“补肾生髓法”基于SDF-1/CXCR4轴及PI3K/Akt-Jak1/STAT信号通路促进骨髓间充质干细胞移植治疗肝硬化的机制研究

Treatment of bone marrow mesenchymal stem cells(BMSCs) transplantation can effectively improve liver function and immune function in patients with hepatic cirrhosis. But insufficient number of stem cells homing to liver after transplantation is one of the problems that affect curative effect in hepatic cirrhosis. Granulocyte colony stimulating factor(GCSF) and Erythropoietin(EPO) are recognized as mobilization agent of BMSCs.GCSF and EPO excite the SDF1 / CXCR4 axis and PI3K/Akt - Jak1 / STAT signal pathway to accelerate the homing of stem cells to liver after BMSCs transplantation, and then significantly improve effect. We have observed that the classical JiSheng ShenQi decoction, which function is " Tonifying kidney and nourishing marrow " , could promote BMSCs proliferation and significantly improve erythrocyte immunity function and leukocyte immunity function in cirrhosis. JiSheng ShenQi decoction also induced transplanted stem cells homing to the liver and help to repair cirrhosis, but the mechanism is underlying. Therefore we would prepare hepatic cirrhosis model rats and treat them with JiSheng ShenQi decoction combined with BMSCs transplantation. Liver function and histology of all cirrhosis rats will be observed before and after treatment. GCSF, EPO, Interleukin-3(IL-3), Interleukin-6(IL–6), Hepatocyte growth factor(HGF), Stem cell factor(SCF), Hypoxia-inducible factor 1(HIF1) and Stromal-derived factor 1(SDF1) in peripheral blood of all rats be detected by ELISA technique. Protein expression of SDF1, CXCR4, PI3K, Akt, Jak1, STAT of liver tissue of all cirrhosis rats be analysed by immune coprecipitation technique. Molecule expression of PI3K/Akt-Jak1/STAT signal pathway in liver of all cirrhosis rats be detected by immunohistochemistry, western blot and RT-PCR technique. Stem cell labeled by DAPI will be count in the liver tissue with fluorescence microscopy after BMSCs transplantation. And to explore the mechanism of homing of transplanted BMSCs to liver promoted by JiSheng ShenQi decoction through excited SDF1/CXCR4 axis and PI3K/Akt-Jak1/STAT signal pathway. Also to reveal scientific of the classical chinese medicine theory of " Tonifying kidney and nourishing marrow ", and enrich traditional chinese medicine theoretical system for the treatment In hepatic cirrhosis.

骨髓间充质干细胞移植可改善肝硬化患者肝脏合成及免疫功能，移植后干细胞归巢不足则降低疗效。GCSF和EPO是公认的BMSCs动员剂，通过影响SDF1/CXCR4轴及PI3K/Akt-Jak1/STAT信号通路促进移植干细胞归巢。我们前期观察到“补肾生髓法”方药济生肾气汤可促进BMSCs增殖，改善肝硬化患者的红细胞免疫及白细胞免疫，并诱导移植干细胞向肝脏归巢，但分子机制不明。我们拟复制肝硬化大鼠模型，用济生肾气汤结合BMSCs移植治疗，观察治疗前后肝功能及肝脏病理，运用ELISA检测外周血GCSF、EPO、IL-3等细胞因子，免疫共沉淀检测肝组织SDF1、CXCR4等蛋白表达，免疫组化、WB及PCR技术检测PI3K/Akt-Jak1/STAT通路各分子表达，荧光显微镜观察肝组织DAPI标记的干细胞计数。探讨“补肾生髓法”促进BMSCs移植治疗肝硬化的作用机制，证实“补肾生髓以养肝“理论的科学性

肝硬化是由各种原因引起的以肝脏结构破坏、肝功能失代偿、肝脏血液动力学障碍及免疫功能异常为特点的临床综合征。中国是肝炎、肝硬化疾病高发国家，目前尚无治疗肝硬化的有效药物。既往课题组应用中药联合BMSCs移植治疗肝硬化，发现补肾方剂济生肾气汤可增加BMSCs向肝脏归巢，促进干细胞分化为功能肝细胞，加速肝再生，恢复肝功能，但具体作用机制尚不明确。本项目通过建立肝硬化模型大鼠，运用形态学、分子生物学、免疫组化、荧光标记法、RT-PCR、Western blot等方法。通过研究结果表明，济生肾气汤联合BMSCs移植治疗可显著改善肝硬化大鼠血清肝功能、凝血功能，血常规，减少炎症反应。经治疗后的大鼠血清中GCSF、EPO、SCF、HGF等干细胞动员因子分泌增加。病理结果显示，经治疗后的大鼠肝小叶得到修复，假小叶数量减少，纤维化程度及炎症活动度改善。荧光显微镜下观察治疗后的大鼠肝组织内BMSCs归巢数量明显增多。此外，治疗后的大鼠肝组织内肝组织 SDF-1、CXCR4、PI3K、Akt、Jak1、STAT 蛋白阳性表达显著上调。因此我们推测济生肾气汤联合BMSCs移植治疗肝硬化的机制为，济生肾气汤通过补肾生髓的作用促进植入的BMSCs在肝脏中定植并自我增殖，以及与其他细胞发生融合或直接分化为肝细胞以代替修复损失的肝脏，增强肝脏再生能力，恢复肝细胞功能。.