CN115029299B - JAK2抑制剂在胰岛β细胞诱导分化中的应用 - Google Patents
JAK2抑制剂在胰岛β细胞诱导分化中的应用 Download PDFInfo
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- CN115029299B CN115029299B CN202210812902.XA CN202210812902A CN115029299B CN 115029299 B CN115029299 B CN 115029299B CN 202210812902 A CN202210812902 A CN 202210812902A CN 115029299 B CN115029299 B CN 115029299B
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Abstract
本发明涉及生物技术领域,尤其涉及JAK2抑制剂在胰岛β细胞诱导分化中的应用。本发明中所述JAK2抑制剂包括选自CEP‑33779、TG101209、TG101348中的至少一种。利用JAK2抑制剂能够大幅度提高内分泌细胞向胰岛β细胞的分化效率,从而可以调控体内胰岛β细胞的分化,应用于疾病治疗领域。
Description
技术领域
本发明涉及生物技术领域,尤其涉及JAK2抑制剂在胰岛β细胞诱导分化中的应用。
背景技术
糖尿病是影响人类健康的主要疾病之一,胰腺内分泌β细胞功能下降所导致的胰岛素分泌不足、糖代谢紊乱是糖尿病的重要原因,在研究发病机理的同时,研究胰岛细胞的功能状态是其中一个很重要的部分。其中,利用胰岛β细胞治疗糖尿病是一种有效的方法。
然而,分化获得胰岛β细胞还需要进一步研究。
发明内容
本发明旨在至少在一定程度上解决相关技术中的技术问题之一。为此,本发明的一个目的在于提出一种JAK2抑制剂在胰岛β细胞诱导分化领域中的用途,以及JAK2抑制剂在制备预防和/或治疗糖尿病药物中用途。
本发明的发明人在研究过程中发现:JAK2抑制剂能够在胰腺前体细胞(Pancreatic progenitors)向胰岛β细胞(pancreaticβ cells)定向分化的过程中发挥重要作用,JAK2抑制剂能够大幅提高内分泌细胞向胰岛β细胞分化的效率,使得被作用细胞表达胰岛β细胞特定的分子标记物胰岛素原(Proinsulin)、胰岛素(Insulin)、C肽(C-Peptide)和NKX6.1。由此通过JAK2抑制剂能够调控体内或者体外胰岛β细胞的定向分化,进一步可以用来预防或者治疗由于胰岛β细胞缺失或者异常所引起的疾病。
为此,根据本发明的第一方面,本发明提供了一种JAK2抑制剂在胰岛β细胞诱导分化领域中的用途。发明人发现,JAK2抑制剂能够大幅度提高内分泌细胞向胰岛β细胞的分化效率,从而可以调控体内胰岛β细胞的分化;或者可以用来在体外诱导分化成胰岛β细胞,用作特定检测。
根据本发明的实施例,以上JAK2抑制剂在胰岛β细胞诱导分化领域中的用途可以进一步附加如下技术特征:
在本发明的一些实施例中,所述JAK2抑制剂为JAK2蛋白激酶抑制剂。
在本发明的一些实施例中,所述JAK2蛋白激酶抑制剂包括选自CEP-33779、TG101209、TG101348中的至少一种。JAK2抑制剂的代表性物质CEP-33779、TG101209、或者TG101348能够抑制JAK2蛋白激酶,使得能够诱导内分泌细胞或者胰腺前体细胞分化为胰岛β细胞,从而可以作为诱导分化剂或者调控因子,介导胰岛β细胞的分化。
在本发明的一些实施例中,所述JAK2抑制剂能够用于促进胰腺内分泌前体细胞(pancreatic endocrine progenitor)向胰岛β细胞的定向分化。本发明中所述“胰腺内分泌前体细胞”指的是具备分化获得胰岛细胞潜能的前体细胞,该前体细胞表达胰腺内分泌的特定分子标记物。
在本发明的一些实施例中,所述JAK2抑制剂能够促进胰腺前体细胞向胰岛β细胞的定向分化。
根据本发明的第二方面,本发明提供了一种JAK2抑制剂在制备预防和/或治疗糖尿病药物中的用途。JAK2抑制剂能够大幅度提高胰腺内分泌前体细胞向胰岛β细胞的分化效率,从而可以调控体内胰岛β细胞的分化,用来治疗与胰岛β细胞异常相关的疾病,例如可以用来预防或者治疗糖尿病。
根据本发明的实施例,以上所述JAK2抑制剂在制备预防和/或治疗糖尿病药物中的用途可以进一步附加如下技术特征:
在本发明的一些实施例中,所述JAK2抑制剂为JAK2蛋白激酶抑制剂。
在本发明的一些实施例中,所述JAK2蛋白激酶抑制剂包括选自CEP-33779、TG101209、TG101348中的至少一种。
在本发明的一些实施例中,所述JAK2抑制剂能够用于促进胰腺内分泌前体细胞向胰岛β细胞的定向分化。
在本发明的一些实施例中,所述JAK2抑制剂能够促进胰腺前体细胞向胰岛β细胞的定向分化。
在本发明的第三方面,本发明提供了一种药物组合物,所述药物组合物能够诱导细胞分化为胰岛β细胞,所述药物组合物包括JAK2蛋白激酶抑制剂。
在本发明的一些实施例中,所述JAK2蛋白激酶抑制剂包括CEP-33779、TG101209、TG101348中的至少一种。
在本发明的一些实施例中,以上所述的胰岛β细胞的诱导分化物进一步包括:其他药学上可接受的载体和/或赋形剂。
在本发明的第四方面,本发明提供了一种试剂盒,所述试剂盒能够用于促进细胞分化为胰岛素β细胞,所述试剂盒包括JAK2抑制剂。
根据本发明的实施例,所述细胞选自胰腺内分泌前体细胞或胰腺前体细胞。
根据本发明的实施例,所述试剂盒中,所述JAK2抑制剂包括选自CEP-33779、TG101209、TG101348中的至少一种。
在本发明的第五方面,本发明提供了一种促进细胞分化成胰岛β细胞的方法,包括:将所述细胞和JAK2抑制剂接触培养,以便获得胰岛β细胞。
在本发明的一些实施例中,以上所述促进细胞分化成胰岛β细胞的方法可以进一步包括如下技术特征:
在本发明的一些实施例中,所述细胞为胰腺内分泌前体细胞或胰腺前体细胞。
在本发明的一些实施例中,将所述细胞和所述JAK2抑制剂接触培养5~7天。通过将细胞和JAK2抑制剂接续培养,连续培养5~7天,可以使得胰腺内分泌前体细胞或胰腺前体细胞分化为胰岛β细胞,从而可以调控体内胰岛β细胞的表达。也可以用来在体外诱导细胞的分化,从而用作诱导剂。
附图说明
图1为根据本发明的实施例提供的加入JAK2信号通路相关的化学小分子抑制剂来提升胰岛β细胞特定的分子标记物Proinsulin/Insulin/C-Peptide和NKX6.1的表达水平的流式细胞图。
图2为根据本发明的实施例提供的对加入JAK2信号通路相关的化学小分子抑制剂对应胰岛β细胞的产生进行显著性分析图。
图3为根据本发明的实施例提供的持续加入TG101348处理6天,获得胰岛β细胞的比例图。
图4为根据本发明的实施例提供的加入TG101348提高胰岛β细胞的生成比例图。
图5为根据本发明的实施例提供的加入TG101348后各分子标记物的含量比,其中5a显示的是加入TG101348后NKX6.1以及胰岛素原、胰岛素、C肽的含量占比,5b显示的是加入TG101348后NKX6.1以及C肽的含量占比。
具体实施方式
下面详细描述本发明的实施例,所述实施例的示例在附图中示出,其中自始至终相同或类似的标号表示相同或类似的元件或具有相同或类似功能的元件。下面通过参考附图描述的实施例是示例性的,旨在用于解释本发明,而不能理解为对本发明的限制。
本发明发现JAK2抑制剂在胰腺前体细胞(Pancreatic progenitors)向胰岛β细胞(pancreaticβcells)定向分化的过程中发挥重要作用,从而提供了一种JAK2抑制剂在胰岛β细胞诱导分化领域中的用途,能够大幅提高胰腺内分泌前体细胞向胰岛β细胞分化的效率,表达胰岛β细胞特定的分子标记物胰岛素原(Proinsulin)、胰岛素(Insulin)、C肽(C-Peptide)和NKX6.1。
为此,本发明提供了JAK2抑制剂在制备预防和/或治疗糖尿病药物中的用途。根据本发明的一种具体实施方式,本发明提供了JAK2蛋白激酶抑制剂在制备预防和/或治疗糖尿病领域中的用途。根据本发明的实施例,所述JAK2蛋白激酶抑制剂可以为CEP-33779、TG101209或者TG101348。JAK2抑制剂尤其是JAK2蛋白激酶抑制剂能够诱导胰腺前体细胞或者胰腺内分泌前体细胞分化为胰岛β细胞,从而使得可以调控血糖的平衡,用来预防或者治疗糖尿病。
本文中,术语“JAK2抑制剂”意指能够作用于JAK2信号通路的所有抑制剂物质,这些物质能够抑制JAK2信号通路中信号分子的表达。
本文中,术语“JAK2蛋白激酶抑制剂”意指以JAK2蛋白激酶作为靶标,能够抑制JAK2蛋白激酶的活性。根据本发明的实施例,所述JAK2蛋白激酶抑制剂包括选自CEP-33779、TG101209、TG101348中的至少一种,如表1所示。根据本发明的实施例,所述JAK2蛋白激酶抑制剂为选自CEP-33779、TG101209、TG101348中的至少两种。
表1 JAK2信号通路相关的化学小分子抑制剂的结构与相关信息
根据本发明的一种具体实施方式,本发明提供了一种药物组合物,所述药物组合物包括JAK2蛋白激酶抑制剂,所述药物组合物能够诱导细胞分化为胰岛β细胞。
根据本发明的实施例,所述药物组合物还进一步包括其他药学上可接受的载体和/或赋形剂。利用不同的赋形剂配合JAK2蛋白激酶抑制剂,可以制备成不同的药物组合物,所述药物组合物根据其目的给药途径可以制备成不同的剂型。给药途径的例子包括但不限于:肠胃外途径,例如静脉内、真皮内、皮下、肌内、皮下、口服、口腔、舌下、吸入、鼻内、透皮、局部、透粘膜、瘤内、滑膜内和直肠给药。在具体的实施方案中,按照常规方法将组合物配制成适于静脉内、皮下、肌内、口服、鼻内或局部对人给药的药物组合物。在一种具体实施方案中,按照常规方法将药物组合物配制用于对人类皮下给药。通常,用于静脉内给药的组合物是用无菌的缓冲液配制的溶液。需要时,药物组合物还可含有增溶剂和局部麻醉剂以减轻注射部位的疼痛。
本发明还提供了一种药物剂型。所述药物剂型的例子包括,但不限于:片剂;囊片;胶囊,如软弹性明胶胶囊;扁囊剂;锭剂;分散剂;栓剂;软膏剂;膏剂;粉剂;敷料剂;霜剂;硬膏剂;溶液;贴剂;气溶胶(例如,鼻喷雾或吸入剂);凝胶;适合给个体口服或粘膜给药的液体剂型,包括悬浮液(例如,水性或非水性液体悬浮液、水包油乳剂或油包水液体乳剂);适合给个体肠胃外给药的液体剂型;以及固体剂型。
以口服剂型为例,适合口服给药的本发明的药物组合物以分散剂型存在,包括但不限于:片剂(例如咀嚼片剂)、囊片、胶囊和液体(例如调味糖浆)。在某些实施方案中,所述口服剂型是固体并且是在无水条件下用无水成分制备的。典型的本发明的口服剂型是按照常规的药物混合技术将活性成分与至少一种赋形剂充分混合而制备的。赋形剂可采取多种形式,这取决于给药所需的制剂形式。例如,适用于口服液体或气溶胶剂型的赋形剂包括但不限于:水、乙二醇、油、醇类、调味剂、防腐剂和着色剂。适用于固体口服剂型(例如,粉剂、片剂、胶囊和囊片)的赋形剂的例子包括但不限于:淀粉、蔗糖、微晶纤维素、稀释剂、成粒剂、润滑剂、粘合剂和崩解剂。
可用于本发明的口服剂型的赋形剂的例子包括但不限于:粘合剂、填料、崩解剂和润滑剂。适用于所述药物组合物和剂型的粘合剂包括但不限于:玉米淀粉、马铃薯淀粉或其它淀粉、明胶、天然及合成树胶如***胶、藻酸钠、藻酸、其它藻酸盐、粉状黄蓍胶、瓜耳胶、纤维素及其衍生物(例如,乙基纤维素、乙酸纤维素、羧甲基纤维素钙、羧甲基纤维素钠)、聚乙烯吡咯烷酮、甲基纤维素、预胶凝淀粉、羟丙基甲基纤维素、微晶纤维素以及它们的混合物。
适用于本文所述药物组合物和剂型的填料的例子包括但不限于:滑石、碳酸钙(例如颗粒或粉末)、微晶纤维素、粉状纤维素、葡聚糖、高岭土、甘露醇、硅酸、山梨糖醇、淀粉、预胶凝淀粉以及它们的混合物。崩解剂被用于本发明的组合物以在片剂接触含水环境时使其崩解。含过多崩解剂的片剂在储藏时即会崩解,而含有过少崩解剂的片剂不会以所需速率或在所需条件下崩解。因此,应使用既不过多也不过少以至于对活性成分的释放造成不良影响的适量的崩解剂以形成本发明的固体口服剂型。可用于本发明的药物组合物和剂型的崩解剂包括但不限于:琼脂、藻酸、碳酸钙、微晶纤维素、交联羧甲基纤维素钠、交聚维酮、聚克立林钾、淀粉羟乙酸钠、马铃薯或木薯淀粉、预胶凝淀粉、其它淀粉、粘土、其它藻胶、其它纤维素、树胶,以及它们的混合物。可用于本发明的药物组合物和剂型的润滑剂包括但不限于:硬脂酸钙、硬脂酸镁、矿物油、轻质矿物油、甘油、山梨糖醇、甘露醇、聚乙二醇、其它乙二醇、硬脂酸、月桂基硫酸钠、滑石、氢化植物油(例如,花生油、棉籽油、葵花籽油、芝麻油、橄榄油、玉米油和大豆油)、硬脂酸锌、油酸乙酯、月桂酸乙酯、琼脂,以及它们的混合物。
下面将结合实施例对本发明的方案进行解释。本领域技术人员将会理解,下面的实施例仅用于说明本发明,而不应视为限定本发明的范围。实施例中未注明具体技术或条件的,按照本领域内的文献所描述的技术或条件或者按照产品说明书进行。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。
实施例1
利用CEP-3379、TG101209以及TG101348分别与胰腺前体细胞悬浮培养6天,然后再继续培养8天,用流式细胞仪检测胰岛β细胞特定的分子标记物胰岛素前体(Proinsulin、胰岛素(Insulin)、C肽(C-Peptide)和NKX6.1。其中,NKX6.1是胰岛β细胞中NKX6.1基因所表达的蛋白。具体实验过程如下:
1、胰腺前体细胞向胰腺内分泌前体细胞分化的方法
采用悬浮培养方法来验证胰腺前体细胞向胰腺内分泌前体细胞的分化,培养6天。分别将CEP-3379、TG101209以及TG101348加入到含有胰腺前体细胞的培养基中,其中CEP-3379的作用浓度(即在培养基中的浓度)为0.05μM,TG101209的作用浓度为0.5μM,TG101348的作用浓度为0.2μM。
其中悬浮培养板为低吸附六孔板(Beaverbio,Cat#40406)。使用的培养基:DMEM(Gibco,Cat#C11965500CP)+50x不含有维他命A的B-27(Gibco,Cat#12587-010)+1%谷氨酸酯(Glutamax,Invitrogen,Cat#35050079)+0.25mM2磷-L-抗坏血酸三钠盐(Phospho-L-ascorbic acid trisodium salt)(Sigma Aldrich;Cat#49752)+10μM ALK5抑制剂II(EnzoLife Sciences;Cat#ALX-270-445-M100),三碘-L-甲状腺原氨酸(5-Triiodo-L–thyronine)(Sigma,Cat#T2877)+0.1μMγ-Secretase inhibitor XX(EMD Millipore;Cat#565789-1MG)。
其中,培养条件:37℃、5%CO2和88%湿度,转速为90rpm。
通过上述悬浮培养,得到胰腺内分泌前体细胞,然后继续加入CEP-3379、TG101209或者TG101348,悬浮培养,促进胰腺内分泌前体细胞向胰岛β细胞分化。
2、胰腺内分泌前体细胞向胰岛β细胞分化的方法
采用悬浮培养方法来验证胰腺内分泌前体细胞向胰岛β细胞分化。将上述胰腺内分泌前体细胞在如下培养基中继续培养8天,检测胰腺内分泌前体细胞是否分化为胰岛β细胞。
其中,悬浮培养板为低吸附六孔板(Beaverbio,Cat#40406)。使用的培养基为:DMEM(Gibco,Cat#C11965500CP)+50X不含有维他命A的B-27(Gibco,Cat#12587-010)+1%Glutamax(Invitrogen,Cat#35050079)+10μM ALK5 Inhibitor II(Enzo Life Sciences;Cat#ALX-270-445-M100)。通过流式细胞检测发现,培养4-8天后即可获得胰岛β细胞。
3、流式细胞检测方法
胰岛β细胞特定的分子标记物胰岛素前体(Proinsulin)、胰岛素(Insulin)、C肽(C-Peptide)和NKX6.1的检测方法如下:
分化的细胞团用预热的Accutase细胞消化液细胞分离液(Sigma,A6964-100ML)消化5-10分钟成单细胞,PBS洗一遍,200μL固定渗透液(Fixation and PermeabilizationSolution,BD;Cat#554722)固定20min。
然后用200μL BD渗透洗涤液(BD Perm/WashTMBuffer,Cat#554723)洗两遍,利用一抗在4℃孵育2小时,其中一抗按照如下进行配制:200μL BD渗透洗涤液(BD Perm/WashTMBuffer)+1μL鼠抗NKX6.1(mouse anti-NKX6.1,DSHB;Cat#F55A12)+1μL兔C肽抗体(Rat C-pep antibody,DSHB;Cat#GN-ID4)。
孵育结束后,利用200μL BD渗透洗涤液(BD Perm/WashTMBuffer)洗两遍,利用荧光二抗在4℃避光孵育1小时,其中荧光二抗按照如下进行配制:200μL BD Perm/WashTMBuffer+带有荧光染料Alexa Fluor 488(Life Technologies;Cat#A-21208)的猴抗兔抗体(Donkey anti-Rat IgG(H+L)Secondary Antibody)+带有荧光染料Alexa647conjugate(Life Technologies;Cat#A-31571)的猴抗鼠抗体(Donkey anti-Mouse IgG(H+L)Secondary Antibody)。
孵育结束后,利用200μL BD Perm/WashTMBuffer洗两遍,然后用200μL BD Perm/WashTMBuffer重悬,转移至流式管中,采用流式细胞仪(BD Calibur)进行检测,用FlowJo软件进行流式结果分析。
实验结果如图1所示。从图1可以看出,与对照相比,加入JAK2信号通路相关的化学小分子抑制剂能有效提高胰岛β细胞特定的分子标记物Proinsulin/Insulin/C-Peptide和NKX6.1的表达水平(主要表现为位于Q2中的分子标记物的含量和荧光强度增强)。作为JAK2的抑制剂,CEP-33779,TG101209和TG101348都能够有效提高胰腺内分泌前体细胞向胰岛β细胞定向诱导的分化效率图1。
同时我们对JAK2的抑制剂在促进胰腺内分泌前体细胞向胰岛β细胞定向分化过程中,生成胰岛β细胞的比例进行了显著性分析,如图2所示。其中TG101209和TG101348均能显著提高胰岛β细胞比例。
而且,我们研究了不同时间节点加入TG101348诱导胰腺内分泌前体细胞产生胰岛β细胞的情况。以TG101348为例,如图3所示,图3中的纵坐标代表不同的处理时间,其中“1”代表加入TG101348处理一天,“1-2”代表在第一天加入TG101348处理,连续处理两天,依次类推“1-3”代表在第一天加入TG101348处理,连续处理三天。同样地,“2-5”代表在第二天加入TG101348处理,直至第五天。从图3给出的结果不难看出,发现持续加入TG101348处理6天,获得胰岛β细胞的比例最高。
实施例2
同时我们评价了JAK2抑制剂对于胰岛素剪切的影响,用不加入JAK2抑制剂作为对照。所用到的胰岛素剪切检测方法如下:
分化的细胞团用预热的Accutase细胞消化液细胞分离液(Sigma,A6964-100ML)消化5-10分钟成单细胞,PBS缓冲液洗一遍,200μL固定渗透液(Fixation andPermeabilization Solution)固定20min。
然后用200μL BD渗透洗涤液(BD Perm/WashTMBuffer)洗两遍,利用一抗在4℃孵育2小时,其中一抗按照如下进行配制:200μL BD渗透洗涤液(BD Perm/WashTMBuffer)+1μL鼠抗NKX6.1(mouse anti-NKX6.1,DSHB;Cat#F55A12)+1μL猪抗C肽抗体(Pig anti-C Peptideantibody,Abcam;Cat#ab30477)。
孵育结束后,利用200μL BD Perm/WashTMBuffer洗两遍,利用荧光二抗在4℃下避光孵育1小时,其中荧光二抗按照如下进行配制:200μL BD Perm/WashTMBuffer+带有荧光染料Alexa488conjugate(Life Technologies;Cat#A-21202)的驴抗鼠抗体(Donkeyanti-Mouse IgG(H+L)Secondary Antibody)+带有荧光染料Alexa/>647conjugate(Life Technologies;Cat#A-21450)的山羊抗猪抗体(Goat anti-Guinea Pig IgG(H+L)Secondary Antibody)。
孵育结束后,利用200μL BD Perm/WashTMBuffer洗两遍,然后用200μL BD Perm/WashTMBuffer重悬,转移至流式管中,采用流式细胞仪(BD Calibur)进行检测,用FlowJo软件进行流式结果分析。
流式结果图如图4和图5所示,经过研究发现,与对照相比,加入TG101348不仅能提高胰岛β细胞的生成比例,还能促进胰岛素原向胰岛素的剪切,使得分化获得的胰岛β细胞更趋于功能成熟的胰岛β细胞。
在本说明书的描述中,参考术语“一个实施例”、“一些实施例”、“示例”、“具体示例”、或“一些示例”等的描述意指结合该实施例或示例描述的具体特征、结构、材料或者特点包含于本发明的至少一个实施例或示例中。在本说明书中,对上述术语的示意性表述不必针对的是相同的实施例或示例。而且,描述的具体特征、结构、材料或者特点可以在任一个或多个实施例或示例中以合适的方式结合。此外,在不相互矛盾的情况下,本领域的技术人员可以将本说明书中描述的不同实施例或示例以及不同实施例或示例的特征进行结合和组合。
尽管上面已经示出和描述了本发明的实施例,可以理解的是,上述实施例是示例性的,不能理解为对本发明的限制,本领域的普通技术人员在本发明的范围内可以对上述实施例进行变化、修改、替换和变型。
Claims (2)
1.JAK2蛋白激酶抑制剂在制备预防和/或治疗糖尿病药物中的用途,所述JAK2蛋白激酶抑制剂促进胰腺内分泌前体细胞或胰腺前体细胞向胰岛β细胞分化,所述JAK2蛋白激酶抑制剂选自CEP-33779、TG101209、TG101348中的至少一种。
2.一种非治疗目的的促进细胞分化成胰岛β细胞的方法,其特征在于,包括:
将所述细胞和JAK2抑制剂接触培养5~7天,以便获得胰岛β细胞;所述JAK2抑制剂是JAK2蛋白激酶抑制剂;
所述细胞为胰腺内分泌前体细胞或胰腺前体细胞;所述JAK2蛋白激酶抑制剂选自CEP-33779、TG101209、TG101348中的至少一种。
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