白的MC3T3-E1小鼠成骨樣細(xì)胞株施以8%形變率的周期性牽張力,作用5、15、30和60分鐘后,分別將細(xì)胞裂解,利用Western blot分析,以轉(zhuǎn)染pCI-neo空載體組為對照,分別用抗pIKKα-Ser180及pIKKβ-Ser181的特異化磷酸化抗體檢測IKK磷酸化水平。 結(jié)果: 一、將獲得的重組載體質(zhì)粒進(jìn)行PCR、雙酶切及測序鑒定,結(jié)果顯示,陽性克隆擴(kuò)增出特異性目的基因大小的條帶;將提取的質(zhì)粒雙酶切,電泳結(jié)果表明,可切出符合載體大小的條帶及符合插入片段大小的特異條帶,另外,經(jīng)寶生物工程有限公司測序鑒定與目的序列完全一致,表明表達(dá)載體pCI-neo-PP-1構(gòu)建正確。 Western blot檢測結(jié)果顯示,在轉(zhuǎn)染組出現(xiàn)了明顯的特異性條帶,目的蛋白表達(dá)水平顯著高于未轉(zhuǎn)染成骨細(xì)胞對照組,PP-1蛋白能在轉(zhuǎn)染pCI-neo-PP-1的MC3T3-E1細(xì)胞中穩(wěn)定高表達(dá)。 二、運用多通道細(xì)胞牽張應(yīng)力加載系統(tǒng)對MC3T3-E1成骨樣細(xì)胞株施以8%形變率的周期性牽張力后,發(fā)現(xiàn)無論哪個時間點,與轉(zhuǎn)染空載體pCI-neo對照相比,過表達(dá)的PP-1均能明顯減少IKK的磷酸化水平。 結(jié)論: 本課題成功構(gòu)建了pCI-neo-PP-1真核表達(dá)載體,并進(jìn)一步建立了穩(wěn)定高表達(dá)PP-1蛋白的MC3T3-E1成骨樣細(xì)胞系;另外,在周期性牽張力作用下誘導(dǎo)的成骨細(xì)胞NF-κB通路中,過表達(dá)的PP-1基因能夠抑制牽張應(yīng)力誘導(dǎo)的IKK的磷酸化水平,為進(jìn)一步深入研究PP-1在細(xì)胞力學(xué)信號轉(zhuǎn)導(dǎo)通路中的功能及作用機(jī)制奠定了實驗基礎(chǔ)。
[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.

【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號】：R346

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