WO2017020691A1 - 一种哌啶基吡唑并嘧啶类化合物的筛选和用途 - Google Patents

一种哌啶基吡唑并嘧啶类化合物的筛选和用途 Download PDF

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WO2017020691A1
WO2017020691A1 PCT/CN2016/089970 CN2016089970W WO2017020691A1 WO 2017020691 A1 WO2017020691 A1 WO 2017020691A1 CN 2016089970 W CN2016089970 W CN 2016089970W WO 2017020691 A1 WO2017020691 A1 WO 2017020691A1
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gene
plasmid
screening
cell line
bac
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杨隽
周芳
宫世强
韦清霞
赵双
司书毅
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中国医学科学院基础医学研究所
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Definitions

  • the invention belongs to the method for screening BMP signal upregulation by using stem cell technology, and the obtained 1H-pyrazole [3,4-d]pyrimidine, 1-phenyl-4-(1-piperidinyl)- in the treatment of cardiopulmonary vascular diseases Applications.
  • Pulmonary hypertension is an increase in pulmonary resistance due to primary lesions of the pulmonary arterioles and is a condition with low incidence but high mortality. Pulmonary vascular resistance progressively increases, eventually leading to death and death in patients with right heart failure.
  • the main pathological features are severe pulmonary vascular remodeling, including endothelial dysfunction and small pulmonary artery intimal fibrosis caused by smooth muscle cell proliferation, mesenteric hypertrophy, adventitial hyperplasia, plexiform lesions and arterial lumen occlusion.
  • endothelial dysfunction and small pulmonary artery intimal fibrosis caused by smooth muscle cell proliferation, mesenteric hypertrophy, adventitial hyperplasia, plexiform lesions and arterial lumen occlusion.
  • prostacyclin and its analogues, endothelin receptor antagonists, and phosphodiesterase inhibitors have been approved for marketing, but the 3-year mortality rate is still around 40%. And these treatments are mainly aimed at reducing blood pressure and achieving
  • Bone morphogenetic protein BMPs are a class of proteins in the TGF[beta] family that play an important role in early development and differentiation, including organ formation and angiogenesis in the lungs.
  • BMP ligands including BMP2, BMP4, BMP6, BMP9 and BMP10
  • BMPRII with serine/threonine kinase activity modulates Smad protein (R-Smad) via a type 1 receptor, phosphorylation receptor.
  • Smad protein Smad protein
  • phosphorylation receptor Among the effector genes that induce transcription by BMP/Smad, the most well-researched is the inhibitor of DNA binding protein (Id).
  • the Id protein belongs to the helix-loop-helix (HLH) transcription factor, and the Id protein competitively binds to the transcription factor (bHLH) with a basic helix-loop-helix structure to inhibit its transcriptional activity.
  • Id plays a role in cell growth, senescence and differentiation. effect.
  • the experimental results clearly suggest that the BMPR-II receptor and its downstream Id gene expression decline play a leading role in the occurrence of pulmonary hypertension.
  • Increasing the concentration of BMP ligand is an effective method to increase the expression level of downstream Id.
  • BMP2 expression upregulation primary screening model - ie containing mouse bmp2 promoter The mouse and cranial cells MC3T3-E1 stably transfected with the reporter plasmid of the luciferase reporter gene were first established in China in 2002 and applied in the discovery of anti-osteoporosis drugs or their lead compounds. to date. After more than ten years of screening for BMP2 up-regulation drugs, 2-acetylbenzothiophene and its analogues were obtained, and studies on anti-osteoporosis activity were carried out. Recently, BMP signaling upregulation has also been considered as a promising drug target for the treatment of pulmonary hypertension. Edda Spiekerkoetter et al.
  • the C2C12-BRE cell line is obtained by stably transfecting C2C12 cells with a BMP response element (BRE) on the promoter region of Id and a firefly luciferase reporter gene [Katagiri T at al Genes Cells 2002 7] : 949-960], luciferase activity induced by low-dose stimulation of pm-grade BMP9 can be detected by bioluminescence detection.
  • BRE BMP response element
  • the invention first adopts the BMP2 up-regulation model, and finds that the obtained BMP2 expression up-regulating agent can improve the BMP signal and is applied to the preliminary screening of pulmonary hypertension drugs, and then utilizes the stem cell line with cardiovascular differentiation ability to construct and specifically improve pulmonary vascular remodeling. Id protein expression upregulator drug screening model.
  • One of the innovations of the present invention is the application of human embryonic stem cells to drug screening: the previously established BMP2 up-regulation drug screening platform is based on the mouse osteosarcoma cell line, and there is also the defect that there is a species difference with human cells.
  • the advantages of applying human embryonic stem cells to drug screening are: 1. The infinite proliferative ability of stem cells, in line with high-throughput drug screening; 2. Based on their multifunctional potential, they can differentiate into related cell types. 3.
  • Induction of pluripotent stem cell technology can reduce the limitation of single cell line lack of effective simulation of individual differences in individual cell lines based on individual patient differences, avoiding sifting and mis-screening [Willard VP at al Arthritis Rheumatol. 2014, 66 ( 11): 3062-72]. Therefore, it is a beneficial choice to replace tumor cells with a cell that can be derived from any individual.
  • Human embryonic stem cells have the advantage that tumor cells do not have, but whether this cell can be successfully used for anti-pulmonary hypertension drug screening has not been reported.
  • the present inventors applied human embryonic stem cells to the screening of anti-pulmonary hypertension drugs, and unexpectedly found that the measurement results are sensitive, stable, simple, and have advantages that are unmatched by the prior art. Therefore, after thorough research on the key signaling pathways of hereditary and idiopathic pulmonary hypertension, we designed the dual-reporter gene into the Id1 genome to construct a recombinant vector and then transferred it to the target gene downstream of the BMP signal.
  • a human embryonic stem cell line obtains a stably expressed cell line, thereby establishing a compound for treating pulmonary hypertension with up-regulated expression of Id1 using luciferase expression as an index. Filter the model. The candidate compounds were tested to achieve the desired technical results.
  • the invention is innovative in that a new type of pulmonary tuberculosis therapeutic drug piperidinylpyrazolopyrimidine is screened by the above method, and the phosphodiesterase activity is weak.
  • the pyrimidinone ring of sildenafil is similar to cGMP, thereby competitively binding to phosphodiesterase type 5 and inhibiting cGMP hydrolyzing ring opening.
  • 1H-pyrazole [3,4-d]pyrimidine, 1-phenyl-4-(1-piperidinyl)- does not have a ketone ring, and the structure with a piperidine ring determines its regulation of BMP signaling.
  • Related activity a new type of pulmonary tuberculosis therapeutic drug piperidinylpyrazolopyrimidine is screened by the above method, and the phosphodiesterase activity is weak.
  • the pyrimidinone ring of sildenafil is similar to cGMP, thereby competitively binding to phosphodiesterase type 5
  • the object of the present invention is to establish a stem cell model for screening drugs for pulmonary hypertension and to find a novel lead compound for the treatment of pulmonary hypertension using BMP signaling.
  • the present invention provides a cell line having the deposited number CGMCC11091.
  • the cell was named ID1-V-LUC hES, and was deposited in: China General Microbial Culture Collection and Management Center (CGMCC). Address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Beichen West Road, Chaoyang District, Beijing, China. : CGMCC No.11091.
  • the present invention provides a model for screening compounds against pulmonary hypertension, the model being characterized by:
  • the double reporter gene was designed to insert into the Id1 genome to construct a recombinant vector, and then transferred into human embryonic stem cell line to obtain a cell line stably expressing CGMCC11091, and the compound to be tested for anti-pulmonary hypertension was stimulated.
  • the cell line of CGMCC11091 was lysed with cell lysate, and the luciferase expression activity was detected by the method described by the gene detection system.
  • the expression of the double reporter gene driven by the Id1 promoter was used as an index to obtain the pulmonary hypertension with up-regulation of Id1 expression. Screening model for compounds.
  • the invention further provides a method of constructing a model, the steps are as follows:
  • the genomic sequence of the Id1 gene was found from the database, the target site was determined to be its exon 1, and the BAC plasmid (5' and 3' upstream and downstream 5Kb sequences) containing the complete sequence of the Id1 gene was searched.
  • the primer for amplifying the recombinant fragment is composed of a universal primer sequence and an Id1 gene sequence, and a resistance screening frame is inserted into a BAC plasmid having induced recombinase activity to obtain a BAC-Id1 plasmid with a screening frame.
  • a plasmid Psc101gbaA based on the Psc101 plasmid framework and tetracycline (Tet)-induced recombination was transformed into an E. coli clone carrying the Id1 gene BAC plasmid (BAC-Id1); the first homologous recombination will be carried out with the gateway R1/ The DNA fragment of the R2 locus and the zeocin negative (pheS) selection marker was inserted into the U5/D3 locus of the BAC-Id1 plasmid; the second homologous recombination will be the pBR322 with two gateway sites of R3 and R4. The plasmid fragment was subjected to a gap-repair reaction with BAC-Id1 to obtain an intermediate vector.
  • BAC-Id1 E. coli clone carrying the Id1 gene BAC plasmid
  • Construction of the destination vector was based on three plasmids of the Gateway system: an intermediate vector constructed in accordance with the invention and a pL1L2_BacT plasmid containing a targeting element and a pL3L4_DTA plasmid containing a negative selection marker. After two gateway conversion systems, a high copy of the desired recombinant vector Id1-Venus-Luc-MC1-DTA was finally produced.
  • the Id1-Venus-Luc-MC1-DTA plasmid was linearized with AsiSI, and the P57 generation human embryonic stem cell H9 was electroporated to obtain an integrated Id1 promoter to drive the human embryonic stem cell line expressing Venus-Luci double reporter gene to obtain a cell line of CGMCC11091.
  • To perform early mesoderm differentiation stimulate the anti-pulmonary hypertension compound to stimulate the cell line of CGMCC11091, and then lyse the cells with cell lysate, and detect the luciferase expression activity according to the method described by the gene detection system, driven by the Id1 promoter.
  • the reporter gene expression level was used as an indicator to obtain a screening model for the treatment of pulmonary hypertension compounds with up-regulated Id1 expression.
  • the invention also encompasses the use of a compound obtained by screening with a model of the invention for the preparation of a medicament for the prevention of pulmonary hypertension, said compound being selected from the group consisting of:
  • Piperidinylpyrazolopyrimidines 1H-pyrazole [3,4-d]pyrimidine, 1-phenyl-4-(1-piperidinyl)-1H-Pyrazolo[3,4-d]pyrimidine,1 -phenyl-4-(1-piperidinyl)-(Cas No:23000-46-6),
  • the application of the invention also includes screening for the following drugs:
  • BMP bone morphogenetic protein
  • the screening method of the present invention mainly comprises the following steps:
  • BMP2 up-regulation can increase BMP signal and is applied to pulmonary hypertension drug screening: BMP2 up-regulation model screens the effect of the compound on myoblast C2C12-BRE (Example 1).
  • the human embryonic stem cell Id1 gene targeting vector was constructed by targeting the key gene Id gene downstream of BMP signal (Example 2).
  • the materials used in the present invention are all commercially available.
  • Figure 8 Comparison of luciferase activity and endogenous Id expression levels in different clones stimulated by BMP4; wherein A: luciferase activity; B: endogenous Id expression level
  • control normal saline
  • MCT monocrotaline
  • sildenafil positive drug sildenafil
  • A piperidinylpyrazolopyrimidine
  • Example 1 Using C2C12-BRE cells to find that BMP2 expression up-regulation can increase BMP signal and be applied to pulmonary artery drug screening
  • Promega enzyme expression system was assayed using Promega's Luciferase Assay System reporter assay using Perkin Elmer's EnVision Model 2104-0010 Multi-Purpose Reader.
  • C2C12-BRE cells a modified myoblast, provided by the Department of Cell Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences
  • inoculated 96-well plates at 50,000 cells/well, until cells grow to 70% ⁇
  • remove the original medium gently rinse the cells once with PBS to remove the interference of various active factors in the serum
  • add an equal amount of serum-free DMEM medium to each well, and then add a certain amount a total of 31 compounds of piperidinylpyrazolopyrimidines BUR1-12 and OUR1-19, such that the final concentration of each compound is 2 ⁇ g / mL and 10 ⁇ g / mL, respectively, and each concentration of each compound is set in parallel.
  • the luciferase expression activity rate of the compound at each concentration was calculated as follows:
  • the Id gene targeting vector was constructed by targeting the key gene Id gene downstream of BMP signal; the Id gene promoter drives the double reporter gene vector and stably obtained human embryonic stem cell line.
  • the targeting of the human stem cell Id gene was carried out by the GATWAY method. The first is to use the established bacterial artificial chromosome library containing the full human genome information to find a suitable length of BAC plasmid containing the gene of interest. Then, the gene sequence and functional region analysis were performed by computer design to determine the key exons of the gene, and then the appropriate target site was selected by AOS (Array Oligo Selector) computer program. Gene-specific recombinant fragments were then obtained using gene-specific recombinant primers and 96-well plate high-throughput PCR.
  • AOS Array Oligo Selector
  • a high-efficiency, Tet-inducible recombinant plasmid was introduced into the BAC clone in a 96-well plate, and the purified recombinant fragment was added, and the recombinant reaction was carried out by two recombination reactions (the first recombination was inserted into the single-copy BAC plasmid,
  • the BAC subcloned with a suitable size homology arm was subcloned into another plasmid framework by a gap-repair reaction to recombine the gene-specific recombinant fragment and the corresponding BAC clone to obtain an intermediate vector library.
  • the genomic sequence of the Id1 gene was found from the database, the target site was determined to be its exon 1, and the BAC plasmid containing the complete sequence of the Id1 gene [provided by the Sanger Institute] (5' and 3' upstream and downstream 5Kb sequences) was searched.
  • the primers used to amplify the recombinant fragment consisted of 70 bases. Among them, 20 bp at the 3' end of the primer is a universal primer for the resistance screening frame, and 50 bp at the 5' end of the primer is the Id1 gene sequence.
  • High-throughput primers are ordered in the form of 96-well plates in a sequence corresponding to gene-specific BAC clones in 96-well plates. The PCR reaction was then completed in a 96-well plate, the PCR product (about 10 kb) was purified by ultrafiltration, and finally 2 ug of the purified fragment was electroporated into a BAC clone that had induced recombinase activity.
  • a plasmid Psc101gbaA (supplied by Sanger Institute) based on the Psc101 plasmid framework, tetracycline (Tet)-induced recombination was transformed into an E. coli clone carrying the Id1 gene BAC plasmid (BAC-Id1).
  • a DNA fragment containing the gateway R1 and R2 sites and the zeocin negative (pheS) selection marker was inserted into the BAC-Id1 plasmid. Insert the U5 and D3 sites.
  • Gap repair subclones a BAC fragment containing a modified key exon and a 5 kb homology arm upstream and downstream of a key exon to another vector in order to remove the BAC fragment flanking the homologous arm.
  • the Gap repair framework is a linearized plasmid based on pBR322 (supplied by the Sanger Institute). This framework contains two gateway sites, R3 and R4, which can interact with L3 and L4gateway sites carrying high-copy plasmids carrying embryonic stem cells (ES), targeting negative screening marker MC1 promoter-diphtheria A subtype (MC1-DTA) A replacement occurs.
  • the resulting vector is referred to as the "intermediate vector”. This intermediate vector can be substituted for different components by the attR1/R2 and R3/R4 sites of the gateway system to obtain the desired target vector.
  • the recombinant vector of interest consists of three Gateway system-based plasmids: an intermediate vector constructed according to the invention and a pL1L2_BacT plasmid containing a targeting element and a pL3L4_DTA plasmid containing a negative selection marker (Sanger Institute) (Fig. 5).
  • This example includes two gateway-based conversion systems, each consisting of two pairs of gateway sites.
  • the intermediate vector comprises four gateway sites (R1/R2 and R3/R4), the vector of the targeting element comprises an L1/L2 site corresponding to R1/R2, and the vector of negative screening marker comprises a corresponding R3/R4 L3/L4 locus.
  • a negative reporter marker (PheS) between the R1/R2 sites of the intermediate vector and a double reporter (Venus-Luc) system element between the L1/L2 sites containing the vector of the targeting element are used in the present invention (SA- Replacement occurs in T2A-H2B_venus-T2A-LUC-pA-promoter-puro-pA) (Sanger Institute) (Fig. 6).
  • the second transformation system will react with R3-L3 and R4-L4 and will contain homology arms and targets.
  • the fragment of the element was transferred to a high-copy plasmid containing the mammalian negative selection marker gene (DTA) (Sanger Institute), and the R3 and R4 sites were located at the end of the homologous arm of the targeting vector, at the L3 and L4 sites.
  • DTA mammalian negative selection marker gene
  • the mammalian negative selection marker gene is included.
  • a high copy of the destination vector designated as Id1-Venus-Luc-MC1-DTA, was generated by reaction of R3/R4 with the L3/L4 site. ( Figure 7).
  • the plasmid of Id1-Venus-Luc-MC1-DTA was electroporated into P57 generation human embryonic stem cells H9 (a passaged human embryonic stem cell, commercially available).
  • the medium containing the serum substitute was cultured on a DR4MEF-trophoblast cell-(US Applied Stem Cell Co., Ltd.) at 37 ° C under a condition of 5% CO 2 per day.
  • the cells were electroporated using a BioRad electroporator (ZAP: 250V, uF 500, TC 8.6), and then 19 clones were obtained by puromycin screening.
  • the green fluorescent integrated plasmid clones (cells with MC1-DTA plasmid type could not survive) were selected and tested. Fluorescein activity was consistent with endogenous Id gene expression (Figure 8).
  • clone 2 was observed to have the same level of luciferase expression as the QPCR-measured Id1 mRNA transcription level under the same amount of BMP4 stimulation, and the enzyme activity measured at 1, 2, 3, and 5 hours after stimulation increased with time. Large, fluorescein expression levels can be used to maximize the effect of external stimuli on Id gene expression. Therefore, clone 2 was designated as ID1-V-LUC hES and the compound was screened (Fig. 9). ID1-V-LUC hES for early mesoderm differentiation, expression of smooth muscle cell marker ⁇ SMA, the vascular cell type of Calponin.
  • the ID1-V-LUC hES-differentiated cell line was stimulated by BMP4 (Peprotech), and a concentration gradient activation effect was observed. After 6.25 ng/mL BMP4 stimulation of ID1-V-LUC, the mediation rate reached 7.5 times.
  • the 31 compounds were compared for activity levels after stimulation at two different concentrations (2 ⁇ g/mL, 10 ⁇ g/mL) for 4 hours ( Figure 10).
  • the Venus-Luci double reporter gene-driven Id1 promoter system human embryonic stem cell line ID1-V-LUC hES model can be applied to high-throughput drug screening.
  • the ID1-V-LUC hES model stem cell strain was deposited in the General Microbiology Center of the China Microbial Culture Collection Management Committee, and the deposit number is CGMCC No:11091.
  • the ID1-V-LUC hES model stem cells were inoculated into 96-well plates. When the cell colonies were over 80% of the whole wells, the original medium was removed, gently rinsed once with PBS, and added to human embryonic stem cell mTeSR medium ( Hangzhou Baitong Biotechnology Co., Ltd.) After 1 h of starvation, 1H-pyrazole [3,4-d]pyrimidine, 1-phenyl-4-(1-piperidinyl)-(Cas No: 23000-46-6) The cells were incubated together.
  • the luciferase expression activity rate of the compound at each concentration was calculated as follows:
  • Endothelial cells were cultured to complete fullness in SFM containing 1% FBS in complete medium and treated with 0.2% FBS SFM starvation medium for 16 hours one day prior to tube formation experiments.
  • Matrigel gel (Corning, 10 mg/ml) was placed on ice overnight and thawed.
  • 50 ul per well was added to the pre-cooled 96-well plate on ice, evenly distributed in the well, and then placed in 37. The incubator was allowed to stand for 30 minutes.
  • the starvation medium was removed, and washed twice with DPBS solution, then accutase was added, digested for 1 minute, and the cells were resuspended by adding SFM starvation medium, and the cells were gently pipetted to prepare a single cell suspension, and centrifuged at 1000 rpm for 4 minutes at room temperature. The cells were resuspended in SFM starvation medium and counted. 1x10 4 cells were seeded per well, the cells were evenly distributed by shaking, placed in a 37-degree incubator, and the cells were observed to form tubes within 2-8 hours.
  • Example 6 Therapeutic effect of piperidinylpyrazolopyrimidine on rat model of monocrotaline pulmonary hypertension (MCT-PAH)
  • MCT-PAH rats in vivo male Sprague-Dawley rats (China Food and Drug Control Research Institute) were given subcutaneous subcutaneous administration of monocrotaline (MCT, 55 mg/kg) or normal saline. After MCT injection for 3 weeks, 4.5 mg was intragastrically administered. /kg piperidinylpyrazolopyrimidine compound (A), and saline and positive drug control sildenafil (Sildenafil). After 2 weeks of dosing, right ventricular systolic pressure was measured and survival was counted: the BL-420E system preserved right ventricular systolic pressure (RVSP) data ( Figure 13). It was shown that piperidinylpyrazolopyrimidine significantly reduced pulmonary artery pressure in rats with pulmonary hypertension (MCT-PAH) and significantly improved the survival rate of model animals (Fig. 14).

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Abstract

提供了保藏编号为CGMCC11091的细胞株及其制备方法，所述方法包括通过以BMP信号下游关键基因Id1基因为靶点，设计双报告基因原件***Id1基因组构建重组载体，并转入人胚胎干细胞株。通过该细胞株筛选出的对Id1表达上调的化合物在制备抗肺动脉高压的药物中的应用。

Description

一种哌啶基吡唑并嘧啶类化合物的筛选和用途 技术领域
本发明属于利用干细胞技术建立BMP信号上调剂筛选方法,得到的1H-吡唑[3,4-d]嘧啶,1-苯-4-(1-哌啶基)-在心肺血管疾病治疗药物中的应用。
背景技术
肺动脉高压(PAH)是由于肺小动脉原发病变而导致的肺动脉阻力增加，是一种少发但高致死率的病症。肺血管阻力进行性升高，最终导致患者右心衰竭而死亡。其主要病理特征是严重肺血管重构，包括内皮功能紊乱和平滑肌细胞增殖所致的小肺动脉内膜纤维化，中膜肥厚，外膜增生，丛样病变及动脉管腔闭塞。在过去20年里包括前列环素和其类似物，内皮素受体拮抗剂，磷酸二酯酶抑制剂在内的几类新药已经批准上市，但3年死亡率仍然在40％左右。并且这些治疗方案主要针对减轻血管紧张而达到治疗效果。因此我们迫切需要研发改善血管重构，延缓肺动脉高压病程，提高患者生存率为目的的药物。
优化设计PAH理性治疗方案应基于对此疾病细胞分子机理的透彻认识。研究表明肺动脉高压中存在着BMP二型受体(BMPRII)表达缺陷和Id基因表达下调[Yang J at al Circulation Reserch 2008,102:1212-1221]。恢复生理水平BMP信号,尤其是血管细胞中Id基因的表达是改善血管重构的肺动脉高压药物的新靶点之一[Yang J at al Am J Physiol Lung Cell Mol Physiol 2013 15:305(4):L 312-21]。
骨形成蛋白BMPs是TGFβ家族中的一类蛋白，它们在早期发育和分化，包括肺的器官形成以及血管生成中起着重要作用。在BMP配体包括BMP2，BMP4，BMP6，BMP9和BMP10存在的情况下，具有丝氨酸/苏氨酸激酶活性的BMPRII，通过一型受体，磷酸化受体调节Smad蛋白(R-Smad)。在BMP/Smad诱导转录的效应基因中，目前研究最清楚的是DNA结合蛋白抑制因子家族成员(inhibitor of DNA binding protein，Id)。Id蛋白属于螺旋-环-螺旋(HLH)转录因子，Id蛋白竞争性结合具基本螺旋-环-螺旋结构的转录因子(bHLH)而抑制其转录活性，Id在细胞生长、衰老及分化中均发挥作用。实验结果明显提示BMPR-II受体以及其下游Id基因表达下降在肺动脉高压发生中起主导作用。提高BMP配体浓度是一种有效的提高下游Id表达水平的方法。BMP2表达上调剂初筛模型-即包含小鼠bmp2启动 子和荧光素酶报告基因的报告质粒稳定转染得到的小鼠颅骨细胞MC3T3-E1是第一发明人2002年首次在国内建立的,并在抗骨质疏松药物或其先导化合物的发现中应用至今。经过十余年的BMP2上调剂药物筛选工作，得到了包括2-乙酰苯并噻吩及其类似物，并开展了抗骨质疏松活性方面的研究。最近BMP信号上调剂也被认为是治疗肺动脉高压的一个有前景的药靶，Edda Spiekerkoetter等人[Edda Spiekerkoetter at al The Journal of Clinical Investigation2013,123(8):3600-3612]通过C2C12-BRE细胞株进行了对BMP信号上调剂的筛选，发现FK506有较好的改善和逆转肺动脉高压的效果，但由于其毒副作用未进入临床。C2C12-BRE细胞株是通过将Id的启动子区上的BMP效应元件(BMP Responsive Element,BRE)与萤火虫荧光素酶报告基因相连，稳定转染C2C12细胞得到的[Katagiri T at al Genes Cells 2002 7：949-960]，通过生物发光检测可检出pm级BMP9低剂量刺激后引起的荧光素酶活性。本发明首先采用BMP2上调剂模型，发现获得的BMP2表达上调剂可提高BMP信号并应用于肺动脉高压药物初筛，继而利用具有心血管分化能力的干细胞系，构建获得特异地改善肺血管重构的Id蛋白表达上调剂药物筛选模型。
本发明的创新点之一在于将人胚胎干细胞应用于药物筛选：之前建立的BMP2上调剂药物筛选平台基于鼠骨肉瘤细胞系进行的，还存在与人类细胞有种属差异这一缺陷。将人胚胎干细胞应用于药物筛选的优势在于1.干细胞的无限增殖能力,符合高通量药物筛选；2.基于其多功能潜性，可以分化形成相关的细胞类型。3.诱导多功能干细胞技术可以根据病人个体差异‘定身量衣’，打破单一细胞系缺乏有效模拟生物个体差异的局限，避免漏筛和错筛[Willard VP at al Arthritis Rheumatol.2014,66(11):3062-72]。因此用一种可以来自任何个体的细胞代替肿瘤细胞是一种有益的选择。
人胚胎干细胞具有肿瘤细胞没有的优势，但该细胞是否能够成功用于抗肺动脉高压药物筛选，目前没有报道。本发明人将人胚胎干细胞应用于抗肺动脉高压药物的筛选，意外的发现测定结果灵敏，稳定，简单，具有现有技术无法比拟的优势。所以在通过对遗传性和特发性肺动脉高压致病关键信号通路进行透彻研究后，我们以BMP信号下游关键基因Id1基因为靶点，设计双报告基因原件***Id1基因组构建重组载体，并转入人胚胎干细胞株，获得稳定表达的细胞株，以此建立以荧光素酶表达量作为指标，获得对Id1表达上调的治疗肺动脉高压的化合物 筛选模型。对候选化合物进行了测试，达到了理想的技术效果。
本发明创新性在于通过以上方法筛选得到一类新型肺动脉高压治疗药物哌啶基吡唑并嘧啶类化合物，抑制磷酸二酯酶活性微弱。西地那非的嘧啶酮环与cGMP相似，从而竞争性的结合5型磷酸二酯酶而抑制cGMP水解开环。但1H-吡唑[3,4-d]嘧啶,1-苯-4-(1-哌啶基)-不具有酮环，而带有哌啶环的结构决定了其对BMP信号的调节和相关的活性。
发明内容
本发明的目的是建立一种用于筛选肺动脉高压治疗药物的干细胞模型，发现新型的提高BMP信号的肺动脉高压治疗药物先导化合物。
为此，本发明提供一种保藏编号为CGMCC11091的细胞株。所述细胞命名为为ID1-V-LUC hES，保藏于：中国普通微生物菌种保藏管理中心(CGMCC)，地址：北京市朝阳区北辰西路1号院3号中国科学院微生物研究所，保藏编号：CGMCC No.11091。
本发明提供一种筛选抗肺动脉高压的化合物的模型，所述模型特征在于:
以BMP信号下游关键基因Id1基因为靶点，设计双报告基因原件***Id1基因组构建重组载体，并转入人胚胎干细胞株，获得稳定表达的CGMCC11091的细胞株，将待测抗肺动脉高压的化合物刺激CGMCC11091的细胞株，再用细胞裂解液裂解细胞，按基因检测***所述方法检测荧光素酶表达活性，以Id1启动子驱动的双报告基因表达量作为指标，获得对Id1表达上调的治疗肺动脉高压化合物的筛选模型。
本发明进一步提供构建模型的方法，步骤如下：
(1)从数据库中找到Id1基因的基因组序列，确定打靶位点为其外显子1，检索包含Id1基因完整序列的BAC质粒(5’和3’上下游5Kb序列)。
(2)生物信息学分析：利用AOS电脑程序，设计并选择最优的位于Id1基因1号外显子ATG下游231bp的U5和D3位点，用于重组打靶。
(3)PCR反应产生基因特异性重组片段：
用于扩增重组片段的引物是由通用引物序列和Id1基因序列共同组成，电转将抗性筛选框***已诱导重组酶活性的BAC质粒中，获得带筛选框的BAC-Id1质粒。
(4)中间载体的构建：
将一个基于Psc101质粒框架、四环素(Tet)诱导重组的质粒Psc101gbaA，转化至携带含Id1基因BAC质粒(BAC-Id1)的E.Coli克隆中；通过第一次同源重组将带有gateway R1/R2位点和细菌正(zeocin)负(pheS)筛选标记的DNA片段***到BAC-Id1质粒U5/D3位点中；第二次同源重组将带有R3和R4两个gateway位点的pBR322质粒片断与BAC-Id1进行gap-repair反应，获得中间载体。
(5)目的重组载体的构建：
目的载体的构建基于Gateway***的三个质粒：本发明构建的中间载体和含有靶向元件的pL1L2_BacT质粒以及含负筛选标记的pL3L4_DTA质粒组成。经两次gateway的转换体系，最终产生高拷贝的目的重组载体Id1-Venus-Luc-MC1-DTA。
(6)筛选细胞株的获得
将Id1-Venus-Luc-MC1-DTA质粒用AsiSI线性化处理，电转P57代人胚胎干细胞H9，获得整合型Id1启动子驱动Venus-Luci双报告基因表达的人胚胎干细胞株，得到CGMCC11091的细胞株，进行早期中胚层分化，将待测抗肺动脉高压的化合物刺激CGMCC11091的细胞株，再用细胞裂解液裂解细胞，按基因检测***所述方法检测荧光素酶表达活性，以Id1启动子驱动的双报告基因表达量作为指标，获得对Id1表达上调的治疗肺动脉高压化合物的筛选模型。
本发明还包括用本发明的模型筛选后得到的化合物在制备抗肺动脉高压的药物中的应用，所述化合物选自：
哌啶基吡唑并嘧啶类化合物:1H-吡唑[3,4-d]嘧啶,1-苯-4-(1-哌啶基)-1H-Pyrazolo[3,4-d]pyrimidine,1-phenyl-4-(1-piperidinyl)-(Cas No:23000-46-6)，
本发明所述的应用还包括对以下药物的筛选：
(1)上调骨形态形成蛋白(BMP)信号的调节剂；
(2)上调骨形态形成蛋白II型受体表达水平的调节剂；
(3)改善肺动脉血流动力学和改善肺血管重构的制剂；
(4)在促进内皮细胞成血管的功能中的应用；
(5)肺动脉高压治疗药物；
(6)心肺血管疾病治疗药物。
本发明的筛选方法主要包括以下步骤：
一、发现BMP2表达上调剂可提高BMP信号并应用于肺动脉高压药物初筛:BMP2上调剂模型筛选所得化合物对成肌细胞C2C12-BRE的作用(实施例一)。
二、肺动脉高压药物干细胞复筛模型的建立和应用
(1)以BMP信号下游关键基因Id基因为靶点，构建人胚胎干细胞Id1基因打靶载体(实施例二)。
(2)干细胞筛选模型的建立：Id1上调剂双报告体系载体构建和稳定表达报告体系的人胚胎干细胞株ID1-V-LUC hES的获得。(实施例三)。
(3)哌啶基吡唑并嘧啶类化合物在人胚胎干细胞株ID1-V-LUC hES上调Id1表达的作用。(实施例四)
三、通过肺动脉高压野百合碱大鼠检测化合物对肺动脉高压的治疗效果(实施例五)
本发明中所用的原料都是可以从市场上购买到的。
附图说明：
附图1、C2C12-BRE报告***对化合物上调BMP2的验证
其中：A OUR系列1-19；B BUR系列1-12。
附图2、打靶载体构建流程
附图3、Id1基因打靶位点设计
附图4、中间载体构建流程
附图5、目的载体的构建组成
附图6、目的载体的构建
附图7、目的载体
附图8、不同克隆在BMP4刺激下荧光素酶活性与内源性Id表达水平的比较；其中A：荧光素酶活性；B：内源性Id表达水平
附图9、BMP4刺激克隆ID1-V-Luc hES后显示浓度依赖性和时间依赖性
附图10、ID1-V-Luc hES在不同化合物刺激后荧光素酶活性
附图11、ID1-V-Luc hES在哌啶基吡唑并嘧啶类化合物刺激后荧光素酶活性；
其中：纵坐标：荧光素酶上调率，横坐标：化合物浓度(μg/ml)
附图12、哌啶基吡唑并嘧啶类化合物促内皮细胞成管作用
附图13、给药后对MCT-PAH大鼠右室收缩压的作用
附图14、给药后对MCT-PAH大鼠存活率的作用
其中：control：生理盐水，MCT：野百合碱，sildenafil：阳性药西地那非，A：哌啶基吡唑并嘧啶类化合物
具体实施方式：
以下通过实施例进一步说明本发明。
本项目是在中国医学科学院基础医学研究所医学分子生物学重点实验室进行,天然与合成化合物样品由中国医学科学院医药生物技术研究所新药(微生物)筛选实验室提供。
一、发现BMP2表达上调剂可提高BMP信号，并应用于肺动脉高压药物初筛:
实施例1利用C2C12-BRE细胞发现BMP2表达上调剂可提高BMP信号并应用于肺动脉高压药物初筛
(1)荧光素酶表达活性的测定
采用Promega公司的Luciferase Assay System报告基因检测***，利用Perkin Elmer公司的EnVision 2104-0010型多功能读板仪测定荧光素酶表达活性。
(2)具有BRE转录激活功能小分子化合物的筛选
消化对数生长期的C2C12-BRE细胞(一种改造的成肌细胞,中国医学科学院基础医学研究所细胞生物学系提供)，以50,000个/孔接种96孔板，待细胞生长至70％～90％汇合时，移去原有培养基，用PBS轻轻漂洗细胞一次，以除去血清中各种活性因子的干扰，向各孔中加入等量的无血清DMEM培养基，再分别加入一定量的哌啶基吡唑并嘧啶类化合物BUR1-12和OUR1-19共31个化合物，使每种化合物的终浓度分别为2μg/mL和10μg/mL，每种化合物的每个浓度均平行设立三个复孔，同时以只加入1‰DMSO的孔作为空白对照。37℃，5％CO2条件下培养18～24h后，用CCLR(Promega)细胞裂解液裂解细胞，按Luciferase Assay System报告基因检测***说明书所述方法检测荧光素酶表达活性。
按如下公式计算化合物在各个浓度下的荧光素酶表达活性调节率：
Figure PCTCN2016089970-appb-000001
结果表明，31个化合物中，除BUR9、OUR1、OUR9外，其余化合物均具有较为明显的(≥1.5倍)BRE转录激活功能，附图1A和B中显示31个化合物上调BRE转录活性的比值。
二、肺动脉高压药物干细胞复筛模型的建立和应用
以BMP信号下游关键基因Id基因为靶点，构建Id基因打靶载体；Id基因启动子驱动双报告基因体系载体和稳定表达的人胚胎干细胞株的获得。
针对人类干细胞Id基因的打靶是通过GATWAY方法进行的。首先是利用已建的包含全人基因组信息的细菌人工染色体文库，寻找包含目的基因的合适长度的BAC质粒。然后通过电脑设计进行基因序列与功能区域分析，确定该基因的关键外显子，然后利用AOS(Array Oligo Selector)计算机程序挑选合适的打靶位点。继而利用基因特异的重组引物以及96-孔板高通量PCR，获得基因特异的重组片段。然后将一个高效的、Tet诱导型的重组质粒导入96孔板中的BAC克隆，加入纯化的重组片段，通过两次重组反应(第一次重组将顺式元件***到单拷贝的BAC质粒，第二次利用gap-repair反应将携带合适大小同源臂的BAC亚克隆到另一个质粒框架中)，实现基因特异重组片段与相对应BAC克隆的重组，获得中间载体文库。最后利用3-way gateway***，将所需元件***中间载体，获得最终的基因打靶载体。最后利用25孔板，将纯化获得的基因打靶载体电转导入胚胎干细胞，通过负筛选获得基因打靶克隆。干细胞高通量基因打靶载体构建及细胞株获得流程示意图见图2。
实施例二：人胚胎干细胞Id1基因打靶载体的构建
1.从数据库中找到Id1基因的基因组序列，确定打靶位点为其外显子1，检索包含Id1基因完整序列的BAC质粒[Sanger研究所提供](5’和3’上下游5Kb序列)。
2.生物信息学分析：利用AOS电脑程序，在箭头所示的候选区，设计并选择最优的核苷酸片段，用于重组打靶。共设计4个重组打靶位点(图3)：U5和D3位点位于Id1基因1号外显子ATG下游231bp；G5位于U5上游约6kb的位置；G3位于D3下游约4kb的位置。
3.PCR反应产生基因特异性重组片段流程：
用于扩增重组片段的引物由70个碱基组成。其中位于引物3’端的20bp为抗性筛选框的通用引物，而引物5’端50bp则为Id1基因序列。高通量的引物是以96孔板的形式订制，其排列顺序与96孔板中基因特异的BAC克隆相对应。然后在96孔板中完成PCR反应，通过超滤纯化PCR产物(约10kb)，最后将2ug纯化的片段电转至已诱导重组酶活性的BAC克隆中。
4.中间载体的构建过程：
高通量Id1基因中间载体的构建在96孔板中进行，主要包括三个步骤(图4)
(1)Bac转化
将一个基于Psc101质粒框架、四环素(Tet)诱导重组的质粒Psc101gbaA(Sanger研究所提供)转化至携带含Id1基因BAC质粒(BAC-Id1)的E.Coli克隆中。
(2)关键外显子上下游序列***
将一段包含gateway R1和R2位点以及细菌正(zeocin)负(pheS)筛选标记的DNA片段(Sanger研究所提供)***到BAC-Id1质粒中。***U5和D3位点。
(3)Gap repair
Gap repair将包含经过修饰的关键外显子和关键外显子上下游5kb同源臂的BAC片段亚克隆到另一个载体上，目的是将同源臂两侧的BAC片段去除。Gap repair框架是一个基于pBR322的线性化质粒(Sanger研究所提供)。这个框架包含R3和R4两个gateway位点，能与携带胚胎干细胞(ES)，打靶负筛选标记MC1启动子-白喉菌素A亚型(MC1-DTA)的高拷贝质粒的L3和L4gateway位点发生置换。最终获得的载体我们称之为“中间载体”，这个中间载体可以通过gateway***attR1/R2和R3/R4位点置换不同的元件，最终获得理想的目的载体。
5.目的重组载体的构建流程：
目的重组载体由3个基于Gateway***的质粒：本发明构建的中间载体和含有靶向元件的pL1L2_BacT质粒以及含负筛选标记的pL3L4_DTA质粒(Sanger研究所)(图5)组成。本实例包括两次基于gateway的转换体系，每个体系主要由2对gateway位点组成。中间载体包含四个gateway位点(R1/R2和R3/R4)，靶向元件的载体包含与R1/R2相对应的L1/L2位点，负筛选标记的载体包含与R3/R4相对应的L3/L4位点。
第一次转换，本发明中采用中间载体R1/R2位点间的负筛选标记(PheS)与含靶向元件载体的L1/L2位点间的双报告(Venus-Luc)***元件(SA-T2A-H2B_venus-T2A-LUC-pA-promoter-puro-pA)(Sanger研究所)发生置换(图6)，第二次转化体系通过R3-L3和R4-L4反应，将含有同源臂和靶向元件的片段转移到一个含有哺乳动物负筛选标记基因(DTA)的高拷贝的质粒(Sanger研究所)中，R3和R4位点位于靶向载体同源臂的末端，L3和L4位点之间包含哺乳动物负筛选标记基因。最终通过R3/R4与L3/L4位点反应产生高拷贝的目的载体，命名为Id1-Venus-Luc-MC1-DTA的重组载体。(图7)。
实施例三：人胚胎干细胞Id1基因打靶双报告基因Id1-Venus-Luc细胞株的构建
在用AsiSI线性化处理后，将Id1-Venus-Luc-MC1-DTA的质粒电转P57代人胚胎干细胞H9(一种经过传代的人胚胎干细胞，可以从市场上购买得到)。在37℃，CO2 5％的条件下，通过含血清替代物的培养基在DR4MEF-滋养层细胞-(美国应用干细胞有限公司)上，每天换液培养。使用BioRad电转仪(ZAP:250V，uF 500，TC 8.6)电转，然后用用puromycin筛选共得到19个克隆，挑选出发绿色荧光整合型质粒克隆(带MC1-DTA质粒型的细胞不能存活)，检测荧光素活性与内源性Id基因表达的一致性(图8)。
我们发现克隆2在同量BMP4刺激下，可观察到荧光素酶表达水平与QPCR测得Id1mRNA转录水平一致，并且在刺激后1，2，3，5小时测得的酶活性随时间增长而加大，可以通过荧光素酶表达水平最大限度地反映外界刺激对Id基因表达的影响。所以选定克隆2定名为ID1-V-LUC hES并进行对化合物的复筛(图9)。ID1-V-LUC hES进行早期中胚层分化，获得表达平滑肌细胞标志α SMA，Calponin的血管细胞类型。ID1-V-LUC hES分化细胞株在BMP4(Peprotech公司)刺激下，可观察到浓度梯度激活效应,6.25ng/mL BMP4刺激ID1-V-LUC 4小时后调解率达到7.5倍。31个化合物在两个不同浓度(2μg/mL,10μg/mL)4小时刺激后比较活性水平(图10)。说明Venus-Luci双报告基因驱动Id1启动子***人胚胎干细胞株ID1-V-LUC hES模型可应用于高通量药物筛选。ID1-V-LUC hES模型干细胞株在中国微生物菌种保藏管理委员会普通微生物中心保藏，保藏编号：CGMCC No：11091
实施例四:Venus-Luci双报告基因驱动Id1启动子***人胚胎干细胞株模型的应用
将ID1-V-LUC hES模型干细胞接种于96孔板，待细胞集落长满全孔的80％左右时，移去原有培养基，用PBS轻轻漂洗一次，加入人胚胎干细胞mTeSR培养基(杭州百通生物技术公司)饥饿6h后，将1H-吡唑[3,4-d]嘧啶,1-苯-4-(1-哌啶基)-(Cas No:23000-46-6)与细胞共孵育。用上述培养基逐级稀释成5,2.5,1.25,0.625,0.3μg/mL，顺次加入接种有模型干细胞的96孔板中，每个浓度平行做三个复孔，同时以只含有0.1‰DMSO的培养基作为空白对照。37℃，5％CO2条件下培养18～24h后，用CCLR(Promega)细胞裂解液裂解细胞，按Luciferase Assay System报告基因检测***说明书所述方法检测荧光素酶表达活性。
按如下公式计算化合物在各个浓度下的荧光素酶表达活性调节率：
Figure PCTCN2016089970-appb-000002
结果表明，哌啶基吡唑并嘧啶类化合物以剂量依赖的方式上调模型干细胞荧光素酶的表达水平，其中2.5μg/mL的上调水平最高，达到3.31倍左右(图11)。表明该化合物可以上调Id基因表达。
实施例五:哌啶基吡唑并嘧啶类化合物促内皮细胞血管形成作用
内皮细胞在SFM含1％FBS的完全培养基中培养至完全长满，成管实验前一天开始用含0.2％FBS SFM饥饿培养基处理16小时。成管实验前一天将Matrigel胶(Corning，10mg/ml)置于冰上过夜融化，成管当天每孔50ul加至冰上预冷的 96孔板中，均匀分布于孔中，然后置于37度培养箱静置30分钟。去除饥饿培养基，并且加入DPBS溶液洗两次，再加入accutase酶，消化1分钟，再加入SFM饥饿培养基重悬细胞，轻轻吹打细胞制成单细胞悬液，室温离心1000rpm，4分钟。SFM饥饿培养基重悬细胞，并计数。每孔种1x104细胞，轻轻晃动使细胞分布均匀，置于37度培养箱，2-8小时内观察细胞成管。
如(图12)所示，与对照组比较，BUR1处理后(1μM和5μM)的EC细胞成管能力明显高于对照组；与阳性对照VEGF处理组比较，BUR1处理后(1μM和5μM)促EC细胞成管能力与VEGF相当。
实施例六：哌啶基吡唑并嘧啶类化合物对野百合碱肺动脉高压(MCT-PAH)造模大鼠的治疗效果
MCT-PAH大鼠体内实验:雄性Sprague-Dawley大鼠(中国食品药品检定研究院)单次皮下给予野百合碱(MCT，55mg/kg)或生理盐水，MCT注射3周后灌胃给予4.5mg/kg哌啶基吡唑并嘧啶类化合物(A)，和生理盐水及阳性药对照西地那非(Sildenafil)。给药2周后，检测右室收缩压并统计存活率：，BL-420E***保存右心室收缩压(right ventricular systolic pressure，RVSP)数据(图13)。表明哌啶基吡唑并嘧啶类化合物显著降低肺动脉高压(MCT-PAH)造模大鼠肺动脉压力，明显提高造模动物的生存率(图14)。

Claims (5)

  1. 一种保藏编号为CGMCC11091的细胞株。
  2. 一种筛选抗肺动脉高压的化合物的模型，所述模型特征在于，
    以BMP信号下游关键基因Id1基因为靶点，设计双报告基因原件***Id1基因组构建重组载体，并转入人胚胎干细胞株，获得稳定表达的CGMCC11091的细胞株，将待测抗肺动脉高压的化合物刺激CGMCC11091的细胞株，再用细胞裂解液裂解细胞，按基因检测***所述方法检测荧光素酶表达活性，以Id1启动子驱动的双报告基因表达量作为指标，获得对Id1表达上调的治疗肺动脉高压化合物的筛选模型。
  3. 构建权利要求2所述模型的方法，其特征在于，步骤如下：
    (1)从数据库中找到Id1基因的基因组序列，确定打靶位点为其外显子1，检索包含Id1基因完整序列的BAC质粒(5’和3’上下游5Kb序列)；
    (2)生物信息学分析：利用AOS电脑程序，设计并选择最优的位于Id1基因1号外显子ATG下游231bp的U5和D3位点，用于重组打靶；
    (3)PCR反应产生基因特异性重组片段：
    用于扩增重组片段的引物是由通用引物序列和Id1基因序列共同组成，电转将抗性筛选框***已诱导重组酶活性的BAC质粒中，获得带筛选框的BAC-Id1质粒；
    (4)中间载体的构建：
    将一个基于Psc101质粒框架、四环素(Tet)诱导重组的质粒Psc101gbaA，转化至携带含Id1基因BAC质粒(BAC-Id1)的E.Coli克隆中；通过第一次同源重组将带有gateway R1/R2位点和细菌正(zeocin)负(pheS)筛选标记的DNA片段***到BAC-Id1质粒U5/D3位点中；第二次同源重组将带有R3和R4两个gateway位点的pBR322质粒片断与BAC-Id1进行gap-repair反应，获得中间载体；
    (5)目的重组载体的构建：
    目的载体的构建基于Gateway***的三个质粒：本发明构建的中间载体和含有靶向元件的pL1L2_BacT质粒以及含负筛选标记的pL3L4_DTA质粒组 成。经两次gateway的转换体系，最终产生高拷贝的目的重组载体Id1-Venus-Luc-MC1-DTA；
    (6)筛选细胞株的获得
    将Id1-Venus-Luc-MC1-DTA质粒用AsiSI线性化处理，电转P57代人胚胎干细胞H9，获得整合型Id1启动子驱动Venus-Luci双报告基因表达的人胚胎干细胞株，得到CGMCC11091的细胞株，进行早期中胚层分化，将待测抗肺动脉高压的化合物和CGMCC11091的细胞株接触，再用细胞裂解液裂解细胞，按基因检测***所述方法检测荧光素酶表达活性，以Id1启动子驱动的双报告基因表达量作为指标，获得对Id1表达上调的治疗肺动脉高压化合物的筛选模型。
  4. 用权利要求2所述模型筛选后得到的化合物在制备抗肺动脉高压的药物中的应用，所述化合物选自：
    1H-吡唑[3,4-d]嘧啶,1-苯-4-(1-哌啶基)-(Cas No:23000-46-6)和其衍生物。
  5. 根据权利要求4所述的应用，所述的应用还包括对以下药物的筛选：
    (1)上调骨形态形成蛋白(BMP)信号的调节剂；
    (2)上调骨形态形成蛋白II型受体表达水平的调节剂；
    (3)在促进内皮细胞形成血管的调节剂；
    (4)改善肺动脉血流动力学和改善肺血管重构的制剂；
    (5)肺动脉高压治疗药物；
    (6)心肺血管疾病治疗药物。
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