PTP-oc抑制剂—熊果酸对破骨细胞分化及正畸牙根吸收的影响

发布时间：2018-05-23 14:16

本文选题：破骨细胞 + 蛋白质酪氨酸磷酸酶　；参考：《吉林大学》2015年博士论文

【摘要】：正畸治疗过程中，正畸力诱导牙周组织和牙槽骨改建，从而使牙齿发生移动。但是，机械压力也会诱发牙齿发生不同程度的牙根吸收，轻度牙根吸收存在自愈的可能，但中重度牙根吸收往往会出现牙根尖部不可复性变短、冠根比例不调、甚至发生牙齿松动的现象，使正畸治疗趋向复杂化。牙根吸收的机制与骨吸收相类似，牙根吸收由与破骨细胞相似的破牙骨质细胞承担主要吸收牙骨质的功能，其中众多信号因子构成复杂的网络调控系统。目前，临床上对正畸力引起的牙根吸收尚无有效的预防措施，而如何减少牙根吸收是每一位正畸医生期待解决的问题。蛋白质酪氨酸磷酸酶(Protein Tyrosine Phosphatases，PTPs)是细胞信号传导过程中的关键酶，与人类生理和病理过程密切相关。破骨细胞分化、粘附及活性的调节是十分复杂的，其信号传导过程大部分都与蛋白质酪氨酸磷酸化密切相关。破骨细胞分化过程中，周围组织细胞中的PTPs参与调控其信号传导途径中的靶蛋白发生磷酸化作用。PTP-oc是一种特异性表达于破骨前体细胞、破骨细胞的蛋白质酪氨酸磷酸酶，对破骨细胞具有正调节作用。因此，我们认为特异性的抑制PTP-oc的活性，进而抑制破骨细胞的生成和分化，最终可以在一定程度上预防或减少正畸致牙根吸收。为了验证这一假设，我们设计了如下实验： 1. PTP-oc蛋白催化结构域的克隆、表达：以含有编码PTP-oc催化结构域(ΔPTP-oc)cDNA的质粒pMD18-T-ΔPTP-oc为模板，通过PCR扩增ΔPTP-oc，然后将其克隆到pET-28a(+)载体中，并将重组质粒转入大肠杆菌BL21(DE3)中，加入0.1mM IPTG16℃诱导24h。 2. PTP-oc蛋白催化结构域的分离纯化及酶学表征：菌体经超声破碎后，利用Ni-NTA agarose亲和层析方法成功的获得了可溶的ΔPTP-oc重组蛋白，纯化后的ΔPTP-oc的纯度达80%以上，且具有相当高的特异性。ΔPTP-oc二级结构预测与圆二色谱分析结果基本一致，证明重组ΔPTP-oc具有正确的空间结构。酶促反应动力学研究表明，当底物为p-NPP时，ΔPTP-oc符合米氏酶的性质，其Km=801.8μmol，Vmax=6.1μmol/min。酶学性质研究表明，ΔPTP-oc的最适反应温度为34℃，最适离子强度为0，这与其他常见PTPs相似，但独特的是ΔPTP-oc最适pH为7.0，这与其他常见PTPs不同。 3. PTP-oc抑制剂的筛选：采用比色法，从69种单体化合物中筛选ΔPTP-oc的抑制剂，在每种单体化合物终浓度为100μmol/L的情况下，抑制效果达到90%以上的单体化合物有四种，其中熊果酸对ΔPTP-oc酶的抑制效果最好，达到了98.3%，测定其对ΔPTP-oc的IC50为6.381±0.56μmol/L。通过双倒数作图法研究发现熊果酸对ΔPTP-oc的抑制类型为竞争性抑制，抑制常数Ki为7.9μmol/L。此外，熊果酸对ΔPTP-oc抑制效果具有很好的专一性。因此，我们选取熊果酸作为进一步的研究对象，研究其对破骨细胞的相关影响。 4.熊果酸对U937细胞诱导的破骨样细胞分化及吸收活性的影响选用U937细胞，使用1×10-7M/L的TPA和1×10-8M/L的1,25(OH)2D3两种诱导剂联合诱导，成功将U937细胞诱导为破骨样细胞。CCK-8实验结果表明，熊果酸用量小于5μmol并不影响U937细胞向破骨样细胞的分化，对细胞无明显毒性，因此，我们选择分别为1μmol、2.5μmol和5μmol三个用药浓度分析熊果酸对细胞的影响。Western blot分析发现，熊果酸并不改变c-Src表达量，但是随着用药浓度的增加，c-Src Tyr527磷酸化水平随之升高。TRAP染色结果显示，与对照组相比，熊果酸可以显著抑制破骨细胞的分化。Real-time PCR检测各标志性基因，与对照组相比，不同浓度熊果酸处理组可以显著抑制破骨细胞标志性基因TRAP、RANK、MMP-9、CK、CTR的表达。 5.熊果酸对大鼠牙根吸收模型的影响我们选用8周龄健康雄性Wistar大鼠，随机分为空白对照组，对照组和给药组，给药组又分为低剂量给药组(0.5mmol)，中剂量给药组(1mmol)和高剂量给药组(2mmol)。50g力近中移动大鼠上颌第一磨牙14d和28d后处死，测量正畸牙移动的距离。与对照组相比，14d各组第一磨牙移动距离无统计学差异，而28d高剂量给药组(2mmol)，牙齿移动距离减小，且具有统计学差异。大鼠牙周组织的形态学结果显示，随着给药浓度的升高，牙根吸收出现的时间延后，牙根吸收程度减弱。免疫组织化学染色结果显示，与对照组相比，，随着给药浓度升高，给药组牙周组织中c-Src Tyr527磷酸化表达呈升高趋势。通过以上实验结果，可以得出以下结论： 1.通过改变诱导条件，我们可以对PTP-oc催化结构域进行可溶性表达，表达量随着诱导温度的降低而增加。 2.与其他的PTPs相比，PTP-oc存在一定的特殊之处，如最适pH为7.0，最适反应温度为34℃。 3.熊果酸能够有效抑制PTP-oc的酶活性，且具有较好的专一性。 4.熊果酸主要通过抑制PTP-oc的酶活性，升高c-Src Tyr527的磷酸化水平，进而抑制c-Src介导的信号通路，影响破骨细胞的分化及活性。 5.熊果酸能够缓解牙根吸收模型的大鼠的牙根吸收程度，其具有剂量依赖性的特点。
[Abstract]:In the process of orthodontic treatment, orthodontic force induces the remodeling of periodontal tissue and alveolar bone to make the tooth move. However, the mechanical pressure can also induce the tooth root absorption to varying degrees and the possibility of self healing in the light root resorption. However, the absorption of the root tip of the teeth tends to shorten the root apex of the present teeth, and the proportion of the crown and root is not adjusted. The mechanism of orthodontic treatment is complicated. The mechanism of root absorption is similar to that of bone absorption. The root absorption is mainly absorbed by the osteoclasts similar to osteoclasts, and many of the signal factors constitute a complex network modulation system. At present, the orthodontic teeth are clinically applied to the teeth. There are no effective preventive measures for root resorption, and how to reduce root resorption is a problem that every orthodontic doctor expects to solve.
Protein tyrosine phosphatase (Protein Tyrosine Phosphatases, PTPs) is a key enzyme in cell signaling, which is closely related to human physiological and pathological processes. The regulation of osteoclast differentiation, adhesion and activity is very complex. Most of the signal transduction processes are closely related to protein tyrosine phosphorylation. Osteoclasts In the process of differentiation, the PTPs in the surrounding tissue cells participates in the regulation of the phosphorylation of the target protein in the signal transduction pathway..PTP-oc is a specific expression of the protein tyrosine phosphatase in osteoclast, and the osteoclast has a positive regulation effect on osteoclasts. In order to verify this hypothesis, we have designed the following experiments to inhibit the formation and differentiation of osteoclasts and ultimately prevent or reduce the root resorption of orthodontic.
1. PTP-oc protein catalyzes the cloning of the domain, and the expression is:
The plasmid pMD18-T- Delta PTP-oc containing the encoded PTP-oc domain (delta PTP-oc) cDNA was used as the template to amplify the delta PTP-oc by PCR, and then cloned into the pET-28a (+) vector, and the recombinant plasmid was transferred into the Escherichia coli BL21 (DE3) and added to the 0.1mM IPTG16 to induce the pET-28a (DE3).
Purification and characterization of 2. PTP-oc protein catalytic domain:
After the strain was broken by ultrasound, the soluble Delta PTP-oc recombinant protein was successfully obtained by Ni-NTA agarose affinity chromatography. The purity of the purified Delta PTP-oc was more than 80%, and was quite high specificity. The delta PTP-oc two structure prediction was basically consistent with the circular two chromatographic analysis results, which proved that the recombinant Delta PTP-oc had the correct spatial structure. The enzymatic reaction kinetics studies show that when the substrate is p-NPP, the delta PTP-oc conforms to the properties of the micelloid enzyme. Its Km=801.8 Mu mol, Vmax=6.1 micron mol/min. properties study shows that the optimum reaction temperature of the delta PTP-oc is 34 and the optimum ionic strength is 0, which is similar to other common PTPs, but the special pH is 7 for Delta PTP-oc, which is the same as other common PTPs. Different.
3. screening of PTP-oc inhibitors:
By using colorimetric method, the inhibitor of delta PTP-oc was screened from 69 monomers. In the case of the final concentration of 100 mu mol/L, there were four kinds of monomers with inhibition effect more than 90%, of which the inhibition effect of ursolic acid on the delta PTP-oc enzyme was the best, and the IC50 of delta PTP-oc was 6.381 + 0.56 micron mol/L.. The inhibitory type of ursolic acid on Delta PTP-oc was found to be competitive, the inhibition constant Ki was 7.9 micron mol/L., and ursolic acid had a good specificity on the inhibition effect of delta PTP-oc. Therefore, ursolic acid was selected as a further research object, and the effects of ursolic acid on osteoclast were studied.
Effects of 4. ursolic acid on differentiation and absorption activity of osteoclast like cells induced by U937 cells
Using U937 cells, using 1 x 10-7M/L TPA and 1 x 10-8M/L 1,25 (OH) 2D3 two inducers, the U937 cells were induced to be osteoclast like cells in.CCK-8 experiment. The results showed that the dosage of ursolic acid less than 5 mu did not affect the differentiation of the osteoclast like cells, and there was no obvious toxicity to the cells. Therefore, we chose 1 micron mol, respectively. The effect of ursolic acid on the cells by three doses of 2.5 mol and 5 mol.Western blot analysis found that ursolic acid did not change the c-Src expression, but with the increase of drug concentration, the phosphorylation level of c-Src Tyr527 increased with the.TRAP staining results, and ursolic acid could significantly inhibit the differentiation of osteoclasts from the control group. -time PCR detected all the marker genes, and compared with the control group, the expression of osteoclast marker gene TRAP, RANK, MMP-9, CK, CTR could be significantly inhibited by the ursolic acid treatment group with different concentrations.
The effect of ursolic acid 5. on the model of root resorption in rats
We selected healthy male Wistar rats of 8 weeks old, randomly divided into blank control group, control group and administration group, and the administration group was divided into low dose group (0.5mmol), middle dose dose group (1mmol) and high dose group (2mmol).50g force near the first molar 14d and 28d in the upper maxillary molar of rats, and measured the distance between orthodontic tooth movement and the control group. There was no significant difference in the moving distance between the first molar of 14d, but the 28d high dose group (2mmol), the tooth movement distance decreased, and there was a statistical difference. The morphological results of the periodontal tissue showed that with the increase of the concentration of the drug, the time of the root resorption was delayed and the degree of root absorption was weakened. The results showed that compared with the control group, the expression of c-Src Tyr527 phosphorylation in the periodontal tissue of the drug delivery group increased with the increase of drug concentration.
Through the above experimental results, we can draw the following conclusions:
1. by changing the induction conditions, we can express soluble expression of PTP-oc catalytic domain, and the amount of expression increases with the decrease of induction temperature.
2. compared with other PTPs, PTP-oc has some special characteristics, for example, the optimum pH is 7, and the optimum reaction temperature is 34 C.
3. ursolic acid can effectively inhibit the enzyme activity of PTP-oc, and has better specificity.
4. ursolic acid mainly inhibits the phosphorylation level of c-Src Tyr527 by inhibiting the enzyme activity of PTP-oc, and then inhibits the signal pathway mediated by c-Src and affects the differentiation and activity of osteoclast.
5. ursolic acid alleviated root resorption in rats with root resorption, which was dose-dependent.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R783.5

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