益气养阴方及其拆方治疗急性白血病的临床依据及诱导KG1a细胞凋亡的实验研究
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摘要
目的：基于数据挖掘技术研究急性白血病的中医常见证候及用药规律，为其辨证论治提供循证数据支持。并以KG1a/NOD-SCID小鼠为研究平台，探讨益气养阴方及其拆方诱导急性白血病KG1a细胞凋亡的作用机制。
方法：检索近30年国内关于急性白血病中医药辨证及治疗的文献，从证候和药物使用角度出发，运用Excel软件建立数据库，应用频数分析，得到常见证候、证候要素、高频药物、药类及药性；聚类分析得到常见证候的四诊信息；通过关联分析，探讨药物的配伍规律。
以NOD-SCID小鼠为实验对象，采用60Co照射，尾静脉注射KG1a细胞，复制急性白血病小鼠模型。将造模后小鼠随机分为空白对照组、扶正组、祛邪组、全方组，应用流式细胞术检测KG1a/NOD-SCID小鼠淋巴结细胞凋亡率、细胞周期分布；采用免疫组化法检测其淋巴结组织中c-myc、survivin、p21蛋白的表达。采用PCR-ELISA法检测小鼠骨髓细胞端粒酶活性，荧光定量PCR法检测小鼠骨髓细胞hTERT-mRNA及其启动子甲基化的表达。
结果：
1、急性白血病的常见证候是：气阴两虚证、热毒炽盛证、气血两虚证、痰热瘀结证；常见证候要素以热、气虚、阴虚为主；常见证候要素靶位是肾、肝和脾。
2、急性白血病常用药物药类有补益药、清热药、活血化瘀药、利水渗湿药和化痰药，其中补虚药、清热药的地位最为重要。常用药物药味多具有甘味及苦味；药性以寒性为主，其次为温性和平性；归经以归肝经的药物最多，其次是归肺、脾、肾经的药物。
3、药物干预后，各用药组小鼠生存时间较模型组延长（P<0.05），细胞凋亡率、G1期停滞细胞百分比较对照组升高（P<0.05），淋巴结中c-myc及survivin蛋白的表达较对照组降低（P<0.05），p21蛋白的表达较对照组升高（P<0.05），骨髓细胞端粒酶活性、hTERT-mRNA及其启动子甲基化的表达较对照组降低（P<0.05）。其中全方组对于各指标的影响均优于扶正组与祛邪组（P<0.05）。
结论：通过应用数据挖掘对现代文献中急性白血病的常见证候及用药规律进行分析，认为本病属本虚标实，本虚以气阴两虚、气血亏虚为主，标实以热毒、瘀血、痰湿为主。治疗上应扶正与祛邪兼顾，治法以补气养阴、清热解毒为主，佐以活血化瘀、化痰祛湿等。
实验研究证明，益气养阴方通过调节细胞周期，降低hTERT-mRNA及其启动子甲基化的表达，下调端粒酶活性，诱导白血病细胞凋亡，抑制白血病细胞增殖，从而延长KG1a/NOD-SCID小鼠生存期，对急性白血病具有治疗作用。
Objective Study on the traditional Chinese medicine syndrome and herbaladministration regularity of acute leukemia based on Data Mining, in order to offer theevidence-based data supporting to guide the TCM differential treatment of diabetic arterialobliterations. KG1a/NOD-SCID mice was used as the study platform,to explore themechanism of leukemia cell apoptosis induced by Tonicing Qi and Nurishing YinDecoction and its decomposed recipes.
Methods The research literature were collected from periodical database withinrecent30years, focus on Chinese medicine syndrome and the medicine that had been used.Database was established with Excel software.To find the common syndrome, influencefactors,the commonly used medicines and medicine category by using frequency analysis.To obtaine information from the TCM four diagnostic methods by using cluster analysis.Toexploer Compatibility Law of herb by using association analysis.
Taking the NOD-SCID mice as the experimental object,and injected with KG1a cellsfrom the tail vein after Irradiation by60Co.Model mices were randomly divided into modelgroup,Fuzheng group,Quxie group,Quanfang group.flow cytometry was applied tomeasure the cell cycle and apoptosis.The immunochemistry method was used to detect theexpression of c-myc,survivin,p21in mices’ lymph node tissue.The PCR-ELISA wasapplied to measure the Telomerase Activity of bone marrow cells.The expression ofhTERT mRNA and methylation of its promoter were measured using RT-PCR.
Results
1. The common syndrome of acute leukemia are deficiency of both qi and yin, heattoxin syndrome, deficiency of both qi and blood, and junction of sputum,thermal andstasis.Influence factors include thermal,qi deficiency and yin deficiency.The target sites ofinfluence factors are kidney,liver and spleen.
2. The commonly used medicine categorise are tonifying herbs,heat-clearingherbs,blood-activating and stasis-resolving herbs, removing dampness and promotingdiuresis herbs,apophlegmatisant herbs.Tonifying herbs and heat-clearing herbs occupy animportant position.The most common Flavors of Drugs are Sweet and bitter.Cold drug inall drug occupies the main position.Secondly it is warm drug,and the next is ping drug.Thechannel-tropism of medicine is mostly liver,secondly lung,spleen and kidney.
3. After treatment,survival time of all treatment groups were longer than those inmodel group（P<0.05）. Apoptosis rate and the proportion of cells in the G1phase in alltreatment groups were increased（P<0.05）. The expression of c-myc、survivin in mices’lymph node tissues as well as hTERT mRNA and methylation of its promoter,and theTelomerase Activity of bone marrow cells in all treatment groups were significantlydeclined compared with model group（P<0.05）.Howere, the expression of p21in mices’lymph node tissues in all treatment groups was higher than model group（P<0.05）.TheEffect of Quanfang group on the all Indices was superior to Fuzheng group and Quxiegroup（P<0.05）.
Conclusion Through studing on the traditional Chinese medicine syndrome andherbal administration Rregularity of acute leukemia based on Data Mining, we obtained acorollary that the basic pathogenesis of acute leukemia was root deficiency and branchexcess.Root deficiency were deficiency of both qi and yin, deficiency of both qi andblood.Branch excess were heat toxin,sputum and stasis.On the treatment,not only ispel evilbut assist the justice.Supplementing qi and nourishing yin, heat-clearing and detoxifyingare the main treatment methods,and combined with water dampness, promoting bloodcirculation and removing blood stasis methods.Experimental studies proved that Tonicing Qi and Nurishing Yin Decoction inducedleukemia cell apoptosis,regulatid cell cycles,declined the expression of hTERT mRNA andmethylation of its promoter,effected the telomerase activity, Induce apoptosis,inhibit theproliferation of leukemia cells,prolonged the survival time of KG1a/NOD-SCID mices.So,Tonicing Qi and Nurishing Yin Decoction has significant therapeutic action on acuteleukemia.
方法：检索近30年国内关于急性白血病中医药辨证及治疗的文献，从证候和药物使用角度出发，运用Excel软件建立数据库，应用频数分析，得到常见证候、证候要素、高频药物、药类及药性；聚类分析得到常见证候的四诊信息；通过关联分析，探讨药物的配伍规律。
以NOD-SCID小鼠为实验对象，采用60Co照射，尾静脉注射KG1a细胞，复制急性白血病小鼠模型。将造模后小鼠随机分为空白对照组、扶正组、祛邪组、全方组，应用流式细胞术检测KG1a/NOD-SCID小鼠淋巴结细胞凋亡率、细胞周期分布；采用免疫组化法检测其淋巴结组织中c-myc、survivin、p21蛋白的表达。采用PCR-ELISA法检测小鼠骨髓细胞端粒酶活性，荧光定量PCR法检测小鼠骨髓细胞hTERT-mRNA及其启动子甲基化的表达。
结果：
1、急性白血病的常见证候是：气阴两虚证、热毒炽盛证、气血两虚证、痰热瘀结证；常见证候要素以热、气虚、阴虚为主；常见证候要素靶位是肾、肝和脾。
2、急性白血病常用药物药类有补益药、清热药、活血化瘀药、利水渗湿药和化痰药，其中补虚药、清热药的地位最为重要。常用药物药味多具有甘味及苦味；药性以寒性为主，其次为温性和平性；归经以归肝经的药物最多，其次是归肺、脾、肾经的药物。
3、药物干预后，各用药组小鼠生存时间较模型组延长（P<0.05），细胞凋亡率、G1期停滞细胞百分比较对照组升高（P<0.05），淋巴结中c-myc及survivin蛋白的表达较对照组降低（P<0.05），p21蛋白的表达较对照组升高（P<0.05），骨髓细胞端粒酶活性、hTERT-mRNA及其启动子甲基化的表达较对照组降低（P<0.05）。其中全方组对于各指标的影响均优于扶正组与祛邪组（P<0.05）。
结论：通过应用数据挖掘对现代文献中急性白血病的常见证候及用药规律进行分析，认为本病属本虚标实，本虚以气阴两虚、气血亏虚为主，标实以热毒、瘀血、痰湿为主。治疗上应扶正与祛邪兼顾，治法以补气养阴、清热解毒为主，佐以活血化瘀、化痰祛湿等。
实验研究证明，益气养阴方通过调节细胞周期，降低hTERT-mRNA及其启动子甲基化的表达，下调端粒酶活性，诱导白血病细胞凋亡，抑制白血病细胞增殖，从而延长KG1a/NOD-SCID小鼠生存期，对急性白血病具有治疗作用。
Objective Study on the traditional Chinese medicine syndrome and herbaladministration regularity of acute leukemia based on Data Mining, in order to offer theevidence-based data supporting to guide the TCM differential treatment of diabetic arterialobliterations. KG1a/NOD-SCID mice was used as the study platform,to explore themechanism of leukemia cell apoptosis induced by Tonicing Qi and Nurishing YinDecoction and its decomposed recipes.
Methods The research literature were collected from periodical database withinrecent30years, focus on Chinese medicine syndrome and the medicine that had been used.Database was established with Excel software.To find the common syndrome, influencefactors,the commonly used medicines and medicine category by using frequency analysis.To obtaine information from the TCM four diagnostic methods by using cluster analysis.Toexploer Compatibility Law of herb by using association analysis.
Taking the NOD-SCID mice as the experimental object,and injected with KG1a cellsfrom the tail vein after Irradiation by60Co.Model mices were randomly divided into modelgroup,Fuzheng group,Quxie group,Quanfang group.flow cytometry was applied tomeasure the cell cycle and apoptosis.The immunochemistry method was used to detect theexpression of c-myc,survivin,p21in mices’ lymph node tissue.The PCR-ELISA wasapplied to measure the Telomerase Activity of bone marrow cells.The expression ofhTERT mRNA and methylation of its promoter were measured using RT-PCR.
Results
1. The common syndrome of acute leukemia are deficiency of both qi and yin, heattoxin syndrome, deficiency of both qi and blood, and junction of sputum,thermal andstasis.Influence factors include thermal,qi deficiency and yin deficiency.The target sites ofinfluence factors are kidney,liver and spleen.
2. The commonly used medicine categorise are tonifying herbs,heat-clearingherbs,blood-activating and stasis-resolving herbs, removing dampness and promotingdiuresis herbs,apophlegmatisant herbs.Tonifying herbs and heat-clearing herbs occupy animportant position.The most common Flavors of Drugs are Sweet and bitter.Cold drug inall drug occupies the main position.Secondly it is warm drug,and the next is ping drug.Thechannel-tropism of medicine is mostly liver,secondly lung,spleen and kidney.
3. After treatment,survival time of all treatment groups were longer than those inmodel group（P<0.05）. Apoptosis rate and the proportion of cells in the G1phase in alltreatment groups were increased（P<0.05）. The expression of c-myc、survivin in mices’lymph node tissues as well as hTERT mRNA and methylation of its promoter,and theTelomerase Activity of bone marrow cells in all treatment groups were significantlydeclined compared with model group（P<0.05）.Howere, the expression of p21in mices’lymph node tissues in all treatment groups was higher than model group（P<0.05）.TheEffect of Quanfang group on the all Indices was superior to Fuzheng group and Quxiegroup（P<0.05）.
Conclusion Through studing on the traditional Chinese medicine syndrome andherbal administration Rregularity of acute leukemia based on Data Mining, we obtained acorollary that the basic pathogenesis of acute leukemia was root deficiency and branchexcess.Root deficiency were deficiency of both qi and yin, deficiency of both qi andblood.Branch excess were heat toxin,sputum and stasis.On the treatment,not only ispel evilbut assist the justice.Supplementing qi and nourishing yin, heat-clearing and detoxifyingare the main treatment methods,and combined with water dampness, promoting bloodcirculation and removing blood stasis methods.Experimental studies proved that Tonicing Qi and Nurishing Yin Decoction inducedleukemia cell apoptosis,regulatid cell cycles,declined the expression of hTERT mRNA andmethylation of its promoter,effected the telomerase activity, Induce apoptosis,inhibit theproliferation of leukemia cells,prolonged the survival time of KG1a/NOD-SCID mices.So,Tonicing Qi and Nurishing Yin Decoction has significant therapeutic action on acuteleukemia.
引文
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