中间型地中海贫血证候规律及方证效应的分子机制

-密 级：密 单位代码： 84502： 学 号： 1318 中国中医科学院二零零五级博士研究生学位论文中间型地中海贫血证候规律与方证效应的分子机制申 请 人：王文娟指导教师：吴志奎 研究员专业：中西医结合根底分子生物学研究方向：肾生髓、髓生血理论与治疗地中海贫血临床实践中国中医科学院门医院2021年4月 原创性声明本人重声明：所呈交的学位论文，是在导师的指导下独立完成的，文中除注明引用的容外，不包含其他已经发表的科研成果。对本文研究做出重要奉献者，已在文中以明确方式说明。本声明的法律责任完全由自己承担。论文作者签名 导师签名 日期 2008-5-1关于学位论文使用授权的声明本人完全知道中国中医科学院有关保存使用学位论文的规定，同意学校保存或向国家有关部门机构送交论文的复印件和电子版，允许被查阅或借阅。本人授权中国中医科学院可以将本学位论文的全部或局部容编入有关数据库进展检索，可以采用影印、缩印或其他复印手段保存和汇编本学位论文。论文在解密后应遵守此规定论文作者签名 日期 2008-5-1. z-目 录中文摘要1ABSTRACT7英文缩略语13文献综述17一、中医五脏生血理论及其临床应用与研究17二、地中海贫血现代医学研究进展23三、地中海贫血中医药研究进展30前言36临床研究38第一局部中间型地中海贫血患者中医证候分布规律研究381 材料与方法382 研究结果403 讨论44第二局部中间型地中海贫血患者遗传背景与中医证候关系的研究47一、中间型地中海贫血患者基因型与中医证候的关系471 材料与方法472研究结果473讨论52二、中间型-地中海贫血患者中医证候的家系调查561材料与方法562研究结果563讨论59第三局部益髓生血颗粒治疗中间型地中海贫血临床研究62一、益髓生血颗粒治疗-HbH病抚慰剂平行对照研究621材料与方法622研究结果643讨论66二、益髓生血颗粒治疗中间型地中海贫血治疗前后自身对照研究681材料与方法682研究结果693讨论71临床研究小结73实验研究：益髓生血颗粒治疗中间型地中海贫血疗效分子机制研究75一、益髓生血颗粒对中间型地中海贫血患者外周血红细胞膜蛋白mRNA表达的影响751 材料与方法752 研究结果783 讨论82二、益髓生血颗粒对-HbH病患者外周血红细胞膜蛋白组分的影响861 材料与方法862 研究结果903 讨论92三、益髓生血颗粒对-HbH病患者外周血红细胞膜过氧化损伤的影响961材料与方法962研究结果983讨论99四、益髓生血颗粒对-HbH病患者外周血红细胞包涵体的影响1041 材料与方法1042 研究结果1053讨论107五、益髓生血颗粒对中间型地中海贫血患者骨髓幼稚红细胞DNA构造的影响1091材料与方法1092研究结果1103讨论111实验研究小结114结语116致121个人简历12311252135. z-中文摘要-地中海贫血属世界上发病率高、危害最大的单基因遗传病之一，至今尚无有效治疗方法，我国南方也是本病的高发区。导师吴志奎教授从七十年代开场一直致力于肾生髓理论现代研究，首次提出“中医肾生髓、髓生血是中医药治疗地中海贫血的理论核心这一假说，采用补肾益髓法的代表方益髓生血颗粒在*高发区治疗-地中海贫血取得了肯定的疗效，并从整体效应基因突变与疗效关系调控珠蛋白基因表达等不同层面深入研究了疗效的分子机制，验证了该理论假说的客观性，使中医药治疗地中海贫血整体水平到达了国际领先。本研究是在前期工作的根底上，以国家自然科学基金重大研究方案资助课题为依托开展的。我们对中间型地中海贫血患者的中医证候分布规律与特点、中医证候与遗传背景的关系等进展了较大样本的研究；并在益髓生血颗粒治疗-地中海贫血的根底上，拓展到治疗-HbH病的临床规研究；在该药取得显著临床疗效的根底上，针对地中海贫血患者红细胞寿命短、溶血性贫血的临床特点，采用透射电镜、激光扫描共聚焦显微镜图像分析、real-time PCR、SDS-PAGE、脂质过氧化物含量及抗氧化酶活力检测等技术，从红细胞膜骨架蛋白组成及表达、红细胞膜氧化损伤、红细胞包涵体、幼稚红细胞DNA荧光强度等方面，从红细胞构造与功能角度探讨了益髓生血颗粒治疗中间型地中海贫血方证效应的分子机制，取得了有重要学术价值的研究结果：1主要研究结论1.1 中间型地中海贫血患者中医证候分布规律研究通过对168例中间型地中海贫血患者中医证候分布规律的研究，初步明确了中间型地贫患者的根本临床表现为：自汗、口干咽燥、面色淡白或萎黄、爪甲色淡、倦怠乏力、潮热盗汗、易于感冒、心悸、舌质淡红、舌苔薄白、脉细数，其根本中医证型为精血亏虚、肝肾阴虚证。同时还发现，中间型地贫患者的中医证候积分与中医证型、血液参数RBC和Hb水平、年龄等存在一定的关系，中医证型的分布与切脾与否亦存在一定的关系。这一研究结果可以为地中海贫血的中医辨证治疗提供一定的参考依据。1.2 中间型地中海贫血患者遗传背景与中医证候关系的研究1通过对166例中间型地中海贫血患者基因突变型和中医证候的关系进展分析，结果说明：临床表型为0的中间型-地贫、带有非缺失型突变的-HbH病患者的中医证候临床表现较为严重，说明中间型地中海贫血患者中医证型的分布、中医证候积分的上下与基因突变型存在密切的关系。2通过对-地中海贫血73个家系共198人进展基因突变型检测与中医证候调查，结果说明：中间型-地贫患者子代其基因型或是与亲代中一人完全一样，或是为两个亲代的双重杂合，其中医证候临床表现都较亲代严重，子代血虚表现明显，而亲代则不明显。这种临床表现的差异可能与患者的基因突变型、遗传修饰成分等都有关系。中医证候与遗传背景关系的研究结果提示我们：中间型地中海贫血患者的中医证候一定程度上受基因突变型的影响，但同时还可能受一些遗传修饰成分的影响。该研究结果为地中海贫血的中医辩证治疗提供了科学依据。1.3 益髓生血颗粒治疗中间型地中海贫血临床研究1通过对59例表现为精血亏虚、肝肾阴虚证的-HbH病患者进展随机、抚慰剂平行对照研究，结果显示：-HbH病患者经益髓生血颗粒治疗后，疗效性血液指标 Hb、RBC均明显提高P0.05；益髓生血颗粒治疗组中医证候量化评分较抚慰剂组明显降低P0.01；益髓生血颗粒治疗组总有效率为77.8%，而抚慰剂组总有效率仅为15.6%，其疗效差异存在显著性意义P0.01；此外，患者服用益髓生血颗粒治疗期间未见任何副作用，且服用方便。平行对照研究结果说明，中药益髓生血颗粒治疗-HbH病用药平安，疗效确定，有很好的应用前景。2通过对68例表现为精血亏虚、肝肾阴虚证的中间型地中海贫血患者-地贫26例、-HbH病42例进展益髓生血颗粒治疗前后自身对照研究，结果显示：68例患者经益髓生血颗粒治疗后，疗效性血液指标Hb、RBC、Ret自第1个月起至3个月疗程完毕均明显升高P0.05，而中医证候积分与治疗前比则明显降低P0.01，患者血液参数的改善与临床表现的改善相一致；按照西医疗效评定标准，中药治疗的总有效率为77.9%，并且患者服药期间未见任何副作用。自身对照研究说明，中药益髓生血颗粒对不同类型的中间型地中海贫血均有较好的疗效。临床研究结果提示我们：益髓生血颗粒对中医证型表现为精血亏虚、肝肾阴虚证的，不同类型的地贫患者均具有较好的临床疗效，且患者用药平安，服用方便，表达了中医方证相应、异病同治的原则，验证了中医肾生髓、髓生血理论的客观性，显示出了中医药治疗疑难病的潜在优势。1.4 益髓生血颗粒治疗中间型地中海贫血疗效分子机制研究1通过对34例中间型地贫患者-地贫13例、-HbH病21例益髓生血颗粒治疗前、后红细胞膜蛋白的mRNA表达水平进展real-time PCR检测，发现：经中药治疗后，中间型地贫患者外周血红细胞膜-收缩蛋白SPTA1、-收缩蛋白SPTB、锚蛋白ANK1、带3蛋白Band3、带4.1蛋白EPB4.1的基因表达均根本呈现上调的趋势，经统计分析，其中-收缩蛋白、-收缩蛋白、带4.1蛋白的mRNA表达上调具有统计学意义均P0.05。提示中药益髓生血颗粒能够有效改善地中海贫血患者的溶血程度，可能也从改善红细胞膜骨架蛋白的mRNA表达这方面一定程度发挥了稳定红细胞膜骨架的作用。2通过对21例-HbH病患者益髓生血颗粒治疗前、后及12例正常人的红细胞膜蛋白组分进展SDS-PAGE检测，发现：-HbH病患者外周血红细胞膜蛋白条带中的带3、带4.1、带4.5+4.9、带6的灰度值比正常人要低P0.05，说明中药益髓生血颗粒能够改善地贫患者的溶血程度不是通过调节红细胞膜蛋白的组分，而是通过影响红细胞膜蛋白的基因表达来实现的。3通过对22例-HbH病患者益髓生血颗粒治疗前、后及22例正常人的红细胞膜脂质过氧化产物MDA含量以及红细胞的抗氧化酶活力进展检测，发现：-HbH病患者与正常人比较，外周血红细胞中总SOD及GSH-P*活力明显升高P0.05，其原因可能有二，一是由于地贫红细胞中氧自由基较多，使SOD及GSH-P*活力均代偿性升高，一定程度抑制了红细胞膜的脂质过氧化损伤；二是由于地贫红细胞寿命明显缩短，仅为正常红细胞寿命的1/4，患者外周血中分布的红细胞多为年轻红细胞，相应地膜过氧化损伤也稍轻。经中药益髓生血颗粒治疗后，-HbH病患者SOD活力较治疗前明显增加P0.01，而GSH-P*活力则无明显变化但较正常人仍明显升高P0.05。实验结果说明，益髓生血颗粒抗红细胞膜过氧化损伤，减轻红细胞溶血主要是通过明显提高-HbH病患者外周血红细胞中SOD活力到达的。4通过对6例-HbH病患者益髓生血颗粒治疗前、后外周血红细胞包涵体进展透射电镜观察，发现：-HbH病患者外周血红细胞中分布有大量由过剩的-珠蛋白链聚合而成的包涵体。经中药益髓生血颗粒治疗后，患者外周血红细胞中包涵体的数量与体积明显减少。研究说明益髓生血颗粒能明显减少-HbH病患者外周血红细胞中的包涵体，从而有效改善患者的溶血程度。5通过对4例中间型地中海贫血患者-地贫2例、-HbH病2例益髓生血颗粒治疗前、后及2例正常人的骨髓幼稚红细胞涂片进展激光扫描共聚焦观察，发现：中间型地中海贫血患者骨髓幼稚红细胞核DNA缺损，与正常人比存在明显的凋亡现象。经中药益髓生血颗粒治疗后，患者骨髓幼稚红细胞核DNA缺损程度明显减轻。说明益髓生血颗粒能够影响骨髓幼稚红细胞的形态与构造，具有保护骨髓、促进造血的作用。通过从红细胞构造与功能这一角度，探讨中药益髓生血颗粒治疗中间型地中海贫血的疗效分子机制，实验结果说明：益髓生血颗粒可通过促进红细胞膜蛋白的基因表达、对抗红细胞膜过氧化损伤、减轻红细胞包涵体变性珠蛋白链的沉积、增强骨髓幼稚红细胞核DNA的荧光强度等途径，有效地延长了地贫红细胞的寿命，减轻了地贫红细胞的溶血程度。该结果与临床研究结果相互印证，进一步验证了中医肾生髓、髓生血理论的客观性，并从分子水平提醒了其科学涵。2主要创新点本研究的创新之处有以下几点：1首次系统地对中间型地中海贫血患者的基因突变型及中医证候分布规律进展较大样本的调查，总结提出了中间型地中海贫血中医证候分布的一般规律和临床特点、中医证候与遗传背景的关系，为地中海贫血的中医辩证治疗提供了科学的依据。在国外地中海贫血的临床研究中未见同类报道。2对73个家系共198人进展子代、亲代基因突变型检测和中医证候调查研究，分析了遗传背景、先天禀赋与中医证候的关系，发现子代比亲代临床血虚表现严重，这种临床表现的差异与基因突变型、遗传修饰成分等有关。该研究属国外首次完成。3在高发区采用抚慰剂平行对照法研究益髓生血颗粒对精血亏虚、肝肾阴虚证的中间型-地贫的临床疗效，证实益髓生血颗粒不仅可以有效治疗-地贫，而且对证型相符的-地贫也有明显的疗效。该研究结果表达了中医方证相应、异病同治的原则，拓展了中医药治疗单基因遗传病的手段，在地中海贫血临床研究中未见同类报道。4从地贫患者红细胞膜骨架蛋白的表达、红细胞膜过氧化损伤、红细胞包涵体、骨髓幼稚红细胞DNA荧光强度等红细胞构造与功能的角度，探讨了中药益髓生血颗粒治疗中间型地中海贫血，促进有效红细胞生成，延长红细胞寿命，减轻红细胞溶血的分子机制，在地中海贫血疗效机制研究中未见同类报道。5本研究从中间型地中海贫血中医证候分布规律，遗传背景与中医证候的关系，应证方剂与不同类型疗效分析，以及证型特点的分子根底研究等方面拓展了中医药治疗地中海贫血的方法手段，从一个侧面提醒了中医药治疗地中海贫血的科学性及其理论的科学涵，使已领先的中医药治疗地中海贫血研究增加了新的亮点，研究为国际单基因遗传病临床治疗提供了新思路。关键词：肾生髓理论；中间型地中海贫血；益髓生血颗粒；中医证候；临床疗效；分子机制；家系ABSTRACTBeta-thalassemia is aninherited disorder of hemoglobin synthesis, which causes a high worldwide morbidity.Todaywe still having no effective means to cure it.In the south provinces of Chinathere are lots of thalassemic patients.My mentor professor ZhikuiWu,from the 70 years to now having engaged in the modern research of “shen sheng sui theory.He presented a hypothesis that “the shen sheng sui theory is the nuclear theory which directing the therapyof thalassemia with TCM methods.The hypothesis was provedto be objective by two ways,one is using YiSui Sheng*ue Granule to treat beta-thalassemia and obtaining positive curative effect,the other is studying the molecular mechanism of curative effect from three different layers: the whole responses,the relation between genotype and curative effect, the gene e*pression and regulation.This research was carried out on the base of the earlier studies,and wassupported by NSFC.We researched the distributingcharacter and regularityof TCM syndromes,the relation of TCM syndromes to genetic backgroundsin Patients with thalassemia intermedia,and developed the TCM methods to treating -HbH disease(another thalassemic type).Base on the marked curative effect,we e*plored the molecular mechanism from the point of red cellsfunction and structure,such as the membrane protein ponents and mRNA e*pression,the membrane o*idative damage,the inclusionbodies, and the intensity of DNAfluorescence inerythroid precursors,by using the techniques of transmission electron microscopy,laser confocal microscopy,real-time PCR,SDS-PAGE,MDA contents and antio*idative enzyme activities detections.We have obtained high academic valuable data.1 The primary research conclusions1.1 Study on distributiveregularity of TCM syndromes in patients with thalassemia intermediaBy e*ploring the distributive regularity of TCM syndromes in 168 patients with thalassemia intermedia,we find that:The basic clinical manifestations in patients with thalassemia intermedia are spontaneous perspiration,dry mouth and tongue,pale or sallow ple*ion,pale fingernails,lassitude,tidal fever and night sweat,frequent attack of cold, palpitation,light red tongue body,thin or white tongue coating,thready or rapid pulse;The main syndromic type of thalassemia intermedia is deficiency of essence, blood, the kidney-yin and the liver-yin;Furthermore,somecorrelation factors such as age,splenectomized or nonsplenectomized,the levels of RBC and Hb canaffecton the distribution of TCM syndromes. 1.2 Study on relation between TCM syndromes and genetic backgrounds in Patients with thalassemia intermedia1By analyzing the relation between TCM syndromes and genetic backgrounds in 166 Patients with thalassemia intermedia,we find that ifthephenotype of patientswith beta-thalassemia intermedia is 0or with alpha-thalassemia intermedia is non-deletional mutations,they will have more severe symptoms.Thisindicatesthat the distribution of TCM syndromic-type and the syndromic-scores have close relation to genotype.2By detecting the genotype of beta-thalassemia in 73 families a total of 198 peoples,investigatingtheir TCM Syndromes,we find that:The offspringsgenotype is the same as one of their parents or is the double heterozygous of their parents,their TCM syndromes (Clinical manifestations) are more severe than their parents;The offsprings have evident deficiency of blood,but their parents are not.The difference of clinical manifestations may be related to the genic mutations and other genetic modified ingredients.The resultsof study on relation between TCM syndromes and genetic backgrounds in patients with thalassemia intermedia indicate that the TCM syndromes of thalassemia intermedia patients are influenced by genic mutations and other genetic modified ingredients.This study providesa scientific basis to TCM dialectical therapy with thalassemia.1.3 Clinical research on treating thalassemia intermedia with YiSui Sheng*ue Granule1By using randomized placebo parallel-controlled trial to treat 59 HbH disease patients with YiSui Sheng*ue Granule,we find that: pared to that of before treatment, after three months treated, the blood inde*es (Hb、RBC) and the TCM symptoms of patients in the YiSui Sheng*ue Granule group are statistically different (P0.05);The total effective rate in the YiSui Sheng*ue Granule group being 77.8%, in the placebo parallel-control group only being 15.6%,the two groups have significant different (P0.01); In the treatment period no adverse effects are monitored.These results indicate that: YiSui Sheng*ue Granule is effective and safe in treating HbH disease.2By using self-controlled trial to observe the clinical effect of YiSui Sheng*ue Granule in treating 68 patients with thalassemia intermidia (26bete-thalassemia,42HbH disease),we find that:The blood inde*es Hb, RBC, Ret are obviously elevated after treated with YiSui Sheng*ue Granule from the lst to 3rd month (all p0.05);The TCM syndromes ofpatients are statistically reduced (P0.01);The improvement of blood inde*es arein accordance with the improvement of TCM syndromes;The total effective rate being77.9%; In the treatment period no adverse effects are monitored.The results indicate that:YiSui Sheng*ue Granule is effective and safe in treating different types of thalassemia.The clinical research shows that: YiSui Sheng*ue Granule is effective and safe in treating different types of thalassemia when the patients TCM syndromic-type is deficiency of essence,blood,the kidney-yin and the liver-yin.Which shows the potential advantages of TCM in treating difficult diseases. 1.4Study onthe molecular mechanism of YiSui Sheng*ue Granule in treating thalassemia intermedia1By real-time PCR,we detected the mRNA e*pression of red cells membrane protein in34patients withthalassemia intermedia before and after the treatment with YiSui Sheng*ue Granule.Wediscoverthat:After treated with YiSui Sheng*ue Granule,the -spectrin,-spectrin,ankyrin,band 3,band 4.1 have the increased trend of gene e*pression,especially the -spectrin,-spectrin,band 4.1has a statistically high e*pressionall P0.05.This indicates that YiSui Sheng*ue Granule maybe by improving the gene e*pression to stabilize the membrane skeleton and reduce the degree of hemolysis.2By SDS-PAGE, we e*amined the ponents of red blood cell membrane protein in 21 patients of HbH disease before and after the treatment with YiSui Sheng*ue Granuleand 12 normal controls.We find that:The volume ofband3,band 4.1,band4.5+4.9,band6 in patients with HbH disease are lower than that in normal controlsP0.05.This indicates to us YiSui Sheng*ue Granulereduce the degree of hemolysis maybe by affecting the gene e*pression , not by the protein e*pression.3By detecting the MDA contents and antio*idative enzyme activities in 22 patients of HbH disease before and after the treatment with YiSui Sheng*ue Granule and 22 normal controls,We find that:The activities of SOD and GSH-P* in patients with HbH disease are more higher than thatin normal controlsP0.05.This indicates that there are pensatory mechanisms in HbH erythrocytes to cope with increased generation of o*ygen free radicals.After treated withYiSui Sheng*ue Granule,the activities of SOD have an apparent increaseP0.05.This result indicates that YiSui Sheng*ue Granule can prevent the red cells from o*idative damage mainly by increasing the activities of SOD.4By transmission electron microscopy,we observed the red cells inclusion bodies in 6 patients of HbH disease before and after the treatment with YiSui Sheng*ue Granule.We discover that:In the red cells there are plenty of inclusion bodies,which are forming by the the unmatched beta-globin chain.After treated with YiSui Sheng*ue Granule,the amount and volume of inclusion bodies have an apparent decrease.This indicates that: YiSui Sheng*ue Granule can reduce the red cells inclusion bodies,improve the intra medullary hemolysis and RBC destruction in the peripheral blood.5By laser confocal microscopy,we observed the intensity of DNA fluorescence in erythroid precursors in 4 patients of thalassemia intermedia before and after the treatment with YiSui Sheng*ue Granule and 2 normal controls.We find that:The intensity of DNA fluorescencein thalassemic erythroid precursors are weaker than that in nomal controls.After treated with YiSui Sheng*ue Granule,the intensity of DNA fluorescence in thalassemic erythroid precursors bees stronger,being similar to that in normal controls.This indicates that:YiSui Sheng*ue Granule can protect the shape and structure of thalassemic erythroid precursors,promote hemopoiesis.From the point of red cellsfunction and structure to e*plore the molecular mechanism of YiSui Sheng*ue Granule in treating patients with thalassemia intermedia,the results indicate that:YiSui Sheng*ue Granule can improve the gene e*pressionof membrane skeleton proteins,increase the activities of SOD,reduce the inclusion bodies of red cells,increase the intensity of DNA fluorescence in thalassemic erythroid precursors.These results are in accordance with the clinical research,prove the TCM theory “shen sheng sui, sui sheng *ue to be objective, and reveal its scientific content from the molecular level.Key Words:Theoryof ShenSheng Sui;Thalassemia Intermedia;YiSuiSheng*ue Granule;TCM Syndromes;Clinical Curative Effect;Molecular Mechanism;Genealogy英文缩略语英文缩写英文全称中文全称AcrAcrylamide丙烯酰胺-HbHAlpha Hemoglobin H Disease-珠蛋白生成障碍性贫血血红蛋白H病ANK1Ankyrin 1锚蛋白AHSPAlpha Hemoglobin Stabilizing Protein-血红蛋白稳定蛋白APS Ammonium Persulfate过硫酸铵BisN,N-Methylene Bis-Acrylamide甲叉双丙烯酰胺CatCatalase过氧化氢酶cDNAplementary DNA互补DNABFU-EBurst Forming Unit -Erythroid红系爆式集落形成单位CFU-EColonyForming Unit-Erythroid红系集落形成单位CRFCase Report Form临床病例报告表CSFColony-Stimulating Factor集落刺激因子CtThreshold Cycle循环阈值ddH2ODistilled Deionized Water蒸馏去离子水DEPC Diethylpyrocarbonate焦碳酸二乙酯DNADeo*yribonucleic Acid脱氧核糖核酸DTNB5,5-dithiobis-nitrobenzoic acid二硫代二硝基苯甲酸EPOErythropoietin促红细胞生成素FEPFree Erythrocyte Protoporphyrin红细胞游离原卟啉GATA-1GATA-1 transcription factor红系特异转录因子-1GATA-2GATA-2 transcription factor红系特异转录因子-2GMGranulocyte Monocyte 粒细胞-单核细胞GMCFUGranulocyte Monocyte-Colony Forming Unit粒细胞-单核细胞集落形成单位GSHGlutathione谷胱甘肽GSH-P*Glutathione Pero*idase谷胱甘肽过氧化物酶HbHemoglobin血红蛋白HbAHemoglobin A血红蛋白AHbA2Hemoglobin A2血红蛋白A2HbF Fetal Hemoglobin胎儿血红蛋白HbHHemoglobin H血红蛋白HHClHydrochloric acid盐酸HCTHematocrit红细胞压积HPFHHereditary Persistence of Fetal Hemoglobin遗传性持续胎儿血红蛋白综合症H2OWater水H2O2Hydrogen Pero*ide过氧化氢HS2Hypersensitive Sites 2高敏位点2MDAMalonyldialdehyde丙二醛mRNAMessenger RNA信使RNANCBINational Center for Biotechnology Information美国国立生物技术信息中心NF-E2Nuclear Factor-Erythroid 2红细胞核因子2O2O*ygen氧O2-Supero*ide Anion Radical超氧阴离子自由基ODAbsorbance吸光度PAGE Polyacrylamide Gel Electrophoresis聚丙烯酰胺凝胶电泳PCRPolymerase Chain Reaction聚合酶链式反响PCPhosphatidylcholine磷脂酰胆碱PEPhosphatidylethanolamine磷脂酰乙醇胺PSPhosphatidylserine磷脂酰丝氨酸PLTPlatelet Count血小板计数RBCRed Blood Cell Count红细胞计数real-time PCR Real-Time Polymerase Chain Reaction实时荧光定量聚合酶链反响RetReticulocyte网织红细胞RNARibonucleic Acid核糖核酸SCFStem Cell Factor干细胞因子SDSSodium Dodecyl Sulphate十二烷基磺酸钠SFSerum Ferritin血清铁蛋白SODSupero*ide Dismutase超氧化物歧化酶SPTA1-spectrin-收缩蛋白SPTB-spectrin-收缩蛋白SMSphingomyelin鞘磷脂TBAThibabituric Acid硫代巴比妥酸TEMEDTetramethylethylenediamineN,N,N,N-四甲基乙二胺文献综述一、中医五脏生血理论及其临床应用与研究血是循行于脉中而富有营养的红色液态物质，是构成人体和维持人体生命活动的根本物质之一1。血液运行于人体的脉道之中，循环不已，流布全身，为全身各脏腑组织器官提供了丰富的营养，因此血对人生命的维持、生理机能的发挥都起到了重要的作用。查阅中医历代文献，古人均从不同角度直接或间接地阐述了五脏在血液化生中的作用，经后世医家不断补充，逐渐形成了中医五脏生血理论。1 五脏生血理论涵1.1脾胃为气血生化之源水谷精微是化生血液的根本物质之一，包括营气和津液。营气化血，?灵枢邪客篇?2说“营气者，泌其津液，注之于脉，化以为血；?金匮要略心典?3也说“营者血之源。津液化血，?读书随笔?4中说“津亦水谷所化，其浊者为血，清者为津。营气和津液都是脾胃将摄入的饮食物进展运化转输而生成的，因此脾胃的运化功能在血液生成的过程中发挥着重要的作用，被称为“气血生化之源。?灵枢.决气?2 云“中焦受气取汁，变化而赤，是谓血。此处“中焦指脾胃，“受气就是脾胃受纳运化饮食物，“取汁就是吸收其中的精微物质，“变化而赤就是精微物质进入脉中化生为血液。?景岳全书?5云“血者，水谷之精气也，源源而来，而实生化于脾。亦指出了脾胃在化生血液中的重要作用。脾胃通过运化功能，为血液的生成不断地提供着物质原料，因此脾胃运化功能的强弱直接影响着血液的化生。假设脾胃运化功能长期失调，则可导致血液生成缺乏而形成血虚。1.2 肺之气化蒸变与血的化生肺主一身之气，参与宗气的生成。脾胃运化的水谷精微要转输到肺，与肺吸入的清气相结合，经肺气的化合，贯注于脉中，才能变化成为红色的血液。?医家秘奥?6中提到“中气上升于肺而为气，从肺回下则化为血。指出了肺在化生血液中的重要作用。?灵枢.营卫生会?2中说“中焦亦并胃中，出上焦之后，此所受气者，泌糟粕，蒸津液，化其精微，上注于肺脉，乃化而为血，即是说中焦水谷精气的化生，起源于脾胃之腐熟运化功能，而所受纳的水谷，必须经过“泌别糟粕和“蒸发津液的消化吸收过程，方能转化成水谷精微，且向上输注于肺脉，经肺的气化、蒸变、升降，将精微与津液合而成为血液。1.3心阳化赤以为血脾胃运化的水谷精微与肺吸入的清气结合后，还要贯注于心脉，在心气的作用下变化而成为红色的血液。?血证论.阴阳水火气血论?7 云“食气入胃，脾经化汁，上奉心火。火得之，变化而赤，是之为血。清.志聪?侣山堂类辨.辨血?8云“血乃中焦之汁，流溢于中以为精，奉心化赤而为血。?医碥.血?9提到“血色独红者，血为心火之化。?血证论.阴阳水火气血论?7指出“血色者，火赤之色也。火者，心之所主，化生血液，以濡周身，火为阳而生血之阴。均说明心脏的生理功能参与血液的生成。1.4肾精骨髓可化血?病机沙篆?10中说“血液之源在于肾。?侣山堂类辨?8亦指出“肾为水脏，主藏精而化血，说明血液的生成与肾密切相关，肾所藏之精是化生血液的重要物质。肾的化血功能具体表现为肾精化血、骨髓化血和肾阳的推动温煦作用。?景岳全书?5中说“血即精之属也，但精藏于肾，所蕴不多，而血富于冲，所至皆是。?类经.脏象类?11云“精足则血足。均说明肾藏精，精能生血。?氏医通.诸血门.诸见血证?12云“经言血之与气，异名同类，虽有阴阳清浊之分，总由水谷精微所化，其始也混然一区，未分清浊，得脾气之鼓运，如雾上蒸于肺而为气；气不耗，归精于肾而为精；精不泄，归精于肝而化清血；血不泄，归精于心，得离火之化而为真血，指出了精气血的密切关系。故肾中精气充盈，则血液化生有源。假设肾精缺乏，化血乏源则往往导致血液生成缺乏。唐宗海在?中西汇通医经精义?13中指出“盖髓者,肾精所生,精足则髓足。?素问.生气通天论?云“肾主骨，骨者髓之府。?素问.生气通天论?2 云“骨髓稳固，气血皆从。说明肾藏精、主骨，精能生髓，髓充养于骨，而骨髓又可化生为血液。此外，肾阳具有推动、激发各脏腑生理功能的作用。脾阳根于肾阳，通过肾阳的温煦、推动可促进脾胃化生水谷精微，进而奉心化赤为血。如?读医随笔?4所说“夫血者，水谷之精微