CN114377013B - 去甲波尔定的应用及其促进间充质干细胞向成骨细胞分化的方法 - Google Patents
去甲波尔定的应用及其促进间充质干细胞向成骨细胞分化的方法 Download PDFInfo
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Abstract
本发明属于医药技术领域,具体涉及去甲波尔定的应用以及使用其诱导人羊膜间充质干细胞(hAMSCs)向成骨细胞分化的方法。本发明通过研究发现去甲波尔定能够诱导体外培养的hAMSCs向成骨细胞分化,包括增加早期成骨标志物碱性磷酸酶的表达和酶活,晚期成骨标志钙化结节的形成,以及上调早期和晚期成骨分化相关基因和蛋白的表达。hAMSCs分化形成的成骨细胞可作为种子细胞,应用于骨缺损领域相关疾病的治疗,如骨质疏松、关节炎和骨折等;去甲波尔定也可作为促进干细胞成骨分化的药物用于骨科相关疾病的治疗。本发明为去甲波尔定和hAMSCs在干细胞移植为基础的再生治疗领域的应用提供了理论和实验基础。
Description
技术领域
本发明属于医药技术领域,具体涉及去甲波尔定的应用及其促进间充质干细胞向成骨细胞分化的方法。
背景技术
人羊膜间充质干细胞(hAMSCs)来源的人胎盘羊膜是胚胎早期产物,属于医疗废弃物,取材方便,也不存在伦理道德争议(Du L,Lv R,Yang X,et al.Hypoxic conditionedmedium of placenta-derived mesenchymal stem cells protects against scarformation.Life Sci.2016,149:51-57)。另外hAMSCs能够表达胚胎干细胞的典型标志物,如SSEA-3、SSEA-4、Oct4和Rex1等(Bilic G,Zeisberger S M,Mallik A S,etal.Comparative characterization of cultured human term amnion epithelial andmesenchymal stromal cells for application in cell therapy.Cell Transplant,2008,17:955-968),具有可塑性和多向分化潜能,并且无致瘤性。因此,hAMSCs被认为是组织工程理想的种子细胞。
目前,hAMSCs已证实在一定条件下能够分化为成骨细胞。成骨细胞是骨形成的主要功能细胞,负责骨基质的合成、分泌和矿化,其形成和功能异常引起的骨组织缺损与一列骨骼疾病相关,如骨关节炎、骨质疏松、骨折等。hAMSCs作为成骨细胞的绝佳来源在骨组织缺损修复领域具有极大的应用前景,但其成骨分化的定向调控及作用机制尚未完全明了,目前尚无理想的诱导策略。
随着化学生物学的发展,小分子化合物在调控干细胞命运方面显示出重要价值。我国传统中草药资源丰富,且有长期的临床应用基础,来自于天然中草药的小分子化合物因其结构的多样性和新颖性,具有多样的药理活性(屠鹏飞,曾克武,廖理曦,宋小敏.天然活性小分子靶标蛋白识别方法学研究进展.中国中药杂志,2016,41(01):6-13)。利用天然的小分子化合物诱导干细胞分化将是很有潜力的一条途径。
去甲波尔定(Laurolitsine,LAU)也称新木姜子碱,属于喹啉生物碱类化合物,是传统中草药乌药的主要有效成分之一。去甲波尔定的分子式为C18H19NO4,分子量为313.35。传统中医理论证明,乌药具有行气止痛,温肾散寒的功效(袁代昌,袁玲,袁盼盼,鲁玉梅,南一.乌药的本草考证.山西中医,2021,37(7):55-58)。现代药理研究表明乌药具有广泛的药理活性,如能增强消化液的分泌,有兴奋和增强运动节律作用,可以显著抑制溃疡的形成,明显对抗乙醇诱发的细胞损伤,具有细胞保护作用(邓桂明,向彪,肖小芹,欧阳林旗,刘景诗,魏凤,朱青,蒋司晨.基于网络药理学的乌药主要化学成分药效作用研究.中草药,2018,49(21):5125-5133)。近来又发现还具有较强的镇痛、抗炎作用和显著的抗风湿作用(张剑,罗人仕,杨瑜,刘冰晶.乌药总生物碱抗炎镇痛药理学研究.中国医院药学杂志,2016,36(24):2187-2190)。
目前,关于去甲波尔定药理活性的研究还较少,有研究报道富含去甲波尔定的生物碱具有降血糖和降血脂的活性(Zhang X,Jin Y,Wu Y,Zhang C,Jin D,Zheng Q,LiY.Anti-hyperglycemic and anti-hyperlipidemia effects of the alkaloid-richextract from barks of Litsea glutinosa in ob/ob mice.Sci Rep.2018,8(1):12646)。但去甲波尔定用于诱导干细胞向成骨细胞分化的研究尚未见报道。
发明内容
本发明针对背景技术中提到的问题,为推进小分子干细胞定向成骨分化诱导剂的研发,以及干细胞在骨科相关疾病临床治疗中的应用,本发明的目的一是提供一种可在体外安全、高效诱导人羊膜间充质干细胞向成骨细胞分化的成骨诱导方法。
为了实现上述目的,本发明采用的技术为:
提供去甲波尔定在制备治疗骨缺损疾病的药物中的应用;去甲波尔定在制备促进间充质干细胞向成骨细胞分化的药物中的应用。
根据上述应用,可将去甲波尔定作为主要活性成分制备成药物,或将其与药学上可接受的载体或赋形剂混合制成的药物组合物后,用于治疗骨缺损疾病,还可用于促进间充质干细胞向成骨细胞分化。
上述赋形剂是指可用于药学领域的稀释剂、黏合剂、润滑剂、崩解剂、助溶剂、稳定剂等以及一些药用基质。上述载体是药物领域中可得到的功能性药用辅料,包括表面活性剂、助悬剂、乳化剂以及一些新型药用高分子材料,如环糊精、壳聚糖、聚乳酸(PLA)、聚乙醇酸聚乳酸共聚物(PLGA)、透明质酸等。
本发明还提供了一种诱导人羊膜间充质干细胞向成骨细胞分化的方法,具体是:取用去甲波尔定并将其稀释到安全浓度后,添加到人羊膜间充质干细胞中进行体外诱导培养至形成钙化结节后,即得到成骨细胞。
所得成骨细胞可作为种子细胞应用于骨组织工程和细胞移植中。
进一步,所述去甲波尔定的安全浓度为0.1~50μM,所述诱导培养的时间至少为5天。
优选去甲波尔定的安全浓度为10μM,所述诱导培养的时间至少为21天。
本发明的有益效果在于:提供了去甲波尔定的新应用,即具有诱导间充质干细胞向成骨细胞分化的作用。本发明的研究显示,去甲波尔定能够诱导体外培养的hAMSCs向成骨分化,包括增加早期成骨标志物碱性磷酸酶的表达和酶活,晚期成骨标志钙化结节的形成,以及上调早期和晚期成骨分化相关基因和蛋白的表达。hAMSCs分化形成的成骨细胞可作为种子细胞,应用于骨缺损领域相关疾病的治疗,如骨质疏松、关节炎和骨折等;去甲波尔定也可制备成促进干细胞成骨分化的药物,用于防治骨科相关疾病。为去甲波尔定和hAMSCs在干细胞移植为基础的再生治疗领域的应用提供了理论和实验基础。
附图说明
图1为人羊膜间充质干细胞(hAMSCs)鉴定结果图:(A)表示倒置显微镜观察培养的细胞形态;(B)表示免疫组化鉴定hAMSCs特征标志物;(C)表示流式细胞术鉴定hAMSCs表面特征分子;
图2为不同浓度的去甲波尔定(LAU)对hAMSCs的细胞毒性作用分析结果图(*P<0.05vs con);
图3为LAU诱导hAMSCs向成骨细胞分化过程中成骨细胞标志物碱性磷酸酶的检测结果图:(A)表示成骨细胞标志物碱性磷酸酶的表达;(B)表示成骨细胞标志物碱性磷酸酶的酶活(*P<0.05,**P<0.01vs con);
图4为LAU诱导hAMSCs形成成骨细胞标志物矿化钙结节的结果图(黑色箭头指示钙结节);
图5为LAU诱导hAMSCs特异性成骨相关基因表达上调的结果图:(A)表示早期成骨分化相关基因表达(5d);(B)表示晚期成骨分化相关基因表达(21d)(*P<0.05,**P<0.01vscon);
图6为LAU诱导hAMSCs特异性成骨相关蛋白表达上调的结果图:(A)表示早期(5d)成骨分化相关蛋白典型蛋白免疫印迹条带结果图;(B)表示图A中各蛋白的灰度分析结果图;(C)表示晚期(21d)成骨分化相关蛋白典型蛋白免疫印迹条带结果图;(D)表示图C中各蛋白的灰度分析结果图(*P<0.05,**P<0.01vs con)。
具体实施方式
下面通过具体实施方式进一步详细说明,在此需要说明的是,对于这些实施方式的说明用于帮助理解本发明,但并不构成对本发明的限定。此外,下面所描述的本发明各个实施方式中所涉及的技术特征只要彼此之间未构成冲突就可以相互组合。
下述实施例中的实验方法,如无特殊说明,均为常规方法,下述实施例中所用的试验材料,如无特殊说明,均为可通过常规的商业途径购买得到的。
以下为下述各实施例所使用到的材料:
(1)人羊膜间充质干细胞(hAMSCs)分离培养:将健康孕妇足月剖宫产新鲜胎盘放于预先准备的无菌手术盘中,用含1%青霉素和链霉素的D-PBS轻轻冲洗胎盘表面,去除血渍。用剪刀镊子钝性剥离胎盘表面羊膜,放入装有D-PBS的广口瓶中。于超净工作台将羊膜剪切为1cm-3cm大小的组织块,装入50mL的离心管中,加入约2倍羊膜体积的含0.02%EDTA-2Na的0.125%胰蛋白酶消化液,封口膜封闭试管口,在200rpm 37℃恒温摇床中消化60min,300目不锈钢滤网过滤,将滤下的组织块倒入D-PBS中反复清洗,装入50mL的离心管,加入等体积的含0.05mg/mL DNase I的0.5mg/mL II型胶原酶,封口膜封闭试管口,200rpm 37℃恒温消化,每20min晃动一次,直到试管中沉淀组织消失,但不要超过90min。300目不锈钢滤网再次过滤,1500rpm,4℃离心10min。超净台中弃去上清,沉淀即为hAMSCs。将细胞传至3代时将细胞冻存备用。
(2)去甲波尔定(LAU)药液:称取LAU粉末(CAS货号为5890-18-6),按照说明书加入无菌DMSO制成浓度为100mM的药物母液,待母液充分溶解后用EP管分装并封口,用液氮速冻后冻存于-20℃备用,在进行成骨诱导处理时,将LAU母液稀释至所需工作浓度(0.1-50μM)后使用。
(3)实验分组:LD-DMEM/F12培养基重悬细胞,均匀接种于6孔板放置培养箱中,待细胞融合度达80%弃去培养基。分别设置空白对照组(CON):人羊膜间充质干细胞中只加入HD-DMEM/F12培养基;阳性对照组(PG):人羊膜间充质干细胞中加入含0.1μM地塞米松、10mMβ-磷酸甘油以及50μM抗坏血酸的HG-DMEM/F12培养基;去甲波尔定组(LAU):人羊膜间充质干细胞中加入含不同浓度LAU的HG-DMEM/F12培养基。每三天天更换一次相应的培养基。
实施例1:hAMSCs的分离培养与鉴定
按前述方法分离培养hAMSCs,待细胞融合度达80%以上进行传代培养,第2-5代细胞用于后续实验。当第2代细胞培养到融合度80%时,收集细胞进行形态分析和表型鉴定。图1(A)显示hAMSCs贴壁生长,P0代细胞含较多杂质,传至P3代后,杂质减少,细胞形态呈梭状纤维样,旋涡样生长状态。图1(B)免疫组化结果显示,hAMSC高表达间充质干细胞表面标志波形蛋白,不表达角蛋白CK19。图1(C)流式细胞术结果显示,hAMSCs高表达间充质干细胞表面分子CD29、CD44、CD73、CD90、CD105,不表达造血干细胞表面标志分子CD34、CD11b、CD19、CD45和HLA-DR,具有典型的间充质细胞特征,其纯度较高。
实施例2:LAU对hAMSCs的细胞毒性分析。
为了说明采用本发明中采用去甲波尔定对hAMSCs的安全性,进行相应测试:
取生长良好的P2代hAMSCs消化离心,LD-DMEM/F12培养基重悬,按2×105cells/孔的密度接种100μL hAMSCs于96孔板,贴壁后,在hAMSCs对数生长期弃去培养基,加药处理。设置CON组和PG组。LAU组分别加入含0.1μM、1μM、10μM、20μM、50μM和100μM的药物的HG-DMEM/F12培养基。在培养24h、48h、72h后终止培养,加入10μL CCK8置于96孔板中,放入37℃培养箱2h,用酶标仪在450nm处检测吸光度值。结果如图2所示:与CON组相比,LAU作用24h、48h和7 2h后,在0.1μM-50μM剂量范围内,对hAMSCs无细胞毒性;但LAU在100μM剂量时,24h和48h未显示细胞毒性,但作用72h后呈抑制细胞增殖作用,有较明显的细胞毒性。
实施例3:LAU诱导hAMSCs高表达碱性磷酸酶(ALP)
为了说明采用本发明采用去甲波尔定诱导hAMSCs发生早期成骨细胞分化的情况,进行相应测试:
ALP是早期成骨细胞成熟分化及矿化的标志指标,为骨骼细胞矿化形成骨骼提供物质基础。本实施例以ALP活性作为指标,评价LAU对hAMSCs早期成骨分化的影响。用不同安全剂量(0.1μM,1μM,10μM,20μM和50μM)LAU添加到hAMSCs中。在加药后第5天采用BCIP/NBT染色法测定成骨细胞的标志物ALP。如图3(A)所示,在剂量1μM~20μM范围内,有较多紫色阳性细胞,尤其是10μM剂量的效果最好。进一步的ALP活力测定,按说明书加样后放孵箱中,37℃孵育5~10min,每孔加入100μL反应终止液终止反应。在405nm波长下检测标本的吸光度值。根据碱性磷酸酶活力定义计算碱性磷酸酶活力。如图3(B)结果所示,其表达情况与酶活的结果基本一致,浓度为0.1μM~10μM剂量时,LAU可浓度依赖性增加ALP的酶活,10μM的LAU显示了最优的诱导分化效果。
实施例4:LAU诱导hAMSCs生成钙化结节
为了说明采用本发明采用去甲波尔定诱导hAMSCs发生晚期成骨细胞分化的情况,进行相应测试:
将hAMSCs分成CON组,PG组和LAU组(10μM)进行成骨分化诱导,诱导21天后,采用显微镜观察细胞可见成骨分化所形成的钙盐结节沉积在细胞表面,将细胞固定后,用PBS清洗细胞3次,然后用茜素红S进行染色,室温孵育30分钟后,用PBS缓慢清洗细胞3次,显微镜下观察钙盐结节的形成情况。图4所示的矿化钙结节染色结果表明,与CON相比,PG组与LAU组可以促进hAMSCs成骨分化钙盐结节的沉积,显示有大量成熟的成骨细胞形成。
实施例5:LAU诱导hAMSCs特异性成骨相关基因表达上调
为了说明采用本发明采用去甲波尔定诱导hAMSCs向成骨细胞分化的情况,进行相应测试:
用最佳剂量10μM LAU添加到hAMSCs中,在加药后第5天和第21天,采用RT-qPCR检测成骨相关基因表达情况,各基因的引物序列见表1。结果如图5所示,与CON组相比,LAU组与PG组结果类似,能促进早期(5天)成骨分化相关基因RUNX2、OSX、ALP和晚期(21天)成骨分化相关基因OCN、OPN、CoL1α1的表达。
表1基因的引物序列
实施例6:LAU诱导hAMSCs特异性成骨相关蛋白表达上调
为了说明采用本发明采用去甲波尔定诱导hAMSCs向成骨细胞分化的情况,进行相应测试:
用最佳剂量10μM LAU添加到hAMSCs中,在加药后第5天和第21天,采用Westernblotting检测成骨相关蛋白表达情况。结果如图6所示,与CON组相比,LAU组与PG组结果类似,能促进早期(5天)成骨分化相关蛋白RUNX2、OSX、ALP和晚期(21天)成骨分化相关蛋白OPN、CoL1-α1、OCN的表达。
综合上述实施例的结果可见,LAU能够诱导体外培养的hAMSCs向成骨分化,包括增加早期成骨标志物碱性磷酸酶的表达和酶活,晚期成骨标志钙化结节的形成,以及上调早期和晚期成骨分化相关基因和蛋白的表达。hAMSCs分化形成的成骨细胞可作为种子细胞,应用于骨缺损领域相关疾病的治疗,如骨质疏松、关节炎和骨折等;LAU也可制备成促进干细胞成骨分化的药物,用于防治骨科相关疾病。为LAU和hAMSCs在干细胞移植为基础的再生治疗领域的应用提供了理论和实验基础。
Claims (5)
1.去甲波尔定在制备在体外促进间充质干细胞向成骨细胞分化的药物中的应用。
2.根据权利要求1所述的应用,其特征在于,所述药物还包括药学上可接受的载体和/或赋形剂。
3.一种诱导人羊膜间充质干细胞向成骨细胞分化的方法,其特征在于:取用去甲波尔定并将其稀释到安全浓度后,添加到人羊膜间充质干细胞中进行体外诱导培养至形成钙化结节后,即得到成骨细胞。
4.根据权利要求3所述的一种诱导人羊膜间充质干细胞向成骨细胞分化的方法,其特征在于:所述去甲波尔定的安全浓度为0.1~50μM,所述诱导培养的时间至少为5天。
5.根据权利要求4所述的一种诱导人羊膜间充质干细胞向成骨细胞分化的方法,其特征在于:所述去甲波尔定的安全浓度为10μM,所述诱导培养的时间至少为21天。
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