【作者】 刘娜；

【导师】 孙伟正；

【作者基本信息】 黑龙江中医药大学 ， 中西医结合临床， 2011， 博士

【摘要】 目的：1.观察以补肾生血法所立之方补髓生血颗粒对慢性再生障碍性贫血(chronic aplastic anemia, CAA)患者的临床疗效。2.探讨FAK/PI3K信号转导通路及Rho GTP酶在CAA造血粘附异常发病机制中的作用,阐明补髓生血颗粒对CAA患者骨髓FAK/PI3K信号通路及Rho GTP酶干预作用。方法：1.按照随机数字表法将108例CAA患者分成两组,试验组55例,使用补髓生血颗粒治疗；对照组53例,使用再造生血片治疗。两组均以3个月为1个疗程,共治疗2个疗程。2.治疗6个月后,对两组患者临床疗效、中医症状、中医证候疗效、外周血象及骨髓象进行比较。3.采用蛋白印迹法检测CAA患者治疗前后骨髓FAK/PI3K信号通路中相关酶类蛋白及其磷酸化水平和Rho GTP酶的表达；采用逆转录PCR法检测CAA患者治疗前后骨髓FAK及PI3K p85 mRNA表达变化。结果：1.补肾生血中药补髓生血颗粒治疗CAA的临床总有效率为74.55%,明显优于对照组(P<0.05)；补髓生血颗粒能在一定程度上改善CAA患者中医症状频次的出现及中医症状平均积分(P均<0.05),试验组中医证候疗效总有效率为87.27%,明显高于对照组(P<0.05)；在改善CAA患者外周血白细胞计数及血红蛋白含量时,试验组优于对照组(P均<0.05)；而CAA患者肾阳虚型的临床疗效优于肾阴虚型(P<0.05)。2. CAA患者骨髓单个核细胞FAK、phospho-FAK Tyr397的蛋白表达与FAK mRNA表达均低于正常对照组(P均<0.05),补髓生血颗粒能够上调三者低水平的异常表达(P均<0.05),且试验组优于对照组(P<0.05)；试验组肾阳虚型中的FAK蛋白表达和FAK mRNA表达改善程度与肾阴虚型比较,具有统计学意义(P均<0.05),其中肾阳虚型FAK mRNA表达基本可达正常对照组水平(P>0.05)。3.CAA患者骨髓单个核细胞PI3K、phospho-PI3K p85的蛋白表达与PI3K p85 mRNA表达均低于正常对照组(P均<0.05),补髓生血颗粒能够改善其表达水平(P均<0.05)；试验组PI3K及phospho-PI3Kp85的蛋白表达水平改善优于对照组(P均<0.05)；疗前试验组肾阳虚型PI3K蛋白表达高于肾阴虚型(P<0.05)；疗后肾阳虚型与肾阴虚型进行比较,仅PI3K p85 mRNA的改善程度存在显著性差异(P<0.05)。4.CAA患者骨髓单个核细胞RhoA、CDC42和Rac的蛋白表达均明显低于正常对照组(P均<0.05),经补髓生血颗粒治疗后,三者的蛋白表达均有所上升(P均<0.05)；试验组中RhoA和CDC42改善程度优于对照组(P均<0.05)；疗前试验组中肾阳虚型CDC42蛋白表达高于肾阴虚型(P<0.05)；治疗前后RhoA和CDC42蛋白表达进行比较,肾阳虚型的改善程度优于肾阴虚型(P均<0.05)。结论：1.补髓生血颗粒治疗CAA的疗效确切,提示治疗CAA应从肾论治,以补肾为主法。2.骨髓单个核细胞FAK异常低表达与CAA发病造血粘附异常机制有一定关系。3.骨髓单个核细胞PI3K及PI3K p85的异常低水平表达可能参与CAA发病机制。4.整合素介导的FAK/PI3K信号转导通路异常可能是CAA造血粘附异常发病机制的重要环节。5.骨髓单个核细胞中Rho GTP相关酶类Rho、CDC42和Rac呈异常低表达,可能影响造血干细胞的归巢和迁移机制。6.补髓生血颗粒可以改善FAK/ PI3K信号转导通路中相关酶类及Rho GTP酶的蛋白表达,使得FAK/PI3K信号通路及Rho GTP酶得以活化,其机制可能是通过改善粘着斑的形成、调节细胞骨架、促进造血细胞的增殖、改善造血微环境、促进造血干细胞与造血微环境相互作用等,从而改善造血干细胞/祖细胞的归巢、移行,增强造血粘附信号转导,使造血功能得以恢复。7.对CAA根据肾虚进行辨证分型治疗,在临床疗效及改善FAK、FAK mRNA、PI3K p85 mRNA、RhoA和CDC42的表达时,肾阳虚型皆优于肾阴虚型。更多还原

【Abstract】 Objectives:1. To observe the clinical efficacy of Busui Shengxue Granule in chronic aplastic anemia (CAA) patients.2. To explore the roles of FAK/PI3K signaling transduction pathway and Rho GTPase in CAA pathogenesis of abnormal hematopoietic adhesion. We observed the influences on FAK/PI3K signaling transduction pathway and Rho GTPase in CAA patients with Busui Shengxue Granule.Methods:1. One hundred and eight patients of CAA were divided into two groups at random, the experimental group contained 55 cases, and the control group contained 53 cases. The experimental group was treated by Busui Shengxue Granule, while the control group was treated by Zaizao Shengxue Tablet. All the patients should be treated for 6 months at least.2. After 6 months of treatment, compared the differences of clinical efficacy, traditional Chinese medicine (TCM) symptoms, the efficacy of TCM syndromes, peripheral blood and bone marrow in the two groups.3. Before and after treatment, the protein expression of some enzymes and theirs phosphorylation levels in FAK/PI3K signaling transduction pathway of CAA patients were examined by Western blot, the protein expression levels of Rho GTPase ware examined by Western blot too, and the mRNA expression levels of FAK and PI3K p85 in bone marrow mononuclear cells of CAA patients were examined by RT-PCR.Results:1. The total clinical effective rate of Busui Shengxue Granule in CAA patients was 74.55%, significantly better than the control group (P<0.05). Busui Shengxue Granule could significantly regulate the frequencies and average score of TCM clinical symptoms significantly (P all<0.05), the total effective rate of TCM syndromes in experimental group was 87.27%, significantly higher than the control group (P<0.05).. While improving WBC, Hb and PLT in CAA patients’peripheral blood, the experimental group was better than control group (P all<0.05). The clinical efficacy of CAA patients in Kidney-Yang deficiency type was superior to Kidney-Yin (P<0.05).2. The protein expression levels of FAK, phospho-FAK Tyr397 and mRNA expression levels of FAK in CAA patients’bone marrow mononuclear cells were lower than the normal control group (P all<0.05), Busui Shengxue Granule could increase the abnormal expression levels of these three (P all<0.05), and the significant difference existed in the experimental group and control group (P<0.05). While regulating the expression levels of FAK and FAK mRNA, the Kidney-Yang deficiency type was better than Kidney-Yin (P all<0.05), and the mRNA expression levels of FAK could be up to the normal control group (P>0.05).3. The protein expression levels of PI3K, phospho-PI3K p85 and the mRNA expression levels of PI3K p85 in CAA patients’bone marrow mononuclear cells were significantly lower than the normal control group (P all<0.05), Busui Shengxue Granule could improve the expression levels all of them (P all<0.05). In improving the protein expression levels of PI3K and phospho-PI3K p85, the experimental group was better than control group (P all<0.05). Before treatment, the protein expression levels of PI3K in Kidney-Yang deficiency type were higher than Kidney-Yin (P<0.05). After treatment, the significant difference existed in regulating PI3K p85 mRNA between the two types (P<0.05).4. The protein expression levels of RhoA, CDC42 and Rac in CAA patients’bone marrow mononuclear cells were lower than the normal control group (P all<0.05), Busui Shengxue Granule could increase the levels all of them (P all<0.05). In improving the protein expression levels of RhoA and CDC42, the experimental group was better than control group (P all<0.05). Before treatment, the protein expression levels of CDC42 in Kidney-Yang deficiency type were higher than Kidney-Yin (P<0.05). After treatment, the significant difference existed in regulating RhoA and CDC42 between the two types (P all<0.05).Conclusion:1. The clinical efficacy of Busui Shengxue Granule in CAA patients was significant, it reminded us that treating CAA should be invigorating kidney based on kidney. 2. The low expression of FAK in bone marrow mononuclear cells might have a certain relationship with CAA pathogenesis of abnormal hematopoietic adhesion.3. The low expression of PI3K and PI3K p85 in bone marrow mononuclear cells might be involved the pathogenesis of CAA.4. The abnormity of FAK/PI3K signal transduction pathway integrin-mediated might be a key link in CAA pathogenesis of abnormal hematopoietic adhesion.5. The low expression of Rho GTPase, including Rho, CDC42 and Rac, might affect the mechanisms of hematopoietic stem cell homing and migration.6. Busui Shengxue Granule could improve some enzymes in FAK/PI3K signal transduction pathway and Rho GTPase family, make FAK/PI3K signal transduction pathway and Rho GTPase family to activate, the mechanisms might improve the formation of focal adhesions, regulate cytoskeleton, promote hematopoietic cell proliferation, differentiation, improve the hematopoietic microenvironment, so as to improve the hematopoietic stem cell/progenitor cell homing, migration, and enhance the adhesion of hematopoietic signal transduction, then the hematopoietic function to resume.7. The clinical efficacy of Busui Shengxue Granule in Kidney-Yang deficiency type is higher than Kidney-Yin in CAA patients. Meanwhile, in Kidney-Yang deficiency type, the protein expression levels of FAK, RhoA, CDC42 and mRNA expression levels of FAK, PI3K p85 also could be better corrected than in Kidney-Yin.更多还原