CN109010800A - PDGF-BB is inhibiting Adipocyte Differentiation, formation and its application of function - Google Patents
PDGF-BB is inhibiting Adipocyte Differentiation, formation and its application of function Download PDFInfo
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- CN109010800A CN109010800A CN201810730670.7A CN201810730670A CN109010800A CN 109010800 A CN109010800 A CN 109010800A CN 201810730670 A CN201810730670 A CN 201810730670A CN 109010800 A CN109010800 A CN 109010800A
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Abstract
本发明涉及一种血小板衍生生长因子‑BB在抑制脂肪细胞分化、形成及其功能至少一种的药物中的应用。同时还涉及一种抑制脂肪细胞分化、形成的方法、一种抑制脂肪细胞功能的方法，以及一种抑制间充质干细胞中Wint/β‑catenin信号通路介导的Pparγ与Runx2表达的方法。本发明披露了PDGF‑BB如hrPDGF‑BB能够用于生活方式相关疾病的预防，治疗，改善；并用于制备科研及临床相关的抑制脂肪细胞分化、形成和功能的试剂和药物，以及用于抑制与间充质干细胞趋向于脂肪细胞分化相关而促进的其他功能趋向，从而实现预防、治疗由脂肪引起的相关健康问题和疾病。
The invention relates to an application of platelet-derived growth factor-BB in a drug for inhibiting at least one of fat cell differentiation, formation and its function. At the same time, it also relates to a method for inhibiting the differentiation and formation of adipocytes, a method for inhibiting the function of adipocytes, and a method for inhibiting the expression of Pparγ and Runx2 mediated by the Wint/β-catenin signaling pathway in mesenchymal stem cells. The invention discloses that PDGF‑BB such as hrPDGF‑BB can be used for the prevention, treatment and improvement of lifestyle-related diseases; and for the preparation of scientific research and clinical related reagents and drugs for inhibiting adipocyte differentiation, formation and function, and for inhibiting Other functional tendencies promoted related to the tendency of mesenchymal stem cells to differentiate into adipocytes, so as to prevent and treat related health problems and diseases caused by fat.
Description
技术领域technical field
本发明属于医药技术领域，具体的是涉及一种PDGF-BB在抑制脂肪细胞分化、形成及其功能的应用。The invention belongs to the technical field of medicine, and in particular relates to the application of PDGF-BB in inhibiting the differentiation, formation and function of fat cells.
背景技术Background technique
脂肪细胞是终末分化细胞，本身不能通过分裂增殖，主要是由间充质干细胞在多种激素的作用下控制细胞内与脂肪生成相关的基因表达，细胞的形态也会发生一定的变化，并最终形成成熟的脂肪细胞，细胞内可见明显的脂肪累积，细胞变大变圆。脂肪细胞的主要功能是储存多余脂质，在机体糖供应不足时脂肪细胞中的脂肪氧化供能。主要由脂肪细胞组成的脂肪组织不仅仅是储存脂肪的场所，也是机体重要的内分泌组织。脂肪组织除了提供能量外，它还具有维持体温，缓冲外界机械冲击，保护内脏器官和调节生理机能等作用。然而，当脂肪代谢出现异常，糖代谢、蛋白质代谢以及核酸代谢均会受到影响，从而导致能量及物质代谢相关的疾病，如肥胖，糖尿病，以及心脑血管疾病。随着人们生活方式的改变，如饮食习惯的转变和缺乏适宜的体育锻炼，肥胖、糖尿病、心脑血管类疾病等在世界范围内的发病率都很高。而这些受生活方式影响的疾病被称为“生活方式相关的疾病”。据报道，肥胖、糖尿病、心脑血管类疾病是目前涉及人口最多，致死率最高，现已严重威胁到人类健康。因此，对脂肪细胞分化，形成及其功能的调控是防治生活方式相关疾病的重要手段之一。Adipocytes are terminally differentiated cells, which themselves cannot proliferate through division. The main reason is that mesenchymal stem cells control the expression of genes related to adipogenesis in cells under the action of various hormones, and the shape of cells will also change to a certain extent. Finally, mature fat cells are formed, and obvious fat accumulation can be seen in the cells, and the cells become larger and rounder. The main function of adipocytes is to store excess lipids and provide energy for fat oxidation in adipocytes when the body's sugar supply is insufficient. Adipose tissue, mainly composed of adipocytes, is not only a place to store fat, but also an important endocrine tissue of the body. In addition to providing energy, adipose tissue also has the functions of maintaining body temperature, buffering external mechanical shocks, protecting internal organs and regulating physiological functions. However, when fat metabolism is abnormal, glucose metabolism, protein metabolism, and nucleic acid metabolism will all be affected, leading to diseases related to energy and substance metabolism, such as obesity, diabetes, and cardiovascular and cerebrovascular diseases. With changes in people's lifestyles, such as changes in eating habits and lack of appropriate physical exercise, the incidence of obesity, diabetes, and cardiovascular and cerebrovascular diseases is very high worldwide. These lifestyle-affected diseases are called "lifestyle-related diseases". According to reports, obesity, diabetes, and cardiovascular and cerebrovascular diseases currently involve the largest population and have the highest fatality rate, which have seriously threatened human health. Therefore, regulating the differentiation, formation and function of adipocytes is one of the important means to prevent and treat lifestyle-related diseases.
血小板衍生生长因子-BB(PDGF-BB)是一类重要的促生长因子，通过自分泌或旁分泌从而影响各细胞功能。其可应用于促进伤口愈合，包括对神经再生和血管再生的促进作用，且安全性较高。近年来，越来越多的研究发现，PDGF-BB可结合骨修复支架材料联合应用于骨缺损修复中，其作用机制是调控缺损部位的血管再生和新骨形成。截至目前，现有发现中没有PDGF-BB参与调控脂肪代谢的相关报道。Platelet-derived growth factor-BB (PDGF-BB) is an important class of growth-promoting factors, which affect the functions of various cells through autocrine or paracrine. It can be applied to promote wound healing, including the promotion of nerve regeneration and blood vessel regeneration, and has high safety. In recent years, more and more studies have found that PDGF-BB can be used in combination with bone repair scaffold materials in bone defect repair, and its mechanism of action is to regulate angiogenesis and new bone formation at the defect site. Up to now, there are no related reports about PDGF-BB participating in the regulation of fat metabolism in the existing findings.
发明内容Contents of the invention
为了实现本发明的上述目的，特采用以下技术方案：In order to realize the above-mentioned purpose of the present invention, special adopt following technical scheme:
本发明的发明人经过大量研究发现，PDGF-BB具有抑制脂肪细胞的分化、形成及其功能的能力。由此发明人完成了本发明。The inventors of the present invention have found through extensive research that PDGF-BB has the ability to inhibit the differentiation, formation and function of adipocytes. The inventors thus completed the present invention.
本发明提供了一种PDGF-BB在制备用于抑制脂肪细胞的分化、形成及其功能至少一种的药物中的应用。The invention provides an application of PDGF-BB in the preparation of a drug for inhibiting at least one of the differentiation, formation and function of adipocytes.
本发明还提供了一种用于抑制脂肪细胞分化、形成及其功能至少一种的药物。所述药物如下组分：The present invention also provides a drug for inhibiting at least one of fat cell differentiation, formation and its function. The drug consists of the following components:
PDGF-BB；PDGF-BB;
药学上可接受的所述PDGF-BB的载体。A pharmaceutically acceptable carrier of the PDGF-BB.
本发明还提供了一种抑制脂肪细胞分化、形成的方法，所述方法包括将有效计量的PDGF-BB或所述药物与间充质干细胞、前体脂肪细胞和成纤维细胞中的至少一种细胞接触的步骤。The present invention also provides a method for inhibiting the differentiation and formation of adipocytes, the method comprising combining an effective amount of PDGF-BB or the drug with at least one of mesenchymal stem cells, preadipocytes and fibroblasts Steps for cell contact.
本发明还提供了一种抑制脂肪分化、形成的方法，所述方法包括使用有效剂量的hrPDGF-BB或所述药物与间充质干细胞、前体脂肪细胞和成纤维细胞中的至少一种细胞接触的步骤。The present invention also provides a method for inhibiting fat differentiation and formation, the method comprising using an effective dose of hrPDGF-BB or the drug and at least one of mesenchymal stem cells, preadipocytes and fibroblasts contact steps.
本发明还提供了一种抑制脂肪细胞功能的方法，所述方法包括将有效计量的PDGF-BB或所述药物与间充质干细胞、前体脂肪细胞和成纤维细胞中的至少一种细胞接触的步骤。The present invention also provides a method for inhibiting the function of adipocytes, the method comprising contacting an effective dose of PDGF-BB or the drug with at least one of mesenchymal stem cells, preadipocytes and fibroblasts A step of.
本发明还提供了一种抑制间充质干细胞趋向脂肪分化而促进其他分化趋向的方法，所述方法包括使用有效剂量的hrPDGF-BB与间充质干细胞接触的步骤。The present invention also provides a method for inhibiting adipose differentiation of mesenchymal stem cells and promoting other differentiation tendencies, said method comprising the step of contacting mesenchymal stem cells with an effective dose of hrPDGF-BB.
与现有技术相比，本发明的有益效果为：Compared with prior art, the beneficial effect of the present invention is:
根据本发明，PDGF-BB能够抑制脂肪细胞分化，且能够抑制脂肪细胞的相关功能，如减少脂肪细胞中油滴的形成及聚集、抑制脂肪细胞分泌脂蛋白的功能等。基于上述效果，所述PDGF-BB可用于生活方式相关疾病的预防、治疗和/或改善；并用于制备科研及临床相关的用于抑制脂肪细胞分化、形成及其功能的试剂/药物。According to the present invention, PDGF-BB can inhibit the differentiation of adipocytes, and can inhibit the related functions of adipocytes, such as reducing the formation and aggregation of oil droplets in adipocytes, inhibiting the function of adipocytes to secrete lipoproteins, and the like. Based on the above effects, the PDGF-BB can be used for the prevention, treatment and/or improvement of lifestyle-related diseases; and for the preparation of scientific research and clinical related reagents/drugs for inhibiting the differentiation, formation and function of adipocytes.
因此，本发明用于抑制脂肪细胞分化、形成及其功能至少一种的药物能够有效抑制分化、形成，并能够抑制脂肪细胞的相关功能，从而可用于生活方式相关疾病的预防、治疗和/或改善。Therefore, the drug used in the present invention for inhibiting at least one of fat cell differentiation, formation and its function can effectively inhibit differentiation, formation, and can inhibit the related functions of fat cells, so it can be used for the prevention, treatment and/or prevention of lifestyle-related diseases improve.
附图说明Description of drawings
为了更清楚地说明本发明具体实施方式或现有技术中的技术方案，下面将对具体实施方式或现有技术描述中所需要使用的附图作简单地介绍。附图中：In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following will briefly introduce the drawings that need to be used in the description of the specific embodiments or the prior art. In the attached picture:
图1为人源重组血小板衍生生长因子-BB对C3H10 T1/2细胞油滴形成及聚集的影响图；其中，图A为油红O染色图；图B为油红O染色定量分析图；图B中***表示P<0.001和Normal组相比有显著统计学差异，###表示P<0.001和Adipo-8d组相比有显著统计学差异，$$$表示P<0.001和Adipo-8d/PDGF-BB-12.5ng/ml组相比有显著统计学差异；Figure 1 is the effect of human recombinant platelet-derived growth factor-BB on the formation and aggregation of oil droplets in C3H10 T1/2 cells; among them, Figure A is the oil red O staining picture; Figure B is the oil red O staining quantitative analysis figure; Figure B Middle *** means P<0.001 is significantly different from Normal group, ### means P<0.001 is significantly different from Adipo-8d group, $$$ means P<0.001 is different from Adipo-8d /PDGF-BB-12.5ng/ml group compared with significant statistical difference;
图2为人源重组血小板衍生生长因子-BB对脂肪细胞表达脂蛋白的影响图；其中，图A为蛋白印迹图，图B1为脂联系(Adiponectin)蛋白表达定量分析图，图B2为leptin蛋白表达定量分析图，图B3为抵抗素(Resistin)蛋白表达定量分析图；**表示P<0.01和Normal组相比有显著统计学差异，#表示P<0.05和Adipo-8d组相比有统计学差异；Figure 2 is a graph showing the influence of human recombinant platelet-derived growth factor-BB on lipoprotein expression in adipocytes; where, Figure A is a Western blot, Figure B1 is a quantitative analysis of the expression of adiponectin protein, and Figure B2 is the expression of leptin protein Quantitative analysis chart, Figure B3 is the quantitative analysis chart of Resistin protein expression; ** indicates that P<0.01 has a significant statistical difference compared with the Normal group, # indicates that P<0.05 has a statistically significant difference compared with the Adipo-8d group difference;
图3为人源重组血小板衍生生长因子-BB对C3H10 T1/2细胞向成脂分化过程的影响；其中，图A为C/ebpαmRNA变化曲线；图B为PparγmRNA变化曲线；图C为Fabp4 mRNA变化曲线，图D为Lpl mRNA变化曲线；*表示P<0.05，**表示P<0.01和Normal组相比有显著统计学差异，#表示P<0.05，##表示P<0.01和Adipo组相比有统计学差异；Figure 3 is the effect of human recombinant platelet-derived growth factor-BB on the adipogenic differentiation process of C3H10 T1/2 cells; among them, Figure A is the change curve of C/ebpα mRNA; Figure B is the change curve of PparγmRNA; Figure C is the change curve of Fabp4 mRNA , Figure D is the change curve of Lpl mRNA; * means P<0.05, ** means P<0.01 has a significant statistical difference compared with the Normal group, # means P<0.05, ## means P<0.01 compared with the Adipo group Statistical difference;
图4为人源重组血小板衍生生长因子-BB抑制C3H10 T1/2细胞趋向成骨成脂双向分化而促进其他分化的影响；图A为Runx2 mRNA变化曲线；图B为PparγmRNA变化曲线；**表示P<0.01，***表示P<0.001和Normal组相比有显著统计学差异；###表示P<0.001和Osteo-8d组相比有显著统计学差异；$表示P<0.05,$$表示P<0.01和Adipo-2d组相比有显著统计学差异；@表示P<0.05和Adipo-4d组相比有显著统计学差异。Figure 4 shows the effect of human recombinant platelet-derived growth factor-BB on inhibiting the dual differentiation of C3H10 T1/2 cells towards osteogenic and adipogenic differentiation and promoting other differentiation; Figure A is the change curve of Runx2 mRNA; Figure B is the change curve of PparγmRNA; ** indicates P <0.01, *** indicates that there is a significant statistical difference between P<0.001 and the Normal group; ### indicates that there is a significant statistical difference between P<0.001 and the Osteo-8d group; $ indicates that P<0.05, and $$ indicates that P<0.01 has significant statistical difference compared with Adipo-2d group; @ means P<0.05 has significant statistical difference compared with Adipo-4d group.
具体实施方式Detailed ways
为了使本发明要解决的技术问题、技术方案及有益效果更加清楚明白，以下结合实施例与附表，对本发明进行进一步详细说明。应当理解，此处所描述的具体实施例仅仅用以解释本发明，并不用于限定本发明。In order to make the technical problems, technical solutions and beneficial effects to be solved by the present invention clearer, the present invention will be further described in detail below in conjunction with the examples and attached tables. It should be understood that the specific embodiments described here are only used to explain the present invention, not to limit the present invention.
本发明的发明人基于大量研究发现，PDGF-BB具有抑制脂肪细胞分化、形成及其功能等相关能力，基于此，本发明实施例提供了PDGF-BB在下文相关方面的应用。Based on a large number of studies, the inventors of the present invention found that PDGF-BB has the ability to inhibit the differentiation, formation and function of adipocytes. Based on this, the embodiment of the present invention provides the application of PDGF-BB in the following related aspects.
一方面，本发明提供了PDGF-BB在制备用于抑制脂肪细胞分化、形成其功能至少一种药物中的应用。如在一优选实施例中，所述PDGF-BB可以在制备抑制脂肪细胞中脂肪蓄积量和脂肪细胞表达脂蛋白至少一种药物中的应用；其中，所述脂蛋白包括脂联素(Adiponectin)、瘦素(Leptin)、抵抗素(Resistin)中的至少一种。在另一实施例中，所述PDGF-BB可以在制备抑制脂肪细胞分化过种中涉及调控相关基因表达水平的药物中的应用。其中，所述相关基因包括C/ebpα、Pparγ、Fabp4、Lpl中的至少一种。In one aspect, the present invention provides the use of PDGF-BB in the preparation of at least one drug for inhibiting fat cell differentiation and forming its function. As in a preferred embodiment, the PDGF-BB can be used in the preparation of at least one drug that inhibits fat accumulation in adipocytes and lipoprotein expression in adipocytes; wherein, the lipoproteins include adiponectin (Adiponectin) , Leptin (Leptin), resistin (Resistin) at least one. In another embodiment, the PDGF-BB can be used in the preparation of drugs involved in regulating the expression levels of related genes involved in the process of inhibiting the differentiation of adipocytes. Wherein, the related genes include at least one of C/ebpα, Pparγ, Fabp4, and Lpl.
基于上述所述PDGF-BB的应用，优选的，所述药物用于预防、治疗或改善生活方式相关疾病。Based on the above-mentioned application of PDGF-BB, preferably, the drug is used to prevent, treat or improve lifestyle-related diseases.
生活方式疾病在本领域中被定义为，生活方式如饮食习惯、健身习惯、休息、吸烟或饮酒影响疾病的发生和发展的一组疾病。所述PDGF-BB所制备的药物特别适用于饮食习惯和/或健身习惯引起其发生和发展的疾病或病症。这些疾病和病症的实例包括肥胖(特别是绝经后妇女引起的肥胖)、血脂异常(特别是高甘油三酯血症)、糖尿病(特别是II型糖尿病)、高血压、动脉硬化(特别是动脉粥样硬化)、代谢综合征等。本发明所述的代谢综合征是指内脏脂肪蓄积伴随高血糖症、高血压和血脂异常中的两种或两种以上同时发生的病症。Lifestyle diseases are defined in the art as a group of diseases in which lifestyle, such as eating habits, exercise habits, rest, smoking or drinking, affects the onset and progression of the disease. The medicine prepared by the PDGF-BB is especially suitable for diseases or diseases whose occurrence and development are caused by eating habits and/or fitness habits. Examples of such diseases and conditions include obesity (especially in postmenopausal women), dyslipidemia (especially hypertriglyceridemia), diabetes (especially type II diabetes), hypertension, arteriosclerosis (especially atherosclerosis), metabolic syndrome, etc. The metabolic syndrome in the present invention refers to a disease in which visceral fat accumulation is accompanied by two or more of hyperglycemia, hypertension and dyslipidemia.
另外，上述各实施例中的PDGF-BB可以但不仅仅为人源重组血小板衍生生长因子-BB(hrPDGF-BB)，所述hrPDGF-BB，英文名：Human recombinant platelet derived growthfactor-BB。通过生物工程和基因工程技术得到，其所用的基本原料与技术已有相关论文和专利，可以参考现有技术，所以不再叙述。另外，所述hrPDGF-BB可以直接购买于Peprotech(USA)，白色粉末，为以二硫键相连的两条beta-多肽链二聚体，含有218个氨基酸，分子量大小为24.3kDa。产品货号：100-14B，批号：071504，纯度：采用SDS-PAGE和HPLC分析其纯度＞98％。本发明所用的hrPDGF-BB用0.1％BSA溶解，配成10μg/ml的储存液，于－40℃冰箱保存，待用。In addition, the PDGF-BB in the above-mentioned embodiments can be, but not limited to, human recombinant platelet-derived growth factor-BB (hrPDGF-BB). The English name of hrPDGF-BB is Human recombinant platelet derived growth factor-BB. Obtained through bioengineering and genetic engineering techniques, the basic raw materials and technologies used have related papers and patents, and the prior art can be referred to, so it will not be described again. In addition, the hrPDGF-BB can be directly purchased from Peprotech (USA), and is a white powder, which is a dimer of two beta-polypeptide chains linked by disulfide bonds, contains 218 amino acids, and has a molecular weight of 24.3 kDa. Product number: 100-14B, batch number: 071504, purity: analyzed by SDS-PAGE and HPLC, the purity is >98%. The hrPDGF-BB used in the present invention is dissolved in 0.1% BSA, prepared as a storage solution of 10 μg/ml, and stored in a -40°C refrigerator until use.
基于上文所述PDGF-BB的在对脂肪细胞相关性能抑制和其作用的基础上，本发明实施例还提供了一种用于抑制脂肪细胞分化、形成及其功能至少一种的药物。所述药物包括以下物质：Based on the above-mentioned inhibitory effect of PDGF-BB on adipocyte-related properties and its effects, the embodiment of the present invention also provides a drug for inhibiting at least one of adipocyte differentiation, formation and its function. The drugs include the following substances:
1).PDGF-BB；1). PDGF-BB;
2).药学上可接受的PDGF-BB的载体。2). A pharmaceutically acceptable carrier of PDGF-BB.
当然，本发明还可以提供一种用于抑制脂肪细胞分化、形成及其功能至少一种的组合物，所述组合物包括以下物质：Of course, the present invention can also provide a composition for inhibiting at least one of fat cell differentiation, formation and its function, said composition comprising the following substances:
1).PDGF-BB；1). PDGF-BB;
2).药学上可接受的PDGF-BB的载体。2). A pharmaceutically acceptable carrier of PDGF-BB.
其中，所述药物或组合物中所含的PDGF-BB可以但不仅仅为人源重组血小板衍生生长因子-BB(hrPDGF-BB)，所述药学上可接受的PDGF-BB的载体适当地含有不会恶化hrPDGF-BB作用的量的通常用于准药(quasi drug)、医药、食品等技术领域中的其他成分。这样的其它成分的实例包括结合剂(糖浆、阿拉伯树胶、明胶、山梨醇、黄芪胶(tragacanth)、聚乙烯吡咯烷酮等)、填充剂(乳糖、蔗糖、淀粉、磷酸钙、山梨糖醇、甘氨酸等)、润滑剂(硬脂酸镁、滑石、聚乙二醇等)、崩解剂(淀粉、微晶纤维素(microcrystallinecellulose)等)、湿润剂(十二烷基硫酸钠(sodium laury1 sulphate)等)、悬浮剂(山梨糖醇、糖浆、甲基纤维素、葡萄糖浆(glucose syrup)、明胶、氢化食用脂肪等)、乳化剂(卵磷脂、山梨醇单油酸酯、阿拉伯树胶等)、非水性载体(杏仁油、分馏椰子油或甘油、丙二醇、乙醇等疏水酯等)、防腐剂(对羟基苯甲酸甲酯或对羟基苯甲酸丙酯、山梨酸等)、芳香剂(合成香料、天然香料等)、甜味剂(蔗糖、甜叶菊、木糖醇等)、pH调节剂(碳酸氢钠、碳酸钾等)、粉剂(色素、染料、树脂等)、增稠剂(阿拉伯树胶、甲基纤维素等)、抗氧化剂(维生素C、维生素E等)等等。当然所述载体还可以包括一些高分子材料，如PLGA，或者一些无机材料，如HA或TCP等。该些载体一方面能够负载所述PDGF-BB，特别是hrPDGF-BB，保持其生物活性；另一方面可以赋予所述药物或组合物出现多种剂型或者存在的形态。Wherein, the PDGF-BB contained in the medicine or composition can be but not only human recombinant platelet-derived growth factor-BB (hrPDGF-BB), and the carrier of the pharmaceutically acceptable PDGF-BB suitably contains Other ingredients commonly used in technical fields such as quasi drugs, medicines, foods, etc. in amounts that worsen the action of hrPDGF-BB. Examples of such other ingredients include binders (syrup, gum arabic, gelatin, sorbitol, tragacanth, polyvinylpyrrolidone, etc.), fillers (lactose, sucrose, starch, calcium phosphate, sorbitol, glycine, etc. ), lubricants (magnesium stearate, talc, polyethylene glycol, etc.), disintegrants (starch, microcrystalline cellulose, etc.), wetting agents (sodium lauryl sulfate, etc. ), suspending agent (sorbitol, syrup, methylcellulose, glucose syrup (glucose syrup), gelatin, hydrogenated edible fat, etc.), emulsifier (lecithin, sorbitol monooleate, gum arabic, etc.), non- Aqueous carrier (almond oil, fractionated coconut oil or hydrophobic esters such as glycerin, propylene glycol, ethanol, etc.), preservatives (methyl or propyl paraben, sorbic acid, etc.), fragrance (synthetic fragrance, natural Spices, etc.), sweeteners (sucrose, stevia, xylitol, etc.), pH regulators (sodium bicarbonate, potassium carbonate, etc.), powders (pigments, dyes, resins, etc.), thickeners (gum arabic, formazan, etc.) cellulose, etc.), antioxidants (vitamin C, vitamin E, etc.) and so on. Of course, the carrier may also include some polymer materials, such as PLGA, or some inorganic materials, such as HA or TCP. On the one hand, these carriers can load the PDGF-BB, especially hrPDGF-BB, and maintain its biological activity; on the other hand, they can endow the drug or composition with multiple dosage forms or existing forms.
基于上文所述的本发明实施例用于抑制脂肪细胞分化、形成及其功能至少一种的药物或组合物，所述药物或组合物对治疗生活方式疾病的施药方法可以是：对给药对象给药适当剂量的如上所述的药物或组合物。Based on the medicament or composition for inhibiting at least one of the differentiation, formation and function of adipocytes according to the embodiments of the present invention described above, the administration method of the medicament or composition for the treatment of lifestyle diseases may be: The subject is administered an appropriate dose of a drug or composition as described above.
所述药物或组合物可以给药对象为包括人类的哺乳动物。特别地，适用于健康受试者和生活方式相关的疾病的患者。所述药物或组合物可以给药给健康受试者用于预防生活方式相关的疾病，以及给药给生活方式相关的疾病的患者用于治疗或改善疾病。The medicine or composition can be administered to mammals including humans. In particular, it is suitable for healthy subjects and patients with lifestyle-related diseases. The medicament or composition can be administered to healthy subjects for preventing lifestyle-related diseases, and to patients with lifestyle-related diseases for treating or ameliorating diseases.
只要PDGF-BB如hrPDGF-BB可以被给药，本发明的组合物可以制成准药品、药品、食品或研究试剂的任何形式，其中优选准药品、药品或食品。As long as PDGF-BB such as hrPDGF-BB can be administered, the composition of the present invention can be made into any form of quasi-drug, medicine, food or research reagent, among which quasi-drug, medicine or food is preferred.
更具体地，准药品或药品的实例包括片剂、丸剂、粉剂、颗粒剂、胶囊剂、液体和溶液、悬浮液、乳液、注射剂和滴剂等。食品的实例包括饮料(营养补充饮料)、食品(包括营养功能食品、特定保健用食品)、补充剂(片剂、胶囊剂、颗粒剂等)和患者的食物(医院饮食、被护理者的食物等)等。所述药物或组合物也可制成食品添加剂(以液体、粉剂、膏剂等形式)，或可添加到现有的调昧料等中作为食品添加剂。上述形式的组合物可根据本领域中公知的方法制造。More specifically, examples of quasi-drugs or pharmaceuticals include tablets, pills, powders, granules, capsules, liquids and solutions, suspensions, emulsions, injections and drops, and the like. Examples of foods include beverages (nutritional supplement drinks), foods (including nutritional functional foods, foods for specific health use), supplements (tablets, capsules, granules, etc.), and foods for patients (hospital diets, foods for care receivers) and many more. The medicine or composition can also be made into a food additive (in the form of liquid, powder, ointment, etc.), or can be added to existing condiments and the like as a food additive. Compositions of the above forms can be manufactured according to methods known in the art.
本发明对所述药物或组合物的给药方法没有任何限制，具体给药方法可以从口服、注射(皮下、皮内、肌内、静脉内、动脉内)、皮肤给药、经皮给药等中进行适当地选择。The present invention does not have any limitation to the administration method of described medicine or composition, and specific administration method can be from oral administration, injection (subcutaneous, intradermal, intramuscular, intravenous, intraarterial), skin administration, transdermal administration etc. to make an appropriate selection.
基于上文所述PDGF-BB的在对脂肪细胞相关性能抑制和其作用的基础上，本发明实施例还涉及一种抑制脂肪分化、形成的方法，所述方法包括使用有效剂量的PDGF-BB或上文所述用于抑制脂肪细胞分化、形成及其功能至少一种的药物或组合物与间充质干细胞、前体脂肪细胞和成纤维细胞中的至少一种细胞接触的步骤。优选的，如上所述的方法，优选将间充质干细胞有效剂量的PDGF-BB接触。其中，PDGF-BB可以但不仅仅为hrPDGF-BB。Based on the inhibition of the above-mentioned PDGF-BB on the related properties of adipocytes and its effects, the embodiment of the present invention also relates to a method for inhibiting fat differentiation and formation, the method comprising using an effective dose of PDGF-BB Or the above-mentioned step of contacting at least one of mesenchymal stem cells, preadipocytes and fibroblasts with the drug or composition for inhibiting at least one of adipocyte differentiation, formation and its function. Preferably, in the above-mentioned method, the mesenchymal stem cells are preferably contacted with an effective dose of PDGF-BB. Wherein, PDGF-BB can be but not only hrPDGF-BB.
本发明实施例还涉及一种抑制脂肪细胞功能的方法。所述方法包括使用有效剂量的PDGF-BB或上文所述用于抑制脂肪细胞分化、形成及其功能至少一种的药物或组合物与间充质干细胞、前体脂肪细胞和成纤维细胞中的至少一种细胞接触的步骤。优选的，所述方法为抑制脂肪细胞相关脂蛋白表达的方法，包括使用有效剂量的PDGF-BB与间充质干细胞、前体脂肪细胞和成纤维细胞中的至少一种细胞接触的步骤。其中，所述相关脂蛋白包括抵抗素(Resistin)、瘦素(Leptin)或脂联素(Adiponectin)等中的至少一种。其中，PDGF-BB可以但不仅仅为hrPDGF-BB。The embodiment of the present invention also relates to a method for inhibiting the function of fat cells. The method comprises using an effective dose of PDGF-BB or the above-mentioned drug or composition for inhibiting at least one of adipocyte differentiation, formation and function thereof in mesenchymal stem cells, preadipocytes and fibroblasts at least one cell contacting step. Preferably, the method is a method for inhibiting the expression of adipocyte-associated lipoprotein, including the step of using an effective dose of PDGF-BB to contact at least one of mesenchymal stem cells, preadipocytes and fibroblasts. Wherein, the related lipoprotein includes at least one of resistin (Resistin), leptin (Leptin) or adiponectin (Adiponectin) and the like. Wherein, PDGF-BB can be but not only hrPDGF-BB.
本发明实施例还涉及一种抑制间充质干细胞趋向脂肪分化而促进其他分化趋向的方法，所述包括使用有效剂量的PDGF-BB或上文所述用于抑制脂肪细胞分化、形成及其功能至少一种的药物或组合物与间充质干细胞接触的步骤。其中，所述抑制间充质干细胞趋向脂肪分化而促进其他分化趋向的方法包括抑制间充质干细胞中Wint/β-catenin信号通路介导的Pparγ与Runx2表达的方法，因此，抑制间充质干细胞中Wint/β-catenin信号通路介导的Pparγ与Runx2表达的方法，所述方法包括使用有效剂量的PDGF-BB或上文所述用于抑制脂肪细胞分化、形成及其功能至少一种的药物或组合物与间充质干细胞接触的步骤。其中，PDGF-BB可以但不仅仅为hrPDGF-BB。The embodiment of the present invention also relates to a method for inhibiting mesenchymal stem cells from tending to fat differentiation and promoting other differentiation tendencies, which includes using an effective dose of PDGF-BB or the above-mentioned method for inhibiting the differentiation, formation and function of fat cells A step of contacting at least one drug or composition with mesenchymal stem cells. Wherein, the method for inhibiting mesenchymal stem cells from tending to fat differentiation and promoting other differentiation tendencies includes the method of inhibiting the expression of Pparγ and Runx2 mediated by the Wint/β-catenin signaling pathway in mesenchymal stem cells, therefore, inhibiting the expression of mesenchymal stem cells A method for expressing Pparγ and Runx2 mediated by the Wint/β-catenin signaling pathway, the method comprising using an effective dose of PDGF-BB or the above-mentioned drugs for inhibiting at least one of adipocyte differentiation, formation and function thereof Or the step of contacting the composition with the mesenchymal stem cells. Wherein, PDGF-BB can be but not only hrPDGF-BB.
下面将结合实施例对本发明的实施方案进行详细描述，但是本领域技术人员将会理解，下列实施例仅用于说明本发明，而不应视为限制本发明的范围。实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。Embodiments of the present invention will be described in detail below in conjunction with examples, but those skilled in the art will understand that the following examples are only for illustrating the present invention, and should not be considered as limiting the scope of the present invention. Those who do not indicate the specific conditions in the examples are carried out according to the conventional conditions or the conditions suggested by the manufacturer. The reagents or instruments used were not indicated by the manufacturer, and they were all commercially available conventional products.
本发明公开了PDGF-BB如hrPDGF-BB在抑制脂肪分化，形成及其功能的新用途。在下述实施例中，采用C3H10 T1/2细胞(一种从CH3小鼠胚胎发展而来的一类间充质干细胞特性的细胞系)评价hrPDGF-BB抑制脂肪形成的作用影响。结果显示，hrPDGF-BB可以显著抑制C3H10 T1/2细胞向脂肪分化促进其向成骨分化。hrPDGF-BB通过下调了在分化过程中成脂的CCAAT-启动子结合蛋白α(C/ebpα)、过氧化物酶体增殖物激活受体γ(Pparγ)，脂肪酸结合蛋白4(Fabp4)，脂蛋白脂肪酶(Lpl)的基因水平，进而抑制脂肪细胞的形成及油滴的聚集，并且降低了脂联系(Adiponectin)和抵抗素(Resistin)的蛋白表达。另外，我们发现PDGF-BB通过调控Wint/β-catenin信号通路介导的Pparγ与Runx2表达的方法来抑制脂肪形成。基于上述结果，确认本发明所述的血小板衍生生长因子-BB的新用途成立。The invention discloses a new application of PDGF-BB such as hrPDGF-BB in inhibiting fat differentiation, formation and function. In the following examples, C3H10 T1/2 cells (a cell line with mesenchymal stem cell characteristics developed from CH3 mouse embryos) were used to evaluate the effect of hrPDGF-BB on adipogenesis inhibition. The results showed that hrPDGF-BB could significantly inhibit the adipogenic differentiation of C3H10 T1/2 cells and promote their osteogenic differentiation. hrPDGF-BB down-regulates CCAAT-promoter-binding protein α (C/ebpα), peroxisome proliferator-activated receptor γ (Pparγ), fatty acid-binding protein 4 (Fabp4), lipid The gene level of protein lipase (Lpl) inhibits the formation of adipocytes and the aggregation of oil droplets, and reduces the protein expression of adiponectin and resistin. In addition, we found that PDGF-BB inhibits adipogenesis by regulating the expression of Pparγ and Runx2 mediated by Wint/β-catenin signaling pathway. Based on the above results, it was confirmed that the new application of the platelet-derived growth factor-BB of the present invention was established.
实施例1Example 1
人源重组血小板衍生生长因子-BB对C3H10 T1/2细胞油滴形成及聚集的影响实验：Effect of human recombinant platelet-derived growth factor-BB on the formation and aggregation of oil droplets in C3H10 T1/2 cells:
C3H10 T1/2细胞来源于ATCC(American Type Culture Collection,Manassas,VA,USA)，细胞用含3％胎牛血清和1％双抗的低糖DMEM培养基培养。细胞按3万细胞每孔接种于12孔板中，培养2天后进行实验。成脂分化实验分为正常组(Normal)，成脂诱导对照组(Adipo-8d)，成脂诱导8d+人源重组血小板衍生生长因子-BB低、中、高剂量组(Adipo-8d/PDGF-BB-12.5ng/ml,Adipo-8d/PDGF-BB-25ng/ml,Adipo-8d/PDGF-BB-50ng/ml)，成脂分化实验持续观察8天，每隔2天更换一次相应的培养液。细胞用4％多聚甲醛(中性)固定30分钟，然后用油红O染色10分钟，磷酸盐缓冲液清洗3次，光学显微镜下拍照。再用异丙醇闭光溶解油红O，然后于520nm处检测吸光度值。实验数据以Mean±SD表示，应用SPSS 19.0软件进行数据处理，采用ANOVA检验进行统计学分析，p<0.05有统计学意义。结果如图1所示，其中，图A为油红O染色图；图B为油红O染色定量分析图；图B中***表示P<0.001和Normal组相比有显著统计学差异，###表示P<0.001和Adipo-8d组相比有显著统计学差异，$$$表示P<0.001和Adipo-8d/PDGF-BB-12.5ng/ml组相比有显著统计学差异。由图1可知，C3H10T1/2细胞可在特定的诱导坏境中可向成脂方向分化，人源重组血小板衍生生长因子-BB可抑制C3H10 T1/2细胞向成脂分化，并抑制细胞分化终端油滴的形成及聚集，且具有显著性差异，PDGF-BB对成脂的抑制作用具有浓度梯度依赖性。C3H10 T1/2 cells were derived from ATCC (American Type Culture Collection, Manassas, VA, USA), and the cells were cultured in low-glucose DMEM medium containing 3% fetal bovine serum and 1% double antibody. Cells were seeded in 12-well plates with 30,000 cells per well, and the experiment was carried out after 2 days of culture. Adipogenic differentiation experiments were divided into normal group (Normal), adipogenic induction control group (Adipo-8d), adipogenic induction 8d + human recombinant platelet-derived growth factor-BB low, medium and high dose groups (Adipo-8d/PDGF- BB-12.5ng/ml, Adipo-8d/PDGF-BB-25ng/ml, Adipo-8d/PDGF-BB-50ng/ml), the adipogenic differentiation experiment was continuously observed for 8 days, and the corresponding culture was replaced every 2 days liquid. Cells were fixed with 4% paraformaldehyde (neutral) for 30 minutes, then stained with Oil Red O for 10 minutes, washed three times with phosphate buffer saline, and photographed under an optical microscope. Oil red O was then dissolved with isopropanol to block light, and then the absorbance value was detected at 520 nm. The experimental data are expressed as Mean±SD, SPSS 19.0 software is used for data processing, and ANOVA test is used for statistical analysis, p<0.05 is considered statistically significant. The results are shown in Figure 1, wherein, Figure A is the oil red O staining diagram; Figure B is the quantitative analysis diagram of Oil Red O staining; in Figure B, *** indicates that P<0.001 has a significant statistical difference compared with the Normal group, ### indicates a significant statistical difference between P<0.001 and Adipo-8d group, $$$ indicates a significant statistical difference between P<0.001 and Adipo-8d/PDGF-BB-12.5ng/ml group. It can be seen from Figure 1 that C3H10T1/2 cells can differentiate into adipogenicity in a specific induction environment, and human recombinant platelet-derived growth factor-BB can inhibit the differentiation of C3H10T1/2 cells into adipogenicity and the terminal differentiation of cells. The formation and aggregation of oil droplets have significant differences, and the inhibitory effect of PDGF-BB on adipogenesis is concentration-gradient dependent.
实施例2Example 2
人源重组血小板衍生生长因子-BB对脂肪细胞表达脂蛋白的影响实验：Effect of human recombinant platelet-derived growth factor-BB on lipoprotein expression in adipocytes:
C3H10 T1/2细胞用含3％胎牛血清和1％双抗的低糖DMEM培养基培养。细胞按6万细胞每孔接种于6孔板中，培养2天后进行实验。成脂分化实验分为正常组(Normal)，成脂诱导对照组(Adipo-8d)，成脂诱导8d+人源重组血小板衍生生长因子-BB中剂量组(Adipo-8d/PDGF-BB-25ng/ml)，成脂分化实验持续观察8天，每隔2天更换一次相应的培养液。蛋白用RIPA lysis buffer(Pierce，USA)进行裂解，收集蛋白提取液。用BCA蛋白试剂盒(Pierce，USA)测定蛋白浓度，并绘制标准曲线。提取液中加入了上样缓冲液使蛋白变性，采用Western Blotting技术测定脂联系(Adiponectin)、瘦素(Leptin)和抵抗素(Resistin)蛋白水平。实验数据以Mean±SD表示，应用SPSS 19.0软件进行数据处理，采用ANOVA检验进行统计学分析，p<0.05有统计学意义。结果如图2所示，其中，图A为蛋白印迹图，图B1为脂联系(Adiponectin)蛋白表达定量分析图，图B2为瘦素(Leptin)蛋白表达定量分析图，图B3为抵抗素(Resistin)蛋白表达定量分析图。由图2可知，人源重组血小板衍生生长因子-BB可抑制脂肪细胞表达脂蛋白，且具有显著性差异。C3H10 T1/2 cells were cultured in low-glucose DMEM medium containing 3% fetal bovine serum and 1% double antibody. Cells were seeded in 6-well plates at the rate of 60,000 cells per well, and experiments were performed after 2 days of culture. Adipogenic differentiation experiments were divided into normal group (Normal), adipogenic induction control group (Adipo-8d), adipogenic induction 8d+human recombinant platelet-derived growth factor-BB medium dose group (Adipo-8d/PDGF-BB-25ng/ ml), the adipogenic differentiation experiment was continuously observed for 8 days, and the corresponding culture medium was replaced every 2 days. The protein was lysed with RIPA lysis buffer (Pierce, USA), and the protein extract was collected. Protein concentration was determined with BCA protein kit (Pierce, USA), and a standard curve was drawn. Loading buffer was added to the extract to denature the protein, and the protein levels of Adiponectin, Leptin and Resistin were measured by Western Blotting technique. The experimental data are expressed as Mean±SD, SPSS 19.0 software is used for data processing, and ANOVA test is used for statistical analysis, p<0.05 is considered statistically significant. The results are shown in Figure 2, wherein, Figure A is a Western blot, Figure B1 is a quantitative analysis of lipid-linked (Adiponectin) protein expression, Figure B2 is a quantitative analysis of Leptin (Leptin) protein expression, and Figure B3 is resistin ( Quantitative analysis chart of Resistin) protein expression. It can be seen from Figure 2 that human recombinant platelet-derived growth factor-BB can inhibit the expression of lipoproteins in adipocytes, and there is a significant difference.
实施例3Example 3
人源重组血小板衍生生长因子-BB对C3H10 T1/2细胞向成脂分化过程的影响实验：Effect of human recombinant platelet-derived growth factor-BB on the adipogenic differentiation of C3H10 T1/2 cells:
C3H10 T1/2细胞用含3％胎牛血清和1％双抗的低糖DMEM培养基培养。细胞按3万细胞每孔接种于12孔板中，培养2天后进行实验。成脂分化实验分为正常组(Normal)，成脂诱导对照组(Adipo)，成脂诱导8d+人源重组血小板衍生生长因子-BB中剂量组(Adipo/PDGF-BB-25ng/ml)，成脂分化实验持续观察6天，每隔2天更换一次相应的培养液。mRNA用AxygenRNA提取试剂盒提取纯化，纯化的mRNA用TaKaRa逆转录试剂盒得到cDNA，cDNA用Green PCR Master Mix试剂盒进行扩增，用进行裂解，扩增引物请参考附表一。该实验检测了成脂分化过程中2天、4天、6天、8天成脂分化相关的CCAAT-启动子结合蛋白α(C/ebpα)、过氧化物酶体增殖物激活受体γ(Pparγ)、脂肪酸结合蛋白4(Fabp4)、脂蛋白脂肪酶(Lpl)的mRNA水平。实验数据以Mean±SD表示，应用SPSS 19.0软件进行数据处理，采用ANOVA检验进行统计学分析，p<0.05有统计学意义。结果如图3所示；其中，图A为C/ebpαmRNA变化曲线；图B为PparγmRNA变化曲线；图C为Fabp4 mRNA变化曲线，图D为LplmRNA变化曲线；*表示P<0.05，**表示P<0.01和Normal组相比有显著统计学差异，#表示P<0.05，##表示P<0.01和Adipo组相比有统计学差异。由图3可知，人源重组血小板衍生生长因子-BB可抑制脂肪细胞分化过种中C/ebpα，Pparγ，Fabp4，Lpl基因水平的表达，且具有显著性差异。C3H10 T1/2 cells were cultured in low-glucose DMEM medium containing 3% fetal bovine serum and 1% double antibody. Cells were seeded in 12-well plates with 30,000 cells per well, and the experiment was carried out after 2 days of culture. Adipogenic differentiation experiments were divided into normal group (Normal), adipogenic induction control group (Adipo), adipogenic induction 8d + human recombinant platelet-derived growth factor-BB medium dose group (Adipo/PDGF-BB-25ng/ml), adult The adipose differentiation experiment was observed continuously for 6 days, and the corresponding culture medium was replaced every 2 days. The mRNA was extracted and purified with the AxygenRNA extraction kit, and the purified mRNA was obtained with the TaKaRa reverse transcription kit to obtain cDNA. The Green PCR Master Mix kit was used for amplification and cleavage. For the amplification primers, please refer to Attached Table 1. This experiment detected CCAAT-promoter binding protein α (C/ebpα), peroxisome proliferator-activated receptor ), fatty acid binding protein 4 (Fabp4), lipoprotein lipase (Lpl) mRNA levels. The experimental data are expressed as Mean±SD, SPSS 19.0 software is used for data processing, and ANOVA test is used for statistical analysis, p<0.05 is considered statistically significant. The results are shown in Figure 3; among them, Figure A is the change curve of C/ebpαmRNA; Figure B is the change curve of PparγmRNA; Figure C is the change curve of Fabp4 mRNA, and Figure D is the change curve of LplmRNA; * indicates P<0.05, ** indicates P <0.01 has significant statistical difference compared with Normal group, # means P<0.05, ## means P<0.01 has statistical difference compared with Adipo group. It can be seen from Figure 3 that human recombinant platelet-derived growth factor-BB can inhibit the expression of C/ebpα, Pparγ, Fabp4, and Lpl gene levels in adipocyte differentiation, and there are significant differences.
实施例4Example 4
人源重组血小板衍生生长因子-BB抑制C3H10 T1/2细胞趋向成脂双向分化而促进其他分化的实验：Human recombinant platelet-derived growth factor-BB inhibits C3H10 T1/2 cells from adipogenic bidirectional differentiation and promotes other differentiation experiments:
C3H10 T1/2细胞用含3％胎牛血清和1％双抗的低糖DMEM培养基培养。细胞按3万细胞每孔接种于12孔板中，培养2天后进行实验。骨髓间充质干细胞按10万细胞每孔接种于12孔板中，培养2天后进行实验。成脂分化实验分为正常组(Normal)，成脂诱导对照组(Adipo-8d)，成脂诱导8d+人源重组血小板衍生生长因子-BB组中剂量组(Adipo-8d/PDGF-BB-25ng/ml)。成脂成骨双向调控实验分为正常组(Normal)、成骨诱导对照组(Osteo-8d)、成脂诱导2天+成骨诱导6天对照组(Adipo-2d)、成脂诱导4天+成骨诱导4天对照组(Adipo-4d),成脂诱导2天+成骨诱导6天+人源重组血小板衍生生长因子-BB组中剂量组(Adipo-2d/PDGF-BB-25ng/ml)，成脂诱导4天+成骨诱导4天+人源重组血小板衍生生长因子-BB组中剂量组(Adipo-4d/PDGF-BB-25ng/ml)。成脂分化实验持续观察8天，每隔2天更换一次相应的培养液。mRNA用AxygenRNA提取试剂盒提取纯化，纯化的mRNA用TaKaRa逆转录试剂盒得到cDNA，cDNA用Green PCR Master Mix试剂盒进行扩增，用进行裂解，扩增引物请参考附表一。该实验检测了成脂分化过程中8天过氧化物酶体增殖物激活受体γ(Pparγ)和成骨特异性转录因子(Runx2)的mRNA水平。实验数据以Mean±SD表示，应用SPSS19.0软件进行数据处理，采用ANOVA检验进行统计学分析，p<0.05有统计学意义。结果如图4所示；其中，图A为Runx2 mRNA变化曲线；图B为PparγmRNA变化曲线；**表示P<0.01，***表示P<0.001和Normal组相比有显著统计学差异；###表示P<0.001和Osteo-8d组相比有显著统计学差异；$表示P<0.05,$$表示P<0.01和Adipo-2d组相比有显著统计学差异；@表示P<0.05和Adipo-4d组相比有显著统计学差异。由图4可知，人源重组血小板衍生生长因子-BB可抑制脂肪细胞分化过种中Pparγ基因水平的表达，并促进Runx2基因水平的表达，且具有显著性差异。C3H10 T1/2 cells were cultured in low-glucose DMEM medium containing 3% fetal bovine serum and 1% double antibody. Cells were seeded in 12-well plates with 30,000 cells per well, and the experiment was carried out after 2 days of culture. Bone marrow mesenchymal stem cells were seeded in a 12-well plate at a rate of 100,000 cells per well, and the experiment was carried out after 2 days of culture. Adipogenic differentiation experiments were divided into normal group (Normal), adipogenic induction control group (Adipo-8d), adipogenic induction 8d+human recombinant platelet-derived growth factor-BB group medium dose group (Adipo-8d/PDGF-BB-25ng /ml). Adipogenic and osteogenic bidirectional regulation experiments were divided into normal group (Normal), osteogenic induction control group (Osteo-8d), adipogenic induction 2 days + osteogenic induction 6 days control group (Adipo-2d), adipogenic induction 4 days + Osteogenic induction 4 days control group (Adipo-4d), adipogenic induction 2 days + osteogenic induction 6 days + human recombinant platelet-derived growth factor-BB group medium dose group (Adipo-2d/PDGF-BB-25ng/ ml), adipogenic induction for 4 days + osteogenic induction for 4 days + human recombinant platelet-derived growth factor-BB group (Adipo-4d/PDGF-BB-25ng/ml). The adipogenic differentiation experiment was continuously observed for 8 days, and the corresponding culture medium was replaced every 2 days. The mRNA was extracted and purified with the AxygenRNA extraction kit, and the purified mRNA was obtained with the TaKaRa reverse transcription kit to obtain cDNA. The Green PCR Master Mix kit was used for amplification and cleavage. For the amplification primers, please refer to Attached Table 1. This experiment examined the mRNA levels of peroxisome proliferator-activated receptor gamma (Pparγ) and osteogenesis-specific transcription factor (Runx2) at 8 days during adipogenic differentiation. The experimental data are expressed as Mean±SD, SPSS19.0 software is used for data processing, and ANOVA test is used for statistical analysis, p<0.05 is considered statistically significant. The results are shown in Figure 4; wherein, Figure A is the Runx2 mRNA change curve; Figure B is the PparγmRNA change curve; ** indicates that P<0.01, *** indicates that P<0.001 has a significant statistical difference compared with the Normal group;# ## indicates that there is a significant statistical difference between P<0.001 and Osteo-8d group; $ indicates that P<0.05, $$ indicates that there is a significant statistical difference between P<0.01 and Adipo-2d group; @ indicates that P<0.05 and Compared with Adipo-4d group, there was significant statistical difference. It can be seen from Figure 4 that human recombinant platelet-derived growth factor-BB can inhibit the expression of Pparγ gene level in adipocyte differentiation and promote the expression of Runx2 gene level, and there is a significant difference.
表一：RT-PCR实验所用的基因引物Table 1: Gene primers used in RT-PCR experiments
以上所述的实施例仅为本发明的较佳实施例而已，并不用以限制本发明，凡在本发明的精神和原则之内所作的任何修改、等同替换和改进等，均应包含在本发明的保护范围之内。The above-described embodiments are only preferred embodiments of the present invention, and are not intended to limit the present invention. Any modifications, equivalent replacements and improvements made within the spirit and principles of the present invention shall be included in the present invention. within the scope of protection of the invention.
序列表sequence listing
<110> 中国科学院深圳先进技术研究院<110> Shenzhen Institute of Advanced Technology, Chinese Academy of Sciences
<120> PDGF-BB在抑制脂肪细胞分化、形成及其功能的应用<120> Application of PDGF-BB in inhibiting the differentiation, formation and function of adipocytes
<130> 1<130> 1
<160> 12<160> 12
<170> PatentIn version 3.3<170> PatentIn version 3.3
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<211> 23<211> 23
<212> DNA<212>DNA
<220><220>
<223> 人工合成，以用作得到M-GAPDH基因的编码序列的上游引物<223> Artificially synthesized to be used as an upstream primer to obtain the coding sequence of the M-GAPDH gene
<400> 1<400> 1
CATGTTCCAG TATGACTCCA CTC 23CATGTTCCAG TATGACTCCA CTC 23
<210> 2<210> 2
<211> 20<211> 20
<212> DNA<212>DNA
<220><220>
<223> 人工合成，以用作得到M-GAPDH基因的编码序列的下游引物<223> Artificially synthesized to be used as a downstream primer to obtain the coding sequence of the M-GAPDH gene
<400> 2<400> 2
GGCCTCACCC CATTTGATGT 20GGCCTCACCC CATTTGATGT20
<210> 3<210> 3
<211> 22<211> 22
<212> DNA<212>DNA
<220><220>
<223> 人工合成，以用作得到M-C/ebpα基因的编码序列的上游引物<223> Artificially synthesized to be used as an upstream primer to obtain the coding sequence of the M-C/ebpα gene
<400> 3<400> 3
GAACAGCAAC GAGTACCGGG TA 22GAACAGCAACGAGTACCGGGTA22
<210> 4<210> 4
<211> 21<211> 21
<212> DNA<212>DNA
<220><220>
<223> 人工合成，以用作得到M-C/ebpα基因的编码序列的下游引物<223> Artificially synthesized to be used as a downstream primer to obtain the coding sequence of the M-C/ebpα gene
<400> 4<400> 4
GCCATGGCCT TGACCAAGGA G 21GCCATGGCCT TGACCAAGGA G 21
<210> 5<210> 5
<211> 20<211> 20
<212> DNA<212>DNA
<220><220>
<223> 人工合成，以用作得到M-Pparγ基因的编码序列的上游引物<223> Artificially synthesized to be used as an upstream primer to obtain the coding sequence of the M-Pparγ gene
<400> 5<400> 5
CGCTGATGCA CTGCCTATGA 20CGCTGATGCA CTGCCTATGA 20
<210> 6<210> 6
<211> 22<211> 22
<212> DNA<212>DNA
<220><220>
<223> 人工合成，以用作得到M-Pparγ基因的编码序列的下游引物<223> Artificially synthesized to be used as a downstream primer to obtain the coding sequence of the M-Pparγ gene
<400> 6<400> 6
AGAGGTCCAC AGAGCTGATT CC 22AGAGGTCCAC AGAGCTGATT CC 22
<210> 7<210> 7
<211>18<211>18
<212> DNA<212>DNA
<220><220>
<223> 人工合成，以用作得到M-Fabp4基因的编码序列的上游引物<223> Artificially synthesized to be used as an upstream primer to obtain the coding sequence of the M-Fabp4 gene
<400> 7<400> 7
CATGGCCAAG CCCAACAT 18CATGGCCAAGCCCAACAT18
<210> 8<210> 8
<211> 20<211> 20
<212> DNA<212>DNA
<220><220>
<223> 人工合成，以用作得到M-Fabp4基因的编码序列的下游引物<223> Artificially synthesized to be used as a downstream primer to obtain the coding sequence of the M-Fabp4 gene
<400> 8<400> 8
CGCCCAGTTT GAAGGAAATC 20CGCCCAGTTTTGAAGGAAATC20
<210> 9<210> 9
<211> 21<211> 21
<212> DNA<212> DNA
<220><220>
<223> 人工合成，以用作得到M-Lpl基因的编码序列的上游引物<223> Artificially synthesized to be used as an upstream primer to obtain the coding sequence of the M-Lpl gene
<400> 9<400> 9
GGGAGTTTGG CTCCAGAGTT T 21GGGAGTTTGG CTCCAGAGTT T 21
<210> 10<210> 10
<211> 21<211> 21
<212> DNA<212>DNA
<220><220>
<223> 人工合成，以用作得到M-Lpl基因的编码序列的下游引物<223> Artificially synthesized to be used as a downstream primer to obtain the coding sequence of the M-Lpl gene
<400> 10<400> 10
TGTGTCTTCA GGGGTCCTTA G 21TGTGTCTTCA GGGGTCCTTA G 21
<210> 11<210> 11
<211> 20<211> 20
<212> DNA<212>DNA
<220><220>
<223> 人工合成，以用作得到M-Runx2基因的编码序列的上游引物<223> Artificially synthesized to be used as an upstream primer to obtain the coding sequence of the M-Runx2 gene
<400> 11<400> 11
AGACTGCAAG AAGGCTCTGG 20AGACTGCAAG AAGGCTCTGG 20
<210> 12<210> 12
<211> 20<211> 20
<212> DNA<212>DNA
<220><220>
<223> 人工合成，以用作得到M-Runx2基因的编码序列的下游引物<223> Artificially synthesized to be used as a downstream primer to obtain the coding sequence of the M-Runx2 gene
<400> 12<400> 12
TTCCTGCATG GACTGTGGTT 20TTCCTGCATGGACTGTGGTT 20
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Application publication date: 20181218 |