本文選題：破骨細胞 + 蛋白質(zhì)酪氨酸磷酸酶　； 參考：《吉林大學》2015年博士論文

【摘要】：正畸治療過程中，正畸力誘導牙周組織和牙槽骨改建，從而使牙齒發(fā)生移動。但是，機械壓力也會誘發(fā)牙齒發(fā)生不同程度的牙根吸收，輕度牙根吸收存在自愈的可能，但中重度牙根吸收往往會出現(xiàn)牙根尖部不可復性變短、冠根比例不調(diào)、甚至發(fā)生牙齒松動的現(xiàn)象，使正畸治療趨向復雜化。牙根吸收的機制與骨吸收相類似，牙根吸收由與破骨細胞相似的破牙骨質(zhì)細胞承擔主要吸收牙骨質(zhì)的功能，其中眾多信號因子構(gòu)成復雜的網(wǎng)絡調(diào)控系統(tǒng)。目前，臨床上對正畸力引起的牙根吸收尚無有效的預防措施，而如何減少牙根吸收是每一位正畸醫(yī)生期待解決的問題。 蛋白質(zhì)酪氨酸磷酸酶(Protein Tyrosine Phosphatases，PTPs)是細胞信號傳導過程中的關(guān)鍵酶，與人類生理和病理過程密切相關(guān)。破骨細胞分化、粘附及活性的調(diào)節(jié)是十分復雜的，其信號傳導過程大部分都與蛋白質(zhì)酪氨酸磷酸化密切相關(guān)。破骨細胞分化過程中，周圍組織細胞中的PTPs參與調(diào)控其信號傳導途徑中的靶蛋白發(fā)生磷酸化作用。PTP-oc是一種特異性表達于破骨前體細胞、破骨細胞的蛋白質(zhì)酪氨酸磷酸酶，對破骨細胞具有正調(diào)節(jié)作用。因此，我們認為特異性的抑制PTP-oc的活性，進而抑制破骨細胞的生成和分化，最終可以在一定程度上預防或減少正畸致牙根吸收。為了驗證這一假設，我們設計了如下實驗： 1. PTP-oc蛋白催化結(jié)構(gòu)域的克隆、表達： 以含有編碼PTP-oc催化結(jié)構(gòu)域(ΔPTP-oc)cDNA的質(zhì)粒pMD18-T-ΔPTP-oc為模板，通過PCR擴增ΔPTP-oc，然后將其克隆到pET-28a(+)載體中，并將重組質(zhì)粒轉(zhuǎn)入大腸桿菌BL21(DE3)中，加入0.1mM IPTG16℃誘導24h。 2. PTP-oc蛋白催化結(jié)構(gòu)域的分離純化及酶學表征： 菌體經(jīng)超聲破碎后，利用Ni-NTA agarose親和層析方法成功的獲得了可溶的ΔPTP-oc重組蛋白，純化后的ΔPTP-oc的純度達80%以上，且具有相當高的特異性。ΔPTP-oc二級結(jié)構(gòu)預測與圓二色譜分析結(jié)果基本一致，證明重組ΔPTP-oc具有正確的空間結(jié)構(gòu)。酶促反應動力學研究表明，當?shù)孜餅閜-NPP時，ΔPTP-oc符合米氏酶的性質(zhì)，其Km=801.8μmol，Vmax=6.1μmol/min。酶學性質(zhì)研究表明，ΔPTP-oc的最適反應溫度為34℃，最適離子強度為0，這與其他常見PTPs相似，但獨特的是ΔPTP-oc最適pH為7.0，這與其他常見PTPs不同。 3. PTP-oc抑制劑的篩選： 采用比色法，從69種單體化合物中篩選ΔPTP-oc的抑制劑，在每種單體化合物終濃度為100μmol/L的情況下，抑制效果達到90%以上的單體化合物有四種，其中熊果酸對ΔPTP-oc酶的抑制效果最好，達到了98.3%，測定其對ΔPTP-oc的IC50為6.381±0.56μmol/L。通過雙倒數(shù)作圖法研究發(fā)現(xiàn)熊果酸對ΔPTP-oc的抑制類型為競爭性抑制，抑制常數(shù)Ki為7.9μmol/L。此外，熊果酸對ΔPTP-oc抑制效果具有很好的專一性。因此，我們選取熊果酸作為進一步的研究對象，研究其對破骨細胞的相關(guān)影響。 4.熊果酸對U937細胞誘導的破骨樣細胞分化及吸收活性的影響 選用U937細胞，使用1×10-7M/L的TPA和1×10-8M/L的1,25(OH)2D3兩種誘導劑聯(lián)合誘導，成功將U937細胞誘導為破骨樣細胞。CCK-8實驗結(jié)果表明，熊果酸用量小于5μmol并不影響U937細胞向破骨樣細胞的分化，對細胞無明顯毒性，因此，我們選擇分別為1μmol、2.5μmol和5μmol三個用藥濃度分析熊果酸對細胞的影響。Western blot分析發(fā)現(xiàn)，熊果酸并不改變c-Src表達量，但是隨著用藥濃度的增加，c-Src Tyr527磷酸化水平隨之升高。TRAP染色結(jié)果顯示，與對照組相比，熊果酸可以顯著抑制破骨細胞的分化。Real-time PCR檢測各標志性基因，與對照組相比，不同濃度熊果酸處理組可以顯著抑制破骨細胞標志性基因TRAP、RANK、MMP-9、CK、CTR的表達。 5.熊果酸對大鼠牙根吸收模型的影響 我們選用8周齡健康雄性Wistar大鼠，隨機分為空白對照組，對照組和給藥組，給藥組又分為低劑量給藥組(0.5mmol)，中劑量給藥組(1mmol)和高劑量給藥組(2mmol)。50g力近中移動大鼠上頜第一磨牙14d和28d后處死，測量正畸牙移動的距離。與對照組相比，14d各組第一磨牙移動距離無統(tǒng)計學差異，而28d高劑量給藥組(2mmol)，牙齒移動距離減小，且具有統(tǒng)計學差異。大鼠牙周組織的形態(tài)學結(jié)果顯示，隨著給藥濃度的升高，牙根吸收出現(xiàn)的時間延后，牙根吸收程度減弱。免疫組織化學染色結(jié)果顯示，與對照組相比，，隨著給藥濃度升高，給藥組牙周組織中c-Src Tyr527磷酸化表達呈升高趨勢。 通過以上實驗結(jié)果，可以得出以下結(jié)論： 1.通過改變誘導條件，我們可以對PTP-oc催化結(jié)構(gòu)域進行可溶性表達，表達量隨著誘導溫度的降低而增加。 2.與其他的PTPs相比，PTP-oc存在一定的特殊之處，如最適pH為7.0，最適反應溫度為34℃。 3.熊果酸能夠有效抑制PTP-oc的酶活性，且具有較好的專一性。 4.熊果酸主要通過抑制PTP-oc的酶活性，升高c-Src Tyr527的磷酸化水平，進而抑制c-Src介導的信號通路，影響破骨細胞的分化及活性。 5.熊果酸能夠緩解牙根吸收模型的大鼠的牙根吸收程度，其具有劑量依賴性的特點。
[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.
【學位授予單位】：吉林大學
【學位級別】：博士
【學位授予年份】：2015
【分類號】：R783.5

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相關(guān)期刊論文 前4條

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3 ;Purification and Characterization of Protein Tyrosine Phosphatase MEG1 and Preparation of Anti-PTPMEG1 Antibody[J];Chemical Research in Chinese Universities;2010年04期

4 范文斌;趙建寧;包倪榮;;磷脂酰肌醇3激酶/蛋白激酶B信號通路對MC3T3-E1細胞增殖和周期調(diào)控的研究[J];醫(yī)學研究生學報;2014年04期

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