本文關(guān)鍵詞：靶向RANKL的抗骨質(zhì)疏松活性多肽的設(shè)計(jì)、合成及生物活性研究　出處：《第二軍醫(yī)大學(xué)》2017年碩士論文　論文類(lèi)型：學(xué)位論文

  更多相關(guān)文章： 抗骨質(zhì)疏松 多肽 RANKL OPG模擬物

【摘要】：隨著人口老齡化的不斷加劇,骨質(zhì)疏松已成為全球范圍內(nèi)越來(lái)越嚴(yán)重的公共健康問(wèn)題。目前臨床上應(yīng)用的以雙膦酸鹽為代表的抗骨質(zhì)疏松藥物都存在著一定的不足之處,尋找新型、高效、低毒的抗骨質(zhì)疏松藥物是當(dāng)前骨科領(lǐng)域的主要目標(biāo)之一。近些年來(lái),破骨細(xì)胞病理生理學(xué)研究發(fā)現(xiàn),RANK-RANKL-OPG骨調(diào)節(jié)軸共同參與調(diào)節(jié)破骨前體細(xì)胞向破骨細(xì)胞分化成熟過(guò)程,被認(rèn)為是開(kāi)發(fā)新型抗骨質(zhì)疏松藥物的熱點(diǎn)靶標(biāo)。RANKL與其靶蛋白R(shí)ANK的結(jié)合可以激活下游NF-κB信號(hào)通路,造成包括骨質(zhì)疏松在內(nèi)的多種病癥發(fā)生。而OPG作為RANKL的天然誘餌受體,能夠有效抑制RANKL誘導(dǎo)的破骨細(xì)胞分化成熟。通過(guò)比較OPG/RANKL與RANKL/RANK復(fù)合物晶體衍射結(jié)構(gòu)發(fā)現(xiàn),OPG的90s loop序列是OPG與RANKL結(jié)合的關(guān)鍵位點(diǎn)。OPG通過(guò)90s loop占領(lǐng)RANKL,使得RANKL喪失與RANK結(jié)合的能力,發(fā)揮其對(duì)骨骼的保護(hù)作用。本課題根據(jù)OPG 90s loop序列,合理設(shè)計(jì)并合成了包括直鏈肽、二硫鍵肽、二硫鍵糖肽、二硒鍵衍生物以及三氮唑鍵衍生物等不同類(lèi)型的OPG模擬物多肽共6條。經(jīng)文獻(xiàn)檢索,所有化合物均為首次報(bào)道。目標(biāo)化合物均經(jīng)過(guò)HR-MS確證,所有中間體都經(jīng)過(guò)ESI-MS,1H-NMR,13C-NMR確證。細(xì)胞水平活性實(shí)驗(yàn)中,我們提取了ICR小鼠破骨前體細(xì)胞,經(jīng)過(guò)目標(biāo)化合物處理后,進(jìn)行了TRAP染色。測(cè)試結(jié)果初步顯示,在30μM化合物濃度下,除OPG-1外,目標(biāo)化合物均表現(xiàn)出不同程度的破骨前體細(xì)胞分化成熟抑制作用。其中,糖基化衍生物OPG-4顯示出最佳的抑制效果,略?xún)?yōu)于陽(yáng)性對(duì)照OP3-4。我們選取OPG-4再次進(jìn)行了濃度梯度的TRAP染色實(shí)驗(yàn),化合物顯示出劑量依賴(lài)性的抑制作用,測(cè)得其半數(shù)抑制濃度(IC50)為28.51μM。表面等離子共振實(shí)驗(yàn)中,我們通過(guò)Biacore T200生物傳感儀測(cè)定了OPG-4與靶標(biāo)蛋白R(shí)ANKL的結(jié)合能力。實(shí)驗(yàn)結(jié)果表明,OPG-4與RANKL的結(jié)合常數(shù)在微摩爾數(shù)量級(jí),與陽(yáng)性對(duì)照相當(dāng)。體內(nèi)活性測(cè)試中,我們選擇了雌性C57BL/6小鼠,通過(guò)卵巢切除造模,成功獲得OVX骨質(zhì)疏松小鼠模型。腹腔注射給予小鼠OPG-4(20 mg/kg/day)處理6周后,取股骨進(jìn)行HE染色和Micro-CT掃描。我們發(fā)現(xiàn)處理組(OVX+OPG)小鼠骨密度以及骨小梁數(shù)目較去卵巢組(OVX)組有明顯增多,具有有統(tǒng)計(jì)學(xué)意義的明顯差異(Student’s t test,p0.05)。本課題設(shè)計(jì)并合成的糖基化OPG模擬物OPG-4在體內(nèi)外活性實(shí)驗(yàn)中都表現(xiàn)出了良好的破骨細(xì)胞抑制活性,可以作為候選化合物開(kāi)展深入研究。通過(guò)對(duì)其進(jìn)行氨基酸殘基隨機(jī)突變或更換糖基化類(lèi)型以及位點(diǎn)等嘗試,我們有理由相信能夠得到體內(nèi)外活性更優(yōu)異的化合物。在后續(xù)研究中,我們還將對(duì)OPG-4/RANKL復(fù)合物繼續(xù)進(jìn)行單晶衍射研究,力圖闡明兩者相互作用的分子機(jī)制。本研究對(duì)開(kāi)發(fā)新型抗骨質(zhì)疏松藥物有著重大意義。
[Abstract]:With the aging of the population. Osteoporosis has become a more and more serious public health problem in the world. At present, the anti-osteoporosis drugs, such as bisphosphonates, which are used in clinic, have some shortcomings. Low-toxicity anti-osteoporosis drugs are one of the main targets in the field of orthopedics. In recent years, the pathophysiology of osteoclasts has been discovered. The RANK-RANKL-OPG bone regulatory axis is involved in the process of regulating the differentiation and maturation of osteoclasts from osteoclasts to osteoclasts. The binding of RANKL to its target protein RANK can activate the downstream NF- 魏 B signaling pathway. Causes many diseases, including osteoporosis, and OPG acts as a natural bait receptor for RANKL. It can effectively inhibit the differentiation and maturation of osteoclasts induced by RANKL. It was found by comparing the crystal diffraction structure of OPG/RANKL and RANKL/RANK complex. The 90s loop sequence of OPG is the key site of OPG binding to RANKL. OPG occupies RANKL through 90 s loop. The ability of RANKL to bind to RANK was lost and its protective effect on bone was exerted. According to the sequence of OPG 90 s loop, the straight chain peptide was reasonably designed and synthesized. There were 6 polypeptides of disulfide bond peptide, disulfide glycopeptide, diselenide bond derivative and triazolium bond derivative. All the compounds were reported for the first time. The target compounds were confirmed by HR-MS and all intermediates were confirmed by ESI-MSX 1H-NMR-13C-NMR. The osteoclast precursor cells of ICR mice were extracted and treated with the target compounds. The results of TRAP staining showed that at the concentration of 30 渭 M compounds, except OPG-1. The target compounds all showed different degrees of inhibition of osteoclast differentiation and maturation. Among them, glycosylation derivative OPG-4 showed the best inhibitory effect. A little better than the positive control OP3-4. We selected OPG-4 again for the concentration gradient of TRAP staining experiment, the compound showed a dose-dependent inhibitory effect. The half inhibitory concentration (IC50) was determined to be 28.51 渭 M. in the surface plasmon resonance experiment. The binding ability of OPG-4 to target protein RANKL was determined by Biacore T200 biosensor. The binding constant of OPG-4 and RANKL was in the order of micromolarity, which was similar to that of the positive control. In vivo activity test, we selected female C57BL / 6 mice and made the model by ovariectomy. The model of OVX osteoporosis mice was successfully obtained. The mice were treated by intraperitoneal injection of OPG-4(20 mg / kg / day for 6 weeks. The bone mineral density and the number of trabeculae in the treated group were significantly higher than those in the ovariectomized group. There was a significant difference in t test between the two groups. In this study, the glycosylated OPG mimics OPG-4 showed good osteoclast inhibitory activity in vitro and in vivo. It can be used as a candidate compound for further research, by random mutation of amino acid residues or by changing glycosylation types and sites. We have reason to believe that we can obtain more active compounds in vivo and in vitro. In the further study, we will continue to study the single crystal diffraction of OPG-4/RANKL complex. This study is of great significance to the development of new anti-osteoporosis drugs.
【學(xué)位授予單位】：第二軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：R91

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