扶正补血食疗方含药血清干预Notch通路调控骨髓造血干细胞化疗后损伤的机制研究

Mechanism of Fuzheng Buxue Shiliao Formula Containing Serum Intervening Notch Pathway to Regulate Damage of Hematopoietic Stem Cells After Chemotherapy

目的:通过扶正补血食疗方含药血清干预5-FU损伤小鼠骨髓间充质干细胞(BMSCs)和造血干细胞(HSCs)体外共培养模型,探讨补血食疗方对化疗后模拟“造血龛”中小鼠HSCs增殖、凋亡以及Notch信号通路的影响。方法:建立小鼠BMSCs和HSCs的共培养模型。共培养细胞随机分为空白组、5-FU组和扶正补血食疗方含药血清组。镜下观察各组细胞形态变化,取上层悬浮HSCs进行检测,CCK-8检测细胞活力、PI/RNase法检测细胞周期、Annexin V/Alexa Fluor 647双重染色法检测细胞凋亡率、qRT-PCR检测Jagged1、Notch1、Notch2基因的表达水平。结果:与空白组相比,5-FU组细胞活性降低(P<0.05),G0/G1期细胞占比增加(P<0.05),凋亡率增加(P<0.05),在基因水平方面,Jagged1、Notch1、Notch2表达呈下调趋势(P<0.05);与5-FU组相比,扶正补血食疗方含药血清组细胞活性增加(P<0.05),G0/G1期细胞占比减少(P<0.05),总凋亡率呈下降趋势(P<0.05),在基因水平方面,Jagged1、Notch1、Notch2表达呈上调趋势(P<0.05)。结论:扶正补血食疗方含药血清可能通过上调Jagged1、Notch1、Notch2基因的表达激活Notch通路,保护造血微环境,促进HSCs增殖、降低凋亡率,缓解HSCs化疗损伤程度。

Objective:To explore the effect of Fuzheng Buxue Shiliao Formula on the HSCs proliferation,apoptosis and Notch signaling pathway under the state of'hematopoietic niche'after chemotherapy,through the in vitro coculture model of bone marrow mesenchymal stem cells(BMSCs)and hematopoietic stem cells(HSCs)of 5-FUinjured mice intervened by Fuzheng Buxue Shiliao Formula containing serum.Methods:The co-culture model of BMSCs and HSCs was established in mice.The co-cultured cells were randomly divided into the blank group,the 5-FU group and the Fuzheng Buxue Shiliao Formula containing serum group.The morphological changes of the cells in each group were observed under the microscope,the upper suspended HSCs was detected,CCK-8 was used for the detection of cell viability,PI/RNase method for cell cycle,Annexin V/Alexa Fluor 647 double staining for cell apoptosis rate,qRT-PCR for the gene expressions of Jagged1,Notch1 and Notch2.Results:Compared to those in the normal group,the cell viability decreased(P<0.05),the proportion of cells in the G0/G1 phase increased(P<0.05),and the total apoptosis rate increased(P<0.05),the expressions of Jagged1,Notch1 and Notch2 showed a down-regulation trend in the 5-FU group(P<0.05).Compared to those in the 5-FU group,the cell viability increased(P<0.05),the proportion of cells in the G0/G1 phase decreased(P<0.05),the total apoptosis rate showed a downward trend(P<0.05),and the expressions of Jagged1,Notch1 and Notch2 showed an upwardregulated trend in the Fuzheng Buxue Shiliao Formula containing serum group(P<0.05).Conclusion:Fuzheng Buxue Shiliao Formula containing serum can protect the hematopoietic microenvironment,promote the HSCs proliferation,reduce the apoptosis rate and alleviate the degree of HSCs chemotherapy damage through activating the Notch pathway by up-regulating the gene expressions of Jagged1,Notch1 and Notch2.