先天性脊柱侧凸正常-患病同卵双生子的表观遗传学研究

Congenital scoliosis is a complicated spinal discord for spine surgens. The genesis of scoliosis relate to the complex interplay of heredity and environment. However, the molecular mechanism underlining its pathogenes is not well known. In a previous study, we have collected 10 monozygotic twins, one was diagnosed with congenital scoliosis and the other one was normal. We used MeDIP-Chip to perform methylation analysis on one of the twins and found the quantity and distribution of the methylation sites in the upstream regulatory region of some genes were significantly higher in CS than the normal one.. Therefore, we presumed that DNA methylation might have an important role in CS. Up to now, there was no study on the epigenetics research about CS. So, the techniques of MeDIP-Chip, specific methylation PCR, siRNA and gene knock-out will be used to detect the difference of methylation between the twins in this study. And verification of the difference will be conducted through the animal models. According to individual-group and globe-cell-animal model studies, we plan to find the related epigenetics factors for the genesis and prognosis of CS. This study will give a new insight into the pathogenesis of CS and provide a new way for the early diagnosis, treatment and prevention of CS.

先天性脊柱侧凸(CS)的诊治是脊柱外科的难题，其发生除与遗传因素相关外,环境因素所致的表观遗传改变也发挥重要作用，但具体机制尚不明确。同卵双生个体由于其遗传背景完全相同成为表观遗传研究的有力对象。前期的临床观察和研究中，我们发现10对CS正常-患病同卵双生子并用MeDIP-Chip对其中1对进行了甲基化芯片分析，结果显示CS患病个体在某些基因启动子上游调控区呈现高甲基化。据此推测，这些基因的甲基化沉默可能与CS的发生密切相关。在此基础上，本课题将应用MeDIP-Chip结合MSP在更多样本中继续考察特定基因的甲基化差异；进一步应用siRNA、基因敲除等技术在细胞水平、动物模型中进行基因表达调控和功能的验证，从个体－群体、整体－细胞－动物模型多个层次，寻找和阐明与CS发病相关的表观遗传因素。目前国内外尚未见CS表观遗传学研究的报道，本研究将为CS 的预防、早期诊治提供新的思路和新的靶点。

先天性脊柱侧凸(CS)的诊治是脊柱外科的难题，其发生除与遗传因素相关外,环境因素所致的表观遗传改变也发挥重要作用，但具体机制尚不明确。同卵双生个体由于其遗传背景完全相同成为表观遗传研究的有力对象。我们利用MeDIP-ChiCS对正常-患病同卵双生子进行了甲基化芯片分析，结果显示CS患病个体在某些基因启动子上游调控区呈现高甲基化。利用甲基化特异性PCR法扩大样本验证关键基因启动子上游调控区甲基化的位点的数量及分布与CS发生发展的关系。分离培养骨髓的间充质干细胞，在细胞水平上观察去甲基化对目的基因的表达及体节发育基因表达，证实基因的甲基化在目的基因的表达及体节发育相关的信号通路如Notch信号通路、Wnt信号通路、FGF信号通路和RA信号通路的表达起重要作用。我们采用全基因组外显子测序法分析了正常-患病同卵双生子、罕见病合并脊柱侧凸的基因表达情况，筛选与脊柱侧凸发生相关的基因突变。利用基因同源重组进行基因敲除建立基因敲除小鼠模型，我们观察到双敲除的转基因鼠部分发生脊柱侧凸畸形。本课题成功的通过实验筛查出与先天性脊柱侧凸发生相关的甲基化基因，并初步探讨分子生物学机制。证实了基因甲基化参与了先天性脊柱侧凸的发生发展。为下一步进行基因编辑以及靶向药物的研究做了良好的前期基础。本项目目前已经在国际SCI期刊杂志上发表论文多篇，部分课题的实验结果尚未完成论文撰写及发表，将在后续陆续完成。并多次在国际及国内会议上做大会发言交流。本研究为CS 的预防、早期诊治提供新的思路和新的靶点。