Genetic information transfer to hematopoietic cells: Role of microvesicles

描述（由申请人提供）：我们之前已经证明，来自小鼠肺细胞的膜封闭微泡含有肺特异性mRNA、微RNA和蛋白质，这些微泡进入小鼠骨髓细胞并通过肺特异性mRNA的表达影响遗传表型的变化；表面活性剂A-D、水通道蛋白和clara细胞特异性蛋白在移植到致死性辐照小鼠肺上皮细胞后转化增加。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。小鼠的心脏、肝脏和大脑也会影响目标骨髓细胞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囊遗传变化的特异性靶细胞，并将研究单个肺细胞诱导骨髓遗传变化的特异性能力；以前的研究是全肺。我们计划在体外和体内评估观察到的骨髓遗传变化的稳定性。最后，我们将扩展我们对重编程潜力的表征，并尝试鉴定可能介导观察到的表型变化的特定转录蛋白或microRNA。为了开展这些研究，我们将采用荧光活化细胞分选、实时实时RT-PCR、免疫组织化学、细胞培养、小鼠骨髓移植、SILAC来研究蛋白质转移、细胞的慢病毒载体转导以及比较二维凝胶电泳和差异电泳（DIGE）。这些研究应该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，并开始揭示这种变化背后的机制。这项工作可能会改变我们对细胞生物学的总体看法，并为处理以组织变性或损伤为特征的许多疾病开辟新的策略。

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.