Stem Cell Therapy for Pulmonary Hypertension

描述（由申请人提供）：拟议研究的广泛长期目标是开发一种新的基于间充质干细胞（MSC）的治疗肺动脉高压的方法。肺动脉高压是一种严重的、通常是致命的疾病，其特征是肺动脉压升高、右心衰和肺血管床重塑。虽然目前的治疗延长了生存期，但长期结果并不乐观。然而，在体内使用非病毒载体或病毒载体会导致低水平的基因转移、随机基因表达和不良反应，包括炎症。间充质干细胞是从骨髓中自我更新的成体干细胞，在培养中容易分离和扩增，并且有可能分化成许多细胞类型。我们的假设是基因修饰的间充质干细胞可能对肺动脉高压的治疗有有益的作用。我们实验室的初步实验结果表明，基因修饰的MSCs和野生型或未修饰的MSCs可以改善老年大鼠的勃起功能。初步研究结果还表明，气管内给药野生型MSCs可减轻单芥碱诱导的肺动脉高压，并恢复因单芥碱治疗而受损的肺血管舒张剂对乙酰胆碱的反应。本申请中提出的实验将验证以下假设：气管内给药MSCs或经eNOS基因或CGRP基因修饰的MSCs将对单芥碱诱导的大鼠肺动脉高压有有益作用。为了验证这一假设，必须制备MSCs，对其进行表征，进行基因修饰，并将其注射到对照大鼠和单苦参碱诱导的肺动脉高压大鼠的肺部。除了确定MSCs和基因修饰MSCs对基线肺压和血流量的影响外，MSCs对肺血管对通气缺氧和血管活性激动剂（包括乙酰胆碱）的反应的影响，以及注射的MSCs在肺中的命运将使用组织学和组织化学技术进行研究。因此，第一个具体目标是从大鼠中分离、表征和基因修饰MSCs。第二个具体目的是利用新开发的右心导管技术，研究注射MSCs对肺血管功能和单芥蓝碱诱导的肺动脉高压的影响。第三个具体目标是使用组织化学技术、去卷积显微镜以及使用PCR和FISH方法定位雌性大鼠肺中的雄性Y染色体来研究注射的MSCs在肺中的命运。这些研究的结果可能会导致新的基于msc的治疗肺动脉高压的疗法。

DESCRIPTION (provided by applicant): The broad long-term objectives of the proposed research are to develop a new mesenchymal stem-cell (MSC) based therapy for the treatment of pulmonary hypertension. Pulmonary hypertension is a serious, often fatal disease characterized by increased pulmonary arterial pressure, right-heart failure, and remodeling of the pulmonary vascular bed. Although current therapy has prolonged survival, the long-term outcome is not favorable. Gene therapy shows promise, however, in vivo administration of non-viral vectors or viral vectors leads to low level gene transfer, random gene expression, and adverse effects, including inflammation. MSCs are self-renewing adult stem cells from bone marrow, are easily isolated and expanded in culture, and have the potential to differentiate into many cell types. It is our hypothesis that gene-modified MSCs may have a beneficial effect in the treatment of pulmonary hypertension. The results of preliminary experiments in our laboratory show that the administration of gene-modified MSCs and wild-type or unmodified MSCs improve erectile function in the aged rat. The results of preliminary studies also show that intratracheal administration of wild-type MSCs attenuated monocrotaline-induced pulmonary hypertension and restored pulmonary vasodilator responses to acetylcholine, which were impaired by monocrotaline treatment. The experiments proposed in this application will test the hypothesis that intratracheal administration of MSCs or MSCs modified with the eNOS gene or the CGRP gene will have a beneficial effect in monocrotaline-induced pulmonary hypertension in the rat. In order to test this hypothesis, MSCs must be prepared, characterized, gene-modified, and injected into the lung of control rats and rats with monocrotaline-induced pulmonary hypertension. In addition to determining the effect of MSCs and gene-modified MSCs on baseline pulmonary pressures and blood flow, the effects of the MSCs on pulmonary vascular responses to ventilatory hypoxia and vasoactive agonists, including acetylcholine, and the fate of the injected MSCs in the lung will be investigated using histologic and histochemical techniques. Therefore, the first specific aim is to isolate, characterize and gene-modify MSCs from the rat. The second specific aim is to investigate the effect of injected MSCs on pulmonary vascular function and on monocrotaline-induced pulmonary hypertension using newly developed right-heart catheterization techniques. The third specific aim is to study the fate of injected MSCs in the lung using histochemical techniques, the deconvoluted microscope, and localization of the male Y chromosome in the lung of female rats using PCR and FISH methodologies. The results of these studies may lead to new MSC-based therapy for the treatment of pulmonary hypertension.