Midkine对SD大鼠COPD模型气道平滑肌细胞增殖影响及其分子机制研究

Chronic obstructive pulmonary disease (COPD) is a leading cause of morbidity and mortality worldwide with no effective treatment, imposing an enormous economic burden on society. A major reason for lung function decline in COPD is airflow obstruction due to airway remodeling which includes an increased airway smooth muscle (ASM) mass. Midkine(MK) is a heparin-binding growth factor involved in systemic inflammation and can promote the expression of alpha-smooth muscle actin and collagen in alveolar epithelial cells through Notch pathway. However, it is not clear whether the Midkine-Notch pathway could work in airway smooth muscle cells. Previous study on COPD rat model has shown that Midkine highly expressed around airway smooth muscle cells (ASMCs), possibly through the Notch pathway, lead to decrease apposis and over proliferation of the ASMCs and replacement of collagen, increasing the speed of airway remodeling. However, it remains elusive whether block of the expression of midkine would decrease the process of airway remodeling. In this study, we explored the proliferation of ASMCs, expression of alpha-smooth muscle actin and collagen and the relationship with Notch pathway using Flow Cytometry, immunofluorescence,PCR and western blot in mice with knockdown of midkine gene expression by means of antisense oligonucleotide, which implicates new strategies to treat COPD.

COPD发病率、死亡率高，缺乏有效治愈手段，给社会带来严重经济负担，其原因在于COPD患者肺功能进行性下降。而气道平滑肌细胞过度增殖导致的气道重塑是其重要原因。MK是一种新型肝素结合生长因子，可趋化炎症细胞，参与内炎症反应，与Notch通路相关，而Notch信号能促进肺泡上皮细胞中α平滑肌肌动蛋白及胶原的表达，MK-Notch是否调参与气道平滑肌细胞增殖过程尚不清楚。前期实验中发现COPD大鼠模型气道平滑肌细胞内及其周围MK高表达，MK很可能与Notch通路共同作用，抑制气道平滑肌细胞凋亡及促进增殖加速气道重塑；阻断MK表达或抑制Notch活性，可能阻止气道重塑进程，改善肺功能状态。本课题拟应用COPDSD大鼠、MK反义寡脱氧核苷酸及MK基因敲除小鼠，采用流式、免疫荧光、PCR及WB技术，观察MK-Notch与气道平滑肌细胞增殖的关系，为降低COPD患者气道重塑，改善肺功能提新的治疗方法。

气道平滑肌细胞（ASMC）在炎症中能产生各种细胞因子，引起细胞外基质组分发生改变与气道重构相关。Midkine（MK）能促进各种炎症细胞趋化并释放炎症因子，Notch是否与Midkine共同影响气道平滑肌细胞的增殖及凋亡上不清楚。.目的 研究Midkine对LPS诱导气道平滑肌细胞引起的急性肺损伤的作用机制.方法 体外培养大鼠气道平滑肌细胞，分为5组，对照组、脂多糖（LPS）处理组、Non-targeted siRNA处理组、MK siRNA处理组、Notch抑制剂（LY411575）处理组。通过CCK-8检测细胞增殖能力，流式细胞术检测细胞凋亡水平，通过Western blot、qPCR及细胞免疫荧光的方法分析Midkine/Notch2信号通路细胞因子的改变。.结果 Midkine、Notch2在LPS组中高表达。MK siRNA可以有效阻断LPS诱导的Midkine表达，同时下调Notch2的表达，这一结果与Notch抑制剂（LY411575）的结果相同。外源性Midkine促进LPS组气道平滑肌细胞的增殖，降低细胞凋亡率。当阻断Midkine表达时，LPS组气道平滑肌细胞增殖明显降低，而细胞凋亡增加。抑制Notch表达，则LPS组气道平滑肌细胞增殖降低，细胞凋亡增加。结论 Midkine/Notch2信号通路在气道炎症中，对气道平滑肌细胞增殖、凋亡有着重要的调节作用。