从Ptpn11调控MSC修复骨髓壁龛的途径探讨补肾解毒通络法抑制白血病复发的作用及机制研究

It is an important route to prevent leukemia relapse in curing leukemia successfully. The main reason of leukemia relapse (residual LSC＜104 per liter) is that leukemia stem cells (LSC) are activated or hematopoietic stem cells (HSCs) are deteriorated into LSCs at LSCs niche in bone marrow. So, rehabilitating LSC niche in bone marrow is critical for the inhibition of leukemia relapse. It is believed in Traditional Chinese Medicine that minimal residual leukemia (hidden pathogen) damage to collaterals in bone marrow (Niche) and lead to leukemia relapse through turning marrow (HSC) into toxic blood (LSC). According to the theory of hidden pathogen and collateral disorder, we want to find a noval way of prohibiting leukemia relapse. Our hypothesis is that LSC niche can be restored via bushenjiedutongluo method. In our project, we will focus on the mesenchymal stem cell (MSC) niche and finish the following studies. Testify the effect of bushenjiedutongluo method on leukemia relapse in vivo and in vitro. Clarify these proteins in bone marrow niche via iTRAQ joint tandem mass spectrometry and IPA biological information analysis. FCM, PCR, Western-blot, electron microscopy and histochemistry were used to analyse CFU, cell cycle and apoptosis of HSC/LSC, to study the critical genes and proteins of Pnpt11 pathway in MSC. Analyse the mechanism of bushenjiedutongluo method in regulating the bone marrow niche clearly.This research will provide pivotal evidence of Traditional Chinese Medicine on preventing leukemia relapse by restoring the LSC niche, and improve the level of leukemia treatment through the cooperation of Traditional Chinese and Western Medicine.

阻止白血病复发是治愈白血病的重要环节之一。白血病复发（残留＜104/L）的主要原因是白血病干细胞（LSC）骨髓壁龛（Niche）活化LSC和/或诱导造血干细胞（HSC）恶变。因而，阻止白血病复发的关键是修复LSC骨髓壁龛。中医认为残留白血病（“伏邪”）伤及骨髓络脉（“Niche”）致使髓生毒血而复发。基于经典的伏邪、络病理论，我们提出通过补肾解毒通络法修复Niche的假说，探讨防治白血病复发的新思路。拟以间充质干细胞（MSC）为Niche研究核心，体内给药修复和体外观察相结合验证该治法的作用，iTRAQ和IPA明确Niche中功能蛋白，FCM、Western-blot、电镜、免疫组化分析HSC/LSC的CFU、细胞周期、凋亡等手段研究MSC的Pnpt11基因及通路蛋白，解析该治法调控Niche的机制。为中医药修复Niche抑制白血病复发提供科学依据，提高中西医结合治疗白血病的水平。

拟补肾解毒通络法对以间充质干细胞（MSC）为核心的Niche有修复作用，且可通过Ptpn11及通路蛋白调控Niche，本课题通过体内给药修复和体外观察相结合验证该治法的作用，iTRAQ与中药复方成分色谱-质谱分析技术结合明确Niche中功能蛋白，FCM、RT-PCR、免疫组化等手段研究分析HSC/LSC的CFU、细胞周期、凋亡及MSC的Ptpn11基因及通路蛋白，对补肾解毒通络法调控Niche的机制进行了初步研究。首先通过对中药复方的液相质谱分析得出补肾解毒通络法的有效成分，将有效成分所筛选的靶点与iTRAQ技术筛选的差异蛋白结合，经生物信息学分析，得出补肾解毒通络法的作用蛋白Cdk1、Dnmt1、Icam1、Fth1、App、Mmp2、Creb1、Stmn1、Rela、Alb；构建急性白血病小鼠模型，给药后提取小鼠骨髓细胞培养以获得间充质干细胞，通过FCM、RT-PCR、免疫组化等手段研究分析HSC/LSC的CFU、细胞周期、凋亡及MSC的Fth1、Icam1、Ptpn11、Creb1、Stmn1、Rela基因的表达情况，结果显示在补肾解毒通络法作用下的HSC细胞活性明增强，HSC表达的Cdk１降低；LSC活性减低，MSC分泌促进LSC与MSC相互黏附基因Icam1、Fth１表达明显减弱，推测补肾解毒通络法可能通过抑制LSC增殖同时保持HSC活性修复Niche，阻止白血病复方。其中通过免疫组化验证补肾解毒通络法在急性白血病小鼠化疗后骨髓修复及蛋白表达的实验正在进行。综合上述结果，本研究为探讨补肾解毒通络法修复Ｎiche通过调控MSC从而抑制白血病复发的机制研究奠定了坚实的工作基础。项目资助发表论文4篇，待发表1篇。培养硕士生4名，其中2名已经取得硕士学位，2名在读。项目投入经费55万元，支出48.8673万元，各项支出基本与预算相符。剩余经费6.1327万元，剩余经费计划用于本项目研究后续支出。