四物汤及其补肾加味方通过骨髓间充质干细胞调节造血干细胞数量和功能的研究

Radiotherapy and chemotherapy always bring about bone marrow suppression in cancer patients.Pre-osteoblasts located near the endosteum and bone marrow mesenchymal stem cells (MSCs)have been demonstrated to be important components of hematopoietic stem cells(HSCs)niche. Radiotherapy and chemotherapy for cancer can inhibit MSCs and HSCs,as well as their microenvironment,thus cause the patients suffer from varying degrees of anemia and osteoporosis..As heat，poisonous invaders in Traditional Chinese Medicine(TCM), radiation and chemotherapy drugs damage the transfusion and digestion works of spleen and stomach,and weaken the function of liver and kidney,as a consequence,leading to deficiency of kidney essence and blood and nourishment lost of tendons and bones..Chinese medicine of Bu shen have been proved to have stimulation effect on differentiation of MSCs.The promotion effect of Si Wu formula on bone marrow hematopoiesis have been primarily illustrated.This project is under the guidence of TCM theory "Kidney manage bone,genetate marrow,deposite kidney essence" and "Blood is generated from the kidney essence".Modern biology techniques such as flow cytometry and competitive bone marrow transplantation will be used to explore the effect of classic Si Wu Formula and its Bu shen formula on number and function of bone marrow HSCs.The correlation of number of MSCs and their differentiated osteoblast with this effect will be also detected.The project is attempted to establish the platform for further illustrating Si Wu Formula and its Bu shen formula's effect on bone marrow hematopoiesis and its mechanism.

放射性以及化学性治疗后肿瘤患者等均导致骨髓抑制。骨内膜表面前成骨细胞和骨髓间充质干细胞是造血干细胞微环境的重要组成部分，肿瘤放化疗均导致骨髓间充质干细胞和造血干细胞及其微环境受到抑制，患者出现不同程度的贫血及骨质疏松。.放化疗属热毒之邪，同时还损害脾胃运化之功及肝肾的功能，导致精血亏虚，筋骨失养。结合补肾中药在调节MSCs分化中的作用，结合前期证明的四物汤促进骨髓造血的作用，提出本课题的科学假设：四物汤及其补肾加味方通过骨髓间充质干细胞及前成骨细胞调节骨髓造血干细胞数量和功能。.本课题在"肾主骨，生髓，藏精"，"血为精所化"理论指导下，采用流式细胞术、竞争性骨髓移植等现代生物学研究方法，考察四物汤及其补肾加味方对小鼠骨髓造血干细胞数量和功能的影响，以及骨髓间充质干细胞和前成骨细胞在此过程中的作用，为进一步阐明四物汤和补肾中药对骨髓造血的影响及其机制奠定基础。

肿瘤放化疗治疗均导致骨髓间充质干细胞（MSC）和造血干细胞(HSC)及其微环境受到抑制，患者出现不同程度的贫血及骨质疏松。结合研究进展和前期工作，提出科学假设：四物汤及其补肾加味方通过骨髓MSC及前成骨细胞调节骨髓HSC数量和功能。 .本项目建立了流式检测HSC、MSC及磁珠分选骨髓细胞方法，观察四物汤及补肾小复方对正常小鼠骨髓HSC数量及外周血常规影响；改良CTX诱导的小鼠骨髓抑制模型，用于CTX致骨髓抑制机制及与干细胞的相关性研究；确定其骨量丢失症状，开展成骨、破骨功能，细胞周期相关分子变化及与Wnt/β-catenin途径相关性的研究；探索了四物汤、补肾方及补肾方有效组分对CTX导致的骨髓抑制贫血和骨量丢失的治疗作用。 .发现四物汤及补肾小复方提高正常小鼠骨髓HSC数量；滋肾阴方降低小鼠外周血白细胞（主要是淋巴和中性粒细胞）的数量；改良的模型外周白细胞、血小板和红细胞显著降低， HSC和MSC显著降低，粒/巨噬细胞分化（CFU-GM）能力降低，胸腺指数显著降低；模型小鼠出现显著的骨量丢失症状，腰椎成骨、破骨分化相关基因都显著降低，骨髓贴壁细胞ALP染色明显减弱，胫骨切片Trap染色也显著降低，骨髓Lin-细胞出现G0/G1期阻滞，周期相关蛋白c-Myc,CyclinD1表达降低；模型小鼠腰椎Wnt1、Wnt4、β-catenin表达下降，股骨active β-catenin和Runx2免疫组化染色显著降低；四物汤+补肾方(BSSW)能提高模型小鼠胸腺指数；四物汤(SWT)、BSSW能提高模型小鼠WBC水平，补骨脂素(Pso)能提高模型小鼠WBC水平，齐墩果酸(Oa)能提高PLT水平； BSSW含药血清能改善CTX干预引起的G0/G1期阻滞，SWT、补肾方(BSF)、BSSW含药血清皆能增加G2/M期比例，改善CTX干预导致的细胞表面CD29、CD44表达量降低；SWT、BSF、BSSW、Pso及Oa均能改善骨髓Lin-细胞G0/G1期阻滞，同时增加G2/M期比例；Pso能增加Tb.N.，降低Tb.Sp.，改善骨量丢失。 .本项目从建立相关技术方法和适合模型进行骨髓抑制和干细胞相关性研究，初步确定化疗导致骨髓抑制与MSC、HSC受损的相关性及机制，发现四物汤补肾方及其有效组分在治疗CTX致贫血和骨质疏松中的潜力，为进一步机制研究和临床治疗奠定基础。