益气活血法抗乳腺癌血道转移的基础与临床转化研究
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摘要
乳腺癌是严重威胁妇女生命健康的常见恶性肿瘤。乳腺癌的转移直接威胁患者生存。中医药在抗乳腺癌转移中疗效肯定。回顾文献报道,乳腺癌血瘀证与乳腺癌的血道转移关系密切。但是学界对肿瘤活血化瘀药的运用存在争议：怎么用、用多少、为什么,莫衷一是。导师孙桂芝教授从事中西结合肿瘤治疗近半个世纪,认为气虚、血瘀、毒凝是乳腺癌转移的根本病机。乳腺癌血瘀证具有肿瘤血瘀证的通性,也有其自身特性,临床施治当整体把握,区别对待,活血药运用当配合益气药。根据既往自然基金研究结果,结合多年临证经验,导师总结出抗乳腺癌转移的“益气活血方”：黄芪、苏木、蜂房、蒲黄、炮山甲。基于导师学术思想,本课题对“益气活血方”抗乳腺癌血道转移的疗效、机制、运用进行深入、系统的研究。
1目的
导师学术思想研究：系统总结导师治疗乳腺癌经验。
基础研究：获得“益气活血方”的最佳配伍“益气活血核心方”,核心方临床运用适宜剂量。从乳腺癌血道转移中上皮间质转变、血管新生两大核心环节探讨导师抗乳腺癌血道转移学术思想的科学内涵。
临床转化研究：制定乳腺癌血瘀证量化诊断标准,并与基础研究进行理论衔接,为导师学术思想的临床运用提供技术支持。
2方法
导师学术思想研究：跟随导师临证,按照中医理论对导师治疗乳腺癌经验进行总结。
基础研究：采用乳腺癌小鼠荷瘤模型,筛选抗乳腺癌体内增殖作用最强的“益气活血核心方”,采用含药血清药理学方法,获得核心方抗乳腺癌细胞体外增殖适宜剂量。采用体外体内相结合的方法,从TGF-β/Smad通路介导上皮间质转变和血管新生角度,探讨核心方抗乳腺癌血道转移的分子机制。
临床转化研究：采用临床流行病学、计量统计学方法,研究乳腺癌血瘀证量化诊断标准,并以JAVA编程实现判别过程的自动化。基于量化标准判别,检测血瘀证与非血瘀证乳腺癌患者血.清TGF-β水平差异,与基础研究衔接。
3结果
3.1孙桂芝教授治疗乳腺癌辨证、遣方、用药规律分析
导师治疗乳腺癌的主要特色有：辩证论治,以证选方；辨病论治,病证结合；参照科研,精细用药；重视经络,回归传统。治疗重点为扶正、疏肝、活血、理气、解毒。对于乳腺癌发生的肺转移、骨转移、肝转移、脑转移,运用益气活血法,并结合脏器自身生理特性予以施治。特色鲜明,疗效卓著。
3.2基础研究
3.2.1益气活血方最佳组合的拆方研究
益气活血方中“黄芪、苏木、蜂房、炮山甲”成分,对乳腺癌EMT6细胞的BALB/C小鼠体内增殖抑制作用最强,与对照组差异具有统计学意义P<0.05。定名为“益气活血核心方”。
3.2.2益气活血核心方适宜剂量的探索
CCK-8法检测“益气活血核心方”不同浓度含药血清,不同作用时间,在体外对人乳腺癌MCF-7细胞活力抑制情况。“黄芪30g、苏木6g、蜂房6g、炮山甲6g”临床经验剂量为基础,2倍用量抑制肿瘤细胞增殖作用最佳,为推荐剂量。4倍及以下剂量可抑制肿瘤细胞增殖的,为适宜剂量。6倍剂量作用72小时,具有一定的促进肿瘤增殖的作用,P<0.05,9倍剂量在24、48、72小时均可促进肿瘤细胞的增殖,P<0.05,为不推荐剂量。
3.2.3益气活血核心方适宜剂量抗小鼠乳腺癌4T1细胞肺转移的研究
确定益气活血核心方2倍临床剂量具有抗4T1细胞BALB/C小鼠体内肺转移的作用,抑制率为58.82%,P<0.05,较紫杉醇组65.88%为优,但差异无统计学意义,P>0.05。
3.2.4益气活血核心方抗乳腺癌血道转移的实验研究
3.2.4.1益气活血核心方抗乳腺癌上皮间质转变的研究
以TGF-β2ng/ml诱导人乳腺癌MCF-7细胞发生的EMT为体外模型。发现益气活血核心方对乳腺癌EMT进程中细胞形态学改变、侵袭和转移能力的增强具有一定的抑制作用。并能抑制此过程中E-钙粘素等上皮细胞标志分子的下调及N-钙粘素等间质细胞标志分子的上调。
报告基因技术结合实时定量PCR发现,核心方可抑制Smad分子入核,降低TGF-β/Smad通路激活,减少及其下游转录因子SNAIL。
对血清TGF-β水平检测发现,益气活血核心方能够降低4T1乳腺癌荷瘤小鼠血清中TGF-β水平,特别对晚期荷瘤小鼠血清TGF-β水平的爆发性增高具有抑制作用,P<0.05,但是较紫杉醇的作用为弱。
3.2.4.2益气活血核心方抗乳腺癌局部新生血管生成的实验研究
以冰冻切片免疫荧光的方法发现,益气活血核心方适宜剂量对肿瘤边缘和内部新生血管具有一定的抑制作用(P<0.05)。进一步PCR法检测血管生成相关细胞因子,发现可能与其下调肿瘤细胞表达VEGF-A、VEGF-D有关。
3.3临床转化研究
通过对乳腺癌病历资料Logistic回归、条件概率换算、ROC曲线判别、前瞻新检验,获得乳腺癌血瘀证量化诊断标准。诊断阈值16分。诊断标准敏感度95.0%、特异度81.8%、准确度90.3%、阳性似然比5.225、阴性似然比6.11%,检验性能优良。开发出乳腺癌血瘀证诊断自动化软件。
以新制定的乳腺癌血瘀证量化诊断标准,发现血瘀证乳腺癌患者血清中TGF-β水平较非血瘀证患者为高,P<0.05。
4.结论
孙桂芝抗乳腺癌经验方最佳组合“益气活血核心方”具有抗乳腺癌血道转移的作用,与阻断Smad分子入核与靶基因启动子结合,抑制TGF-β/Smad通路介导的乳腺癌EMT进程,同时阻断乳腺癌局部血管新生的机制有关。
结合乳腺癌血瘀证量化诊断标准及孙桂芝教授治疗乳腺癌经验,益气活血核心方可按剂量推荐在临床使用。
孙桂芝教授益气活血法抗乳腺癌血道转移学术思想具有丰富的理论内涵和科学内涵,值得学习、继承、研究和发扬。
5.主要创新点
4.1采用现代生物信息学和分子生物学前沿技术,体内外试验相结合,通过对益气活血核心方抑制乳腺癌细胞增殖,抗乳腺癌血道转移及其机制研究。首次揭示了益气活血法抗乳腺癌治则、治法科学内涵,为传承与挖掘名老中医经验提供了新思路。
4.2研究发现促转移活性因子TGF-β在晚期乳腺癌荷瘤小鼠爆发性高表达与临床血瘀证乳腺癌患者血清中TGF-β升高存在关联性,为临床实时观察及药物干预治疗乳腺癌提供给了新的依据。
4.3采用计量统计学分析技术,对乳腺癌血瘀证的定量诊断标准进行研究,制定乳腺癌血瘀证诊断相关条目及其定量标准。为名老中医经验的推广提供临床转化依据和实践操作工具。
Breast cancer is a malignant tumor that seriously threat women's lives and health. The metastasis of breast cancer is one of the direct causes which deprivate patients'lives. Traditional Chinese Medicine has curative effect in anti breast cancer metastasis. We find that there is a closely relationship between Xueyu syndrome and hematogenus in breast cancer disease basing on the reviewing of literature reports. Also there is large academic disputation in using of Huoxue huayu herbs in cancer disease:how to use, the dosage and the mechanism haven't been explicated. In the past half century, my mentor Professor Sun Guizhi has beening treated malignant tumor patients with integrative oncology medicine methods and has accumulated rich experience of breast cancer treatment. She points out that Qixu, Xueyu, Duning are the fundamental pathogenesis of breast cancer invasion and metastasis, she found that the performance of Xueyu in breast cancer not only has the common characters in all the tumor disease, but also has it's own specialty which based on the meridian patrolling and the situation of Qixue in the meridian. According to the results of basic research funded by national science foundation combined with the experience, Professor Sun Guizhi designed a formula for Xueyu symptom of breat cancer patients which contains five herb medicines:Huangqi, Sumu, Fengfang, Puhuang, Paoshanjia. Under the guidance of my mentor's academic theory in anti breast cancer metastasis, this deviation, we force on efficacy, mechanism and usage of Traditional Chinese Medicine during the deep and systematic research.
1Purpose
The research of my mentor's academic theory
Summarize professor's experience in breast cancer treatment.
Basic research
Gain Yiqi huoxue core formula's best compatibility of "Yiqi huoxue formula" and it's appropriate clinical dose. Investigate the scientific connotation of mentor's academic theory in the two major area at the point of anti breast cancer hematogenesis:Epithelial to mesenchymal transition and Angiogenesis.
Clinical translational research
Formulate the quantify diagnostic criteria of Xueyu Syndrome as the converhence of theoretical and basic research which provide technical support for mentor's clinical academic theory.
2Method
The research of mentor's academic theory
summarize professor Sun Guizhi's experiences basing on the experience of learning clinical skill under Professor's guidance in the outpatient.
Basic research
Using the tumor bearing mouse model, filter the "Yiqi huoxue core formula" which has the strongest activation in anti breast cancer proliferation in vivo; Using drug containing serum pharmacological method, gain the appropriate dose of core formula. Using the methods of vitro and in vivo, explore the molecular mechanism of core formula's function in anti breast cancer hematogenes metastasis via the regulation of TGF-β/Smad pathway mediated epithelial to mesenchymal transition and angiogenesis.
Clinic translational research
Using clinical epidemiology and statistical methods, study the quantify diagnostic criteria of Xueyu syndrome in breast cancer and automate the process of discrimination by JAVA.Test the differences of TGF-β level in breast cancer patients' serum with "Xueyu syndrome" or not. Converge with the basic research.3.Result
3.1The experience of Professor Sun Guizhi in treating breast cancers
The main features of the mentor:treating disease according syndrome diagnosis, choosing famous formula according with the exact syndrome, different kinds of disease different treatment, combining the disease with syndrome, carefully choosing herbs according to the results of base medical researchs, emphasizing the meridian and returning to the tradition. And Fuzhegn, Shugan, Liqi, Huoxue, Jiedu are the key points in professor's treatment experience. Her treatment of the lung, bone, brain, liver metastasis of breast cancer often refers to the physiological characteristics of the organ itself. Obeying these distinctive laws, the treatment efficacy usually becomes outstanding.
3.2Basic research
3.2.1The best combination study of Yiqi huoxue formula by apart prescription method
We find that the core Yiqi huoxue formula consists of "Huangqi, Sumu, Fengfang, Paoshanjia" has the strongest activity in preventing EMT6cells proliferation in vivo, the difference with control group has statistical significance, P<0.05.
3.2.2The appropriate dose of Yiqi huoxue formula reseach
The formula dose "Huangqi30g, Sumu6g, Fengfang6g, Paoshanjia6g" and the2times dose can inhibit the MCF-7well proliferation, especially the2time dose serum containing herb drugs has more powerful activity.4times dose has the inhibiton tendency, but the differences has no statistical significance. At the72hours point, the6dose serum has the activity of promote the cell proliferation, P<0.05. At the24,48,72hours point,9dose serum will promote the cell proliferation, P<0.05.
3.2.3The anti mouse breast cancer cell4T1metastasis to lung research under the treatment of Yiqi huoxue core formula
The model used in this research in BALB/C mouse bearing4T1breast cancer cells. We found that the Yiqi huoxue core formula can prevent4T1cells'metastasis to lung. The inhibitory rate was58.82%, less than the Taxel group whose inhibitory rate was65.88%. But the differences between formula group and Taxel group has no statistical significance.
3.2.4The basic research of Yiqi huoxue formula's function in anti breast cancer hematogenous metastasis
3.2.4.1The basic research of Yqi huoxue formula's function in preventing epithelial to mesenchymal transition progress of beast cancer
The Yiqi huoxue formula could prevent the EMT process in MCF-7cells which were under going the EMT induced by2ng/ml TGF-β. We checked the preventive function at the protein level and the transcription level by identified the changes of several EMT markers and the difference ability of invasion and migration of different treated cells. These results well supported the Yiqi huoxue formula's anti metastasis function at the prevention the deterioration process of breast cancer cell itself.
3.2.4.2The research of Yiqi huoxue formula function in inhibiting the TGF-β activit during the TGF-β induced EMT process
We use dual reciferase report assay combined with real time PCR technology to identify theSmad molecular's entering cell nucleus process, down regulated the activity of TGF-β/smad pathway, reduced the expression of transcription factor SNAIL.
The TGF-β level in the4T1breast cancer cells bearing mouse was low than that in the control group. And the differences has stastistical significance. But this type of function is some lower than the Taxel. The conclusion was that Yiqi huoxue formula may inhibit the EMT progress via down regulating the TGF-β level in serum.
3.2.4.3the research of anti breast cancer angiogenesis function of Yiqi huoxue core formula
We compared the angiogenesis situation in the edge of the lesion and the internal location of the tumor tissue coming from the4T1breast cancer cell bearing mouse via the frozen biopsy organization immunofluorescence techiques. We found that the Yiqi huoxue fonnula had the inhibitory effects of angiogenesis in both the edge and the internal location. Real time PCR examination claimed the this function may be associated with the down regulation of VEGF-A.
3.3Clinic transflational research
Quantify diagnosis criteria is established. The diagnostic threshold is16point, whose sensitivity is95.0%, specificity is81.8%, accuracy is90.3%, positive likelihood ratio is5.225, negative likelihood ratio is6.11%. The test performance is excellent.
The TGF-β level in breast cancer patient with "Xueyu syndrom" is higher than that with out "Xueyu syndrom",P<0.05.
4Conclusions
The "Yiqi huoxue core formula" based on Professor Sun Guizhi's clinical experience has the function of anti-breast cancer hematogenes metastasis via the inhibition of molecular Smads'entering nuclear and binging to the promoter area of target genes which can prevent the EMT process of breast cancer. Also the prevention of angiogenesis is it's mechanism.
"Yiqi huoxue core formula" can be used in breast cancer cases at the recommended dose referring to the breast cancer'Xueyu syndrome'
Professor SunGuizhi's experience of treating breast cancer hematogenes metastasis under the law of 'Yiqi huoxue' owns rich theoretical content and scientific connotation which is worthing learning, inheritance, research and development.
4The main innovation
4.1Reveal the scientific connotation of "Yiqi huoxue" law in preventing breast cancer hematogenes by the research of anti breast cancer cell proliferation and angiogenesis in vitro via the bioinformation and molecular biological techniques. Provide new ideas in inheriting and mining clinical experience of famous Chinese medicine master.
4.2The increased TGF-β level is similar with that in the serum of breast cancer patients."Yiqi huoxue core formula" can decrease the TGF-β level in the serum of tumor bearing mouse model which afford new basis for real time observation and drug intervention treatment in clinical breast cancer cases.
The established quantify diagnostic criteria of breast cancer " Xueyu syndrome" with the measuring statistical analysis techniques afford clinical conversion basis and practical tools for the promotion of famous Chinese medicine masters' experience.
1目的
导师学术思想研究：系统总结导师治疗乳腺癌经验。
基础研究：获得“益气活血方”的最佳配伍“益气活血核心方”,核心方临床运用适宜剂量。从乳腺癌血道转移中上皮间质转变、血管新生两大核心环节探讨导师抗乳腺癌血道转移学术思想的科学内涵。
临床转化研究：制定乳腺癌血瘀证量化诊断标准,并与基础研究进行理论衔接,为导师学术思想的临床运用提供技术支持。
2方法
导师学术思想研究：跟随导师临证,按照中医理论对导师治疗乳腺癌经验进行总结。
基础研究：采用乳腺癌小鼠荷瘤模型,筛选抗乳腺癌体内增殖作用最强的“益气活血核心方”,采用含药血清药理学方法,获得核心方抗乳腺癌细胞体外增殖适宜剂量。采用体外体内相结合的方法,从TGF-β/Smad通路介导上皮间质转变和血管新生角度,探讨核心方抗乳腺癌血道转移的分子机制。
临床转化研究：采用临床流行病学、计量统计学方法,研究乳腺癌血瘀证量化诊断标准,并以JAVA编程实现判别过程的自动化。基于量化标准判别,检测血瘀证与非血瘀证乳腺癌患者血.清TGF-β水平差异,与基础研究衔接。
3结果
3.1孙桂芝教授治疗乳腺癌辨证、遣方、用药规律分析
导师治疗乳腺癌的主要特色有：辩证论治,以证选方；辨病论治,病证结合；参照科研,精细用药；重视经络,回归传统。治疗重点为扶正、疏肝、活血、理气、解毒。对于乳腺癌发生的肺转移、骨转移、肝转移、脑转移,运用益气活血法,并结合脏器自身生理特性予以施治。特色鲜明,疗效卓著。
3.2基础研究
3.2.1益气活血方最佳组合的拆方研究
益气活血方中“黄芪、苏木、蜂房、炮山甲”成分,对乳腺癌EMT6细胞的BALB/C小鼠体内增殖抑制作用最强,与对照组差异具有统计学意义P<0.05。定名为“益气活血核心方”。
3.2.2益气活血核心方适宜剂量的探索
CCK-8法检测“益气活血核心方”不同浓度含药血清,不同作用时间,在体外对人乳腺癌MCF-7细胞活力抑制情况。“黄芪30g、苏木6g、蜂房6g、炮山甲6g”临床经验剂量为基础,2倍用量抑制肿瘤细胞增殖作用最佳,为推荐剂量。4倍及以下剂量可抑制肿瘤细胞增殖的,为适宜剂量。6倍剂量作用72小时,具有一定的促进肿瘤增殖的作用,P<0.05,9倍剂量在24、48、72小时均可促进肿瘤细胞的增殖,P<0.05,为不推荐剂量。
3.2.3益气活血核心方适宜剂量抗小鼠乳腺癌4T1细胞肺转移的研究
确定益气活血核心方2倍临床剂量具有抗4T1细胞BALB/C小鼠体内肺转移的作用,抑制率为58.82%,P<0.05,较紫杉醇组65.88%为优,但差异无统计学意义,P>0.05。
3.2.4益气活血核心方抗乳腺癌血道转移的实验研究
3.2.4.1益气活血核心方抗乳腺癌上皮间质转变的研究
以TGF-β2ng/ml诱导人乳腺癌MCF-7细胞发生的EMT为体外模型。发现益气活血核心方对乳腺癌EMT进程中细胞形态学改变、侵袭和转移能力的增强具有一定的抑制作用。并能抑制此过程中E-钙粘素等上皮细胞标志分子的下调及N-钙粘素等间质细胞标志分子的上调。
报告基因技术结合实时定量PCR发现,核心方可抑制Smad分子入核,降低TGF-β/Smad通路激活,减少及其下游转录因子SNAIL。
对血清TGF-β水平检测发现,益气活血核心方能够降低4T1乳腺癌荷瘤小鼠血清中TGF-β水平,特别对晚期荷瘤小鼠血清TGF-β水平的爆发性增高具有抑制作用,P<0.05,但是较紫杉醇的作用为弱。
3.2.4.2益气活血核心方抗乳腺癌局部新生血管生成的实验研究
以冰冻切片免疫荧光的方法发现,益气活血核心方适宜剂量对肿瘤边缘和内部新生血管具有一定的抑制作用(P<0.05)。进一步PCR法检测血管生成相关细胞因子,发现可能与其下调肿瘤细胞表达VEGF-A、VEGF-D有关。
3.3临床转化研究
通过对乳腺癌病历资料Logistic回归、条件概率换算、ROC曲线判别、前瞻新检验,获得乳腺癌血瘀证量化诊断标准。诊断阈值16分。诊断标准敏感度95.0%、特异度81.8%、准确度90.3%、阳性似然比5.225、阴性似然比6.11%,检验性能优良。开发出乳腺癌血瘀证诊断自动化软件。
以新制定的乳腺癌血瘀证量化诊断标准,发现血瘀证乳腺癌患者血清中TGF-β水平较非血瘀证患者为高,P<0.05。
4.结论
孙桂芝抗乳腺癌经验方最佳组合“益气活血核心方”具有抗乳腺癌血道转移的作用,与阻断Smad分子入核与靶基因启动子结合,抑制TGF-β/Smad通路介导的乳腺癌EMT进程,同时阻断乳腺癌局部血管新生的机制有关。
结合乳腺癌血瘀证量化诊断标准及孙桂芝教授治疗乳腺癌经验,益气活血核心方可按剂量推荐在临床使用。
孙桂芝教授益气活血法抗乳腺癌血道转移学术思想具有丰富的理论内涵和科学内涵,值得学习、继承、研究和发扬。
5.主要创新点
4.1采用现代生物信息学和分子生物学前沿技术,体内外试验相结合,通过对益气活血核心方抑制乳腺癌细胞增殖,抗乳腺癌血道转移及其机制研究。首次揭示了益气活血法抗乳腺癌治则、治法科学内涵,为传承与挖掘名老中医经验提供了新思路。
4.2研究发现促转移活性因子TGF-β在晚期乳腺癌荷瘤小鼠爆发性高表达与临床血瘀证乳腺癌患者血清中TGF-β升高存在关联性,为临床实时观察及药物干预治疗乳腺癌提供给了新的依据。
4.3采用计量统计学分析技术,对乳腺癌血瘀证的定量诊断标准进行研究,制定乳腺癌血瘀证诊断相关条目及其定量标准。为名老中医经验的推广提供临床转化依据和实践操作工具。
Breast cancer is a malignant tumor that seriously threat women's lives and health. The metastasis of breast cancer is one of the direct causes which deprivate patients'lives. Traditional Chinese Medicine has curative effect in anti breast cancer metastasis. We find that there is a closely relationship between Xueyu syndrome and hematogenus in breast cancer disease basing on the reviewing of literature reports. Also there is large academic disputation in using of Huoxue huayu herbs in cancer disease:how to use, the dosage and the mechanism haven't been explicated. In the past half century, my mentor Professor Sun Guizhi has beening treated malignant tumor patients with integrative oncology medicine methods and has accumulated rich experience of breast cancer treatment. She points out that Qixu, Xueyu, Duning are the fundamental pathogenesis of breast cancer invasion and metastasis, she found that the performance of Xueyu in breast cancer not only has the common characters in all the tumor disease, but also has it's own specialty which based on the meridian patrolling and the situation of Qixue in the meridian. According to the results of basic research funded by national science foundation combined with the experience, Professor Sun Guizhi designed a formula for Xueyu symptom of breat cancer patients which contains five herb medicines:Huangqi, Sumu, Fengfang, Puhuang, Paoshanjia. Under the guidance of my mentor's academic theory in anti breast cancer metastasis, this deviation, we force on efficacy, mechanism and usage of Traditional Chinese Medicine during the deep and systematic research.
1Purpose
The research of my mentor's academic theory
Summarize professor's experience in breast cancer treatment.
Basic research
Gain Yiqi huoxue core formula's best compatibility of "Yiqi huoxue formula" and it's appropriate clinical dose. Investigate the scientific connotation of mentor's academic theory in the two major area at the point of anti breast cancer hematogenesis:Epithelial to mesenchymal transition and Angiogenesis.
Clinical translational research
Formulate the quantify diagnostic criteria of Xueyu Syndrome as the converhence of theoretical and basic research which provide technical support for mentor's clinical academic theory.
2Method
The research of mentor's academic theory
summarize professor Sun Guizhi's experiences basing on the experience of learning clinical skill under Professor's guidance in the outpatient.
Basic research
Using the tumor bearing mouse model, filter the "Yiqi huoxue core formula" which has the strongest activation in anti breast cancer proliferation in vivo; Using drug containing serum pharmacological method, gain the appropriate dose of core formula. Using the methods of vitro and in vivo, explore the molecular mechanism of core formula's function in anti breast cancer hematogenes metastasis via the regulation of TGF-β/Smad pathway mediated epithelial to mesenchymal transition and angiogenesis.
Clinic translational research
Using clinical epidemiology and statistical methods, study the quantify diagnostic criteria of Xueyu syndrome in breast cancer and automate the process of discrimination by JAVA.Test the differences of TGF-β level in breast cancer patients' serum with "Xueyu syndrome" or not. Converge with the basic research.3.Result
3.1The experience of Professor Sun Guizhi in treating breast cancers
The main features of the mentor:treating disease according syndrome diagnosis, choosing famous formula according with the exact syndrome, different kinds of disease different treatment, combining the disease with syndrome, carefully choosing herbs according to the results of base medical researchs, emphasizing the meridian and returning to the tradition. And Fuzhegn, Shugan, Liqi, Huoxue, Jiedu are the key points in professor's treatment experience. Her treatment of the lung, bone, brain, liver metastasis of breast cancer often refers to the physiological characteristics of the organ itself. Obeying these distinctive laws, the treatment efficacy usually becomes outstanding.
3.2Basic research
3.2.1The best combination study of Yiqi huoxue formula by apart prescription method
We find that the core Yiqi huoxue formula consists of "Huangqi, Sumu, Fengfang, Paoshanjia" has the strongest activity in preventing EMT6cells proliferation in vivo, the difference with control group has statistical significance, P<0.05.
3.2.2The appropriate dose of Yiqi huoxue formula reseach
The formula dose "Huangqi30g, Sumu6g, Fengfang6g, Paoshanjia6g" and the2times dose can inhibit the MCF-7well proliferation, especially the2time dose serum containing herb drugs has more powerful activity.4times dose has the inhibiton tendency, but the differences has no statistical significance. At the72hours point, the6dose serum has the activity of promote the cell proliferation, P<0.05. At the24,48,72hours point,9dose serum will promote the cell proliferation, P<0.05.
3.2.3The anti mouse breast cancer cell4T1metastasis to lung research under the treatment of Yiqi huoxue core formula
The model used in this research in BALB/C mouse bearing4T1breast cancer cells. We found that the Yiqi huoxue core formula can prevent4T1cells'metastasis to lung. The inhibitory rate was58.82%, less than the Taxel group whose inhibitory rate was65.88%. But the differences between formula group and Taxel group has no statistical significance.
3.2.4The basic research of Yiqi huoxue formula's function in anti breast cancer hematogenous metastasis
3.2.4.1The basic research of Yqi huoxue formula's function in preventing epithelial to mesenchymal transition progress of beast cancer
The Yiqi huoxue formula could prevent the EMT process in MCF-7cells which were under going the EMT induced by2ng/ml TGF-β. We checked the preventive function at the protein level and the transcription level by identified the changes of several EMT markers and the difference ability of invasion and migration of different treated cells. These results well supported the Yiqi huoxue formula's anti metastasis function at the prevention the deterioration process of breast cancer cell itself.
3.2.4.2The research of Yiqi huoxue formula function in inhibiting the TGF-β activit during the TGF-β induced EMT process
We use dual reciferase report assay combined with real time PCR technology to identify theSmad molecular's entering cell nucleus process, down regulated the activity of TGF-β/smad pathway, reduced the expression of transcription factor SNAIL.
The TGF-β level in the4T1breast cancer cells bearing mouse was low than that in the control group. And the differences has stastistical significance. But this type of function is some lower than the Taxel. The conclusion was that Yiqi huoxue formula may inhibit the EMT progress via down regulating the TGF-β level in serum.
3.2.4.3the research of anti breast cancer angiogenesis function of Yiqi huoxue core formula
We compared the angiogenesis situation in the edge of the lesion and the internal location of the tumor tissue coming from the4T1breast cancer cell bearing mouse via the frozen biopsy organization immunofluorescence techiques. We found that the Yiqi huoxue fonnula had the inhibitory effects of angiogenesis in both the edge and the internal location. Real time PCR examination claimed the this function may be associated with the down regulation of VEGF-A.
3.3Clinic transflational research
Quantify diagnosis criteria is established. The diagnostic threshold is16point, whose sensitivity is95.0%, specificity is81.8%, accuracy is90.3%, positive likelihood ratio is5.225, negative likelihood ratio is6.11%. The test performance is excellent.
The TGF-β level in breast cancer patient with "Xueyu syndrom" is higher than that with out "Xueyu syndrom",P<0.05.
4Conclusions
The "Yiqi huoxue core formula" based on Professor Sun Guizhi's clinical experience has the function of anti-breast cancer hematogenes metastasis via the inhibition of molecular Smads'entering nuclear and binging to the promoter area of target genes which can prevent the EMT process of breast cancer. Also the prevention of angiogenesis is it's mechanism.
"Yiqi huoxue core formula" can be used in breast cancer cases at the recommended dose referring to the breast cancer'Xueyu syndrome'
Professor SunGuizhi's experience of treating breast cancer hematogenes metastasis under the law of 'Yiqi huoxue' owns rich theoretical content and scientific connotation which is worthing learning, inheritance, research and development.
4The main innovation
4.1Reveal the scientific connotation of "Yiqi huoxue" law in preventing breast cancer hematogenes by the research of anti breast cancer cell proliferation and angiogenesis in vitro via the bioinformation and molecular biological techniques. Provide new ideas in inheriting and mining clinical experience of famous Chinese medicine master.
4.2The increased TGF-β level is similar with that in the serum of breast cancer patients."Yiqi huoxue core formula" can decrease the TGF-β level in the serum of tumor bearing mouse model which afford new basis for real time observation and drug intervention treatment in clinical breast cancer cases.
The established quantify diagnostic criteria of breast cancer " Xueyu syndrome" with the measuring statistical analysis techniques afford clinical conversion basis and practical tools for the promotion of famous Chinese medicine masters' experience.
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