CN116656601A - β-羟基丁酸在促进脂肪细胞分化中的用途 - Google Patents
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Abstract
本发明公开了β‑羟基丁酸在促进脂肪细胞分化中的用途。β‑羟基丁酸作为血液中主要的酮体之一，既是脂肪分解出的代谢产物，又参与了脂肪细胞中脂质的合成，是机体能量代谢的关键性分子。本发明发现在诱导3T3‑L1前脂肪细胞向脂肪细胞分化的过程中添加β‑羟基丁酸，可显著提高3T3‑L1细胞向脂肪细胞的分化能力。该作用扩展了β‑羟基丁酸作为能量代谢物的功能，同时有助于预防和治疗脂肪形成缺陷所引发的代谢性疾病。
Description
技术领域
本发明属于药物研发技术领域，具体涉及β-羟基丁酸在促进脂肪细胞分化中的用途。
背景技术
从17-19胚龄的Swiss小鼠中分离建立的3T3-L1细胞系是用于体外研究脂肪分化的经典细胞系。当3T3-L1细胞中有甘油三酯积累并表达脂肪细胞特异性蛋白时，表明它已经分化为脂肪细胞。脂肪细胞正常的分化过程主要包括成纤维细胞、前脂肪细胞、不成熟的脂肪细胞和成熟的脂肪细胞四个阶段，当脂肪细胞无法正常分化时会造成脂肪营养不良，从而引发严重的胰岛素抵抗、血脂异常和非酒精性脂肪性肝病等代谢性疾病，对人类的身体健康造成了严重的困扰。因此，寻找到促进脂肪细胞分化的方法，将有助于为脂肪细胞的异常分化提供新的治疗方案，对预防和缓解脂肪功能障碍至关重要。
β-羟基丁酸(β-hydroxybutyrate acid，β-OHB)，又称3-羟基丁酸，化学式：CH3CH(OH)CH2COOH，分子量约为104.1kD，CAS号150-83-4。其结构式为如下所示：
β-OHB在体内主要是由脂肪酸代谢生成的乙酰乙酸经肝脏降解生成，并且血液中的β-OHB浓度受饮食结构和人体生理状态的影响。正常人体血液中β-OHB浓度小于0.7mmol/L，长期禁食后血浆中β-OHB甚至能达到6-8mM。β-OHB可抑制结肠癌的发生与生长；β-OHB能穿过血脑屏障，代替葡萄糖作为大脑神经元的能量来源，维持神经元的完整性，并且通过降低活性氧生成、提高NAD+/NADH比率来应对谷氨酸诱导的氧化应激反应进而保护神经元；β-羟基丁酸在临床上已用于镇静，实验室还将其作为组蛋白去乙酰化酶(HDAC)的抑制剂使用。但是，β-OHB是否能够促进脂肪细胞分化目前尚未见报道。
发明内容
本发明的目的在于提供β-羟基丁酸在促进脂肪细胞分化中的用途。
β-羟基丁酸在制备促进脂肪细胞分化的制剂中的用途。
具体的，通过在培养基中添加β-羟基丁酸，诱导3T3-L1前脂肪细胞向脂肪细胞分化。
β-羟基丁酸在制备促进脂肪细胞分化的药物中的用途。
一种促进脂肪细胞分化的药用组合物，包括β-羟基丁酸和其药学上可接受的盐。
优选的，还包括一种或多种药学上可接受的辅料或载体。
所述辅料或载体包括稀释剂、赋形剂、填充剂、粘合剂、湿润剂、崩解剂、吸收促进剂、表面活性剂、吸附载体或润滑剂。
所述药用组合物可制成的剂型为片剂、胶囊剂、泡腾片、颗粒剂、散剂、分散片、口服液、丸剂或注射剂。
本发明的有益效果：本发明发现β-羟基丁酸具有加速脂肪细胞分化的作用，可改善脂肪发育不良的症状。该作用可应用于预防、治疗和改善脂肪功能障碍所导致的胰岛素抵抗、血脂异常等代谢性疾病。
附图说明
图1是采用Western blot方法检测所诱导的脂肪细胞在β-羟基丁酸处理后脂肪分化标志分子Pparγ和Adipsin的蛋白表达水平。
图2是采用油红O染色检测所诱导的脂肪细胞在β-羟基丁酸处理后脂滴大小和含量的变化情况。
具体实施方式
为了便于理解本发明，下面将对本发明进行更全面的描述。但是，本发明可以以许多不同的形式来实现，并不限于本文所描述的实施例。相反地，提供这些实施例的目的是使对本发明的公开内容的理解更加透彻全面。
实施例1β-羟基丁酸对3T3-L1细胞向脂肪细胞分化的作用
1.β-羟基丁酸的配制
β-羟基丁酸购自美国MCE公司，产品目录号：HY-113378，CAS号300-85-6，取100mgβ-羟基丁酸粉末，加入9.6061ml H2O，震荡使其充分溶解，混匀，此储液终浓度为100mM，分装后保存于-80℃。
2.诱导3T3-L1细胞向脂肪细胞分化的方案
将3T3-L1细胞按照3x105/ml的密度铺于60mm培养皿中，待细胞长满，更换新鲜培养基继续培养48h使细胞接触抑制。按表1的培养基配方配制相应的分化培养基和维持培养基，细胞接触抑制后更换分化培养基培养48h，再更换维持培养基培养，之后每48h更换一次培养基。
表1成脂诱导培养基配方
3.细胞的处理与实验分组
取诱导分化5天的3T3-L1细胞并按如下组别进行干预：
对照组：对照组为阴性对照，培养基中不添加β-羟基丁酸；
β-羟基丁酸干预组：在培养基中加入β-羟基丁酸并使终浓度为2mM。
4.Western Blot实验
按照常规方法收集并裂解诱导分化6天的3T3-L1细胞，提取总蛋白：
(1)β-羟基丁酸干预24h后2000r/min离心5min，收集3T3-L1细胞，弃上清。
(2)加入适量RIPA Lysis Buffer(含蛋白酶抑制剂，磷酸酶抑制剂A、B、C，PMSF，DTT)。
(3)冰上放置30min，使细胞充分裂解，每5min涡旋震荡一次。
(4)12,000r/min离心10min，转移上清液至新离心管中，加入Loading buffer充分混合，100℃水浴10min后进行下一步分析。
(5)蛋白免疫印迹分析，检测增脂肪分化标志分子Pparγ和Adipsin的表达变化。
实验所用抗体如表2所示：
表2
5.油红O染色
3T3-L1细胞诱导分化6天后，缓慢吸去细胞培养液，用PBS洗涤1次，加入4％多聚甲醛固定10min，PBS漂洗2次。根据样品数量及每个样品所需的染色工作液的体积，按照油红O溶液和油红O稀释液3:2的比例配制油红O染色工作液，混匀后静置10min，然后用0.45μm针头滤器过滤，需在两小时内使用。加入适量染色洗涤液覆盖细胞20秒，吸除染色洗涤液，加入适量油红O染色工作液均匀覆盖细胞，染色10-20分钟。去除油红O染色工作液，加入适量染色洗涤液，静置30秒，然后再去除染色洗涤液，用PBS洗涤20秒。加入适量PBS均匀覆盖细胞，在光学显微镜下观察和拍照。
实验结果：
如图1所示，在3T3-L1细胞向脂肪细胞分化过程中施加β-羟基丁酸，与空白对照组相比，β-羟基丁酸显著促进了脂肪分化标志性分子Pparγ和Adipsin蛋白的表达。同时，油红O染色的结果也显示，β-羟基丁酸处理后细胞中脂质含量也明显增加(图2)。以上结果说明，β-羟基丁酸促进了3T3-L1细胞向脂肪细胞的分化。
以上所述实施例仅表达了本发明的几种实施方式，其描述较为具体和详细，但并不能因此而理解为对发明专利范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干变形和改进，这些都属于本发明的保护范围。因此，本发明专利的保护范围应以所附权利要求为准。
Claims (7)
1.β-羟基丁酸在制备促进脂肪细胞分化的制剂中的用途。
2.根据权利要求1所述β-羟基丁酸在制备促脂肪细胞分化的制剂中的用途，其特征在于，通过在培养基中添加β-羟基丁酸，诱导3T3-L1前脂肪细胞向脂肪细胞分化。
3.β-羟基丁酸在制备干预脂肪细胞分化的药物中的用途。
4.一种促进脂肪细胞分化的药用组合物，其特征在于，包括β-羟基丁酸和其药学上可接受的盐。
5.根据权利要求4所述促进脂肪细胞分化的药用组合物，其特征在于，还包括一种或多种药学上可接受的辅料或载体。
6.根据权利要求5所述促进脂肪细胞分化的药用组合物，其特征在于，所述辅料或载体包括稀释剂、赋形剂、填充剂、粘合剂、湿润剂、崩解剂、吸收促进剂、表面活性剂、吸附载体或润滑剂。
7.根据权利要求4所述促进脂肪细胞分化药用组合物，其特征在于，所述药用组合物可制成的剂型为片剂、胶囊剂、泡腾片、颗粒剂、散剂、分散片、口服液、丸剂或注射剂。
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