研究背景和目的
 紫绀型心脏病患者长期遭受着慢性缺氧的影响，机体为了适应慢性缺氧会发生一系列适应性改变。这些改变在帮助机体行使正常功能的同时也会带来损伤。骨髓环境是一个生理低氧环境，慢性缺氧会导致骨髓微环境中氧气含量的进一步降低，而影响到骨髓中细胞的功能。骨髓间充质干细胞存在于骨髓腔中，是一群具有体外分化为多种其他类型细胞能力的成体干细胞群，其增殖和分化特性的维持对机体有重要意义。紫绀型先天性心脏病患者来源的骨髓间充质干细胞的多向分化潜能受到损伤，其相关的机制还未揭示，值得进一步深究。以往的研究指出，Notch通路与多能性细胞分化紧密相关。所以本文以Notch通路为目标，探究了其与骨髓间充质干细胞多谱系分化异常的关系和潜在机制。
研究内容
 1、对比紫绀型先天性心脏病来源的骨髓间充质干细胞在分化潜能方面与非紫绀型先天性心脏病来源的差异。
 2、检测Notch4个受体mRNA水平，及Notch1通路和其靶基因表达的差异。
 3、探究Notch1发生改变的分子机制。
 4、通过体外实验，研究慢性缺氧和D-半乳糖两个因素共同作用对Notch1信号通路的影响。
 5、通过使用Notch1激动剂添加的培养基，探究notch通路在紫绀型先天性心脏病患者来源的骨髓间充质干细胞分化中的作用。
研究方法
 体外培养紫绀先心病和非紫绀先心病来源的骨髓间充质干细胞，诱导其向成脂、成骨、成软骨细胞方向分化，比较三种分化能力。通过Real-Time PCR定量分析Notch1-4四个基因mRNA水平，并使用Western blot定量Notch1和下游靶基因Hes1和Hey1的蛋白表达水平。通过对Notch1启动子区重亚硫酸盐测序PCR（Bisulfite sequencing PCR，BSP）获得Notch1启动子区域的DNA甲基化程度。通过体外低氧培养并在培养基中添加D-半乳糖来模拟紫绀型先心病患者骨髓微环境来培养对照组细胞，检测对照组细胞Notch1及靶基因Hes1的蛋白表达量。进一步通过加入Notch1的激动剂Jagged-1激活Notch通路，比较NCHD组、CCHD组、NCHD添加D-半乳糖组、CCHD添加Jagged-1组和NCHD添加D-半乳糖和Jagged-1组诱导分化成脂、成骨、成软骨细胞的能力。
研究结果
 紫绀型先天性心脏病来源的骨髓间充质干细胞与非紫绀型相比，其分化成脂、成骨、成软骨的能力受到损害。在紫绀型组，Notch1、Notch2和Notch4的mRNA水平显著下调。同时，Notch1、及其下游靶基因Hes1和Hey1的蛋白下调。进一步发现紫绀组Notch1的甲基化程度升高。体外培养非紫绀组骨髓间充质干细胞，发现低氧和D-半乳糖组Notch1和Hes1的蛋白水平显著下降。通过Jagged-1激动剂处理后，紫绀组的骨髓间充质干细胞分化成脂、成骨、成软骨能力部分恢复。
研究结论
受到慢性缺氧的影响，紫绀型先天性心脏病来源的骨髓间充质干细胞的多向分化潜能受到损害。这是由于骨髓微环境中低氧环境和D-半乳糖异常蓄积共同作用引起Notch1通路异常导致的。通过激活Notch1通路可以部分挽救紫绀组先心病骨髓间充质干细胞的多向分化潜能，进一步证明了Notch通路在其中的重要作用。
 

background and ive
patients with cyanotic congenital heart disease (cchd) will suffer from long-term chronic hypoxia if they are not corrected in time. in order to cope with the hypoxic environment, the body will inevitably undergo a series of adaptive changes. the bone marrow microenvironment is a hypoxic environment under physiological conditions, and chronic hypoxia leads to a further decrease in oxygen concentration in the bone marrow microenvironment. bone marrow mesenchymal stem cells could differentiate into a variety of other cells. the effects of hypoxia and high d-galactose accumulation on the differentiation potential of the bone marrow mesenchymal stem cells have not yet been explored.
contents
1. the differences in the differentiation potential of bone marrow mesenchymal stem cells between cyanotic congenital heart disease and non-cyanotic congenital heart disease were compared.
2 notch receptors mrna levels were detected, and the differences between the notch pathway and its target gene expression were also compared.
3. the level of dna methylation of the notch promoter region of bone marrow mesenchymal stem cells in cyanotic congenital heart disease and non-cyanotic congenital heart disease was explored.
4. by in vitro experiments, the effects of chronic hypoxia and d-galactose on the notch pathway of bone marrow mesenchymal stem cells were studied.
5. the role of the notch pathway in the differentiation of bone marrow mesenchymal stem cells derived from patients with cyanotic congenital heart disease was investigated by supplying medium with a notch agonist.
methods
bone marrow mesenchymal stem cells derived from cyanotic congenital heart disease and non-cyanotic congenital heart disease were cultured separately. the adipogenic, osteogenic and chondrogenic differentiation potential of the bmscs from patients with cchd or non-cyanotic congenital heart disease (nchd) were assessed respectively. the expression level of notch1 and its target genes hes1 and hey1 were measured by western blot. real-time pcr analysis was performed to investigate the mrna level of notch1-4. bisulfite sequencing pcr (bsp) was used to analyze the methylation level of the notch1 promoter region. the bmscs from nchd patients were cultured in 4% o2 with d-galactose supply for 2 weeks to simulate the state of bmscs derived from cchd patients, then the protein level of the notch1, hes1 and hey1 was analyzed by western blot. by adding jagged-1 (agonist of notch1) to rescue the deficient bmscs, the multi-lineage differentiation potential was evaluated.
results
bone marrow mesenchymal stem cells derived from cyanotic congenital heart disease have an inferior multipotential differentiation potential compared with non-cyanotic chd. and the mrna level of notch1, notch2, and notch4 were significantly down-regulated in cchd. meanwhile the the protein level of notch1 and downstream target genes hes1 and hey1 were also downregulated. further results showed that the methylation level in the notch1 promoter region was raised, especially in site cpg24 and site cpg32. exposure bmscs from nchd patients with d-galactose under hypoxia decreased the expression of notch1-4 and hes1, but not hey1. and activating notch1 by adding jagged-1 partially restored the deficient bmscs of cchd patients.
conclusion
under the influence of chronic hypoxia, the multi-directional differentiation potential of bone marrow mesenchymal stem cells derived from cyanotic congenital heart disease is continuously damaged, which is caused by d-galactose accumulation, is owing to the decreased notch1 level with a remarkable hypemethylation in its’ promoter region. activated notch1 signaling pathway could partially restore the deficient bmscs in the cchd patients, further demonstrating the important role of the notch pathway.