【摘要】：糖皮質(zhì)激素性骨質(zhì)疏松（GIOP）是最常見的繼發(fā)性骨質(zhì)疏松癥，其本質(zhì)是骨形成的能力降低。雖然目前用于臨床的抗骨質(zhì)疏松（OP）藥物較多，但大部分藥物的療效一般，同時(shí)還伴有較大毒副作用，因此研究高療效、低毒性的抗OP藥物已成為現(xiàn)代醫(yī)學(xué)新藥研發(fā)中的一項(xiàng)熱點(diǎn)工作。 靈芝免疫調(diào)節(jié)蛋白（LZ-8）于1989年被分離提取并得到，屬真菌免疫調(diào)節(jié)蛋白。近年來(lái)，多項(xiàng)研究證實(shí)LZ-8具有調(diào)節(jié)免疫、抑制腫瘤生長(zhǎng)、激活白細(xì)胞介素6（IL-6）和腫瘤壞死因子（TNF-α）等細(xì)胞因子的表達(dá)等活性。本論文中使用的重組靈芝免疫調(diào)節(jié)蛋白（rLZ-8），是經(jīng)過(guò)畢赤酵母重組表達(dá)LZ-8后得到的，旨在探索rLZ-8是否對(duì)OP具有保護(hù)作用。 本論文采用肌肉注射地塞米松（DEX）2.5mg/kg，每周2次，連續(xù)給藥13次的方法建立GIOP大鼠模型。于給藥結(jié)束后檢測(cè)大鼠股骨中的骨礦物質(zhì)含量（BMC）、骨礦物質(zhì)密度（BMD）。實(shí)驗(yàn)結(jié)果顯示，與生理鹽水組相比，模型組大鼠的BMC、BMD均明顯降低（P＜0.01，P＜0.01），證明GIOP模型成功建立。 在GIOP模型成功建立的基礎(chǔ)上，為研究rLZ-8對(duì)GIOP模型大鼠的作用，，本實(shí)驗(yàn)選用Wistar大鼠60只，隨機(jī)分為6組，即生理鹽水組、模型組、陽(yáng)性藥組、rLZ-8低劑量組、rLZ-8中劑量組、rLZ-8高劑量組，每組10只動(dòng)物。于開始造模前3天，對(duì)生理鹽水組和模型組大鼠腹腔注射生理鹽水，陽(yáng)性藥組大鼠灌胃給予1mg/kg阿侖膦酸鈉，rLZ-8低、中、高劑量組大鼠分別腹腔注射rLZ-828μg/kg、56μg/kg、112μg/kg，持續(xù)給藥48天。在最后一次給藥結(jié)束后，深度麻醉大鼠，獲取大鼠血清、骨組織以及各臟器組織，并小心保存以待檢測(cè)。 有研究表明骨小梁可直接反應(yīng)骨的強(qiáng)度和穩(wěn)定性，因此本論文將上述所得骨組織經(jīng)脫鈣等方法處理后，采用HE染色法，在光鏡下觀察其骨小梁的結(jié)構(gòu)變化。實(shí)驗(yàn)結(jié)果顯示模型組大鼠股骨骨小梁數(shù)目相比生理鹽水組顯著減少，并且出現(xiàn)斷裂等現(xiàn)象；而給予rLZ-8干預(yù)后，大鼠股骨骨小梁結(jié)構(gòu)呈現(xiàn)出不同程度的改善。由此可初步斷定rLZ-8對(duì)糖皮質(zhì)激素（GCs）引起的骨小梁結(jié)構(gòu)退化具有一定的保護(hù)作用。 由于GCs在對(duì)骨代謝產(chǎn)生影響的同時(shí)，還參與某些炎癥因子的分泌，因此本論文通過(guò)比色法和ELISA法對(duì)已獲得的大鼠血清進(jìn)行檢測(cè)，得到結(jié)果如下：模型組大鼠血清堿性磷酸酶（ALP）活力和血清磷含量明顯升高（P＜0.01，P＜0.01），血鈣下降（P＜0.01），血清TNF-α和IL-6的含量顯著升高（P＜0.05，P＜0.05）；rLZ-8三個(gè)劑量組中，以上現(xiàn)象得到改善。由此證實(shí)rLZ-8可調(diào)節(jié)GCs引發(fā)的骨代謝紊亂，抑制機(jī)體對(duì)相關(guān)炎癥因子的過(guò)量分泌。 為深入研究rLZ-8對(duì)GIOP保護(hù)作用的機(jī)制，本論文通過(guò)Western blot方法檢測(cè)前述所得的大鼠骨組織中的骨保護(hù)蛋白（OPG）和核因子-κB受體活化因子配體（RANKL）的蛋白表達(dá)量。實(shí)驗(yàn)結(jié)果證實(shí)GCs可通過(guò)OPG/RANKL/RANK系統(tǒng)影響骨吸收和骨形成的平衡，而rLZ-8可抑制此過(guò)程，起到保護(hù)作用。 總之，rLZ-8對(duì)GIOP模型大鼠具有保護(hù)作用，是一種潛在的可用于治療OP的有效藥物。
[Abstract]:Glucocorticoid osteoporotic (GIOP) is the most common secondary osteoporosis, which is essentially a reduction in the ability of bone formation. Although there are many anti-osteoporosis (OP) drugs for clinical use at present, most of the drugs have a general curative effect and have a large toxic and side effect. Therefore, the study of high curative effect and low toxicity OP medicine has become a hot spot in the research and development of modern medicine. Ganoderma immunomodulatory protein (LZ-8) was isolated and extracted in 1989 and was a fungus immunomodulatory egg. In recent years, several studies have shown that LZ-8 has the functions of regulating immunity, inhibiting tumor growth, activating the expression of interleukin-6 (IL-6) and tumor necrosis factor (TNF-1), etc. The recombinant Ganoderma immunomodulatory protein (rLZ-8) used in this paper is obtained by the recombinant expression of LZ-8 in Pichia pastoris, and is designed to explore whether the rLZ-8 has a protective effect on the OP. GIOP was established by intramuscular injection of dexamethasone (DEX) of 2.5 mg/ kg, twice a week, and 13 times a week. Rat model. The bone mineral content (BMC), bone mineral density (B, The results showed that the BMC and BMD in the model group were significantly lower than that of the normal saline group (P <0.01, P <0.01). Based on the successful establishment of the GIOP model,60 Wistar rats were randomly divided into 6 groups, namely the normal saline group, the model group, the positive group, the rLZ-8 low dose group, the rLZ-8 medium dose group and the rLZ-8 high dose group. Ten animals were injected intraperitoneally in the saline group and the model group 3 days before the start of the model. The rats of the positive group were given 1 mg/ kg of sodium alamelate, and rLZ-8 was low. In the middle and high dose group, the rats were injected with rLZ-828. mu.g/ kg,56. mu.g/ kg,112. mu.g/ kg, respectively. The drug was administered for 48 days. At the end of the last dose, the rats were anesthetized with deep anesthesia to obtain the rat serum, bone tissue, and the tissues of the organs and to be carefully protected. The results showed that the bone trabecula can directly reflect the strength and stability of the bone, so this paper, after the above-mentioned bone tissue has been treated with the method of decalcification and the like, uses the HE staining method to observe the bone in the light microscope. The results showed that the number of the trabecular bone trabeculae in the model group was significantly reduced compared with that of the normal saline group, and the fracture and other phenomena were observed; and after the rLZ-8 intervention, the trabecular structure of the femoral bone of the rat It can be concluded that the degeneration of the bone trabecula caused by the rLZ-8 to the glucocorticoid (GCs) has a different degree of improvement. Because of the effect of GCs on bone metabolism, it is also involved in the secretion of some inflammatory factors. The results were as follows: the serum alkaline phosphatase (ALP) activity and the serum phosphorus content in the model group were significantly higher (P <0.01, P <0.01), and the levels of serum TNF-1 and IL-6 increased significantly (P <0.05, P <0.05); rLZ-8 three dose groups The above phenomenon is improved. This confirms that rLZ-8 can regulate the bone metabolism disorder induced by GCs, and inhibit the body-to-body correlation. In order to study the mechanism of the protective effect of rLZ-8 on GIOP, the bone protective protein (OPG) and the nuclear factor-B receptor activating factor ligand (R) in the bone tissue of the rat were detected by Western blot. The experimental results confirm that the GCs can influence the balance of bone resorption and bone formation through the OPG/ RANKL/ RANK system, while rLZ-8 can be inhibited. In conclusion, rLZ-8 has a protective effect on the GIOP model rats and is a potential
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R285.5

【參考文獻(xiàn)】

相關(guān)期刊論文 前10條

1 曾曉,謝林,姚共和;去勢(shì)雌性大鼠骨質(zhì)疏松癥模型的復(fù)制及相關(guān)指標(biāo)測(cè)定[J];浙江中醫(yī)學(xué)院學(xué)報(bào);1999年04期

2 宋敏;李晶;;大鼠骨質(zhì)疏松模型的復(fù)制方法及意義[J];當(dāng)代醫(yī)學(xué);2010年15期

3 張?jiān)路?韓景獻(xiàn);骨質(zhì)疏松動(dòng)物模型研究進(jìn)展[J];動(dòng)物醫(yī)學(xué)進(jìn)展;2005年03期

4 梁重陽(yáng);徐蔚青;曹焱鑫;劉立俠;張淑芹;劉志屹;李泓睿;李柏志;孫非;;FITC標(biāo)記重組靈芝免疫調(diào)節(jié)蛋白(rLz-8)在NB4細(xì)胞中的動(dòng)態(tài)定位[J];高等學(xué)校化學(xué)學(xué)報(bào);2009年03期

5 渠海波;張朝;吳剛;;骨質(zhì)疏松的研究進(jìn)展[J];包頭醫(yī)學(xué)院學(xué)報(bào);2013年03期

6 崔軼;雷偉;吳子祥;劉達(dá);賀強(qiáng);劉緒立;;骨質(zhì)疏松大動(dòng)物模型的研究進(jìn)展[J];脊柱外科雜志;2010年05期

7 高莉莉;任憲輝;富宏然;;骨源性堿性磷酸酶在骨代謝疾病診斷中的應(yīng)用[J];牡丹江醫(yī)學(xué)院學(xué)報(bào);2009年01期

8 呼和;李哲海;武宇赤;;糖皮質(zhì)激素性骨質(zhì)疏松的治療研究進(jìn)展[J];內(nèi)蒙古醫(yī)學(xué)雜志;2010年01期

9 梁重陽(yáng);張淑芹;劉志屹;孫非;;靈芝免疫調(diào)節(jié)蛋白(Lz-8)在畢赤酵母中的表達(dá)及其免疫活性鑒定(英文)[J];生物工程學(xué)報(bào);2009年03期

10 安敏;高福;齊建勛;李鋒;劉杏忠;;靈芝免疫調(diào)節(jié)蛋白LZ-8的制備和晶體分析[J];生物工程學(xué)報(bào);2010年11期

本文編號(hào)：2501273