WO2017008375A1 - 珍珠母蛋白n16在制备抑制破骨活性药物中的应用 - Google Patents

珍珠母蛋白n16在制备抑制破骨活性药物中的应用 Download PDF

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WO2017008375A1
WO2017008375A1 PCT/CN2015/088432 CN2015088432W WO2017008375A1 WO 2017008375 A1 WO2017008375 A1 WO 2017008375A1 CN 2015088432 W CN2015088432 W CN 2015088432W WO 2017008375 A1 WO2017008375 A1 WO 2017008375A1
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mother
protein
pearl
cells
osteoclast
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苏薇薇
李沛波
邵鹏柱
王永刚
彭维
马结仪
吴忠
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Sun Yat Sen University
Shenzhen Research Institute of Sun Yat Sen University
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Shenzhen Research Institute of Sun Yat Sen University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans

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  • the present invention relates to a new use of the mother-of-pearl protein N16 in the pharmaceutical field.
  • osteoblasts In human bones, there are osteoblasts and osteoclasts. Osteoblasts promote the formation of new bones, while osteoclasts digest old bones. However, when the balance between bone cells changes and the activity of osteoclasts is greater than that of osteoblasts, the bone mass will be accelerated and the bone will become looser. Increased osteoclast activity can lead to decalcification of bone, increased bone fragility, suppressed growth, thickened bone and bone and joint pain. Background technique
  • mother-of-pearl protein promotes osteogenic activity, promotes bone damage repair, significantly increases bone mineral density in ovariectomized rats, and improves primary cultured cells and MRC-5 (human fetal fibroblast cell line) of neonatal rat skull.
  • the alkaline phosphatase activity of the cells stimulates the differentiation of osteoblasts and produces mineralized nodules that promote bone formation.
  • the mother-of-pearl protein has good biocompatibility and high safety. At home and abroad, mother-of-pearl powder is considered to be a good substitute for bone tissue. Mother-of-pearl transplantation or transdermal injection into animals without immune rejection or obvious side effects.
  • the mother-of-pearl protein N16 belongs to the mother-of-pearl protein and has a molecular weight of 16 kDa. It was found in 1999 by Samata et al. in the insoluble component of the mother-of-pearl. Studies have shown that N16 can affect the shape of calcium carbonate crystals. However, the role of mother-of-pearl protein N16 in inhibiting osteoclast activity has not been reported in the literature. Summary of the invention
  • the object of the present invention is to provide a new way for preparing a drug for inhibiting osteoclast activity, that is, to provide a mother protein N16 for use in preparing a drug for inhibiting osteoclastic activity.
  • the osteoclast inhibiting active drug can be formulated into any clinically applicable pharmaceutical preparation such as a tablet, a capsule, a granule, an injection or the like.
  • the present invention is the first to apply the mother-of-pearl protein N16 to a drug for inhibiting osteolysis. It provides a new choice and idea for inhibiting osteoclast active drugs, and broadens the field of choice for inhibiting osteoclast active drugs.
  • Figure 1 is a 15% SDS-PAGE diagram of the genetically recombinant naphtha protein N16 of the present invention
  • Figure 2 is a graph showing the proliferative results of the mother protein N16 inhibiting the precursor osteoclast RAW 264.7;
  • FIG. 3 shows the results of N16 inhibition of precursor RAW 264.7 differentiation of osteoclasts
  • Figure 4 shows the results of inhibition of osteoclast actin ring by nacre protein N16.
  • the solids in the solid mixture, the liquid in the liquid, and the percentage of solids in the liquid in the respective examples are calculated in wt/wt, vol/vol, wt/vol, respectively, unless otherwise stated.
  • the preparation method of the mother-of-pearl protein N16 specifically includes the following steps:
  • Primer mother-of-pearl protein N16F (5'-GGAATTC) was designed according to the gene sequence of naphtha protein N16 in NCBI database.
  • GCTGT CCAT TATAAGTGC-3' and mother-of-pearl protein N16R (5'-CG TTAATTGTCAAACCGTTC-3'), wherein the bases underlined are NdeI and BamHI restriction endonuclease sites, respectively.
  • the mother-of-pearl protein N16 gene was amplified by PCR, and the reaction procedure was set at 95 ° C for 1 minute, 50 ° C for 20 seconds, and 72 ° C for 1 minute for a total of 25 cycles, and finally 72 ° C for 10 minutes.
  • the amplified naphtha protein N16 gene was ligated to the expression vector pET3 ⁇ and introduced into BL21(DE3)plysS competent Escherichia coli for expression of the N16 protein.
  • BL21(DE3)plysS competent Escherichia coli was grown to log phase, 0.4 mM IPTG was added and the expression of naphtha protein N16 was induced at 37 °C. After 16 h, the cells were collected.
  • the N16 was subjected to preliminary purification and concentration on a DEAE column, wherein the loading buffer was 8 M urea, 20 mM Tris, 40 mM beta-mercaptoethanol, pH 7.0, and the eluent was 8 M urea, 20 mM Tris, 40 mM beta-mercaptoethanol, 1 M NaCl, At pH 7.0, the elution method was a gradient elution. The mother-of-pearl protein N16 was detected by 15% SDS-PAGE.
  • the eluate containing the mother-of-pearl protein N16 was collected, concentrated by ultrafiltration, and subjected to gel exclusion chromatography on a Superdex 75 column to separate and purify the mother-of-pearl protein N16.
  • the purity of the mother-of-pearl protein N16 was determined by 15% SDS-PAGE.
  • the mother-of-pealin protein N16 obtained by recombinant expression has a molecular weight of about 16 kDa, which is in agreement with the predicted results. High purity, no visible visible protein bands, can be used in subsequent experiments.
  • the mother-of-pearl protein N16 inhibits the proliferation of precursor osteoclast RAW 264.7 cells in a time- and concentration-dependent manner. At 48 hours and 72 hours, the concentration of the mother egg N16 on the RAW264.7 cell proliferation inhibition rate was about 20 ⁇ M and 12 ⁇ M, respectively.
  • Example 3 Naphtha N16 inhibits RANKL-induced differentiation of RAW 264.7 cells into osteoclasts
  • RAW264.7 cells with good growth in the logarithmic growth phase were seeded in 96-well culture plates at a rate of 1 to 3 ⁇ 10 3 cells/well, 180 ⁇ l per well. Incubate for 24 hours at 37 ° C in a 5% CO 2 incubator. After the cells were adherent, the original medium was discarded and replaced with ⁇ -MEM medium containing 50 ng/ml RANKL. Different concentrations of mother-of-pearl protein N16 were added to the experimental group, and 4 replicate wells were set for each concentration.
  • a negative control group (containing 50 ng/ml RANKL, containing no mother-of-pearl protein N16) and a normal control group (excluding 50 ng/ml RANKL) were provided.
  • Acid phosphatase staining was used to detect osteoclasts
  • acid phosphatase activity assay kit was used to measure TRAP activity
  • phalloidin-FITC fluorescence staining was used to determine the formation of osteoclast actin ring.
  • FIGs 3 and 4 The results are shown in Figures 3 and 4.
  • A is the result of TRAP staining.
  • B is the statistical result of the number of multinucleated osteoclasts.
  • C is the result of TRAP activity assay.
  • the black arrow shows the multinucleated osteoclast.
  • the scale is 200 ⁇ m.
  • One-way ANOVA *: P ⁇ 0.05, **: P ⁇ 0.01, ***: P ⁇ 0.001, compared with the negative control group (RANKL).
  • FIG. 3A shows the results of TRAP staining.
  • RAW 264.7 cells can differentiate into multinucleated osteoclasts (nucleus number >3) under the induction of RANKL. The cell volume increases, TRAP expression increases, and TRAP staining is purple-red.
  • TRAP is a hallmark enzyme for osteoclast differentiation.
  • the TRAP activity was measured and the results are shown in Figure 3C.
  • Actin ring is a structure that must be formed when osteoclasts phagocytose old bone, and it is important for bone resorption of osteoclasts. Fluorescence staining of phalloidin-FITC showed that a distinct actin ring was formed in RANKL-induced RAW 264.7 cells without the mother-of-pearl protein N16.
  • the mother-of-pearl protein N16 inhibits the formation of actin rings.
  • the number of actin ring formation was reduced and the volume became small.
  • the above experiments show that the mother-of-pearl protein N16 has an inhibitory effect on the differentiation of osteoclasts.
  • the white arrow indicates the actin ring.
  • the scale is 200 ⁇ m.
  • Example 4 Naphtha N16 inhibits the expression of genes involved in osteoclast differentiation
  • RAW264.7 cells grown in a logarithmic growth phase were seeded in 6-well culture plates at 3 to 5 ⁇ 10 4 cells/well. Incubate for 24 hours at 37 ° C in a 5% CO 2 incubator. After the cells were adherent, the original medium was discarded and replaced with ⁇ -MEM medium containing 50 ng/ml RANKL. Different concentrations of mother-of-pealin protein N16 were added to the experimental group. At the same time, a negative control group (containing 50 ng/ml RANKL, containing no mother-of-pearl protein N16) and a blank control group (excluding 50 ng/ml RANKL) were provided. Continue to train for 72 hours. The original medium was discarded, the mRNA was extracted by Trizol method, the cDNA was reverse transcribed, and the expression of osteoclast differentiation-related genes was determined by real-time quantitative PCR (qPCR).
  • qPCR real-time quantitative PCR
  • TRAP is a marker enzyme for osteoclast differentiation. Lack of c-Src and Cathepsin K can damage the bone resorption function of osteoclasts.
  • the transcription factor NFATc1 is an important factor in RANKL-induced osteoclast maturation pathway. RANKL activates the transcription factor NFATc1 through the MAPK cell signaling pathway, which leads to the expression of a series of downstream osteoclast differentiation-related factors, thereby inducing osteoclast differentiation and maturation.
  • the data in Table 2 is a multiple of the corresponding gene expression levels of the experimental group and the blank control group.
  • TRAP, c-Src, CathepsinK and the transcription factor NFATc1 were increased under the induction of RANKL.
  • the addition of naphtha N16 inhibited the expression of TRAP, c-Src, Cathepsin K and the transcription factor NFATc1, indicating that the mother-of-pearl protein N16 can inhibit the differentiation and maturation of osteoclasts.

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Abstract

本发明公开了珍珠母蛋白N16在制药领域中的一种新用途，具体为公开了珍珠母蛋白N16在制备抑制破骨活性药物中的应用。

Description

珍珠母蛋白N16在制备抑制破骨活性药物中的应用
技术邻域
本发明涉及珍珠母蛋白N16在制药领域中的一种新用途。
背景技术
在人的骨头里，有成骨细胞和破骨细胞。成骨细胞是促进新骨的形成，破骨细胞则是消化旧骨。但当骨细胞间的平衡发生变化，破骨细胞活性大于成骨细胞功能时，骨量就会加速丢失，骨质也就变得日渐疏松。破骨细胞活性增强可导致骨质脱钙、骨脆性增加、生长受抑、长骨变粗及骨关节疼痛。背景技术
研究报道，珍珠母蛋白具有促进成骨活性，能促进骨损伤修复，显着提高去卵巢大鼠的骨密度，提高新生鼠颅骨的原代培养细胞和MRC-5(人类胎儿成纤维细胞系)细胞的碱性磷酸酶活性，刺激成骨细胞的分化和产生矿化结节，促进骨形成。珍珠母蛋白具有良好的生物相容性，安全性高。在国内外，珍珠母粉被认为是一种良好的骨组织替代材料。珍珠母移植或透皮注射到动物体内无免疫排斥反应或明显毒副作用。
珍珠母蛋白N16是属于珍珠母蛋白，分子量为16kDa，在1999年由Samata等人在珍珠母不溶于水的组分中发现。研究表明，N16能影响碳酸钙结晶的形状。但珍珠母蛋白N16抑制破骨活性的作用，尚未见文献报道。发明内容
发明内容
本发明的目的是为制备抑制破骨活性药物提供一种新途径，即提供珍珠母蛋白N16在制备抑制破骨活性药物中的应用。
所述抑制破骨活性药物可以制成任何临床上适用的药物制剂，如片剂、胶囊剂、颗粒剂、注射剂等。
本发明是首次将珍珠母蛋白N16应用于抑制破骨活性药物中。为抑制破骨活性药物提供了一种全新的选择和思路，拓宽了抑制破骨活性药物的选择领域。
附图说明
图1为本发明中基因重组的珍珠母蛋白N16的15％SDS-PAGE图；
图2为珍珠母蛋白N16抑制前体破骨细胞RAW 264.7的增殖结果图；
图3为N16抑制前体破骨细胞RAW 264.7分化实验的结果；
图4为珍珠母蛋白N16抑制破骨细胞actin ring的形成结果。
具体实施方式
以下通过具体的实施例进一步说明本发明的技术方案。
各实施例中所涉及的固体混合物中之固体，液体中之液体，以及液体中之固体的百分比分别是以wt/wt、vol/vol、wt/vol计算，除非另有说明。
实施例1：珍珠母蛋白N16的制备
珍珠母蛋白N16的制备方法，具体包括以下步骤：
从马氏珠母贝Pinctada fucata中提取总mRNA，经逆转录，产生单链cDNA。根据NCBI数据库珍珠母蛋白N16的基因序列，设计引物珍珠母蛋白N16F(5'-GGAATTC
Figure PCTCN2015088432-appb-000001
GCTGT CCAT TATAAGTGC-3')和珍珠母蛋白N16R(5'-CG
Figure PCTCN2015088432-appb-000002
TTAATTGTCAAACCGTTC-3')，其中下划线部位碱基分别为NdeI和BamHI限制性内切酶位点。利用PCR法扩增珍珠母蛋白N16基因，反应程序设为95℃1分钟，50℃20秒，72℃1分钟，共25个循环，最后72℃10分钟。扩增所得珍珠母蛋白N16基因连接到表达载体pET3α上，并导入BL21(DE3)plysS competent Escherichia coli，用于表达N16蛋白。当BL21(DE3)plysS competent Escherichia coli生长到对数期，加入0.4mM IPTG，37℃诱导珍珠母蛋白N16的表达。16h后，收集菌体。
珍珠母蛋白N16的纯化：将表达菌体分散在20mM Tris-HCl(pH 7.0)中，超声破碎，离心，弃上清。不溶物以washing buffer(2M urea,20mM Tris,0.1％Tween 20,pH 7.0)超声溶解。离心，收集不溶物，再以solubilizing buffer(8M urea,20mM Tris,40mM beta-mercaptoethanol,pH 7.0)室温溶解过夜。离心，取上清，0.45μm滤膜过滤。以DEAE柱子对N16进行初步纯化以及浓缩，其中，上样缓冲液为8M urea,20mM Tris,40mM beta-mercaptoethanol,pH 7.0，洗脱液为8M urea,20mM Tris,40mM beta-mercaptoethanol,1M NaCl，pH 7.0，洗脱方式为梯度洗脱。15％SDS-PAGE检测珍珠母蛋白N16。收集含珍珠母蛋白N16的洗脱液，超滤浓缩，以Superdex 75柱子进行凝胶排阻层析，分离纯化珍珠母蛋白N16。15％SDS-PAGE检测珍珠母蛋白N16的纯度。
结果见图1。重组表达所得到的珍珠母蛋白N16，分子量约为16kDa，与预测结果相吻合。纯度高，无明显可见杂蛋白条带，可用于后续实验。
实施例2：珍珠母蛋白N16抑制前体破骨细胞增殖
采用小鼠前体破骨细胞株RAW 264.7，于100U/mL青霉素，100g/mL链霉素的DMEM培养液中培养，培养细胞置于37℃、饱和湿度5％C02浓度下，2～3天换液1次。取对数生长期生长状态良好的细胞以3～5×103个/孔接种于96孔培养板，每孔180μl。37℃、5％C02培养箱中孵育24小时。细胞贴壁生长后，实验组加入不同浓度的珍珠母蛋白N16，每个浓度设4个复孔。同时设阴性对照组(N16＝0μM)。继续培养24、48、72小时。MTT法检测细胞增殖情况。
结果见图2。图2中设阴性对照组(珍珠母蛋白N16＝0μM)增殖率为100％。(One-way ANOVA,*:P<0.05,**:P<0.01,***:P<0.001，与阴性对照组比较。)
珍珠母蛋白N16能抑制前体破骨细胞RAW 264.7细胞的增殖，其抑制作用具有时间依赖性和浓度依赖性。作用48小时和72小时，珍珠母蛋白N16对RAW264.7细胞增殖半数抑制率的浓度分别约为20μM和12μM。
实施例3：珍珠母蛋白N16抑制RANKL诱导RAW 264.7细胞向破骨细胞分化
采用对数生长期生长状态良好的RAW264.7细胞，以1～3×103个/孔接种于96孔培养板，每孔180μl。37℃、5％C02培养箱中孵育24小时。细胞贴壁生长后，弃去原培养基，更换为含50ng/ml RANKL的α-MEM培养基。实验组加入不同浓度的珍珠母蛋白N16，每个浓度设4个复孔。同时设阴性对照组(含50ng/ml RANKL，不含珍珠母蛋白N16)和正常对照组(不含50ng/ml RANKL)。继续培养72小时。酸性磷酸酶染色法检测破骨细胞，酸性磷酸酶活性测定试剂盒测定TRAP活性，phalloidin-FITC荧光染色法测定破骨细胞actin ring的形成。
结果见图3、图4。图3中，A为TRAP染色结果。B为多核破骨细胞数目统计结果。C为TRAP活性测定结果。黑色箭头所示为多核破骨细胞。标尺为200μm。(Non-paired Student’s t-test,#:P<0.05,##:P<0.01,###:P<0.001,阴性对照组(RANKL)与空白对照组(RANKL-)比较；One-way ANOVA,*:P<0.05,**:P<0.01,***:P<0.001,与阴性对照组(RANKL)比较。
图3A为TRAP染色结果。RAW 264.7细胞在RANKL的诱导下，能分化成为多核破骨细胞(核数目>3)，细胞体积增大，TRAP表达增多，TRAP染色呈紫红色。珍珠母蛋白N16能抑制RANKL诱导RAW 264.7细胞分化成为成熟的破骨细胞。统计每组多核破骨细胞的数目并比较每组差异，统计结果见图3B。可见珍珠母蛋白N16对多核破骨细胞形成的抑制作用具有浓度依赖性(IC50＝0.5μM)。TRAP是破骨细胞分化的标志性酶。测定TRAP活性，结果见图3C。珍珠母蛋白N16抑 制RAW 264.7细胞TRAP的活性(IC50＝0.6μM)，该抑制作用具有浓度依赖性。Actin ring是破骨细胞吞噬旧骨时所必需形成的结构，对破骨细胞的进行骨吸收具有重要意义。phalloidin-FITC荧光染色结果显示，在不含珍珠母蛋白N16的RANKL诱导的RAW 264.7细胞中形成了明显的actin ring。珍珠母蛋白N16抑制actin ring的形成。在含珍珠母蛋白N16的实验组中，actin ring形成的数目减少，体积变小。上述实验表明，珍珠母蛋白N16对破骨细胞的分化具有抑制作用。图4中，白色箭头所示为actin ring。标尺为200μm。
实施例4：珍珠母蛋白N16抑制破骨细胞分化相关基因的表达
采用对数生长期生长状态良好的RAW264.7细胞，以,3～5×104个/孔接种于6孔培养板。37℃、5％C02培养箱中孵育24小时。细胞贴壁生长后，弃去原培养基，更换为含50ng/ml RANKL的α-MEM培养基。实验组加入不同浓度的珍珠母蛋白N16。同时设阴性对照组(含50ng/ml RANKL，不含珍珠母蛋白N16)和空白对照组(不含50ng/ml RANKL)。继续培养72小时。弃去原培养基，Trizol法提取mRNA，逆转录得cDNA，实时定量PCR法(qPCR)测定破骨细胞分化相关基因的表达情况。
表1实施例2中所用到的引物
Figure PCTCN2015088432-appb-000003
TRAP、c-Src、Cathepsin K和转录因子NFATc1与破骨细胞分化相关。TRAP是破骨细胞分化标志性酶，缺乏c-Src、Cathepsin K会损伤破骨细胞的骨吸收功能。转录因子NFATc1是RANKL诱导破骨细胞成熟通路中的重要因子。RANKL通过MAPK细胞信号通路，激活转录因子NFATc1，从而引起一系列下游破骨细胞分化相关因子的表达，从而诱导破骨细胞分化成熟。表2中的数据为实验组与空白对照组相应基因表达量比较的倍数。阴性对照组中，TRAP、c-Src、CathepsinK和转录因子NFATc1在RANKL的诱导下，表达量增高。加入珍珠母蛋白N16后，能抑制TRAP、c-Src、Cathepsin K和转录因子NFATc1的表达，表明珍珠母蛋白N16能抑制破骨细胞的分化成熟。
表2 N 16对破骨细胞分化相关基因的表达影响(mean±SD)
Figure PCTCN2015088432-appb-000004
(One-way ANOVA,*:P<0.05,**:P<0.01,***:P<0.001，与阴性对照组比较).

Claims (2)

  1. 珍珠母蛋白N16在制备抑制破骨活性药物中的应用。
  2. 如权利要求1所述的应用，其特征在于，所述药物的剂型是片剂、胶囊剂、颗粒剂、注射剂或鼻吸入剂。
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