WO2022041311A1 - 2-溴棕榈酸在制备防治骨丢失相关疾病的药物中的应用 - Google Patents

2-溴棕榈酸在制备防治骨丢失相关疾病的药物中的应用 Download PDF

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WO2022041311A1
WO2022041311A1 PCT/CN2020/113660 CN2020113660W WO2022041311A1 WO 2022041311 A1 WO2022041311 A1 WO 2022041311A1 CN 2020113660 W CN2020113660 W CN 2020113660W WO 2022041311 A1 WO2022041311 A1 WO 2022041311A1
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bone loss
medicine
bone
drug
related diseases
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陈建权
章礼炜
杨惠林
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Suzhou University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/20—Carboxylic acids, e.g. valproic acid having a carboxyl group bound to a chain of seven or more carbon atoms, e.g. stearic, palmitic, arachidic acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • A61P19/10—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease for osteoporosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]

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  • the invention relates to the application of 2-bromopalmitic acid in the preparation of a medicine for preventing and treating bone loss-related diseases, and belongs to the technical field of medicine.
  • Bone remodeling occurs throughout human life, and most diseases related to bone metabolism affect the process of bone remodeling. Bone remodeling in normal body depends on the direct dynamic balance between osteoclast-mediated bone resorption and osteoblast-mediated bone formation. The imbalance between the two will lead to excessive bone formation and sclerosis-related diseases. Or bone insufficiency, excessive resorption related diseases. Osteoporosis (Osteoporosis) is a systemic bone disease that leads to the decrease of bone density and bone quality, the destruction of bone microstructure, and the increase of bone fragility, which is prone to fracture.
  • osteoporosis is the occurrence of osteoporotic fractures, which is one of the main causes of disability and death in elderly patients.
  • Epidemiological surveys based on imaging show that the prevalence of vertebral fractures in women over the age of 50 in my country is about 15%, and the prevalence of vertebral fractures increases with age after the age of 50.
  • the incidence of osteoporotic vertebral fractures can be as high as 36.6%; while hip fractures are the most serious osteoporotic fractures, studies have shown that within 1 year after hip fractures, 20% of patients will die of various complications, About 50% of the patients are disabled, the quality of life is significantly reduced, and a heavy burden is placed on the family and society.
  • osteoclasts and their mediated bone resorption not only play an important role in the occurrence and development of osteoporosis, but also play an important role in bone loss diseases such as periprosthetic osteolysis, rheumatoid osteoarthritis, and tumor bone metastasis. It is also crucial in the prevention and treatment of such diseases.
  • the prevention of osteoporosis mainly includes adjustment of lifestyle, calcium and vitamin D, and its treatment mainly focuses on inhibiting osteoclast-mediated bone resorption or/and promoting osteoblast-mediated bone formation.
  • Bisphosphonates are the most widely used anti-osteoporosis drugs in clinical practice. They have high affinity with skeletal hydroxyapatite and can specifically bind to the bone surface with active bone remodeling, inhibiting the function of osteoclasts and thus inhibiting Bone resorption.
  • the use of bisphosphonates is associated with gastrointestinal adverse effects, transient "flu-like" symptoms, renal toxicity, and the risk of osteonecrosis of the jaw and atypical femoral fractures.
  • Anti-osteoporosis drugs such as calcitonin, estrogen receptor modulators (raloxifene), menopausal hormone therapy, parathyroid hormone analogs (teriparatide), and RANKL (denosumab) are all available.
  • estrogen receptor modulators raloxifene
  • menopausal hormone therapy a parathyroid hormone analogs
  • RANKL adenosumab
  • the present invention provides an application of 2-bromopalmitic acid in the preparation of a medicine for preventing and treating bone loss-related diseases.
  • the first object of the present invention is to provide an application of 2-bromopalmitic acid in the preparation of a medicine for preventing and treating bone loss-related diseases.
  • the bone loss-related disease is osteoporosis, wear particle-mediated osteolysis, rheumatoid arthritis or tumor bone destruction.
  • the medicine for preventing and treating bone loss-related diseases is a medicine for inhibiting the differentiation and maturation of osteoclasts.
  • the medicine for preventing and treating bone loss-related diseases is a medicine for inhibiting the expression level of characteristic genes in the process of osteoclast differentiation.
  • the characteristic genes include one or more of Nfatc1, C-fos, Ctsk, Acp5, Oscar, Dc-stamp, Atp6v0a3, and Atp6v0d2.
  • the drug for preventing and treating bone loss-related diseases is a drug for inhibiting the activation of the c-Fos/NFATc1 signaling pathway in the process of osteoclast differentiation.
  • the dosage forms of the medicine for preventing and treating bone loss-related diseases are injections, capsules, oral preparations, and microcapsule preparations.
  • the dosage of the medicine for preventing and treating bone loss-related diseases is 1-10 mg/kg.
  • the present invention utilizes various experimental methods such as tartrate-resistant acid phosphatase staining, phalloidin fluorescent staining, evaluation of bone resorption function of bone plate, reverse transcription real-time fluorescent quantitative PCR, western blotting and other experimental methods, and for the first time it is clear that 2-BP can inhibit BMMs in RANKL. Osteoclast differentiation process under stimulation. And by constructing a mouse model of ovariectomized osteoporosis, it is clear that 2-BP also has the function of inhibiting the differentiation and activation of osteoclasts in vivo, which can significantly alleviate the pathological process of a large number of bone loss caused by estrogen deficiency, and provide a mechanism for preventing and treating bone loss. Loss of the in vivo experimental data base for related diseases.
  • Figure 1 shows the cytotoxicity of 2-BP on monocytes and macrophages detected by CCK-8 method
  • Figure 2 shows the effect of 2-BP on the osteoclast differentiation of monocyte-macrophages detected by tartrate-resistant acid phosphatase staining
  • Figure 3 shows the effect of 2-BP on the production of the characteristic F-actin ring during the osteoclast differentiation of monocyte-macrophages detected by fluorescent staining of phalloidin;
  • Figure 4 is a reverse transcription real-time fluorescence quantitative PCR detection of the expression of related characteristic genes in the process of osteoclast differentiation
  • Figure 5 shows the effect of 2-BP on the expression of transcription factors related to the osteoclast differentiation process detected by western blot
  • Figure 6 is a three-dimensional image of tibial cancellous bone and analysis of related bone mass parameters.
  • Example 1 2--BP inhibits osteoclast differentiation without obvious cytotoxicity
  • Mononuclear macrophages were seeded into 96-well plates with a cell density of 5 ⁇ 10 3 cells/well, and each plate had a total of 24 wells (1 control group, 7 drug-added groups with different concentrations, 3 duplicate wells in each group), The highest concentration of 2-BP was 200 ⁇ M; the medium was complete alpha MEM containing 30 ng/ml M-CSF, cultured for 48, 72, and 96 hours, respectively, and the medium was changed every other day.
  • control group and 3 drug-treated groups were set up, each group had 3 replicate wells, the mononuclear macrophages were seeded in 24-well plates (10 ⁇ 10 4 cells/well), and the medium was changed to containing 50ng/ml every other day.
  • Complete Alpha MEM medium of RANKL and 30ng/ml M-CSF, 6.25, 12.5 and 25 ⁇ M 2-BP were added to the drug treatment group respectively, and the control group was added with the same amount of dimethyl sulfoxide as the 25 ⁇ M 2-BP group. , the medium was changed every other day and cultured for 6 days. When the control group had obvious mature and hypertrophic osteoclasts, it was terminated.
  • the experimental results showed that the number of osteoclasts and the area of each osteoclast were significantly reduced under the treatment of 6.25 ⁇ M 2-BP, the degree of inhibition was more obvious under the treatment of 12.5 ⁇ M, and the differentiation of osteoclasts was basically not formed under the treatment of 25 ⁇ M.
  • control group and 3 drug-treated groups were set up, each group had 3 replicate wells, the mononuclear macrophages were seeded in 24-well plates (10 ⁇ 10 4 cells/well), and the medium was changed to containing 50ng/ml every other day.
  • Complete Alpha MEM medium of RANKL and 30ng/ml M-CSF, 6.25, 12.5 and 25 ⁇ M 2-BP were added to the drug treatment group respectively, and the control group was added with the same amount of dimethyl sulfoxide as the 25 ⁇ M 2-BP group. , the medium was changed every other day and cultured for 6 days. When the control group had obvious mature and hypertrophic osteoclasts, it was terminated.
  • the phosphate buffered saline solution was washed once, and 500ul/well of 4% paraformaldehyde was added to fix it. Cells were incubated for 20 minutes, washed twice with phosphate-buffered saline, and then drained.
  • Example 2 2-BP inhibits the expression of related characteristic genes during osteoclast differentiation
  • the control group and 3 drug-treated groups were set up, each group had 3 replicate wells, and the monocyte-macrophages were seeded in 6-well plates (40 ⁇ 10 4 cells/well), and the medium was changed to containing 50ng/ml every other day.
  • Complete Alpha MEM medium of RANKL and 30ng/ml M-CSF, 6.25, 12.5 and 25 ⁇ M 2-BP were added to the drug treatment group respectively, and the control group was added with the same amount of dimethyl sulfoxide as the 25 ⁇ M 2-BP group. , the medium was changed every other day, cultured for 6 days, and terminated when the control group had obvious mature and hypertrophic osteoclasts.
  • the total RNA was extracted with an RNA extraction kit, and after the concentration was determined, 500 ng of RNA was taken from the reaction system to synthesize cDNA.
  • the reaction system includes reverse transcription buffer, primers, dNTPs, reverse transcriptase, DEPC water, and RNA template (total volume 10 ⁇ l). After the synthesis, the samples were diluted with ultrapure water to 100 ⁇ l and stored in a -80°C refrigerator for later use.
  • a 20 ⁇ l reaction system was used for reverse transcription real-time fluorescent quantitative PCR, and 2 ⁇ l of cDNA, 0.25 ⁇ l of upstream and downstream primers, 10 ⁇ l of SYBR Green qPCR Mix, and 7.5 ⁇ l of ddH2O were added to each system.
  • Example 3 2-BP inhibits the expression of important downstream transcription factors NFATc1 and c-Fos in the signaling pathway during osteoclast differentiation
  • the mononuclear macrophages were seeded in 6-well plates (40 ⁇ 10 4 cells/well, 30ng/ml M-CSF in complete Alpha MEM medium), and divided into control group and drug-treated group. After overnight, the medium was changed to Complete Alpha MEM medium containing 50ng/ml RANKL and 30ng/ml M-CSF, 25 ⁇ M 2-BP was added to the drug treatment group, and the control group was added with the same amount of dimethyl sulfoxide solvent, and the medium was changed every other day, respectively. Protein samples were collected on days 0, 1, 3, and 5.
  • Extraction and concentration determination of protein samples After culturing to the corresponding time point, the supernatant was quickly discarded, washed once with phosphate buffered saline at 4°C, and the 6-well plate was placed on ice. 100 ⁇ l of RIPA cell lysate containing enzyme inhibitor, fully pipetted and lysed on ice for 20 minutes, transfer the lysate to a 1.5 ml EP tube, pre-cool the centrifuge at 4°C in advance, centrifuge at 13,000 rpm for 15 minutes, and collect the supernatant into a new EP tube , use the BCA protein concentration kit to measure the protein concentration of each sample, and store it in a -80 °C refrigerator.
  • Example 4 2-BP prevents and treats osteoporosis induced by ovariectomy in mice
  • Muscle, the fallopian tube and ovary were clipped, and the ovary was cut off after ligation below the ovary, and the muscle, fascia, and skin were sutured. After 4 weeks of spraying, they were sacrificed by dislocation of the neck. The tibia was dissected and fixed in 4% paraformaldehyde for 2 days, and then stored in 75% alcohol.

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Abstract

本发明公开了一种2-溴棕榈酸在制备防治骨丢失相关疾病的药物中的应用，属于医药技术领域。本发明利用抗酒石酸酸性磷酸酶染色、鬼笔环肽荧光染色、骨板骨吸收功能评价、逆转录实时荧光定量PCR、蛋白质免疫印迹等多种实验手段，首次明确2-BP可抑制BMMs在RANKL刺激下破骨细胞分化过程。并通过构建小鼠去卵巢骨质疏松模型，明确2-BP在体内也具有抑制破骨细胞分化、活化的功能，可显著缓解雌激素缺失导致的大量骨丢失病理过程，提供了其可防治骨丢失相关疾病的体内实验数据基础。

Description

2-溴棕榈酸在制备防治骨丢失相关疾病的药物中的应用 技术领域
本发明涉及一种2-溴棕榈酸在制备防治骨丢失相关疾病的药物中的应用，属于医药技术领域。
背景技术
骨作为运动系统的重要组成部分，为机体提供结构依托及保护作用，参与体内造血、钙代谢、内分泌调节等生理过程。骨重建贯穿人的一生，并且大部分骨代谢相关疾病均影响骨重建过程。正常机体的骨重建依赖于破骨细胞(Osteoclast)介导的骨吸收与成骨细胞(Osteoblast)介导的骨形成直接的动态平衡，两者之间失平衡将引起骨过度形成、硬化相关疾病或骨形成不全、过度吸收相关疾病。骨质疏松症(Osteoporosis)是多种原因导致的骨密度和骨质量下降，骨微结构破坏，造成骨脆性增加，从而容易发生骨折的全身性骨病，与机体衰老密切相关。2006年我国骨质疏松症患者近7000万，骨量减少者已超过2亿人，尽管缺乏最新的流行病学数据，但估测我国骨质疏松症和骨量减少人数已远超过以上数字。
骨质疏松症造成的严重后果是骨质疏松性骨折的发生，其危害巨大，是老年患者致残和致死的主要原因之一。基于影像学的流行病学调查显示，我国50岁以上女性椎体骨折患病率约为15％，50岁以后椎体骨折的患病率随增龄而渐增，80岁以上的老年女性骨质疏松性椎体骨折发生率可高达36.6％；而髋部骨折是最严重的骨质疏松性骨折，研究显示发生髋部骨折后1年之内，20％患者 会死于各种并发症，约50％患者致残，生活质量明显下降，给家庭和社会造成沉重的负担。此外，破骨细胞及其介导的骨吸收不仅在骨质疏松症发生发展过程中发挥着重要作用，而且在诸如假体周围骨溶解、风湿性骨关节炎、肿瘤骨转移等骨量丢失疾病中也至关重要，是防治该类疾病的重要靶点。
目前骨质疏松症的预防主要为调整生活方式、钙剂、维生素D，其治疗主要集中在抑制破骨细胞介导的骨吸收或(和)促进成骨细胞介导的骨形成。双膦酸盐类药物是目前临床上应用最为广泛的抗骨质疏松症药物，与骨骼羟磷灰石的亲和力高，能够特异性结合到骨重建活跃的骨表面，抑制破骨细胞功能从而抑制骨吸收。然而，双膦酸盐类药物的应用存在胃肠道不良反应、一过性“流感样”症状、肾脏毒性及发生下颌骨坏死、非典型股骨骨折的危险。降钙素、雌激素受体调节剂(雷洛昔芬)、绝经激素治疗、甲状旁腺素类似物(特立帕肽)以及RANKL单抗(狄诺塞麦)等抗骨质疏松药物都有不同的适应症、用法、用量、副作用，且部分药物价格较高而难以被广泛应用。因此，开发针对抑制破骨细胞分化、成熟的新药物显得尤为重要。
发明内容
为解决上述问题，本发明提供一种2-溴棕榈酸在制备防治骨丢失相关疾病的药物中的应用。
本发明的第一个目的是提供一种2-溴棕榈酸在制备防治骨丢失相关疾病的药物中的应用。
进一步地，所述的骨丢失相关疾病是骨质疏松、磨损颗粒介导骨溶解、类风湿性关节炎或肿瘤骨破坏。
进一步地，所述的防治骨丢失相关疾病的药物是抑制破骨细胞分化成熟的药物。
进一步地，所述的防治骨丢失相关疾病的药物是抑制破骨细胞分化过程中特征基因表达水平的药物。
进一步地，所述的特征基因包括Nfatc1、C-fos、Ctsk、Acp5、Oscar、Dc-stamp、Atp6v0a3、Atp6v0d2中的一种或多种。
进一步地，所述的防治骨丢失相关疾病的药物是抑制破骨细胞分化过程中c-Fos/NFATc1信号通路活化的药物。
进一步地，所述的防治骨丢失相关疾病的药物的剂型为注射剂、胶囊剂、口服制剂、微囊制剂。
进一步地，所述的防治骨丢失相关疾病的药物的给药剂量为1-10mg/kg。
本发明的有益效果：
本发明利用抗酒石酸酸性磷酸酶染色、鬼笔环肽荧光染色、骨板骨吸收功能评价、逆转录实时荧光定量PCR、蛋白质免疫印迹等多种实验手段，首次明确2-BP可抑制BMMs在RANKL刺激下破骨细胞分化过程。并通过构建小鼠去卵巢骨质疏松模型，明确2-BP在体内也具有抑制破骨细胞分化、活化的功能，可显著缓解雌激素缺失导致的大量骨丢失病理过程，提供了其可防治骨丢失相关疾病的体内实验数据基础。
附图说明
图1为CCK-8法检测2-BP对单核巨噬细胞的细胞毒性；
图2为抗酒石酸酸性磷酸酶染色检测2-BP对单核巨噬细胞破骨分化过程的影响；
图3为鬼笔环肽荧光染色检测2-BP对单核巨噬细胞破骨分化过程特征性F-actin ring生成的影响；
图4为逆转录实时荧光定量PCR检测破骨分化过程中相关特征基因的表达；
图5为蛋白质免疫印迹检测2-BP对破骨细胞分化过程相关转录因子表达的影响；
图6为胫骨骨松质三维立体图像并分析相关骨量参数。
具体实施方式
下面结合附图和具体实施例对本发明作进一步说明，以使本领域的技术人员可以更好地理解本发明并能予以实施，但所举实施例不作为对本发明的限定。
实施例1：2--BP抑制破骨细胞分化且无明显细胞毒性
(1)原代骨髓来源单核巨噬细胞BMMs的分离及培养
取5到8周的C57BL/6小鼠一只，拉颈处死后75％医用酒精浸泡3分钟，于超净工作台中解剖小鼠双下肢，分离皮肤、筋膜及膝关节髋关节关节囊，得到小鼠股骨和胫骨，置于事先加入灭菌磷酸缓冲盐溶液的细菌培养皿中。使用无菌剪刀剪开股骨上端及胫骨下端，利用10000rpm瞬时离心法分离出骨髓，加入2ml红细胞裂解液重悬骨髓，静置5分钟裂解红细胞。随后室温下水平离心(1000rpm，5分钟)，弃掉液体后加10ml含30ng/ml M-CSF的完全Alpha MEM培养液，轻柔吹打混匀，移至10cm无菌细胞培养皿中，于37℃、5％CO 2培养箱中孵育。第2天换液以去除未贴壁的杂细胞，继续培养直至骨髓来源的单核巨噬细胞长至80％密度以上。
(2)原代骨髓来源单核巨噬细胞的消化及计数
弃去培养基，无菌磷酸缓冲盐溶液清洗两遍后加入3ml胰酶消化10分钟，显微镜下可见细胞稍微皱缩后加入含血清的培养基1ml，缓慢轻柔吹打细胞后收集培养液，室温下水平离心(1000rpm，5分钟)。弃去上清，加入适量含血清的培养基重悬细胞，轻轻吹打混匀；同时取100μl细胞悬液至EP管，加入1900μl 0.4％台盼蓝染液，3min内显微镜下观察，数出计数板4大格中没有被染液染上色的细胞数目。细胞悬液细胞数/ml＝(4大格细胞总数/4)×稀释倍数(20倍)×10 4。(注：死细胞会被染为蓝色，活细胞不会被染色)
(3)CCK-8法检测2-BP对单核巨噬细胞的细胞毒性(图1)
将单核巨噬细胞种入96孔板，细胞密度为5×10 3细胞/孔，每板共24孔(1个对照组，7个不同浓度加药组，每组3个复孔)，2-BP最高浓度为200μM；培养基为含30ng/ml的M-CSF的完全alpha MEM，分别培养48、72、96小时，隔天换液。待到相应时间点后，每孔加入10μl CCK-8试剂，黑暗条件下在细胞培养箱孵育1小时后取出，酶标仪上充分震荡并测定450nm处吸光度值(活性细胞数目越多，吸光度越高)，实验结果显示2-BP浓度在200μM以下均对单核巨噬细胞增殖无明显抑制作用。
(4)抗酒石酸酸性磷酸酶染色检测2-BP对单核巨噬细胞破骨分化过程的影响(图2)
设对照组及3个药物处理组，每组3个复孔，将单核巨噬细胞种于24孔板中(10×10 4细胞/孔)，隔天将培养基换为含50ng/ml RANKL、30ng/ml M-CSF的完全Alpha MEM培养液，并向药物处理组中分别加入6.25、12.5、25μM 2-BP，对照组加入与25μM 2-BP组等量的二甲基亚砜溶剂，隔天换液，培养6天，当对照组有明显成熟肥大的破骨细胞形成时终止，将培养基吸掉后，磷酸缓冲盐溶液清洗1遍，加入4％多聚甲醛500ul/孔固定细胞20分钟，使用试剂盒进行抗酒石酸酸性磷酸酶染色，倒置显微镜下拍照并后期计算多于3个细胞核、抗酒石酸酸性磷酸酶染色阳性的破骨细胞的数量和每个破骨细胞铺展面积，明确2-BP对破骨细胞分化有抑制作用。实验结果显示在6.25μM 2-BP处理下破骨细胞数量及每个破骨细胞面积明显减少，12.5μM处理下抑制程度更明显，25μM时基本无破骨细胞分化形成。
(5)鬼笔环肽荧光染色检测2-BP对单核巨噬细胞破骨分化过程特征性F-actin ring生成的影响(图3)
设对照组及3个药物处理组，每组3个复孔，将单核巨噬细胞种于24孔板中(10×10 4细胞/孔)，隔天将培养基换为含50ng/ml RANKL、30ng/ml M-CSF的完全Alpha MEM培养液，并向药物处理组中分别加入6.25、12.5、25μM 2-BP，对照组加入与25μM 2-BP组等量的二甲基亚砜溶剂，隔天换液，培养6天，当 对照组有明显成熟肥大的破骨细胞形成时终止，将培养基吸掉后，磷酸缓冲盐溶液清洗1遍，加入4％多聚甲醛500ul/孔固定细胞20分钟，磷酸缓冲盐溶液清洗2遍后吸尽。加入0.1％Triton X-100 300μl/孔孵育5分钟，磷酸缓冲盐溶液清洗2遍后吸尽，1％牛血清白蛋白300μl/孔孵育5分钟后吸尽，加入1％鬼笔环肽孵育20分钟，吸尽后DAPI复染，最后去除染色液并加入磷酸缓冲盐溶液，倒置荧光显微镜下拍照。实验结果提示2-BP可浓度依赖性地抑制单核巨噬细胞破骨分化过程中特征性F-actin ring的生成。
实施例2：2-BP抑制破骨细胞分化过程中相关特征基因的表达
(1)细胞处理及RNA收集分离
设对照组及3个药物处理组，每组3个复孔，将单核巨噬细胞种于6孔板中(40×10 4细胞/孔)，隔天将培养基换为含50ng/ml RANKL、30ng/ml M-CSF的完全Alpha MEM培养液，并向药物处理组中分别加入6.25、12.5、25μM 2-BP，对照组加入与25μM 2-BP组等量的二甲基亚砜溶剂，隔天换液，培养6天，当对照组有明显成熟肥大的破骨细胞形成时终止。应用RNA抽提试剂盒提取总RNA，测定浓度后，取500ng RNA反应体系中合成cDNA。该反应体系包括逆转录buffer、引物、dNTP、逆转录酶、DEPC水、RNA模版(总体积10μl)，合成结束后将样本使用超纯水稀释至100μl保存于-80℃冰箱备用。
(2)逆转录实时荧光定量PCR检测破骨分化过程中相关特征基因的表达(图4)
逆转录实时荧光定量PCR选用20μl反应体系，每个体系分别加入cDNA 2μl、上下游引物各0.25μl、SYBR Green qPCR Mix 10μl、ddH2O 7.5μl。在Bio-Rad CFX96仪器上按照以下反应条件进行：预变性95℃ 3分钟，然后95℃ 10秒、60℃ 30秒循环40次，根据溶解曲线判断每个体系反应是否正常，最后使用荧光定量PCR仪提供的软件分析结果得到CT值，使用2 -ΔΔCT法分析并比较每个样品直接对应基因的mRNA表达量并进行统计分析。实验结果显示破骨细胞相 关特征基因Nfatc1、c-Fos、Ctsk、Acp5、Oscar、Dc-stamp、Atp6v0a3、Atp6v0d2的表达随着2-BP浓度的升高而呈浓度依赖性减少。
实施例3：2-BP抑制破骨细胞分化过程中信号通路下游重要转录因子NFATc1、c-Fos的表达
(1)蛋白样本处理及提取
将单核巨噬细胞种于6孔板中(40×10 4细胞/孔，30ng/ml M-CSF的完全Alpha MEM培养液)，分为对照组药物处理组，过夜后将培养基换为含50ng/ml RANKL、30ng/ml M-CSF的完全Alpha MEM培养液，并向药物处理组中加入25μM 2-BP，对照组加入等量的二甲基亚砜溶剂，隔天换液，分别于0、1、3、5天收集蛋白样本。
蛋白样本提取及浓度测定：待培养至对应时间点后，快速吸弃上清，4℃磷酸缓冲盐溶液清洗1遍，6孔板置于冰上，每孔加入事先配制含蛋白酶抑制剂、磷酸酶抑制剂的RIPA细胞裂解液100μl，充分吹打后冰上裂解20分钟，裂解物移至1.5ml EP管，提前4℃预冷离心机，离心13000rpm，15分钟，收集上清至新EP管中，使用BCA蛋白浓度试剂盒进行每管样本蛋白浓度测定，-80℃冰箱保存。
(2)蛋白质免疫印迹检测2-BP对破骨细胞分化过程相关转录因子表达的影响(图5)
利用蛋白质免疫印迹检测长时程蛋白样本破骨分化过程中NFATc1、c-Fos转录因子蛋白表达水平的变化，以进一步验证2-BP调控破骨细胞分化的作用。实验结果显示2-BP可通过影响抑制NFATc1、c-Fos转录因子在RANKL刺激下的表达水平从而抑制破骨细胞分化。
实施例4：2-BP防治小鼠去卵巢所致骨质疏松
(1)药物溶解、小鼠分组及OVX造模后处理：配制20％DMSO+80％PBS溶剂，加入2-BP配成终浓度为1mg/ml的溶液。C57BL/6小鼠28只，随机分 为4组，每组7只，空白对照组常规饲养，阳性OVX对照组造模成功后只给予上述溶剂腹腔注射，低浓度药物组和高浓度药物组在造模成功后，每日分别给予2-BP 2.5mg/kg和5mg/kg腹腔注射一次。
(2)OVX小鼠去卵巢骨质疏松模型：假手术组麻醉后行背部纵行切口并钝性分离皮下筋膜、肌肉，于左右背侧肾脏投影处剪开肌肉，不进行去卵巢操作直接缝合肌肉、筋膜、皮肤。OVX对照组、低浓度药物组和高浓度药物组实施造模手术，麻醉成功后，在无菌条件下行背部纵行切口并钝性分离皮下筋膜、肌肉，于左右背侧肾脏投影处剪开肌肉，夹取输卵管、卵巢，并于卵巢下方结扎后剪除卵巢，缝合肌肉、筋膜、皮肤。经过4周打药后脱颈处死，解剖取胫骨并放于4％多聚甲醛固定2天后，放入75％酒精中保存。
(3)Micro-CT扫描各组颅骨并通过2D构建、3D重建等操作，得到胫骨骨松质三维立体图像并分析相关骨量参数。结果显示OVX对照组较假手术组出现明显骨量减少的表型，模型构造成功，同时低浓度药物组和高浓度药物组中骨量减少的情况均较OVX对照组得到明显改善，2-BP可显著缓解去卵巢所致骨丢失的过程(图6)。
以上所述实施例仅是为充分说明本发明而所举的较佳的实施例，本发明的保护范围不限于此。本技术领域的技术人员在本发明基础上所作的等同替代或变换，均在本发明的保护范围之内。本发明的保护范围以权利要求书为准。

Claims (8)

  1. 一种2-溴棕榈酸在制备防治骨丢失相关疾病的药物中的应用。
  2. 根据权利要求1所述的应用，其特征在于，所述的骨丢失相关疾病是骨质疏松、磨损颗粒介导骨溶解、类风湿性关节炎或肿瘤骨破坏。
  3. 根据权利要求1所述的应用，其特征在于，所述的防治骨丢失相关疾病的药物是抑制破骨细胞分化成熟的药物。
  4. 根据权利要求1所述的应用，其特征在于，所述的防治骨丢失相关疾病的药物是抑制破骨细胞分化过程中特征基因表达水平的药物。
  5. 根据权利要求4所述的应用，其特征在于，所述的特征基因包括Nfatc1、C-fos、Ctsk、Acp5、Oscar、Dc-stamp、Atp6v0a3、Atp6v0d2中的一种或多种。
  6. 根据权利要求1所述的应用，其特征在于，所述的防治骨丢失相关疾病的药物是抑制破骨细胞分化过程中c-Fos/NFATc1信号通路活化的药物。
  7. 根据权利要求1所述的应用，其特征在于，所述的防治骨丢失相关疾病的药物的剂型为注射剂、胶囊剂、口服制剂、微囊制剂。
  8. 根据权利要求1所述的应用，其特征在于，所述的防治骨丢失相关疾病的药物的给药剂量为1-10mg/kg。
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