基于红系分化相关lncRNA表达调控探讨益髓生血颗粒治疗β-地贫的分子机制

Thalassemia is a typical single gene hereditary disease，and there is no effective treatment in domestic and overseas. Yi Sui Sheng Xue Granul can nourish the kidney as well as the liver, tonify spleen to generate the blood and curative effect can be repeated in the treatment of thalassemia in the high incidence area of Guangxi.We found Yi Sui Sheng Xue Granul can regulte globin mRNA expression level in the treatment of β - thalassemia in the former study.Recently, some studies found lncRNA are involved in human and mouse erythroid development.However, there is no reports of the molecular mechanism of Yisui Shengxue granules in the treatment of β - thalassemia based on erythroid differentiation related lncRNA expression. With the effect of Fang Zheng intervention, thalassemia patients from a high incidence area will be studied with the help of advanced molecular biology technology . We will studied the intermediate type of β - thalassemia patients by screening and identifing the lncRNA expressed in reticulocytes (or marrow Nucleated Red Blood Cells) before and after treatment of Yisui Shengxue granule.The molecular mechanisms of lncRNA regulation and the targets of lncRNA will also be revealed through in vitro and in vivo tests. The molecular mechanism of Yisui Shengxue granules in the treatment of β - thalassemia based on erythroid differentiation related lncRNA expression will further reveal the theoretical connotation of “kidney producing marrow, marrow producing blood” .

地中海贫血是一种典型的单基因遗传病，国内外尚无有效的治疗办法。以滋肾养肝、益精生血、健脾补气、逍痞退黄为治疗原则，用补肾益髓法代表方－－益髓生血颗粒在广西高发区治疗β-地贫取得可重复的肯定疗效。课题组前期从珠蛋白mRNA表达层面初步探讨了中药治疗β-地贫作用靶点的分子基础。近期研究发现，人和小鼠红系发育过程中受到lncRNA的调控。目前尚无从红系分化相关lncRNA表达调控角度探讨益髓生血颗粒治疗β-地贫的分子机制的报道。本课题以高发区地贫患者为研究对象，采用分子生物学相关的前沿技术，对中间型β-地贫患者益髓生血颗粒治疗前后患者网织红细胞（和/或骨髓有核红细胞）差异表达的lncRNA筛选和鉴定，并通过体外和体内试验等多方面寻找红系分化相关lncRNA调控的分子机制和作用靶点，从红系分化相关lncRNA的表达调控这一角度探讨益髓生血颗粒治疗β-地贫的分子机制，进一步揭中医肾藏精生髓、髓生血治疗地中海贫血理论内涵。

研究背景：益髓生血颗粒在治疗β-地贫取得可重复的肯定疗效。lncRNA在红细胞分化成熟及凋亡等方面发挥着重要作用。本课题从红系分化相关lncRNA的表达调控这一角度探讨益髓生血颗粒治疗β-地贫的分子机制，进一步揭示中医肾藏精生髓、髓生血治疗地中海贫血理论内涵。.主要研究内容：本课题以地贫患者和K562细胞系为研究对象，利用lncRNA 芯片技术检测中间型β-地贫患者益髓生血颗粒治疗前后患者红细胞（含网织红细胞）和PBMC表达谱以及大黄素作用K562细胞后长链非编码RNA(lncRNA)表达谱，.重要研究结果、关键数据及其科学意义：.1）使用lncRNA 芯片技术分别检测3例中间型β-地贫患者益髓生血颗粒治疗前后患者红细胞（含网织红细胞）和PBMC表达谱，与用药前的lncRNA表达比较，红细胞中变化2倍以上lncRNA共6条，PBMC中变化2倍以上且p<0.05的lncRNA共260条。.2）利用lncRNA 芯片技术检测大黄素作用K562细胞后长链非编码RNA(lncRNA)表达谱，大黄素作用K562细胞后变化4倍以上lncRNA共11条。经过荧光定量PCR检测，大黄素作用K562细胞后CDR1AS和PROX1-AS1表达上调与芯片结果一致，为进一步深入研究大黄素抑制K562细胞增殖和促进K562细胞红细分化的分子机制，进而揭示中医肾藏精生髓、髓生血治疗地中海贫血理论内涵打下基础。.3）本研究首次发现大黄素作用K562细胞24小时后能够促使自噬标记蛋白P62增多。p62作为自噬底物连接桥梁，其表达上调可能是促进自噬发生，也有可能是自噬被抑制导致增多（研究结果待发表）。此研究结果将有助于我们深入的揭示大黄素通过调控自噬参与红系造血的调控机制，本课题的研究对我们进一步从分子生物学角度拓展益髓生血颗粒治疗地贫的分子机制具有重要的理论意义。