CN112813132B - 用于筛选治疗器官纤维化的胶原转录抑制剂的高通量筛选方法 - Google Patents

用于筛选治疗器官纤维化的胶原转录抑制剂的高通量筛选方法 Download PDF

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CN112813132B
CN112813132B CN202011638647.9A CN202011638647A CN112813132B CN 112813132 B CN112813132 B CN 112813132B CN 202011638647 A CN202011638647 A CN 202011638647A CN 112813132 B CN112813132 B CN 112813132B
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王阳
柏旭
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Abstract

本发明公开了一种用于筛选治疗器官纤维化的胶原转录抑制剂的高通量筛选方法，其特征在于，包括以下步骤：克隆人Col1A1、Col1A2、Col3A1启动子‑2000‑100区域，单独或联合插入pGL3‑basic报告基因载体，经转化DH5α菌和质粒提取得到用于转染的质粒；将得到的质粒分别与内参pRL‑TK质粒转染成纤维细胞，后加入TGF‑β1激活；或同时加入TGFβR抑制剂；激活后按照荧光素酶报告基因检测试剂盒进行处理，酶标仪读取化学发光数据，得出对TGF‑β1诱导的胶原合成敏感的质粒，以上结果证实了该种筛选方法的实用性。还公开了利用该方法在测定抑制I/III型胶原转录药物的药效定量分析中的应用。

Description

用于筛选治疗器官纤维化的胶原转录抑制剂的高通量筛选 方法
技术领域
本发明涉及生物医药领域，特别是涉及一种用于筛选治疗器官纤维化的胶原转录抑制剂的高通量筛选方法。
背景技术
任何原因引起的组织细胞损伤，均可导致组织细胞发生变性、坏死和炎症反应。如果损伤很小，损伤细胞周边正常实质细胞将发生增生修复，这种修复可完全恢复正常的结构和功能。然而如果损伤较大或反复损伤超出了损伤周围实质细胞的再生能力时，细胞外基质将大量增生对缺损组织进行修复，即发生纤维化的病理改变。因此本质上纤维化是组织遭受损伤后的修复反应，以保护组织器官的相对完整性。增生的纤维结缔组织虽然修复了缺损，但却不具备原来器官实质细胞的结构和功能。如果这种修复反应过度、过强和失控时，就会引起器官的纤维化和导致器官的功能下降。
在全世界范围内，组织纤维化是许多疾病致残、致死的主要原因，据有关统计资料证明，因各种疾病而致死的病人中，接近45％可以归于组织纤维增生疾病，例如肝纤维化所致肝衰竭或肝癌、肺纤维化所致呼吸衰竭，肾纤维化所致肾功能衰竭和尿毒症等等，都是致死性高的并发症。
器官损伤后，一些可以产生胶原的细胞在因子刺激下向成纤维细胞类型转化，并合成大量胶原，例如肝内的星状细胞、肝窦内皮细胞等，肺内的原始间叶细胞、肺泡II型上皮细胞、动脉血管内皮及平滑肌细胞等，肾内系膜细胞、肾间质成纤维细胞等细胞被活化并产生胶原纤维、可造成纤维化。此类胶原纤维的类型主要是I、III和IV型。所以I型和III型胶原分子的合成步骤可作为抗器官纤维化药物的靶点。
人们发现器官纤维化发生过程总是有一些细胞因子参与，例如转化生长因子TGF-β、血小板生长因子PDGF、结缔组织生长因子CTGF、γ-干扰素(IFN-γ)、肿瘤坏死因子TNF-α等等，其中TGF-β、PDGF和CTGF是实验室及临床常用的器官纤维化检测指标，也是实验室中用来激活成纤维样细胞合成胶原的常用因子。TGF-β增加胶原基因转录通过与其受体结合后启动下游信号通路，例如TGFβ-TGFβR-Smad2/3通路正向调控I型胶原基因(Col1A1和Col1A2)和III型胶原基因(Col3A1)转录，而TGFβR抑制剂Galunisertib和SB-431542被报道可以有效抑制胶原合成，缓解实验动物模型的器官纤维化。
基于以上机制，需要开发一种用于筛选治疗器官纤维化的胶原转录抑制剂的高通量筛选方法。
发明内容
本发明所要解决的技术问题是提供一种用于筛选治疗器官纤维化的胶原转录抑制剂的高通量筛选方法，用荧光素酶报告基因方法检测Col1A1-Col1A2或Col3A1基因启动子受细胞因子-转录因子调控情况。
为解决上述技术问题，本发明采用的第一个技术方案是：提供一种用于筛选治疗器官纤维化的胶原转录抑制剂的高通量筛选方法，包括以下步骤：
步骤1：克隆人Col1A1、Col1A2、Col3A1启动子-2000-100区域，单独或联合插入pGL3-basic报告基因载体，得到4个质粒：pGL3-Col1A1Prom、pGL3-Col1A2Prom、pGL3-Col3A1Prom和pGL3-Col1A1Prom-Col1A2Prom，经转化DH5α菌和质粒提取步骤得到用于转染的质粒；
步骤2：将步骤1中得到的质粒分别与内参pRL-TK质粒转染NIH-3T3细胞，转染12h后培养基中加入TGF-β1激活；
步骤3：TGF-β1激活24h后，按照荧光素酶报告基因检测试剂盒进行处理(插入荧光素酶报告基因载体)，酶标仪读取化学发光数据，得出对TGF-β1诱导的胶原合成敏感的质粒。
为解决上述技术问题，本发明采用的第二个技术方案是：提供一种用于筛选治疗器官纤维化的胶原转录抑制剂的高通量筛选方法，包括以下步骤：
步骤1：克隆人Col1A1、Col1A2、Col3A1启动子-2000-100区域，单独或联合插入pGL3-basic报告基因载体，得到4个质粒：pGL3-Col1A1Prom、pGL3-Col1A2Prom、pGL3-Col3A1Prom和pGL3-Col1A1Prom-Col1A2Prom，经转化DH5α菌和质粒提取步骤得到用于转染的质粒；
步骤2：将步骤1中得到的质粒分别与内参pRL-TK质粒转染NIH-3T3细胞，转染12h后培养基中加入TGF-β1激活，同时加入浓度为0.01nM-10mM的TGFβR抑制剂Galunisertib和SB-431542；
步骤3：TGF-β1激活24h后，按照标准双荧光素酶报告基因检测，酶标仪读取化学发光数据，得出化合物Galunisertib和SB-431542对TGF-β1诱导的胶原合成转录抑制的IC50曲线。
为解决上述技术问题，本发明采用的第三个技术方案是：提供一种利用该筛选方法在测定抑制I/III型胶原转录药物的药效定量分析中的应用。
本发明的有益效果是：本发明胶原转录抑制剂筛选基于I型胶原α1的启动子片段联合I型胶原α2的启动子片段插入荧光素酶报告基因载体，或III型胶原α1的启动子片段插入荧光素酶报告基因载体，得出pGL3-Col1A1Prom-Col1A2Prom对TGF-β1诱导的胶原合成最敏感，测得TGFβR抑制剂Galunisertib和SB-431542抑制I型胶原转录的IC50最低，以上结果证实了该种筛选方法的实用性。
附图说明
图1是不同胶原启动子区对10ng/ml TGF-β激活的响应图；
图2是不同浓度TGFβ处理细胞后EC50的测定图；
图3是TGFβR抑制剂Galunisertib与SB-431542抑制I型胶原转录EC50的测定图；
图4是TGFβR抑制剂Galunisertib与SB-431542抑制III型胶原转录EC50的测定图。
具体实施方式
下面结合附图对本发明的较佳实施例进行详细阐述，以使本发明的优点和特征能更易于被本领域技术人员理解，从而对本发明的保护范围做出更为清楚明确的界定。
实施例1：
一种用于筛选治疗器官纤维化的胶原转录抑制剂的高通量筛选方法，包括以下步骤：
步骤1：克隆人Col1A1、Col1A2、Col3A1启动子-2000-100区域，单独或联合插入pGL3-basic报告基因载体(购自Promega公司)，得到4个质粒：pGL3-Col1A1Prom(简称1A1)，pGL3-Col1A2Prom(简称1A2)，pGL3-Col3A1Prom(简称3A1)和pGL3-Col1A1Prom-Col1A2Prom(简称1A12)，经转化DH5α菌和质粒提取步骤得到用于转染的质粒；
步骤2：将步骤1中得到的质粒3μg分别与内参pRL-TK质粒1μg(购自Promega)转染6孔板内NIH-3T3细胞(105cells/孔)，转染12h后培养基中加入5ng/ml TGF-β1激活；
步骤3：TGF-β1激活24h后，按照荧光素酶报告基因检测试剂盒(南京凯基)说明进行处理，酶标仪读取化学发光数据，结果如图1，可见pGL3-Col1A1Prom-Col1A2Prom对TGF-β1诱导的胶原合成最敏感，效果显著好于pGL3-Col1A1Prom或pGL3-Col1A2Prom。可测出pGL3-Col1A1Prom-Col1A2Prom以及pGL3-Col3A1Prom受到TGF-β1激活的EC50曲线，结果见图2，EC50分别为3.452ng/ml和5.563ng/ml。
实施例2：
一种用于筛选治疗器官纤维化的胶原转录抑制剂的高通量筛选方法，包括以下步骤：
步骤1：克隆人Col1A1、Col1A2、Col3A1启动子-2000-100区域，单独或联合插入pGL3-basic报告基因载体(购自Promega公司)，得到4个质粒：pGL3-Col1A1Prom(简称1A1)，pGL3-Col1A2Prom(简称1A2)，pGL3-Col3A1Prom(简称3A1)和pGL3-Col1A1Prom-Col1A2Prom(简称1A12)，经转化DH5α菌和质粒提取步骤得到用于转染的质粒；
步骤2：将步骤1中得到的质粒3μg分别与内参pRL-TK质粒1μg(购自Promega)转染6孔板内人皮肤成纤维细胞BJ(105cells/孔)，转染8h后培养基中加入5ng/ml TGF-β1激活，同时加入不同浓度的TGFβR抑制剂Galunisertib和SB-431542；
优选的，所述TGFβR抑制剂Galunisertib和SB-431542的浓度为0.01nM-10mM。
步骤3：TGF-β1激活24h后，按照标准双荧光素酶报告基因检测，酶标仪读取化学发光数据，结果如图3和图4。Galunisertib和SB-431542抑制I型胶原转录的IC50分别为65.88nM和209.8nM，Galunisertib和SB-431542抑制III型胶原转录的IC50分别为129.5nM和775.1nM。
实施例3：药物BRL-2021101抑制胶原转录
以上两种方法自有化合物库中筛选出化合物BRL-2021101。其抑制I型胶原转录IC50＝25μM，最大效能为Galunisertib的94％。
以上所述仅为本发明的实施例，并非因此限制本发明的专利范围，凡是利用本发明说明书及附图内容所作的等效流程变换，或直接或间接运用在其他相关的技术领域，均同理包括在本发明的专利保护范围内。

Claims (3)

1.一种用于筛选治疗器官纤维化的胶原转录抑制剂的高通量筛选方法，其特征在于，包括以下步骤：
步骤1：克隆人Col1A1、Col1A2、Col3A1启动子-2000-100区域，单独插入pGL3-basic报告基因载体，得到3个质粒：pGL3-Col1A1Prom、pGL3-Col1A2Prom 、pGL3-Col3A1Prom，和联合插入pGL3-basic报告基因载体，得到1个质粒pGL3-Col1A1Prom-Col1A2Prom，经转化DH5α菌和质粒提取步骤得到用于转染的质粒；
步骤2：将步骤1中得到的质粒分别与内参pRL-TK质粒转染成纤维细胞，转染6—24h后培养基中加入TGF-β1激活，同时加入待筛选的胶原转录抑制剂；
步骤3：TGF-β1激活24h后，按照荧光素酶报告基因检测试剂盒说明进行处理，酶标仪读取化学发光数据，得出对TGF-β1诱导的胶原合成敏感的质粒及所述待筛选的胶原转录抑制剂对TGF-β1诱导的胶原合成转录抑制的IC50曲线。
2.一种用于筛选治疗器官纤维化的胶原转录抑制剂的高通量筛选方法，其特征在于，包括以下步骤：
步骤1：克隆人Col1A1、Col1A2、Col3A1启动子-2000-100区域，单独插入pGL3-basic报告基因载体，得到3个质粒：pGL3-Col1A1Prom、pGL3-Col1A2Prom 、pGL3-Col3A1Prom，和联合插入pGL3-basic报告基因载体，得到1个质粒pGL3-Col1A1Prom-Col1A2Prom，经转化DH5α菌和质粒提取步骤得到用于转染的质粒；
步骤2：将步骤1中得到的质粒分别与内参pRL-TK质粒转染成纤维细胞，转染6—24h后培养基中加入TGF-β1激活，同时加入待筛选的胶原转录抑制剂和浓度为0.01nM-10mM的TGFβR抑制剂Galunisertib和SB-431542；
步骤3：TGF-β1激活24h后，按照标准双荧光素酶报告基因检测，酶标仪读取化学发光数据，得出所述待筛选的胶原转录抑制剂对TGF-β1诱导的胶原合成转录抑制的IC50曲线及化合物Galunisertib和SB-431542对TGF-β1诱导的胶原合成转录抑制的IC50曲线。
3.一种利用如权利要求1或2所述用于筛选治疗器官纤维化的胶原转录抑制剂的高通量筛选方法在测定抑制I型或III型胶原转录药物的药效定量分析中的应用。
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