CN112546199A - Lpar1协调皮层发育过程中的纤毛发生和神经发生 - Google Patents
Lpar1协调皮层发育过程中的纤毛发生和神经发生 Download PDFInfo
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Abstract
本发明公开了LPAR1协调皮层发育过程中的纤毛发生和神经发生。本发明提供了LPAR1蛋白或其相关生物材料在如下任一中的应用：协调皮层发育过程中的纤毛发生和/或神经发生；制备用于协调皮层发育过程中的纤毛发生和/或神经发生的产品；所述相关生物材料为能够表达所述LPAR1蛋白的核酸分子或含有所述核酸分子的表达盒、重组载体、重组菌或转基因细胞系。本发明研究发现，LPAR1对小鼠RG细胞上纤毛的正常发生、VZ/SVZ生成和新皮质形成都至关重要。LPAR1可能通过精确调控纤毛组装和去组装的动态平衡来调节神经发生。
Description
技术领域
本发明涉及生物医学领域，具体涉及LPAR1协调皮层发育过程中的纤毛发生和神经发生。
背景技术
初级纤毛是存在于大多数哺乳动物细胞表面一根天线状细胞器。它能感受胞外的许多物理和化学信号，并在多种生物学过程如胚胎发育和组织稳态中发挥关键作用。纤毛功能的异常与肿瘤发生和许多人类发育疾病有关。
在哺乳动物的大脑皮层发育过程中，神经细胞主要发生在脑室区(VZ)和脑室下区(SVZ)两个区域。皮层上的所有的神经元都是来自这两个区域的神经祖细胞(NPC)。VZ中的放射状胶质细胞(RG)具有初级纤毛，这些纤毛延伸到侧脑室内并作为细胞天线以检测脑脊液(CSF)中存在的各种信号，进而调节神经发生。这些纤毛只存在于间期的RG细胞中，并且在细胞进入有丝分裂之前纤毛必须需要发生去组装。RG细胞会通过有丝分裂来协调其自我更新分裂和不对称分裂，进而以形成SVZ和神经细胞。然而，细胞外调控RG细胞的纤毛去组装以及随后的有丝分裂进入过程的因子尚不清楚。
溶血磷脂酸受体1(LPAR1)是G-蛋白偶联受体家族、EDG亚家族成员(EDG family)，其配体LPA(Lysophosphatidic Acid，溶血磷脂酸)主要来源于被活化的血小板。LPAR1在机体正常组织中普遍表达并与Gi、Gq、G12/13偶联介导诸多的细胞生理病理反应，诸如细胞增殖、分化、血小板凝集以及在肿瘤细胞中促进细胞的增殖、迁移等生物功能。
目前尚未有LPAR1协调皮层发育过程中的纤毛发生和神经发生的相关报道。目前尚未有LPAR1通过调控纤毛发生进而调控神经发生的相关报道。
发明内容
本发明的目的是提供LPAR1协调皮层发育过程中的纤毛发生和神经发生。
第一方面，本发明要求保护LPAR1蛋白或其相关生物材料在如下(A1)或(A2)中的应用：
(A1)协调皮层发育过程中的纤毛发生和/或神经发生；
(A2)制备用于协调皮层发育过程中的纤毛发生和/或神经发生的产品；
所述相关生物材料为能够表达所述LPAR1蛋白的核酸分子或含有所述核酸分子的表达盒、重组载体、重组菌或转基因细胞系。
进一步地，在所述应用中，所述LPAR1蛋白通过调控纤毛组装和去组装的动态平衡来调节神经发生。
进一步地，所述纤毛为初级纤毛。
第二方面，本发明要求保护LPAR1蛋白或其相关生物材料在如下任一中的应用：
(B1)促进神经祖细胞发生，或制备用于促进神经祖细胞产生的产品；
(B2)促进神经祖细胞分裂，或制备用于促进神经祖细胞分裂的产品；
(B3)促进脑室区(VZ)和/或脑室下区(SVZ)生成，或制备用于促进脑室区(VZ)和/或脑室下区(SVZ)生成的产品；
(B4)促进新皮质形成，或制备用于促进新皮质形成的产品。
所述相关生物材料为能够表达所述LPAR1蛋白的核酸分子或含有所述核酸分子的表达盒、重组载体、重组菌或转基因细胞系。
第三方面，本发明要求保护能够促进LPAR1蛋白表达的物质在如下任一中的应用：
(B1)促进神经祖细胞发生，或制备用于促进神经祖细胞产生的产品；
(B2)促进神经祖细胞分裂，或制备用于促进神经祖细胞分裂的产品；
(B3)促进脑室区(VZ)和/或脑室下区(SVZ)生成，或制备用于促进脑室区(VZ)和/或脑室下区(SVZ)生成的产品；
(B4)促进新皮质形成，或制备用于促进新皮质形成的产品。
在上述第一方面至第三方面中，所述产品均可为药品。
在实际应用过程中，可针对由于LPAR1蛋白表达低下所引起的具有“纤毛发生和/或神经发生出现障碍、神经祖细胞发生和/或分裂出现障碍、VZ和/或SVZ生成出现障碍、新皮质形成出现障碍”这些症状中至少一种的个体给予所述产品(药品，即前文所述的LPAR1蛋白或其相关生物材料或所述能够促进LPAR1蛋白表达的物质)，以改善相关症状。
第四方面，本发明要求保护能够抑制LPAR1蛋白表达的物质在如下任一中的应用：
(C1)抑制神经祖细胞发生，或制备用于抑制神经祖细胞产生的产品；
(C2)抑制神经祖细胞分裂，或制备用于抑制神经祖细胞分裂的产品；
(C3)抑制脑室区(VZ)和/或脑室下区(SVZ)生成，或制备用于抑制脑室区(VZ)和/或脑室下区(SVZ)生成的产品；
(C4)抑制新皮质形成，或制备用于抑制新皮质形成的产品。
进一步地，所述产品可为动物模型。
在实际应用中，可以通过抑制正常动物中LPAR1蛋白表达(如通过基因编辑手段敲除LPAR1蛋白编码基因)，制备得到具有“纤毛发生和/或神经发生出现障碍、神经祖细胞发生和/或分裂出现障碍、VZ和/或SVZ生成出现障碍、新皮质形成出现障碍”这些症状中至少一种的动物模型。该动物模型可用于筛药，如筛选能够治疗或缓解由于LPAR1蛋白表达低下所引起的具有“纤毛发生和/或神经发生出现障碍、神经祖细胞发生和/或分裂出现障碍、VZ和/或SVZ生成出现障碍、新皮质形成出现障碍”这些症状中至少一种。
第五方面，本发明要求保护LPAR1蛋白表达量降低的动物模型在筛选能够缓解由于LPAR1蛋白表达低下所引起的具有“纤毛发生和/或神经发生出现障碍、神经祖细胞发生和/或分裂出现障碍、VZ和/或SVZ生成出现障碍、新皮质形成出现障碍”这些症状中至少一种的药物中的应用。
在第四和第五方面中，所述动物可为小鼠。
在所述各方面中，所述神经祖细胞可为放射状胶质细胞(radial glial cells，RG)或中间祖细胞(intermediate progenitor cells，IP)等。
本发明适用于哺乳动物，具体如人或小鼠。
在本发明中，所述LPAR1蛋白均为溶血磷脂酸受体1。
另外，基于本发明的研究成果，还可进一步深入研究，如进行基因疗法。基因治疗(gene therapy)是指将外源正常基因导入靶细胞，以纠正或补偿缺陷和异常基因引起的疾病。具体到本发明，为将外源正常的LPAR1基因导入靶细胞，以纠正或补偿由于LPAR1基因缺陷或异常引起的疾病。
本发明研究发现，LPAR1对小鼠RG细胞上纤毛的正常发生、VZ/SVZ生成和新皮质形成都至关重要。LPAR1可能通过精确调控纤毛组装和去组装的动态平衡来调节神经发生。
附图说明
图1为LPAR1协调皮层发育过程中的纤毛发生和神经发生。a为Lpar1+/+与Lpar1-/-小鼠胚胎14.5天(E14.5)大脑皮层Pax6与Tbr2阳性细胞数目；b为Lpar1+/+与Lpar1-/-小鼠胚胎出生后第0天(P0)大脑皮层Pax6与Tbr2阳性细胞数目；c为Lpar1+/+与Lpar1-/-小鼠胚胎14.5天(E14.5)大脑皮层p-H3阳性细胞比例；d为Lpar1+/+与Lpar1-/-小鼠胚胎出生后第0天(P0)大脑皮层RG细胞上初级纤毛的长度。*表示p<0.05；**表示p<0.01；***表示p<0.001。
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下述实施例中所使用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材料、试剂等，如无特殊说明，均可从商业途径得到。
1、Lpar1+/+和Lpar1-/-小鼠为Jerold Chun教授赠送。即为记载于“JamesJ.A.Contos,et al.Requirement for the lpA1 lysophosphatidic acid receptor genein normal suckling behavior.Proc Natl Acad Sci U S A.2000Nov 21；97(24):13384-9.”一文中的“lpA1 (+/+)”和“lpA1 (-/-)小鼠”，公众可从申请人处获得，尽可用于重复本发明实验使用，不得他用。lpA1 (+/+)小鼠表达LPAR1(正常的野生型小鼠)，lpA1 (-/-)为敲除小鼠，不表达LAPR1(与正常的野生型小鼠相比，仅是敲除了LAPR1基因)。两种小鼠均为129SvJ通过与C57BL/6J小鼠交配洗背景至C57BL/6J。
2、试剂：
Pax6抗体Covance公司，货号PRB-278P；Tbr2抗体Thermo公司，货号14-4875-82；p-H3抗体CST公司，货号9701s；ARL13B抗体Proteintech公司，17711-1-AP。
实施例1、Lpar1协调皮层发育过程中的纤毛发生和神经发生的相关研究
一、实验方法
(1)分别在E14.5或者P0天取Lpar1+/+和Lpar1-/-小鼠胚胎，处死后解剖取大脑，PBS洗去血液。
(2)小鼠大脑置于4％PFA中固定24h。
(3)梯度酒精脱水，然后石蜡包埋。
(4)包埋之后切片，每次3μm。
(5)梯度酒精脱蜡，脱蜡后使用柠檬酸钠进行抗原修复。
(6)进行免疫荧光染色。待测抗原涉及Pax6、Tbr2、p-H3和ARL13B。
二、结果与分析
本发明检查了Lpar1+/+和Lpar1-/-小鼠发育中大脑皮层上的神经祖细胞的分布模式。如图1中a和b所示，本发明分别用Pax6和Tbr2指示RG细胞和IP细胞，本发明发现与胚胎第14.5天(E14.5)和出生后第0天(P0)的Lpar1+/+小鼠对照相比，Lpar1-/-小鼠的大脑皮层中Pax6和Tbr2阳性细胞的数量明显减少。这些数据表明LPAR1是神经祖细胞的产生或者维持所必须的。
接着，本发明利用p-H3指示有丝分裂的细胞，发现Lpar1-/-小鼠的VZ/SVZ细胞表现出较低的有丝分裂指数，这表明神经祖细胞正常分裂增殖依赖于LPAR1(图1中c)。并且，本发明用ARL13B指示初级纤毛，发现Lpar1-/-小鼠RG细胞中纤毛的长度明显长于野生小鼠(即Lpar1+/+小鼠)(图1中d)。
总之，本发明的数据表明LPAR1可能通过精确调控纤毛组装和去组装的动态平衡来调节神经发生。
Claims (10)
1.LPAR1蛋白或其相关生物材料在如下(A1)或(A2)中的应用：
(A1)协调皮层发育过程中的纤毛发生和/或神经发生；
(A2)制备用于协调皮层发育过程中的纤毛发生和/或神经发生的产品；
所述相关生物材料为能够表达所述LPAR1蛋白的核酸分子或含有所述核酸分子的表达盒、重组载体、重组菌或转基因细胞系。
2.根据权利要求1所述的应用，其特征在于：在所述应用中，所述LPAR1蛋白通过调控纤毛组装和去组装的动态平衡来调节神经发生。
3.根据权利要求1或2所述的应用，其特征在于：所述纤毛为初级纤毛。
4.LPAR1蛋白或其相关生物材料在如下任一中的应用：
(B1)促进神经祖细胞发生，或制备用于促进神经祖细胞产生的产品；
(B2)促进神经祖细胞分裂，或制备用于促进神经祖细胞分裂的产品；
(B3)促进脑室区和/或脑室下区生成，或制备用于促进脑室区和/或脑室下区生成的产品；
(B4)促进新皮质形成，或制备用于促进新皮质形成的产品；
所述相关生物材料为能够表达所述LPAR1蛋白的核酸分子或含有所述核酸分子的表达盒、重组载体、重组菌或转基因细胞系。
5.能够促进LPAR1蛋白表达的物质在如下任一中的应用：
(B1)促进神经祖细胞发生，或制备用于促进神经祖细胞产生的产品；
(B2)促进神经祖细胞分裂，或制备用于促进神经祖细胞分裂的产品；
(B3)促进脑室区和/或脑室下区生成，或制备用于促进脑室区和/或脑室下区生成的产品；
(B4)促进新皮质形成，或制备用于促进新皮质形成的产品。
6.根据权利要求1-5中任一所述的应用，其特征在于：所述产品均为药品。
7.能够抑制LPAR1蛋白表达的物质在如下任一中的应用：
(C1)抑制神经祖细胞发生，或制备用于抑制神经祖细胞产生的产品；
(C2)抑制神经祖细胞分裂，或制备用于抑制神经祖细胞分裂的产品；
(C3)抑制脑室区和/或脑室下区生成，或制备用于抑制脑室区和/或脑室下区生成的产品；
(C4)抑制新皮质形成，或制备用于抑制新皮质形成的产品。
8.根据权利要求7所述的应用，其特征在于：所述产品为动物模型。
9.LPAR1蛋白表达量降低的动物模型在筛选能够缓解由于LPAR1蛋白表达低下所引起的具有“纤毛发生和/或神经发生出现障碍、神经祖细胞发生和/或分裂出现障碍、脑室区和/或脑室下区生成出现障碍、新皮质形成出现障碍”这些症状中至少一种的药物中的应用。
10.根据权利要求1-9中任一所述的应用，其特征在于：所述神经祖细胞为放射状胶质细胞或中间祖细胞。
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| WO2013070879A1 (en) * | 2011-11-10 | 2013-05-16 | Bristol-Myers Squibb Company | Methods for treating spinal cord injury with lpa receptor antagonists |
| WO2016103269A1 (en) * | 2014-12-23 | 2016-06-30 | Ramot At Tel-Aviv University Ltd. | Populations of neural progenitor cells and methods of producing and using same |
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