亚低温对颅脑创伤高张微环境中移植温敏干细胞的生物力学调控

Most researches focus on the investigation of chemical microenvironment, such as low level of oxygen or growth factors, whereas little is known about physical microenvironment. The recent reports demonstrate that biomechanics plays key roles in the behavior of grafted stem cells, implying the possible role of biomechanics on grafted stem cells after traumatic brain injury (TBI). According to our last National Nature Scientific Fund results that therapeutic combination of mild hypothermia and temperature sensitive mesenchymal stem cells could raise the survival and neural differentiation of stem cells grafted into post-TBI injured area, and the common pathophysiologic phenomenon of intracranial hypertension and hydrocephalus after TBI, we speculate the change of biomechanics of brain tissue after traumatic brain injury which could lead to high tension of grafted microenvironment, and mild hypothermia may be involved in the regulation of biomechanics. We attempt to evaluate the mechanical force of single cells and tissue elastic modulus in the grafted area after experimental TBI with intracranial pressure monitoring, atomic force microscope and tensile machine. Thereafter, to simulate the biomechanical change of experimental TBI, temperature sensitive mesenchymal stem cells will be cultured in two-dimension or three-dimension manner with different compressive strain and substance elasticity. The growth and differentiation of stem cells under 33 C or 37 C will be observed, and corresponding mechanical signal transmission pathway will be examined to find key factors involving it. On the basis of the above study, we would like to further enhance validity of transplanted stem cells by improving both physical and chemical post-TBI microenvironment.

迄今为止，多数"移植微环境"的研究聚焦于缺氧缺生长因子等化学微环境，对物理微环境知之甚少。最新报道，力学物理微环境可能对移植干细胞的生长分化起着极为重要的作用。本项目旨在延续上一科学基金课题 "亚低温联合温敏干细胞可提高创伤性颅脑损伤（TBI）后干细胞存活率和分化率"的研究结果，依据临床TBI后出现颅内高压、脑水肿等病理生理现象，推测TBI后生物力学发生改变，提出"TBI形成高张移植微环境"的概念以及"亚低温相关力学信号转导调控可提高干细胞移植效率"的假设。研究拟用颅内压监测、原子力显微镜和弹力拉伸等方法评估大鼠TBI后移植处细胞所受的机械力和组织弹性变化；体外模拟分别采用不同压应力和弹性基质加载进行二维或三维培养，观察温敏干细胞在33℃或37℃的生长分化，寻找亚低温相关力学信号转导机制，以期从力学干预的角度出发，与化学因素联合，更为全面的改善移植微环境和促进干细胞在TBI的临床应用。

迄今为止，多数"移植微环境"的研究聚焦于缺氧缺生长因子等化学微环境，对物理微环境知之甚少。最新报道，力学物理微环境可能对移植干细胞的生长分化起着极为重要的作用。本项目依据临床TBI后出现颅内高压、脑水肿等病理生理现象，推测 TBI 后生物力学发生改变，提出"TBI形成高张移植微环境"的概念以及"亚低温相关力学信号转导调控可提高干细胞移植效率"的假设。本研究采用原子力显微镜和弹力拉伸等方法评估模拟大鼠TBI细胞损伤所受的机械力和弹性模量的变化；体外模拟分别采用不同压应力和弹性基质加载进行二维或三维培养，观察温敏干细胞在 33℃或 37℃的生长分化；在体检测亚低温联合温敏干细胞对TBI大鼠神经功能的修复作用。结果显示通过原子力显微镜和弹力拉伸检测得出损伤后细胞力学性能发生改变，弹性模量显著增加，而TBI联合亚低温可逆转因细胞损伤导致的弹性模量的增加，同时可以抑制细胞的进一步损伤；亚低温联合温敏干细胞在模拟脑组织弹性模量的聚丙烯酰胺水凝胶上具有向神经元分化的能力，提示亚低温治疗可以通过降低损伤细胞的弹性模量来改善TBI后颅内物理微环境，从而促进TBI后移植的温敏脐带间充质干细胞向神经元分化；温敏干细胞联合亚低温可逆转TBI后认知障碍，改善TBI后突触可塑性，降低神经学缺损评分，减轻大鼠脑水肿程度和大鼠脑组织损伤体积，亚低温可改善TBI脑组织的高张物理微环境，从而提高温敏干细胞的存活与分化能力，从而促进TBI后大鼠神经功能恢复。本研究中生物力学的引入为干细胞治疗脑损伤带来新的切入点，也首次为亚低温有效治疗重型 TBI提供力学机制，而且物理和化学因素相联合，亚低温与温敏干细胞相联合，可以更为全面的改善移植微环境和促进干细胞在TBI的临床应用。