Cell Specific Localization of Altered Gene Expression in Pulmonary Hypertension
Cell Specific Localization of Altered Gene Expression in Pulmonary Hypertension
批准号:
8335473
负责人:
Marlene Rabinovitch
金额:
$7.9万
依托单位:
依托单位国家:
美国
项目类别:
财政年份:
2011
资助国家:
美国
项目状态:
已结题
起止时间:
2011-09-23 至 2013-07-31
关键词:
7-(N-(3-aminopropyl)amino)heptan-2-oneAddressAntibodiesArchivesArteriesBiological AssayBlood VesselsCardiacCell Culture TechniquesCellsColony-Stimulating Factor ReceptorsCommunitiesCompanionsConfocal MicroscopyCultured CellsDevelopmentDiseaseDissectionEndothelial CellsFluorescence MicroscopyFreezingFunctional disorderGene ExpressionGenesGoalsHarvestImmunofluorescence ImmunologicImmunohistochemistryInflammationInflammatoryInfusion proceduresInheritedLasersLesionLocationLungMedicalMicrofluidic Analytical TechniquesMicrofluidicsModificationMusMutationNaturePECAM1 genePathologyPatientsPenetrancePlatelet-Derived Growth Factor ReceptorPopulationPositioning AttributeProliferatingProteinsPulmonary HypertensionPulmonary artery structureRNAReactionResourcesSample SizeScanningSiteSorting - Cell MovementSourceStructure of parenchyma of lungTNF geneTechnologyTissue SampleTissuesTranscriptVascular remodelingbone morphogenetic protein receptor type IIcohortcytokinedisease diagnosisgranulocyteinterestlaser capture microdissectionmacrophagemonocytemonocyte colony stimulating factornext generationprotein expressionpulmonary arterial hypertensionpulmonary artery endothelial cellresponseselective expressiontherapy developmenttool
中文摘要
摘要
肺动脉高压(PAH)是目前诊断为晚期的不治之症。
当心脏功能经常受损时。开发逆转药物的主要缺点是
这种疾病一直缺乏可以从患者身上研究的肺组织和细胞。PHBI网络
因此为调查界开发了一种资源,通过收集和归档组织和
特发性肺动脉高压患者及与其他疾病相关的肺动脉高压患者的血管细胞
(APAH),以及来自未使用的供体肺作为对照。我们的小组作为PHBI肺采购贡献
地点。我们还采集和培养了IPAH患者的CD31阳性(+)(内皮)细胞。
我们使用包括RNA在内的下一代工具来研究这些细胞中的基因表达。
序列号。在IPAH与CD31+CEL中发现显著上调或下调的转录本。
为了控制肺部,我们在这个提案中的第一个目标是将这些转录本定位到肺内皮细胞中的表达
肺组织。CD31+PAECs将通过激光捕获显微切割从腺泡内动脉中分离出来
在IPAH患者和对照组中发现病变的地方,将通过RNA提取和qRT-
聚合酶链式反应。这些研究将使我们能够验证这些变化是由文化引起的差异,但
在存在PAH血管病变的血管中表达。然后我们将确定是否会有一些
在组织中观察到的IPAH CD31+细胞基因表达的变化也存在于CD31+细胞中
APAH患者的肺。这将使我们将重点放在这些异常的功能意义上
它们与IPAH的高级病理学有关。在其他研究中,我们分析了基因表达的变化
用微流控技术检测新分离的CD31+细胞,其中单个细胞被分选并进行48
聚合酶链式反应。我们在IPAH和对照肺中发现了扩张的血管细胞亚群
CD31+和GM-CSFR+或CD31+和PDGFR+双阳性。这些CD31+GM-CSFR?+细胞
平均分为主要表达EC标记的细胞和表达单核/巨噬细胞的细胞
记号笔。扩增的IPAH患者肺CD31+PDGFR+细胞主要表达
单核/巨噬细胞标志物。因此,我们的第二个目标是在肺中定位这些亚群
通过共聚焦显微镜观察血管系统,并确定它们是否与特定的血管相关
大小,或有特定的病变。然后,我们可以通过微流体和免疫荧光进一步评估
的亚群正在活跃增殖,以及他们是否选择性地表达异常
特异靶向CD31+细胞中鉴定的转录本I.通过鉴定异常扩增的亚群
我们可以在体外进一步询问这些细胞,以确定它们是否处于一种状态
转化或去分化,以及它们是否在产生湮灭和
丛状病变,可能是新兴疗法的良好靶点。
英文摘要
ABSTRACT
Pulmonary arterial hypertension (PAH) is currently an incurable disease diagnosed at an advanced state and
when there is often compromise of cardiac function. The major drawback in developing therapies to reverse
this disease has been the lack of lung tissue and cells that could be studied from patients. The PHBI Network
has therefore developed a resource for the investigative community by harvesting and archiving tissue and
vascular cells from patients with idiopathic PAH (IPAH) and from PAH associated with other medical conditions
(APAH), as well as from unused donor lungs, as controls. Our group contributes as a PHBI lung procurement
site. We have also harvested and cultured CD31 positive(+) (endothelial) cels from patients with IPAH and
from control lungs, and we study gene expression in these cells using 'next generation' tools including RNA-
Seq. Having identified transcripts that are significantly up- or downregulated in CD31+ cels from IPAH vs.
control lungs, our first aim in this proposal is to localize expression of these transcripts to the PAECs in the
lung tissue. The CD31+ PAECs will be isolated by laser-capture microdissection from intra-acinar arteries
where lesions are found in the IPAH patients vs. controls, and will be analyzed by RNA extraction and qRT-
PCR. These studies will allow us to verify that the changes are differentially induced by culture but are
expressed in vessels where the vascular lesions of PAH are present. We will then determine whether some of
the changes in gene expression observed in IPAH CD31+ cells in tissue are also present in CD31+ cells from
lungs in APAH patients. This will position us to focus on the functional significance of these abnormalities as
they relate to the advanced pathology of IPAH. In other studies, we analyzed changes in gene expression in
freshly isolated CD31+ cells using microfluidics assays, in which single cels are sorted and subjected to 48
PCR reactions. We identified expanded subpopulations of vascular cells in IPAH vs control lungs that are
double positive for CD31+ and GM-CSFR¿+, or CD31+ and PDGFR¿+. Those CD31+GM-CSFR¿+ cells are
equally subdivided into cells that express primarily EC markers, and those that express monocyte/macrophage
markers. The expanded CD31+PDGFR¿+ cells from IPAH patient lungs express primarily
monocyte/macrophage markers. Our second aim is therefore to localize these subpopulations in the lung
vasculature by confocal microscopy, and to determine whether they are associated with a vessel of a particular
size, or with a specific lesion. We can then further assess, by microfluidics and by immunofluorescence, which
of the identified subpopulations are actively proliferating, and whether they selectively express the abnormal
transcripts identified in CD31+ cells in Specific Aim I. By identifying the abnormally expanded sub-populations
of cells in IPAH lesions, we can further interrogate these cells ex-vivo to determine whether they are in a state
of transformation or de-differentiation, and whether they are giving rise to the cels in the obliterative and
plexiform lesions, and could be good targets for emerging therapies.
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