CN117482213B - 一种促进成骨细胞增殖的乳源多肽fppqsvl及其应用 - Google Patents
一种促进成骨细胞增殖的乳源多肽fppqsvl及其应用 Download PDFInfo
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- CN117482213B CN117482213B CN202311327636.2A CN202311327636A CN117482213B CN 117482213 B CN117482213 B CN 117482213B CN 202311327636 A CN202311327636 A CN 202311327636A CN 117482213 B CN117482213 B CN 117482213B
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Abstract
本发明公开了一种促进成骨细胞增殖的乳源多肽FPPQSVL及其应用，生物活性肽领域。本发明提供的乳源生物活性多肽FPPQSVL具有促进成骨细胞增殖的作用，并能促进成骨细胞矿化，有望用于制备促进成骨细胞增殖和预防骨质疏松的药物。本发明的乳源生物活性多肽FPPQSVL合成方便，可工业化生产，在医药及化妆品等领域具有良好的应用前景。
Description
技术领域
本发明涉及一种促进成骨细胞增殖的乳源多肽FPPQSVL及其应用，生物活性肽领域。
背景技术
骨质疏松症(Osteoporosis)是一种全身代谢性骨病，其主要特征是全身骨量减少、骨组织显微结构被破坏。在健康的个体中可通过骨吸收与骨形成之间的平衡维持骨代谢平衡。然而，内分泌失调、钙吸收不良、营养、激素调节、衰老和许多病理过程破坏了这种平衡，最终引起骨质疏松症。现阶段，临床上治疗骨质疏松症仍以化学药物为主，但它们往往具有一定的风险，长期服用可能引起乳腺癌和心血管疾病等疾病。因此，寻找能够促进骨形成和逆转骨结构损伤的更安全、食物源的天然替代品正受到越来越多的关注。
多肽是蛋白质降解产物，已报道有多种生物活性肽具有调节免疫、预防心血管疾病、促进骨骼健康等作用。这些多肽可以通过微生物发酵、消化酶解、基因工程的人工合成，化学合成等途径获得。大多数生物活性肽是由2-20个氨基酸残基组成，易于被人体消化吸收。2007年Huttunen等人报道了乳三肽IPP可以促进骨细胞增殖和矿化，但是相比于丰富的乳源多肽，研究报道还是比较有限的。因此挖掘促进骨骼健康和预防骨质疏松的乳源生物活性肽具有重要意义。
发明内容
本发明的目的在于提供一种生物活性肽在促进骨骼矿化和/或预防骨质疏松中的新用途。
本发明提供一种生物活性肽FPPQSVL（氨基酸序列为Phe-Pro-Pro-Gln-Ser-Val-Leu），在制备促进骨骼矿化和/或预防骨质疏松的药物中的应用。
在一种实施方式中，所述生物活性多肽为乳源多肽，来源于β-酪蛋白第157-163位的氨基酸残基；所述β-酪蛋白的氨基酸序列如SEQ ID NO:2所示。
在一种实施方式中，所述药物还含有药学上可接受的载体。
在一种实施方式中，所述应用包括但不限于促进骨细胞增殖，促进骨细胞矿化。
本发明还提供了生物活性肽FPPQSVL在制备有助于改善骨密度的保健品中的应用。
在一种实施方式中，所述保健品包括但不限于功能性发酵乳制品。
本发明还提供所述生物活性肽FPPQSVL在制备仿生矿化或骨修复材料方面中的应用。
本发明还提供了一种促进骨细胞增殖产品，包括生物活性肽FPPQSVL或所述生物活性多肽FPPQSVLS的衍生物；所述的促进骨细胞增殖产品包括促进骨密度提高、促进骨细胞增殖或矿化的药物；所述生物活性多肽FPPQSVL的衍生物是指在生物活性多肽FPPQSVL的氨基酸侧链基团上、氨基端或羰基端进行羟基化、羰基化、羧基化、甲基化、乙酰化、磷酸化、酯化或糖基化等修饰，得到的多肽衍生物。
有益效果：本发明的乳源生物活性多肽FPPQSVL具有显著的促进成骨细胞增殖的作用，且能够显著促进成骨细胞矿化，有望用于制备促进成骨细胞增殖和预防骨质疏松的药物。本发明FPPQSVL合成方便，可工业化生产，在医药及化妆品等领域具有良好的应用前景。
附图说明
图1为生物活性肽FPPQSVL的HPLC鉴定结果。
图2为生物活性肽FPPQSVL的LC-MS鉴定结果。
图3为生物活性肽FPPQSVL对成骨细胞的增殖。测试了浓度为0.02、0.2和2 µM的生物活性肽FPPQSVL对成骨细胞增殖的情况；通过酶标仪测量450 nm处的吸光度来量化CCK-8测定。n=3,数据表示为平均值土SEM,并通过单向ANOVA分析，然后进行Tukey多重比较测试（p<0.05）。
图4为生物活性肽FPPQSVL对成骨细胞矿化的影响：（a）用含有抗坏血酸和β-甘油磷酸钠的培养及不同浓度(0.02、0.2和2 µM)的生物活性肽FPPQSVL处理细胞。然后，用茜素红对细胞进行染色，并拍摄图像；（b）用氯化十六烷基吡啶对细胞进行脱色后测量562 nm处的吸光度；数据是来自3个独立实验的平均值土SEM。*p<0.05，**p<0.01，***p<0.001和****p<0.0001与对照组比较。
具体实施方式
实施例1 乳源生物活性肽FPPQSVL的人工合成
1. 树脂活化：从C端的第一个氨基酸，选取0.5mmol/g的Fmoc-An-Resin保护氨基酸，加入到150 mL的固相反应器中，然后添加50 mL的二氯甲烷（DCM）溶胀树脂30min。
2. 脱保护：树脂浸泡30分钟后，抽掉溶液，然后用氮-二甲基酰胺（DMF）洗涤三遍，加入体积比为20％的六氢吡啶DMF溶液，反应5min，再次加入20％的六氢吡啶DMF溶液反应10分钟，中间DMF洗涤一次，反应结束抽干，DMF洗涤3遍，再用哌啶进行脱保护反应30分钟。
3. 洗脱保护：脱保护30分钟后，抽掉哌啶然后用DMF洗6遍，洗完6遍，然后用茚三酮检测脱保护颜色，如果有颜色说明脱保护成功。
5. 加料：洗完脱保护，检测完，按照FPPQSVL肽的顺序，依次加入称量好的Phe和1-胫基-苯骈三唑(HOBT)，然后加入20 ml的DMF将其溶解，再加3ml的N,N二异丙基碳二亚胺(DIC)，混匀后加入到反应器中反应30分钟。
6. 检测：反应30分钟后，用一定量的醋酸酐封头（醋酸酐：DIE A：DCM=1：1：2 ,v：v：v)30分钟，抽掉反应液，用DMF洗3遍后检测，检查否反应完全。
7. 检测已连上后，即重复2~6步骤，依次连接Arg-His-Pro-His-Pro-His-Leu-Ser-Phe，如果检测未过的，用DCM浸泡起来，然后不脱保护，继续连接本步的氨基酸，直到连上为止。待接上最后一个氨基酸后，脱去保护，用DMF洗涤四次，然后用甲醇将树脂抽干。在0℃下缓慢加入配置好的裂解液（TFA：苯甲硫醚：苯酚：三异丙基硅烷：水=82.5：7.5：5：3：2）缓慢搅拌，低温下反应0.5小时，室温下反应2小时，抽滤得到裂解液，将裂解液缓慢加入无水冰乙醚中并搅拌，过滤分离粗品多肽，冰乙醚洗涤3遍，得到粗品肽。
8. 纯化：取少量样进行超声溶解，溶解完成后取20微升样品。放分析型高效液相色谱仪分析，梯度为10-100，时间是0~25分钟，A泵是100%乙腈加0.1%TFA，B泵100%水加0.1%TFA,进样分析，搜取目标峰以后把质谱确定是否正确，确定目标峰以后给出相应的梯度进行制备，制备型液相色谱仪的流动相和分析的相同时间也相同在目标峰出来后进行质谱确认，确认好质谱后给出相应的梯度进行分析，等分析合格后冻干。
采用UPLC和LC-MS对合成的生物活性肽进行鉴定。
UPLC条件如下：
仪器：Wasters 超高效液相色谱串联四极杆飞行时间质谱联用仪；
色谱柱：Gemini-NX 5μ C18 110A，4.6*250㎜；
流速：1.0 mL/min；
温度：50℃；
紫外检测波长：220 nm；
进样体积：20 μL；
梯度条件：A液：含有0.1%甲酸（v/v）的乙腈，B液：含有0.1%甲酸（v/v）的水；
（2）液质鉴定
LC-MS条件如下：
仪器：Agilent 液质联用仪；
检测器：ESI；
雾化器流速：1.5 L/min；
CDL: -20.0v；
CDL温度：250℃；
Block温度：200℃；
Probe电压：+4.5 kv；
检测器电压：1.5 kv；
流动相：50%水/50%乙腈；
流速：0.2 mL/min；
利用超高效液相色谱串联四极杆飞行时间质谱联用仪和液质联用仪，对生物活性肽FPPQSVL进行色谱分析和质谱分析，其质量色谱提取图如图1所示，保留时间如图2所示，可得此峰的多肽质荷比为787.5Da, 保留时间是16.57 min。
实施例2乳源生物肽FPPQSVL对成骨细胞增殖的影响
将小鼠胚胎成骨细胞前体细胞MC3T3-E1细胞接种在96孔板中，2×104个细胞/孔。37℃培养24h后，分别加入含有0、0.02、0.2、2 μmol/L按实施例1的方法制备的生物活性肽FPPQSVL和IPP的a-MEM完全培养基，于37℃培养24h后，无菌条件下每孔加入10 μL CCK-8，培养箱培养30 min，摇匀，用酶标仪在450 nm处测定吸光值。
结果如图3所示，乳源肽FPPQSVL可以促进成骨细胞增殖，且随着剂量的增加而提升，并增殖效果显著高于IPP对MC3T3-E1的增殖作用。当乳源肽FPPQSVL浓度为0.2、2 μmol/L时增值率分别达到129%和134%。
实施例3乳源生物肽FPPQSVL对成骨细胞矿化的影响
钙沉积量反映了细胞成骨分化水平，钙沉积量越多成骨分化水平越高。茜素红与钙离子螯合生成深红色或紫红色复合物，通常用于观察成骨细胞中的矿化钙结节。茜素红染料溶液和钙离子络合物可以用10%氯化十六烷基吡啶溶解，从而对钙结节进行定量分析。染色的钙结节和提取的染色吸收值是成比例的。
将MC3T3-E1细胞按照1×105/孔接种在12孔板中，每孔加入1 mL的a-MEM完全培养基，第二天将培养基更换为含有不同浓度（0、0.02、0.2、2 μmol/L）的诱导培养基a-MEM（10%FBS，50μg/mL的L-抗坏血酸和10mM的β-甘油磷酸钠）。在21天后，用4%多聚甲醛固定细胞，加入1%茜素红S染色液，37℃孵育30 min后终止，冲洗，显微镜下拍照观察。每孔加入l ml 10%氯代十六烷基吡啶，室温孵育1小时后，测562nm处OD值。
结果如图4所示，乳源肽FPPQSVL可以促进成骨细胞中矿化结节的产生，且随着剂量的增加而提升，0.02、0.2、2 μmol/L的乳源肽FPPQSVL分别增加了35%，97%和133%。
虽然本发明已以较佳实施例公开如上，但其并非用以限定本发明，任何熟悉此技术的人，在不脱离本发明的精神和范围内，都可做各种的改动与修饰，因此本发明的保护范围应该以权利要求书所界定的为准。
Claims (7)
1.生物活性肽FPPQSVL在制备预防骨质疏松的药物中的应用。
2.根据权利要求1所述的应用，其特征在于，所述药物还含有药学上可接受的载体。
3.根据权利要求1或2所述的应用，其特征在于，所述应用包括促进骨细胞增殖。
4.根据权利要求1或2所述的应用，其特征在于，所述应用包括促进骨细胞矿化。
5.生物活性肽FPPQSVL在制备有助于改善骨密度的保健品中的应用。
6.根据权利要求5所述的应用，其特征在于，所述保健品包括功能性发酵乳制品。
7.生物活性肽FPPQSVL在制备仿生矿化或骨修复材料方面中的应用。
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