超声协同纳米微泡提高帕金森小鼠移植干细胞存活率的实验研究

Stem cell transplantation was identified to be effective in the Parkinson animal models but the clinic therapeutic effect was not very stable. One of the reasons was the low survival rate of the transplantated cells. Ultrasound-targeted microbubble destruction could enhance the survival rate of stem cells in ischemic animal models. Our previous study suggested that nano-scale microbubble destruction induced by low-frequency ultrasound could open the localized blood-brain barrier in irradiation area. If this technique could be used to force the stem cells through the blood-brain barrier after intravenous infusion and improve the cell survival rate in the brain, it will promote the clinical application of neural stem cell therapy in the Parkinson's disease. The aims of this research are to evaluate the feasibility of the intravenous neural stem cells (NSCs) migrating into the brain through the opened midbrain blood-brain barrier by ultrasound-mediated nano-scale microbubble destruction, and to observe the effect of the technical parameters to the cell retention in the brain and their safety thresholds. Based on these, the optimized technique will be further used to force the intravenous neural stem cell to the brain to treat Parkinson's rat models. The survival and differentiation of the transplanted NSCs and the efficacy of the treatment will be assessed by the behavioral tests, immunohistochemical method, pathology and electron microscopic observation, PCR and high performance liquid chromatography to evaluate the clinical value of intravenous stem cells transplantation in the treatment of Parkinson's disease compared with the stereotactic injection transplantation in the striatum.

干细胞移植对帕金森动物有肯定疗效，但临床应用效果不稳定，原因之一是移植细胞存活率低。超声协同微泡能增加缺血动物模型干细胞存活率。本课题组前期研究表明，低频超声协同纳米级微泡可开放超声辐照区血脑屏障，如能应用该技术使干细胞经静脉输注后通过血脑屏障并提高脑内存活率，将促进帕金森病干细胞疗法的临床应用。本课题旨在研究应用低频超声协同纳米级微泡开放中脑血脑屏障后使经静脉输注神经干细胞（NSCs）迁移入脑的可行性，观察不同技术参数对脑内移植细胞存留率的影响及其安全阈值，在此基础上，应用优化后技术介导经静脉输注干细胞治疗帕金森小鼠模型，并与纹状体立体定向注射移植方式对比，通过行为学检测、免疫组化、病理和电镜观察、PCR、高效液相色谱法等观察两种移植途径脑内NSCs存活、分化情况以及疗效，以评估经静脉途径移植干细胞治疗帕金森病的临床应用价值。

帕金森病的病理机制为中脑黑质多巴胺能神经元进行性变性、缺失，导致状体中多巴胺含量减少。干细胞是治疗帕金森病潜在的有效方法。然而由于血脑屏障的存在，经静脉注射的干细胞无法在病变脑组织中定植，制约其疗效。本研究采用超声击破微泡无创开放血脑屏障，促进神经干细胞穿过血脑屏障定植于黑质区，从而实现对帕金森的治疗。我们以磷脂作为膜材料制备微泡造影剂，制得的微泡呈中央半透亮球形，浓度为（3.63±0.11）×109 MBs/ml，平均粒径为1546.8±114.2nm，制备后6小时内粒径和浓度相对稳定，能够显著持续增强小鼠心腔显影，半衰期为2.97±0.54分钟。随后我们对超声联合微泡开放小鼠血脑屏障的参数进行优化，并研究其机制。发现随着微泡剂量、机械指数（MI）和辐照时间增加，脑组织中伊文氏兰渗出量增加，蓝染程度增加，同时对脑血管及神经元的损伤也增加。最终筛选出的最佳参数为：微泡剂量1.0×107个，MI 0.8，辐照时间3分钟。血脑屏障开放后三种紧密连接相关蛋白ZO-1、Occludin与Claudin-5表达水平均明显下降，透射电镜观察到脑血管内皮紧密连接开放。超声辐照后0小时，血脑屏障通透性最高，脑皮质及纹状体区分别于6小时和4小时后血脑屏障恢复正常。将干细胞与超声微泡联合注入小鼠尾静脉，超声辐照后28天，黑质区脑组织酪氨酸羟化酶免疫荧光染发现，干细胞联合超声微泡组的多巴胺能神经元数量明显高于单纯干细胞组。同时，干细胞联合超声微泡组脑组织中的多巴胺-β-羟化酶、神经生长因子和多巴胺转运体均高于单纯干细胞组和生理盐水对照组。说明超声击破微泡可显著增强神经干细胞在黑质区定植，定植的干细胞一方面分化为多巴胺神经元，分泌多巴胺，刺激多巴胺-β-羟化酶和多巴胺转运体反应性表达增加；另一方面，干细胞也能分泌神经生长因子，诱导神经元修复，减少神经细胞死亡。本项目为干细胞治疗帕金森提供了一种安全有效的方式，同时也为细胞、大分子药物或基因颅内递送供了新思路。