【作者】 张静；

【导师】 陈泽涛；

【作者基本信息】 山东中医药大学 ， 中西医结合临床（专业学位）， 2019， 博士

【摘要】 目的本研究应用多种统计学方法对众医家以当归补血汤为主治疗老年非重型再障的病例从证型、症状、黄芪-当归用药剂量、用药频率、用药聚类、辨证用药、对症用药进行分析,以系统总结当归补血汤为主治疗老年非重型再障的用药经验。并从线粒体自噬的角度探讨当归补血汤对非重型再障模型小鼠的骨髓造血保护作用机制,为非重型再障的治疗提供新的思路和方法。方法1.临床研究:将搜集到的109例以当归补血汤为主治疗的老年非重型再障病例输入中医验案分析系统,利用中医验案分析系统、SPSS及SAS统计软件对其进行统计分析,分析内容包括:（1）当归补血汤中黄芪、当归的剂量统计以及加药的药物、药味、药类、归经的频数统计及聚类分析;（2）证型、证素统计及主要证型、证素与当归补血汤加药的关联性分析;（3）主症统计及主症与当归补血汤加药的关联性分析。2.实验研究:选取健康雄性ICR小鼠105只,随机分为正常组、模型组、低剂量组、中剂量组、高剂量组,每组各21只。除正常组外,其余各组小鼠以环磷酰胺和苯作用30天制备非重型再障小鼠模型。验证成模后每组当即（d30）处死6只小鼠用于检测。剩余小鼠给予药物,其中低剂量组、中剂量组、高剂量组分别予以2.7g·kg^-1·d^-1、5.4 g·kg^-1·d^-1、10.8g·kg^-1·d^-1当归补血汤灌胃,正常组、模型组以等体积(0.11ml·10g^-1·d^-1)生理盐水灌胃,各组连续用药14天,次日（d45）定为取材时间。观察不同剂量当归补血汤对非重型再障小鼠一般症状、外周血白细胞、血红蛋白、血小板和单侧股骨骨髓单个核细胞数量的影响;光学显微镜下观察其对非重型再障小鼠骨髓病理的影响,估算造血组织增生度;透射电镜下观察其对非重型再障小鼠骨髓单个核细胞线粒体自噬泡数量的影响;Western Blot法检测自噬相关蛋白LC3-II、p62及线粒体自噬相关蛋白parkin的表达。结果1.对以当归补血汤为主治疗的109例老年病例的临床研究结果示:（1）黄芪的用量平均值（30.48±13.40）g,众数30g;当归的用量平均值（12.33±4.68）g,众数15g;黄芪:当归的平均值为（2.77±1.60）:1,众数3:1。（2）以当归补血汤为主治疗的109例老年病例中共用中药207种,其中温性药、甘味药、脾经药、补虚（补气、补血、补阳、补阴）药最多。（3）除黄芪、当归外用药频次>10次的加药有40种,运用varclus聚类分析可将其分为12大类,其分类符合临床实际。（4）证型证素统计得出中医证型30种、证素12种。结合临床实际,对肾阳虚证、肾阴阳两虚证、肾阴虚证、气血两虚证此四种证型及精亏、火毒、血瘀此三种证素与40种高频加药进行logistic回归分析,得出各自的logistic回归方程符合临床实际。（5）主症统计得出除舌苔脉象外共有54种症状,分别对肌衄、纳差、腰膝酸痛、形寒怕冷、水肿、便溏、眠差、发热这8种主症与40种高频加药进行logistic回归分析,得出各自的logistic回归方程符合临床实际。（6）舌象以淡舌最常见,脉象以弱脉、弦细脉、沉细脉最常见。2.实验研究结果（1）低、中、高剂量当归补血汤均能改善非重型再障小鼠的一般症状。（2）低、中、高剂量当归补血汤均能不同程度的提高非重型再障小鼠外周血白细胞、血红蛋白、血小板及骨髓单个核细胞数量（p均<0.01）,低剂量起效,高剂量效果最佳。（3）非重型再障模型组小鼠骨髓造血组织结构改变,造血组织增生度较正常组显著减少（p<0.01）,当归补血汤低、中、高剂量组小鼠骨髓造血组织结构较模型组均有不同程度的恢复,造血组织增生度依次增加（与模型组比较p均<0.01,低、中、高组组间两两比较p均<0.01）。（4）模型组细胞中线粒体自噬泡较正常组明显减少（p<0.01）。用药后低、中、高剂量组线粒体自噬泡较模型组依次增加,且与模型组比较差异均具有统计学意义（p均<0.05）;低、中、高剂量组组间两两比较,差异有统计学意义（p<0.05）。（5）经western blot检测发现,与正常组比较,模型组小鼠骨髓单个核细胞中LC3-II蛋白表达显著下降、p62蛋白表达显著增加（p均<0.01）;与模型组相比,中、高组当归补血汤均可上调LC3-II蛋白表达,下调p62蛋白表达（p<0.05）;低剂量组较模型组差异不明显（p>0.05）;中、高剂量组组间差异不明显（p>0.05）。（6）经western blot检测发现,与正常组比较,模型组小鼠骨髓单个核细胞中parkin蛋白表达显著下降（p<0.01）;低、中、高组当归补血汤均可显著上调parkin蛋白表达（p<0.05）;且作用随剂量增加依次增强（组间比较p均<0.05）。结论（1）本研究运用频数统计、varclus聚类分析、logistic回归分析等数据挖掘方法对当归补血汤为主治疗老年非重型再障的病例进行了客观系统的分析,从证型、症状、黄芪-当归用药剂量、用药频率、用药聚类、辨证用药、对症用药等方面进行了系统总结,可用以指导临床。（2）非重型非重型再生障碍性贫血致病的理论机制之一在于存在线粒体自噬缺陷,这种线粒体自噬稳态失衡可能是parkin介导的。当归补血汤可以通过调节parkin介导的线粒体自噬对非重型再障模型小鼠的骨髓造血功能进行调节恢复,且作用与剂量成正相关。更多还原

【Abstract】 ObjectiveIn this study,a variety of statistical methods were used to analyze the cases of senile non-severe aplastic anemia treated mainly by Danggui Buxue decoction from the aspects of syndrome type,symptom,dosage of astragalus membranaceus and Angelica sinensis,drug use frequency,drug clustering,syndrome differentiation and symptomatic drug use.To systematically summarize the experience of Danggui Buxue decoction in the treatment of senile non-severe aplastic anemia.From the point of view of mitochondrial autophagy,the protective mechanism of Danggui Buxue decoction on bone marrow hematopoiesis in mice with non-severe aplastic anemia was discussed,which provided a new idea and method for the treatment of non-severe aplastic anemia.Method1.Clinical study:109 cases of senile non-severe aplastic anemia treated mainly by Danggui Buxue decoction were input into the traditional Chinese medicine case analysis system,and the statistical analysis was carried out by using the traditional Chinese medicine case analysis system,SPSS and SAS statistical software.The analysis contents include:（1）the dose statistics of Astragalus membranaceus and Angelica sinensis in Danggui Buxue decoction,as well as the frequency statistics and cluster analysis of drugs,taste,drugs and meridians;（2）syndrome type,syndrome element statistics and main syndrome types,syndrome elements and Angelica sinensis tonifying blood.The correlation analysis of decoction;（3）the statistics of the main symptoms and the correlation analysis between the main symptoms and the addition of Danggui Buxue decoction.2.Experimental study:105 healthy male ICR mice were randomly divided into normal group,model group,low dose group,middle dose group and high dose group,with 21 mice in each group.In addition to the normal group,the other groups of mice were treated with cyclophosphamide and benzene for 30days to establish non-severe aplastic anemia mouse model.Six mice in each group were killed immediately（D30）after verification of the model for detection.The remaining mice were treated with Danggui Buxue decoction(2.7g·kg^-1·d^-1,5.4g·kg^-1·d^-1,10.8g·kg^-1·d^-1)in the low dose group,middle dose group and high dose group,respectively,and the mice in the low dose group,middle dose group and high dose group were treated with Danggui Buxue decoction（Danggui Buxue decoction）.Normal group and model group were given equal volume 0.11ml·10g^-1·d^-1normal saline intragastrically,and each group was treated continuously for 14 days.The next day（D45）was set as the sampling time.To observe the effects of different doses of Danggui Buxue decoction on general symptoms,peripheral blood leukocytes,hemoglobin,platelets and the number of bone marrow mononuclear cells in unilateral femur of non-severe aplastic anemia mice,and to observe the effect of Danggui Buxue decoction on bone marrow pathology of non-severe aplastic anemia mice under optical microscope.The effect of hematopoietic tissue proliferation on the number of mitochondrial autophagy vesicles in bone marrow mononuclear cells of non-severe aplastic anemia mice was observed under transmission electron microscope,and the expression of autophagy associated proteins LC3-II,p62and mitochondrial autophagy associated protein parkin was detected by;Western Blot method.Result1.The results of clinical study on 109 elderly cases treated mainly with Danggui Buxue decoction showed that:The main results were as follows:（1）the average dosage of Astragalus membranaceus was（30.48±13.40g）g,the number of Angelica sinensis was 30g,the average dosage of Angelica sinensis was（12.33±4.68）g,the average number of Angelica sinensis was 15g,the average value of Astragalus membranaceus:Angelica sinensis was（2.771.60）:1,the number of Angelica sinensis was 3:1.（2）A total of 207 kinds of traditional Chinese medicine were used in 109cases of senile patients treated mainly with Danggui Buxue decoction,among which warm medicine,sweet medicine,spleen meridian medicine and deficiency tonifying（invigorating qi,blood,yang and yin）were the most.（3）there were 40 kinds of drugs used more than 10 times except Astragalus membranaceus and Angelica sinensis,which could be divided into 12 categories by varclus cluster analysis,and their classification accorded with clinical practice.（4）according to the statistics of syndrome elements,30 kinds of TCM syndromes and 12 kinds of syndrome elements were obtained.Combined with clinical practice,logistic regression analysis was carried out between kidney yang deficiency syndrome,kidney yin deficiency syndrome,qi and blood deficiency syndrome,essence deficiency,fire toxin and blood stasis syndrome and 40 kinds of high frequency drugs added.It is concluded that the respective logistic regression equations are in line with the clinical practice.（5）according to the statistics of the main symptoms,there were 54 kinds of symptoms except tongue coating pulse.logistic regression analysis was carried out between the eight main symptoms of muscle bleeding,anorexia,soreness of waist and knee,cold and cold,edema,loose stool,sleeplessness and fever,and 40 kinds of high frequency drugs.It is concluded that the respective logistic regression equations are in line with the clinical practice.（6）the light tongue is the most common tongue,and the weak pulse,string pulse and heavy pulse are the most common pulse images.2.Experimental results.The main results were as follows:（1）low,middle and high doses of Danggui Buxue decoction could improve the general symptoms of non-severe aplastic anemia mice.（2）low,middle and high doses of Danggui Buxue decoction could increase the number of white blood cells,hemoglobin,platelets and bone marrow mononuclear cells in peripheral blood of non-severe aplastic anemia mice to varying degrees（p<0.01）.（3）the hematopoietic tissue structure of bone marrow in the non-severe aplastic anemia model group was significantly lower than that in the normal group（p<0.01）,and the degree of hematopoietic tissue proliferation in the non-severe aplastic anemia model group was significantly lower than that in the normal group（p<0.01）.The hematopoietic tissue structure of bone marrow in the low,middle and high dose groups of Danggui Buxue decoction was recovered to some extent compared with the model group.The degree of hematopoietic tissue proliferation increased in turn p<0.01 compared with the model group,and p<0.01 between the low,middle and high groups).（4）the number of mitochondrial autophagy vesicles in the model group was significantly lower than that in the normal group（p<0.01）.Compared with the model group,the mitochondrial autophagy vesicles in the low,middle and high dose groups increased in turn,and the difference was statistically significant（p<0.05）.There was significant difference among the low,middle and high dose groups（p<0.05）.（5）compared with the normal group,the expression of LC3-II protein in bone marrow mononuclear cells of the model group was significantly decreased and the expression of p62 protein was significantly increased by western blot（p<0.05）,and the expression of p62 protein in bone marrow mononuclear cells of the model group was significantly higher than that of the normal group（p<0.01）.Compared with the model group,Danggui Buxue decoction could up-regulate the expression of LC3-II protein in both middle and high groups.The expression of p62 protein was down-regulated（p<0.05）,there was no significant difference between the low dose group and the model group（p>0.05）,but there was no significant difference between the middle and high dose groups（p>0.05）.（6）compared with the normal group,western blot showed that,The expression of parkin protein in bone marrow mononuclear cells of model group was significantly decreased（p<0.01）,and the expression of parkin protein was significantly up-regulated in low,middle and high groups of Danggui Buxue decoction（p<0.05）,and the expression of Danggui Buxue decoction was significantly up-regulated in low,middle and high groups.The effect increased with the increase of dose（p<0.05）.ConclusionThe main results are as follows:（1）in this study,frequency statistics,varclus cluster analysis,logistic regression analysis and other data mining methods were used to analyze the cases of senile non-severe aplastic anemia treated mainly by Danggui Buxue decoction.This paper makes a systematic summary from the aspects of syndrome type,symptom,dosage,frequency,clustering,syndrome differentiation and symptomatic medication of Astragalus membranaceus and Angelica sinensis,which can be used to guide clinical practice.（2）one of the theoretical mechanisms of non-severe aplastic anemia lies in the existence of mitochondrial autophagy deficiency,which may be mediated by parkin.Danggui Buxue decoction can regulate and restore the bone marrow hematopoietic function of non-severe aplastic anemia model mice by regulating parkin-mediated mitochondrial autophagy,and the effect is positively correlated with the dose.更多还原