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基底硬度调控中性粒细胞曳尾结构的力学-生物学耦合机制
英文题名Mechano-biological coupling mechanism of substrate stiffness regulating neutrophil’s trail formation
郭盼
导师章燕
2018-05-28
学位授予单位中国科学院大学
学位授予地点北京
培养单位中国科学院力学研究所
学位专业一般力学与力学基础
关键词关键词:基底硬度 中性粒细胞 曳尾结构 Arp2/3复合物 血流剪切
摘要

动脉粥样硬化是慢性的血管炎症,以炎症细胞浸润动脉血管壁为主要特征。在动脉粥样硬化过程中,白细胞的募集遵循着明确的炎症级联反应,首先粘附并且在激活的内皮细胞表面滚动,然后稳定粘附,最后跨内皮迁移到下层组织。上述过程受到趋化因子、粘附分子和生理力学因素(血管硬度、流体剪切)的共同调控,是一个典型的力学-生物学耦合过程。中性粒细胞是帮助器官启动和维持免疫反应的关键参与者,也是第一个穿过血管内皮细胞进入组织的细胞类型,通过向树突状细胞、单核细胞和T淋巴细胞发出信号来形成整体免疫应答。特别是在动脉粥样硬化中,由于血管的硬度和血流动力学的改变,中性粒细胞的跨内皮迁移增多。本课题组前期的研究发现中性粒细胞爬行过程中会形成曳尾结构,并通过这种曳尾结构促进单核细胞的募集。这个结果提示我们,作为免疫“急先锋”的中性粒的运动受到组织环境的影响,同时也伴随着对局部组织环境的重建,将导致和调控后续的免疫细胞募集,但是目前对于生理力学因素(血管硬度)是否会影响这种曳尾结构的形成及其调控的分子机制目前未见报导。

因此,本文以动脉粥样硬化中中性粒细胞的募集的力学-生物学耦合机制为切入点,采用生物力学、生物材料和活细胞成像相结合的技术方法,研究两种关键力学因素——基底(血管)硬度和流体剪切协同调控中性粒细胞曳位结构形成的规律及其调控的分子机制。为此,本文设计了一种基底硬度与流体剪切耦合的微流道实验体系,选择5 kPa的聚丙烯酰胺凝胶(PA gel)模拟正常血管硬度,34.88 kPa的聚丙烯酰胺凝胶模拟发生动脉粥样硬化的血管硬度,并以玻璃基底作为极硬基底作为对照,与此同时,以0.5 dyn/cm2的流体剪切模拟血液中血管壁受到的血流剪切。主要研究内容及结果包括两个方面:

1)  生理力学因素调控中性粒性细胞曳尾结构形成的规律:量化了0.5 dyn/cm2剪切应力和基底硬度(5 kPa、34.88 kPa和玻璃基底)条件下中性粒细胞曳尾形成的比例、长度、面积,中性粒细胞爬行的速度、方向等参数,考察了环境力学因素调控中性粒细胞曳尾结构形成的规律。结果发现,曳尾结构形成的比例、长度,面积随着基底硬度的增加而增加,基底硬度不影响中性粒细胞的黏附和爬行速度,但是增加中性粒细胞的爬行方向性。

2)  基底硬度调控中性粒性细胞曳尾结构形成的分子机制:本文探索了整合素CD11b对曳尾结构的调控作用,阻断CD11b后,曳尾结构比例、面积和长度均显著降低,爬行的速度也显著降低。采用CytoD破坏微丝细胞骨架后曳尾结构的比例、面积、长度也显著降低,证实了细胞骨架在曳尾结构形成中的决定性作用。接着本文分别探索了细胞骨架伪足的生成和骨架收缩对曳尾结构形成的调控,发现抑制Arp2/3的actin成核作用,曳尾结构形成的比例显著降低、且长度和面积也明显减少;抑制myosin II的细胞骨架收缩作用之后,中性粒细胞曳尾结构的形成比例和长度没有变化,但是曳尾结构的形态发生改变、面积也显著减少。Arp2/3 和Myosin II 都对中性粒细胞爬行爬行方向性有作用,但是只有Arp2/3复合物会影响中性粒细胞的速度。免疫荧光检测发现,曳尾结构形成时,CD11b、黏着斑蛋白(talin-1, pFAK, paxillin, vinculin)和微丝细胞骨架(F-actin)有着明显的共定位关系,说明细胞骨架连接蛋白在中性粒细胞曳尾结构形成中发挥着重要作用。

本文的研究对于从细胞分子水平深入了解心血管活动和疾病(特别是动脉粥样硬化)发生的本质具有重要的科学意义和潜在的应用前景。

英文摘要

Atherosclerosis is a chronic inflammation of blood vessels, characterized by inflammatory cells infiltrating the artery walls. In atherosclerosis, leukocyte recruitment is presented as the cascade of successive interactions between leukocytes and endothelial cells. Leukocytes tether to, roll on, firmly adhere to, and crawl on the endothelium before transmigration.This is a typical mechanical-biological coupling process regulated by chemokine, adhesion molecules and many physiological-mechanical factors (eg. blood vessel stiffness and blood flow). Neutrophils are key player that help organs initiate and maintain immune reactions and that shape the overall immune response by signaling to dendritic cells, monocytes, and T cells. Under most inflammatory conditions, neutrophils are the first cell type crossing the blood vessel endothelium into the tissue. Especially in atherosclerosis, neutrophils trans-endothelial migration increase with increased blood vessel stiffness and blood flow. Our previous study found that the neutrophils formed long membrane tethers during migration along blood vessel and subsequently left behind long-lasting trails which result in more monocyte recruitment. This finding indicates that the local tissue microenvironment is remodeled following with the movement of neutrophils, leaving the trails to induce and regulate the monocyte recruitment, but wehter the blood vessel stiffness regulates the trail’s formation and the underlying molecular mechanism remains unclear.

Here, using a coordinated approach based on biochemical, biomechanical, biomaterial and living cell imaging methods, we explored the laws and the underlying mechano-biological coupling mechanisms of neutrophil trail foramtion on varying physiologic stiffness (5 kPa, 34.88 kPa and glass) and blood flow (0.5 dyn/cm2). We modeled blood vessels of varying mechanical properties using ICAM-1-coated polyacrylamide gels. The main results include:

1)        The laws of varying physiologic stiffness regulating neutrophil trail formation. We quantified the formation ratio, length and area of neutrophil’s long-lasting trails, and also quantified the speed and direction index of neutrophil migration on substrate, found that the formation ratio, length and area of trails increase with increased substrate stiffness. And the varying substrate stiffness did not affect the adhesion ratio and migration speed of the neutrophils, but can affect the neutrophil’s direction index of migration.

2)        The molecular mechanism of substrate stiffness regulating neutrophil’s trail formation. We explored the role of CD11b for the trail formation, and found that blocking the CD11b dramatically decrease trail formation ratio, length, area and speed. After disrupting the cyotoskeleton by CytoD, we found the trail formation ratio, length and area are all dramatically decreased, and confirmed the cyotoskeleton played a crucial role in the formation of trail. Inhibiting of Arp2/3 complex dramatically decrease trail formation ratio, length and area, while the ratio and length remained unchanged and area decreased after inhibiting of myosin II. The migration direction index of neutrophil is sensitive to both Arp2/3 complex and myosin II, and only Arp2/3 complex affect speed of neutrophil migration. The immune fluorescence staining study showed that CD11b、focal adhesion molecules (tailin, pFAK, paxillin, vinculin) and F-actin are highly colocalized, indicating that actin-binding protein play critical role in stiffenss dependent neutrophil trail formation.

These results provide an insight for understanding the occurrence and development of atherosclerosis and intervening for the potential prevention or attenuation of cardiovascular disease progress.

语种中文
文献类型学位论文
条目标识符http://dspace.imech.ac.cn/handle/311007/73166
专题国家微重力实验室
作者单位中国科学院力学研究所
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郭盼. 基底硬度调控中性粒细胞曳尾结构的力学-生物学耦合机制[D]. 北京. 中国科学院大学,2018.
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