本文關鍵詞：靶向RANKL的抗骨質疏松活性多肽的設計、合成及生物活性研究　出處：《第二軍醫(yī)大學》2017年碩士論文　論文類型：學位論文

  更多相關文章： 抗骨質疏松 多肽 RANKL OPG模擬物

【摘要】：隨著人口老齡化的不斷加劇,骨質疏松已成為全球范圍內越來越嚴重的公共健康問題。目前臨床上應用的以雙膦酸鹽為代表的抗骨質疏松藥物都存在著一定的不足之處,尋找新型、高效、低毒的抗骨質疏松藥物是當前骨科領域的主要目標之一。近些年來,破骨細胞病理生理學研究發(fā)現,RANK-RANKL-OPG骨調節(jié)軸共同參與調節(jié)破骨前體細胞向破骨細胞分化成熟過程,被認為是開發(fā)新型抗骨質疏松藥物的熱點靶標。RANKL與其靶蛋白RANK的結合可以激活下游NF-κB信號通路,造成包括骨質疏松在內的多種病癥發(fā)生。而OPG作為RANKL的天然誘餌受體,能夠有效抑制RANKL誘導的破骨細胞分化成熟。通過比較OPG/RANKL與RANKL/RANK復合物晶體衍射結構發(fā)現,OPG的90s loop序列是OPG與RANKL結合的關鍵位點。OPG通過90s loop占領RANKL,使得RANKL喪失與RANK結合的能力,發(fā)揮其對骨骼的保護作用。本課題根據OPG 90s loop序列,合理設計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型的OPG模擬物多肽共6條。經文獻檢索,所有化合物均為首次報道。目標化合物均經過HR-MS確證,所有中間體都經過ESI-MS,1H-NMR,13C-NMR確證。細胞水平活性實驗中,我們提取了ICR小鼠破骨前體細胞,經過目標化合物處理后,進行了TRAP染色。測試結果初步顯示,在30μM化合物濃度下,除OPG-1外,目標化合物均表現出不同程度的破骨前體細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,略優(yōu)于陽性對照OP3-4。我們選取OPG-4再次進行了濃度梯度的TRAP染色實驗,化合物顯示出劑量依賴性的抑制作用,測得其半數抑制濃度(IC50)為28.51μM。表面等離子共振實驗中,我們通過Biacore T200生物傳感儀測定了OPG-4與靶標蛋白RANKL的結合能力。實驗結果表明,OPG-4與RANKL的結合常數在微摩爾數量級,與陽性對照相當。體內活性測試中,我們選擇了雌性C57BL/6小鼠,通過卵巢切除造模,成功獲得OVX骨質疏松小鼠模型。腹腔注射給予小鼠OPG-4(20 mg/kg/day)處理6周后,取股骨進行HE染色和Micro-CT掃描。我們發(fā)現處理組(OVX+OPG)小鼠骨密度以及骨小梁數目較去卵巢組(OVX)組有明顯增多,具有有統(tǒng)計學意義的明顯差異(Student’s t test,p0.05)。本課題設計并合成的糖基化OPG模擬物OPG-4在體內外活性實驗中都表現出了良好的破骨細胞抑制活性,可以作為候選化合物開展深入研究。通過對其進行氨基酸殘基隨機突變或更換糖基化類型以及位點等嘗試,我們有理由相信能夠得到體內外活性更優(yōu)異的化合物。在后續(xù)研究中,我們還將對OPG-4/RANKL復合物繼續(xù)進行單晶衍射研究,力圖闡明兩者相互作用的分子機制。本研究對開發(fā)新型抗骨質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.
【學位授予單位】：第二軍醫(yī)大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R91

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