RGD-PLGA微泡介导的干细胞靶向治疗在肝损伤-再生中的作用

Though the liver has powerful regeneration capacity，the residual liver cells’ lacking of proliferation signal result in the disorder of liver regeneration. Bone marrow mesenchymal stem cells（BMSCs）can differentiate into liver cells and secrete cytokines to promote the liver regenation. However, low efficiency of transplantation and poor targeting of the organs are the main problem in this area. So, how to increase the homing rate of BMSCs is the key point waited to be solved. Ultrasound mediated microbubble blasting can increase cell permeability and promote BMSCs trans-endothelial migration into the parenchyma by its cavitation and mechanical action. RGD peptides are recognition sites of integrin ligand proteins’ interaction. It can specifically bound to the αvβ3 on the hepatic sinusoidal endothelium cells. Therefore, this study intends to prepare the polymer ultrasound contrast agent(RGDS-PLGA) targeting hepatic sinusoidal endothelium cells. Blast the targeted microbubbles and using their cavitation and mechanical action to improve transplantation efficiency of BMSCs. Observe the BMSCs’ homing and the targeting location in the liver tissue by imaging, biochemistry, immunology and pathology. To definite the function of targeted microbubbles blasting in promoting liver regeneration and clarify the molecular mechanism. Thereby, we can lays a important theoretical foundation to stem cell transplantation therapy for liver diseases.

虽然肝脏本身再生能力很强，但残余肝细胞缺乏必要的增殖信号导致肝再生障碍。骨髓间充质干细胞(BMSCs)通过直接转化为肝细胞及分泌细胞因子间接促进损伤后肝组织再生，但移植效率低、靶向性差是目前该领域的主要问题。因此，提高BMSCs移植后肝组织归巢率成为关键。超声造影剂微泡在一定强度的超声辐照下，可瞬间爆破产生空化效应和机械作用，使内皮细胞通透性增加，有利于BMSCs跨内皮游出。RGD肽是整合素配体蛋白相互作用的识别位点，可与肝血窦内皮整合素αvβ3特异性的相互结合。因此，本研究拟构建携RGD的靶向超声微泡，利用微泡爆破效应，增加肝血窦内皮通透性，采用生化、分子生物学、细胞学及病理学等手段观察移植后BMSC在外科肝损伤动物模型中再生肝组织内的靶向定位和归巢情况，明确靶向微泡爆破在BMSCs移植促进肝再生中的作用，并阐明其分子作用机制，从而为干细胞移植干预、治疗肝脏疾病奠定重要理论依据。

肝脏本身具很强的再生能力，但残余肝细胞增殖的异常导致肝再生障碍。骨髓间充质干细胞可转化为肝细胞，并分泌细胞因子促进损伤后肝组织再生，但其存在移植效率低、靶向性差等问题。因此，如何提高BMSCs移植后肝组织归巢率，是亟待解决的科学问题。本研究拟构建携RGD的靶向超声微泡，利用微泡爆破效应，增加肝血窦内皮通透性，有利于BMSCs跨内皮游出。然而，实验前期用碳二亚胺法构建携带RGD短肽的PLGA靶向造影剂微泡，由于RGD短肽的微泡携带率过低，影响之后靶向造影剂的造影效果 。因此，对原实验设计做出了调整。以金纳米棒作为输送递质，将合成的H3短肽输送入间充质细胞内，实验证实金纳米棒可以增加H3短肽的通透性。进入细胞内的短肽通过与赖氨酸特异去甲基化酶（LSD1）的作用位点结合，而起到抑制赖氨酸特异的去甲基化酶（LSD1）活性的作用，通过体外细胞实验等证实，可抑制LSD1的活性，从而促进间充质干细胞向肝细胞分化。本实验为干细胞移植干预、治疗肝脏疾病奠定一定的理论依据。