The Brain and Maternal Microchimerism

项目摘要/摘要 微嵌合体(Mc)指的是拥有少量细胞或来自基因不同个体的DNA。 在母亲和孩子的身体结合结束多年后，在她的后代中发现了母亲的Mc(MMC)，并且 胎儿起源于母亲的MC。在儿童和成人的血液和器官中发现了MMC，包括心脏， 肝、脾、肾、胰腺。在实验和人体研究中，Mc似乎是不同的，创造了 例如，心肌中的丝裂原细胞、胰腺中的胰岛细胞和肝脏中的肝细胞。 尽管大脑对人类的健康和功能很重要，但人们对大脑的认识存在着根本的差距 大脑中的MMC。该建议总体目的是生成关于MMC细胞的基础知识 人脑中MMC的类型、数量和转录组。将Mc的起源确定为特定于 母亲需要母亲的参与，这在儿童尸检中通常是不具备的。目标1有两个 零件。Aim 1的第一部分将调查因药物而接受手术切除的儿科患者的MMC。 难治性癫痫，母亲可以参与。人类白细胞抗原和其他多态基因分型 从口腔拭子样本中提取的母体DNA。在对患者和母亲进行基因分型后， 审查母子对以识别非传播、非共享的多态，即母亲独有的多态。 接下来，从一组人类白细胞抗原和其他多态特异的定量分析中选择一种母体特异的检测方法 我们为此而开发的聚合酶链式反应(QPCR)检测方法。然后从切除的脑组织中提取DNA 使用为每个母子对选择的定制化验来询问MMC。类似的方法将是 被用来研究年龄相仿的脑胶质瘤手术患者的脑切除 母性参与也可以包括在内。Aim 1方法的第二部分将从男性身上选择脑组织进行研究 通过与X和Y染色体特异性探针的荧光原位杂交(FISH)；这方面的目的1 方法将允许包括来自谁的尸检的没有神经疾病的儿童的脑组织 产妇参与不是必需的。带有两个X染色体信号的雌性细胞，被认为是母体 与同一地区计数的XY雄性细胞一起计数。将增加免疫荧光(IF)来评估 细胞表型。在目标2中，我们将对脑组织进行单核rna测序(snrna-seq)分析。 样本。SnRNA-seq研究将全面评估大脑中哪些类型的细胞来自 MMC并将评估MMC转录组。MMC影响大脑的方式是多方面的， 根据儿童的潜在利益和/或不利因素而变化。除了癫痫的信息性之外，如果自然的话 获得的MMC是生物学的一个基本方面，正如我们假设的那样，拟议的工作将为 从中可以研究人类大脑的各种疾病，包括发育性疾病， 自身免疫性、退行性或肿瘤性，强调重要性。

PROJECT SUMMARY/ABSTRACT Microchimerism (Mc) refers to harboring a small amount of cells or DNA from a genetically distinct individual. Many years after the physical union of mother and child ends maternal Mc (MMc) is found in her offspring, and Mc of fetal origin in the mother. MMc has been found in children and adults in blood and organs, including heart, liver, spleen, kidney and pancreas. In experimental and human studies Mc appears to be differentiated, creating for example MMc as cardiac myocytes in the heart, islet  cells in the pancreas and hepatocytes in the liver. Despite the importance of the brain to human health and function a fundamental gap of knowledge exists for MMc in the brain. The overall purpose of this proposal is to generate foundational knowledge about MMc cell types, quantities and the MMc transcriptome in human brain. To establish the origin of Mc as specific to the mother requires maternal participation which is generally not available for childhood autopsies. Aim 1 has two parts. Part 1 of Aim 1 will investigate MMc in pediatric patients who have surgical excision for medication refractory epilepsy, for whom mothers are available to participate. HLA and other polymorphism genotyping is done from maternal DNA extracted from a buccal swab sample. After genotyping patients and mothers, each mother-child pair is reviewed to identify a non-transmitted, non-shared polymorphism i.e. unique to the mother. A maternal-specific assay is next selected from a panel of HLA- and other polymorphism-specific quantitative PCR (qPCR) assays we have developed for this purpose. DNA extracted from excised brain tissue is then interrogated for MMc using the selected custom assay for each mother-child pair. A similar approach will be employed to study brain resected from age comparable patients undergoing surgery for gliomas for which maternal participation can be included. Part 2 of the Aim 1 approach will select brain tissues from males to study by fluorescence in situ hybridization (FISH) with X- and Y-chromosome specific probes; this aspect of the Aim 1 approach will permit including brain tissue from children without neurologic disease from autopsy from whom maternal participation is not required. Female cells with two X-chromosome signals, presumed maternal, will be counted with XY male cells enumerated in the same area. Immunofluorescence (IF) will be added to evaluate cell phenotypes. In Aim 2 we will conduct single nuclei RNA sequencing (snRNA-seq) analysis on brain tissue samples. The snRNA-seq studies will comprehensively evaluate what type of cells in the brain are derived from MMc and will assess the MMc transcriptome. The ways in which MMc may affect the brain are multiple and diverse to the potential benefit and/or detriment of a child. In addition to informativeness for epilepsy, if naturally acquired MMc is a basic aspect of biology as we hypothesize, the proposed work will have created a foundation from which diverse disorders of the human brain can be investigated including conditions that are developmental, autoimmune, degenerative, or neoplastic, underscoring significance.