Genetic information transfer to hematopoietic cells: Role of microvesicles

描述（由申请人提供）：我们先前已经证明，源自鼠肺细胞的膜封闭的微泡含有肺特异性mRNA、微小RNA和蛋白质，并且这些微泡进入鼠骨髓细胞并影响通过肺特异性mRNA的表达而表现的遗传表型的变化;表面活性剂A-D、水通道蛋白和clara细胞特异性蛋白，并且在功能上表现为在移植到致死辐射的小鼠中后向上皮肺细胞的转化增加。观察到的mRNA变化似乎是由于mRNA和组织特异性转录蛋白的转移。小鼠心脏、肝脏和脑也影响靶骨髓细胞中的组织特异性遗传变化。目前的建议是这些现已发表的研究的自然延伸。我们计划阐明骨髓中微泡遗传变化的特定靶细胞，并研究单个肺细胞诱导骨髓遗传变化的特异性能力;以前的研究是用整个肺进行的。我们计划在体外和体内评估骨髓中观察到的遗传变化的稳定性。最后，我们将扩展我们的表征的潜在重编程，并试图确定特定的转录蛋白或microRNA可能介导观察到的表型变化。为了进行这些研究，我们将采用荧光激活细胞分选的细胞群，实时RT-PCR，免疫组化，细胞培养，小鼠骨髓移植，SILAC研究蛋白质转移，慢病毒载体转导的细胞和比较2-D凝胶电泳和差异电泳（DIGE）。这些研究将为微泡的研究建立一个更精确的平台，建立微泡诱导的遗传变化的稳定性，并开始解开这些变化背后的机制。这项工作可能会改变我们对细胞生物学的总体看法，并为处理一些以组织变性或损伤为特征的疾病开辟新的策略。 公共卫生相关性：该项目的重点是确定肺细胞脱落的囊泡是否能够影响骨髓细胞的身份，这些骨髓细胞消耗它们，使它们表现得像肺细胞。这种方法可能导致各种肺部疾病的组织再生和修复的新方法。或者，通过防止恶性行为从癌细胞转移到非癌细胞，可以开发癌症的新疗法。

DESCRIPTION (provided by applicant): We have previously demonstrated that membrane enclosed microvesicles derived from murine lung cells contain lung-specific mRNA, micro RNA and protein and that these microvesicles enter murine marrow cells and effect a change in genetic phenotype manifest by expression of the lung-specific mRNAs; surfactants A-D, aquaporins and clara cell specific protein and in function manifest by increased conversion to epithelial lung cells after transplantation into lethally irradiated mice. The observed mRNA changes appear to be due to transfer of both mRNA and of a tissue specific transcriptional protein. Murine heart, liver and brain also effect tissue specific genetic changes in target marrow cells. The present proposal is a natural extension of these now published studies. We plan to elucidate the specific target cells in marrow for microvesicle genetic changes and will also study individual lung cells for their specific capacity to induce genetic change in marrow; previous studies were with whole lung. We plan to evaluate the stability of the observed genetic changes in marrow both in vitro and in vivo. Lastly, we will extend our characterization of the potential for reprogramming and attempt to identify specific transcriptional proteins or microRNA which may mediate the observed phenotype changes. To carry out these studies we will employ fluorescent activated cell sorting of cell populations, Real-time RT-PCR, immunohistochemistry, cell culture, murine marrow transplantation, SILAC to study protein transfer, lente viral vector transduction of cells and comparative 2-D gel electrophoresis and difference electrophoresis (DIGE). These studies should establish a more precise platform for studies of microvesicles, establish the stability of microvesicle induced genetic changes and begin to unravel the mechanisms behind such changes. This work could alter our thinking toward cell biology in general and open new strategies for approaching a number of diseases characterized by tissue degeneration or damage. PUBLIC HEALTH RELEVANCE: The focus of this project is to determine if vesicles shed by lung cells are able to influence the identity of bone marrow cells that consume them to the point that they behave like lung cells. Such an approach could lead to new approaches for tissue regeneration and repair for a variety of lung diseases. Alternatively, by preventing the transfer of malignant behavior from cancerous cells to non-cancerous cells, novel therapies for cancer could be developed.