重组灵芝免疫调节蛋白对骨质疏松大鼠的保护作用及机制研究

发布时间：2019-06-17 21:18

【摘要】：糖皮质激素性骨质疏松（GIOP）是最常见的继发性骨质疏松症，其本质是骨形成的能力降低。虽然目前用于临床的抗骨质疏松（OP）药物较多，但大部分药物的疗效一般，同时还伴有较大毒副作用，因此研究高疗效、低毒性的抗OP药物已成为现代医学新药研发中的一项热点工作。灵芝免疫调节蛋白（LZ-8）于1989年被分离提取并得到，属真菌免疫调节蛋白。近年来，多项研究证实LZ-8具有调节免疫、抑制肿瘤生长、激活白细胞介素6（IL-6）和肿瘤坏死因子（TNF-α）等细胞因子的表达等活性。本论文中使用的重组灵芝免疫调节蛋白（rLZ-8），是经过毕赤酵母重组表达LZ-8后得到的，旨在探索rLZ-8是否对OP具有保护作用。本论文采用肌肉注射地塞米松（DEX）2.5mg/kg，每周2次，连续给药13次的方法建立GIOP大鼠模型。于给药结束后检测大鼠股骨中的骨矿物质含量（BMC）、骨矿物质密度（BMD）。实验结果显示，与生理盐水组相比，模型组大鼠的BMC、BMD均明显降低（P＜0.01，P＜0.01），证明GIOP模型成功建立。在GIOP模型成功建立的基础上，为研究rLZ-8对GIOP模型大鼠的作用，，本实验选用Wistar大鼠60只，随机分为6组，即生理盐水组、模型组、阳性药组、rLZ-8低剂量组、rLZ-8中剂量组、rLZ-8高剂量组，每组10只动物。于开始造模前3天，对生理盐水组和模型组大鼠腹腔注射生理盐水，阳性药组大鼠灌胃给予1mg/kg阿仑膦酸钠，rLZ-8低、中、高剂量组大鼠分别腹腔注射rLZ-828μg/kg、56μg/kg、112μg/kg，持续给药48天。在最后一次给药结束后，深度麻醉大鼠，获取大鼠血清、骨组织以及各脏器组织，并小心保存以待检测。有研究表明骨小梁可直接反应骨的强度和稳定性，因此本论文将上述所得骨组织经脱钙等方法处理后，采用HE染色法，在光镜下观察其骨小梁的结构变化。实验结果显示模型组大鼠股骨骨小梁数目相比生理盐水组显著减少，并且出现断裂等现象；而给予rLZ-8干预后，大鼠股骨骨小梁结构呈现出不同程度的改善。由此可初步断定rLZ-8对糖皮质激素（GCs）引起的骨小梁结构退化具有一定的保护作用。由于GCs在对骨代谢产生影响的同时，还参与某些炎症因子的分泌，因此本论文通过比色法和ELISA法对已获得的大鼠血清进行检测，得到结果如下：模型组大鼠血清碱性磷酸酶（ALP）活力和血清磷含量明显升高（P＜0.01，P＜0.01），血钙下降（P＜0.01），血清TNF-α和IL-6的含量显著升高（P＜0.05，P＜0.05）；rLZ-8三个剂量组中，以上现象得到改善。由此证实rLZ-8可调节GCs引发的骨代谢紊乱，抑制机体对相关炎症因子的过量分泌。为深入研究rLZ-8对GIOP保护作用的机制，本论文通过Western blot方法检测前述所得的大鼠骨组织中的骨保护蛋白（OPG）和核因子-κB受体活化因子配体（RANKL）的蛋白表达量。实验结果证实GCs可通过OPG/RANKL/RANK系统影响骨吸收和骨形成的平衡，而rLZ-8可抑制此过程，起到保护作用。总之，rLZ-8对GIOP模型大鼠具有保护作用，是一种潜在的可用于治疗OP的有效药物。
[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
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R285.5

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