活血药、益气活血药对肿瘤生长转移过程中调节性T细胞介导的免疫逃逸影响研究
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摘要
活血类中药在肿瘤治疗中的作用一直存在争议,临床经验表明活血药可通过合理的配伍增强其改善荷瘤机体高凝状态的作用,同时逆转(或减弱)其可能的促肿瘤转移的趋势。导师的既往课题研究观察了不同类型活血药和配伍不同剂量的益气药黄芪后对肿瘤转移的影响,结果显示:高剂量的益气药配伍活血药不但可以提高单纯活血药对小鼠Lewis肺癌的肿瘤抑制率,并且具有抑制某些单纯活血药促进肿瘤转移的趋势。对其作用机制研究显示:益气活血药与单纯的活血药比较可以降低CD4~+CD25~+调节性T细胞表达,但其分子机制有待进一步深入探讨。
本研究是在导师前期工作的基础上,以国家自然科学基金课题为依托开展的。研究对原发性非小细胞肺癌患者外周血CD4~+CD25~+Foxp3~+调节性T细胞与健康志愿者的差异、与肺癌主要临床特征及主要单证的关系进行了小样本的研究;从丹参、苏木、水蛭三种活血药及其与益气药黄芪配伍后的三种益气活血药中选择出一组对小鼠Lewis肺癌肺转移干预作用差异最大的药物,即苏木和苏木+黄芪作为活血药与益气活血药的代表进行更深入观察;动物实验研究中主要通过免疫磁珠细胞分选、流式细胞术、实时定量PCR、荧光显微镜图像分析、ELISA、CBA flexset等技术,从Lewis肺癌荷瘤小鼠肿瘤组织和T淋巴细胞两方面观察苏木、苏木+黄芪对CD4~+CD25~+调节性T细胞、相关活化和调控分子、效应因子、Jagged1-Notch信号转导通路中主要基因表达及树突状细胞表面分子的干预作用;体外实验研究中主要通过MTT、transwell小室法检测苏木、苏木+黄芪含药血清对PG细胞增殖、运动、侵袭能力的干预作用,并运用共培养技术,建立人外周血DC、外周血CD4~+细胞、PG细胞培养上清混合培养体系,观察苏木、苏木+黄芪含药血清对T细胞分化的影响。研究从影响肿瘤免疫逃逸角度探讨活血药、益气活血药对肿瘤生长转移干预作用差异的分子机制及关键环节,取得了具有一定价值的研究结果:
1主要研究结论
1.1原发性非小细胞肺癌患者外周血CD4~+CD25~+Foxp3~+调节性T细胞检测及临床意义
通过对30例原发性非小细胞肺癌患者和12例健康志愿者外周血CD4~+CD25~+Foxp3~+Treg流式细胞仪检测和对肺癌患者的临床资料分析证实,肺癌患者外周血CD4~+CD25~+Foxp3~+细胞百分比高于健康志愿者,与文献报道相一致;肺癌患者CD4~+CD25~+Foxp3~+细胞百分比升高与病期进展和肿瘤负荷增加有关,在不同性别、年龄、病理、KPS及肿瘤标志物正常与否之间比较无差异;肺癌患者CD4~+CD25~+Foxp3~+细胞百分比升高与患者出现主要单证中的气虚证和血瘀证关系最为密切。这一研究结果在CD4~+CD25~+Foxp3~+Treg与肺癌关系方面为动物和体外实验研究提供了临床数据依据,并对人外周血CD4~+CD25~+Foxp3~+细胞百分比流式检测作为一项新的免疫功能监测指标的临床意义和方法可行性作了初步探讨。
1.2不同类型活血药、益气活血药对小鼠Lewis肺癌生长转移的干预作用观察
选取肿瘤科常用的和血、活血、破血代表药丹参、苏木、水蛭及其与益气药代表黄芪配伍的组方用于抑瘤实验,结果表明:①丹参有促进Lewis肺癌生长及转移的趋势,丹参与黄芪配伍干预后不能逆转这种趋势;苏木、苏木+黄芪、水蛭、水蛭+黄芪在抑制小鼠Lewis肺癌生长转移方面有良好的疗效,提示不同类型活血药、益气活血药对肿瘤生长转移的干预作用不尽相同;②丹参、苏木、水蛭三种不同类型活血药配伍益气药后在增强活血药抑瘤、抗转移作用方面有共同的趋势。通过以上实验结果,选取对小鼠Lewis肺癌肺转移干预作用差异最大的一组即苏木和苏木+黄芪作为活血药和益气活血药的代表进行后续研究。
1.3活血药、益气活血药对荷瘤小鼠脾CD4~+CD25~+Foxp3~+Treg表达干预作用动态观察
通过对苏木、苏木+黄芪作用后Lewis肺癌荷瘤小鼠体重、瘤重、小鼠脾CD4~+CD25~+Foxp3~+细胞表达的动态观察及小鼠肺转移病理观察、生存期的分析,结果显示:①苏木、苏木+黄芪的抑瘤作用随荷瘤天数的增加总体呈降低趋势,提示中药在肿瘤早期,瘤负荷较小时抑瘤作用更明显;②苏木在肿瘤早期抑瘤作用较强,但苏木+黄芪在提高肺转移抑制率、延长生存期方面作用均优于苏木;③荷瘤对照组小鼠脾CD4~+CD25~+Foxp3~+细胞表达随荷瘤天数的增加呈上升趋势;④苏木、苏木+黄芪对荷瘤小鼠脾CD4~+CD25~+Foxp3~+细胞表达的变化趋势有不同影响,20d苏木+黄芪组小鼠脾CD4~+CD25~+Foxp3~+细胞百分比显著低于荷瘤对照组和苏木组。以上结果显示,中药可通过调控荷瘤小鼠脾CD4~+CD25~+Treg表达改善其体内存在的免疫耐受状态,益气活血药苏木+黄芪作用优于其单纯活血药成分,这可能是益气活血中药抑制小鼠Lewis肺癌生长转移、延长生存的作用靶点之一。
1.4活血药、益气活血药对荷瘤小鼠脾CD4~+细胞培养上清中细胞因子及Jagged1-Notch1信号转导通路主要基因的影响
通过对苏木、苏木+黄芪作用后Lewis肺癌荷瘤小鼠脾CD4~+细胞培养上清中细胞因子及Jagged1-Notch1信号转导通路主要基因检测,发现:①苏木+黄芪组小鼠脾CD4~+细胞培养上清IL-2、IFN-γ水平显著高于荷瘤对照组,而TGF-β1水平低于荷瘤对照组;②苏木、苏木+黄芪对荷瘤小鼠脾CD4~+细胞Notch1、Jagged1mRNA表达无明显干预作用。说明中药可通过调控荷瘤小鼠体内细胞因子水平改善体内存在的免疫耐受状态,苏木+黄芪组优于苏木组,而二种中药对Jagged1-Notch1信号转导通路的影响有待进一步研究。
1.5活血药、益气活血药对荷瘤小鼠免疫逃逸相关基因表达的影响
通过对苏木、苏木+黄芪作用后Lewis肺癌荷瘤小鼠脾CD4~+CD25~+细胞和小鼠肿瘤组织Foxp3、CTLA-4基因的表达的同步检测,发现:苏木+黄芪能减低脾CD4~+CD25~+分选后细胞Foxp3及荷瘤小鼠瘤组织CTLA-4、Foxp3 mRNA表达,作用优于苏木。说明苏木+黄芪可通过降低CTLA-4、Foxp3 mRNA的表达而减低CD4~+CD25~+Treg的活性,并且在淋巴组织和肿瘤微环境都表现出作用,为中药的全身调节特点提供了佐证。
1.6活血药、益气活血药对荷瘤小鼠脾树突状细胞表型的影响
通过对正常小鼠和苏木、苏木+黄芪作用后Lewis肺癌荷瘤小鼠脾树突状细胞表型CD11c、CD80、CD86百分比检测发现:①荷瘤机体存在明显的成熟DC数量减少;②苏木+黄芪干预能够显著增加荷瘤小鼠脾成熟DC表型,作用优于苏木。说明苏木+黄芪具有改善荷瘤机体中成熟DC数量减少的作用,同时为体外实验中研究DC成熟程度与T细胞分化的关系提供动物实验的基础。
1.7活血药、益气活血药含药血清对PG细胞增殖及运动、侵袭能力干预作用观察
为体外实验研究需要,制备了苏木、苏木+黄芪大鼠含药血清,并检测其对PG细胞增殖及运动、侵袭能力的干预作用,结果显示:①苏木、苏木+黄芪含药血清对PG细胞增殖无明显抑制作用;②苏木+黄芪含药血清对PG细胞趋化运动、侵袭能力的抑制作用显著高于对照大鼠血清和苏木含药血清。说明中药能够在多个靶点、从多种途径发挥抗肿瘤生长转移作用,对肿瘤细胞的直接抑制杀灭作用只是其中的一个方面。
1.8 PG细胞培养上清对体外培养树突状细胞成熟的影响
本实验从人外周血分离单个核细胞,并采用不同细胞因子体外诱导其分化为不同成熟状态的树突状细胞,以PG细胞培养上清加入DC培养体系,在体外模拟体内肿瘤微环境,并检测PG细胞培养上清干预组和无干预组中DC表型CD11c、CD86百分比,结果证实:PG细胞培养上清干预能显著抑制体外培养DC的成熟。本实验为后续体外实验提供肿瘤细胞培养上清干预抑制体外培养DC成熟的前提结论和方法学铺垫。
1.9 PG细胞培养上清对DC、T细胞混合培养体系的影响及活血药、益气活血药对其干预作用
本实验建立了人外周血DC、外周血CD4~+细胞、PG细胞培养上清混合培养体系,并观察混合培养体系中DC、T细胞分布,检测混合培养体系中CD4~+CD25~+Foxp3~+细胞百分比,细胞培养上清IL-12水平,细胞Jagged1、Notch1 mRNA表达,结果显示:①PG细胞培养上清干预后人外周血DC、外周血CD4~+细胞混合培养体系与无PG干预组相比具有细胞CD4~+CD25~+Foxp3~+表达显著增高、细胞Jagged1、Notch1 mRNA表达明显增高、细胞培养上清IL-12水平明显下降的特点;②苏木、苏木+黄芪含药血清对无PG细胞培养上清干预的人外周血DC、外周血CD4~+细胞混合培养体系中CD4~+T细胞向Treg的分化无干预作用,对细胞培养上清IL-12水平也无影响;③苏木+黄芪含药血清在体外人外周血DC、外周血CD4~+细胞混合培养体系中有抑制CD4~+CD25~-T细胞向CD4~+CD25~+ Treg转化的作用;④苏木+黄芪含药血清在体外有改善肿瘤细胞培养上清干预体系中IL-12水平降低作用;⑤苏木、苏木+黄芪含药血清对人外周血DC、外周血CD4~+细胞混合培养体系中细胞Jagged1、Notch1 mRNA表达无明显干预作用。说明PG细胞培养上清干预体系中Treg的产生与肿瘤细胞分泌大量免疫抑制性细胞因子、DC成熟状态和肿瘤抗原刺激量有关,是多方因素共同作用的结果,而苏木+黄芪含药血清对Treg产生的抑制及对T细胞活化的促进可能与上述因素有关。
本研究分别以非小细胞肺癌患者、Lewis肺癌荷瘤小鼠、高转移性人巨细胞肺癌细胞PG干预的DC、T细胞共培养体系为研究对象,从临床实验、动物实验、体外实验三个不同层次探讨了CD4~+CD25~+Foxp3~+Treg与肺癌生长转移关系,从肿瘤组织和T淋巴细胞两方面观察苏木、苏木+黄芪对CD4~+CD25~+Treg、相关活化和调控分子、效应因子、Jagged1-Notch信号传导通路中主要基因表达及树突状细胞表面分子的干预作用,从整体-细胞-分子基因水平较系统地探讨了活血药、益气活血药对肿瘤生长转移干预作用差异的分子机制及关键环节,为临床更加合理有效地应用活血药和益气活血药提供了科学依据。
2主要创新点
本研究的创新之处主要有以下几点:
2.1本研究分别以非小细胞肺癌患者、Lewis肺癌荷瘤小鼠、高转移性人巨细胞肺癌细胞PG干预的DC、T细胞共培养体系为研究对象,从整体-细胞-分子基因水平探讨了活血药苏木、益气活血药苏木+黄芪对肺癌生长转移过程中CD4~+CD25~+Treg介导的肿瘤免疫逃逸影响差异的分子机制和关键作用环节,为肿瘤临床治疗中更加合理有效地应用活血药、益气活血药提供了科学依据。
2.2在既往研究基础上,通过动态观察Lewis肺癌荷瘤小鼠在肿瘤自然病程中不同时间点脾CD4~+CD25~+Foxp3~+细胞表达,并分析其与小鼠体重、生存期等的关系以及活血药苏木、益气活血药苏木+黄芪的影响作用,提出干预CD4~+CD25~+Treg及相关活化和调控分子、效应因子表达是活血药、益气活血药在肿瘤生长转移过程中作用差异产生的重要机制,首次结合肿瘤免疫编辑学说中免疫监视和免疫逃逸理论,从影响T淋巴细胞和肿瘤微环境两方面评价不同中医治法各自特点和治疗结果差异的原因。
2.3本研究从小样本临床研究切入,证实肺癌患者CD4~+CD25~+Treg检测的重要临床意义;继而通过动物抑瘤实验从三组不同活血药、益气活血药中筛选出一组对Lewis肺癌转移影响差异最大的药物苏木、苏木+黄芪作为代表进行深入研究;然后采用细胞分选、流式细胞术、实时定量PCR、荧光显微镜图像分析、ELISA、CBAflexset等技术从动物实验角度研究活血药、益气活血药对CD4~+CD25~+Treg及相关活化和调控分子、效应因子表达量的影响;最后采用MTT、transwell小室、细胞共培养等技术从体外实验角度观察活血药、益气活血药对Treg的产生的干预作用。围绕CD4~+CD25~+Treg作用机制的主线,通过不同层次研究,较为全面地观察了中药对肺癌生长转移过程中免疫逃逸相关分子表达和功能的干预作用。
2.4临床实验研究中将免疫学新指标与中医基础理论相结合,首次探讨了原发性非小细胞肺癌患者外周血CD4~+CD25~+Foxp3~+Treg表达与肺癌中医证候的关系。
The role that blood activating drugs play in tumor therapy is always in dispute. The clinical experience showed that we can enhance the effect of blood activating drugs on improving the high blood coagulation state of tumor-bearing body and reverse(or decrease) the possible trend of promoting tumor metastasis by reasonable compatibility.The earlier studies of my mentor have observed influence of different types of blood activating drugs compatibility with qi-deficiency drug(Radix Astragali) in different doses on tumor metastasis.The results showed that high dosage qi-deficiency drug compatibility with blood activating drugs could improve the tumor inhibiting rate and reverse the trend of promoting tumor metastasis of simple blood activating drugs.The results of mechanism study showed that the supplementing qi and activating blood circulation drugs could down-regulate the expression of CD4~+CD25~+ Treg.The study needs further research.
The study was carried out on the base of the earlier studies,and was supported by NSFC.We carried out a small sample clinical research on the difference of the expression of CD4~+CD25~+Foxp3~+ Treg in peripheral blood between patients with NSCLC and healthy volunteers and its relationship with main features and TCM syndrome of lung cancer.After the difference comparison studying on the inhibiting rate of lung metastasis in Lewis lung cancer model in mice,we chose a pair of representative drugs(Caesalpinina sappan(SM) and Caesalpinina sappan+Radix Astragali(SMHQ)) from three types of blood activating drugs(Salvia Miltiorrhiza Bunge,Caesalpinina sappan,Hirudo) and three types of supplementing qi and activating blood circulation drugs(blood activating drugs compatibility with Radix Astragali).In animal experiments we observed the expressions of CD4~+CD25~+Treg and its related activated molecules,regulatory molecules,effector molecules,main genes in Jagged1-Notch signal transduction pathway and cell surface molecules of DC intervened by Caesalpinina sappan(SM) and Caesalpinina sappan+Radix Astragali(SMHQ),using the main techniques of MACS,FCM,real-time PCR, fluorescence microscope,ELISA and CBA flexset.In vitro experiments we observed the ability of cell proliferation,movement,invasion in PG intervened by Caesalpinina sappan(SM) and Caesalpinina sappan+Radix Astragali(SMHQ) medicated serum, using the main techniques of MTT and transwell.We also established the mixed culture system of DCs,CD4~+T cells and culture supernatant of PG to study the T cell differentiation intervened by Caesalpinina sappan(SM) and Caesalpinina sappan+ Radix Astragali(SMHQ) medicated serum.The study explored the molecular mechanisms and key links of the different effects of blood activating drugs and supplementing qi and activating blood circulation drugs on tumor growth and metastasis from the angle of tumor immune escape.We have obtained high academic valuable data.
1 The primary research conclusions
1.1 Detection of CD4~+CD25~+Foxp3~+ regulatory T cells in peripheral blood of patients with NSCLC and its clinical significance
By detecting CD4~+CD25~+Foxp3~+ regulatory T cells in peripheral blood of 30 patients with NSCLC and 12 healthy volunteers and analyzing their clinical information,we found that:The population of CD4~+CD25~+Foxp3~+ Treg in peripheral blood from patients with NSCLC was higher in comparison with that in healthy volunteers,which conformed the documental reports.The proportion of CD4~+CD25~+Foxp3~+ Treg in the patients with stageⅣlung cancer was higher than those in stageⅠ-Ⅲ.The proportion of CD4~+CD25~+Foxp3~+ Treg in the patients with tumor was higher than those in the patients without tumor.Treg expression was not obviously correlated with different genders,ages,pathological types,KPS and level of tumor markers.The increase of the proportion of CD4~+CD25~+Foxp3~+ Treg in the patients most closely correlated with Qi deficiency syndrome and blood stasis syndrome.These results had provided data basis for animal and vitro experiments on the relationship between CD4~+CD25~+Foxp3~+Treg and lung cancer.
1.2 The effects on Lewis lung cancer growth and metastasis intervened by different types of blood activating drugs and supplementing qi and activating blood circulation drugs
By studying on the inhibiting rate of lung metastasis in Lewis lung cancer model in mice,we observed the intervention effect of three types of blood activating drugs (Salvia Miltiorrhiza Bunge,Caesalpinina sappan,Hirudo) and three types of supplementing qi and activating blood circulation drugs(blood activating drugs compatibility with Radix Astragali).We found that:①Salvia Miltiorrhiza Bunge had showed the trend of promoting tumor metastasis while Salvia Miltiorrhiza Bunge+Radix Astragali could not reverse it.Caesalpinina sappan,Caesalpinina sappan+Radix Astragali,Hirudo,Hirudo+Radix Astragali had showed good curative effect on decrease tumor metastasis,which suggested that different types of blood activating drugs and supplementing qi and activating blood circulation drugs had different effects.②Supplementing qi and activating blood circulation drugs had better effect on decrease tumor growth and metastasis than blood activating drugs.So we chose a pair of representative drugs(Caesalpinina sappan(SM) and Caesalpinina sappan+Radix Astragali(SMHQ)) for further research.
1.3 Continuous observation on the expression of CD4~+CD25~+Foxp3~+ regulatory T cells in spleen of tumor-bearing mice intervened by blood activating drugs and supplementing qi and activating blood circulation drugs
By Continuous observing on the body weight,tumor weight,expression of CD4~+CD25~+Foxp3~+ regulatory T cells in spleen of tumor-beating mice intervened by Caesalpinina sappan(SM) and Caesalpinina sappan+Radix Astragali(SMHQ) and their survival time,we found that:①Traditional Chinese medicine had showed a more obvious tumor inhibiting effect in the early stage.②Caesalpinina sappan(SM) had showed a better tumor inhibiting effect in the early stage,while the advantages of Caesalpinina sappan+Radix Astragali(SMHQ) was in the inhibiting rate of lung metastasis and survival time.③The expression of CD4~+CD25~+Foxp3~+ Treg in spleen of tumor-beating mice in control group was increased followed with the tumor progression.④Caesalpinina sappan(SM) and Caesalpinina sappan+Radix Astragali (SMHQ) had different effect on the changing tendency of the expression of CD4~+CD25~+Foxp3~+ Treg.The decreased of CD4~+CD25~+Foxp3~+ Treg(20d) was obvious in SMHQ group.These results showed that the supplementing qi and activating blood circulation drugs could improve the tumor-bearing mouse immune tolerance condition by Treg down-regulation and this effect was better than that of blood activating drugs.
1.4 The effects on the levels of cytokines in CD4~+ cell culture supernatant in spleen of tumor-bearing mice and the expression of main genes in Jagged1-Notch signal transduction pathway intervened by blood activating drugs and supplementing qi and activating blood circulation drugs
By detecting the levels of cytokines in CD4~+ cell culture supernatant in spleen of tumor-bearing mice and the expression of main genes in Jagged1-Notch signal transduction pathway intervened by Caesalpinina sappan(SM) and Caesalpinina sappan+Radix Astragali(SMHQ),we found that:①The levels of IL-2,IFN-γin CD4~+ cell culture supernatant in spleen of tumor-bearing mice in SMHQ group was obviously higher than that in control group,while the levels of TGF-β1 was lower than that in control group.②Caesalpinina sappan(SM) and Caesalpinina sappan+Radix Astragali (SMHQ) had no significant effect on the expression of Notch1,Jagged1 mRNA.These results showed that the supplementing qi and activating blood circulation drugs could improve the tumor-bearing mouse immune tolerance condition by regulating the levels of cytokines in tumor-bearing mice and this effect was better than that of blood activating drugs.
1.5 The effects on the expressions of genes related with immune escape in tumor-bearing mice intervened by blood activating drugs and supplementing qi and activating blood circulation drugs
By real-time RT-PCR,we detected the expressions of Foxp3 and CTLA-4 mRNA in tumor tissues and in CD4~+CD25~+cells selected from spleen cells of tumor-bearing mice by magnetic activated cell sorting system.We discovered that:The expressions of Foxp3 and CTLA-4 mRNA in tumor tissues and the expression of Foxp3 mRNA in CD4~+CD25~+cells selected from spleen cells of tumor-bearing mice in SMHQ group were lower than that in control group.These results showed that the supplementing qi and activating blood circulation drugs could improve the tumor-bearing mouse immune tolerance condition by down-regulating the expressions of genes related with immune escape and this effect was showed in both lymphoid tissue and tumor micro-environment.
1.6 The effects on the expressions of phenotypes of DC in spleen of tumor-bearing mice intervened by blood activating drugs and supplementing qi and activating blood circulation drugs
By flow cytometry,we detected the expressions of phenotypes of DC in spleen of tumor-bearing mice.We discovered that:①There was a significantly decrease of the expressions ofphenotypes of DC in tumor-bearing bodies.②Radix Astragali(SMHQ) could increase the expressions of mature phenotypes of DC in spleen of tumor-bearing mice and this effect was better than that of blood activating drugs.These results had provided data basis for vitro experiments on the relationship between the mature degree of DC and differentiation of T cells.
1.7 The effects on the ability of cell proliferation,movement,invasion in PG intervened by Caesalpinina sappan(SM) and Caesalpinina sappan+Radix Astragali(SMHQ) medicated serum
By detecting the effects on the ability of cell proliferation,movement,invasion in PG intervened by Caesalpinina sappan(SM) and Caesalpinina sappan+Radix Astragali (SMHQ) medicated serum,we found that:①Caesalpinina sappan(SM) and Caesalpinina sappan+Radix Astragali(SMHQ) had no significant effect on the ability of cell proliferation in PG.②Caesalpinina sappan+ Radix Astragali(SMHQ) medicated serum had significant inhibitory effect on the ability of cell movement,invasion in PG.These results showed that traditional Chinese medicine could inhibit tumor growth and metastasis in many different targets and many different ways.Directly killing the tumor cells was only one side of these effects.
1.8 The effects on the expressions of phenotypes of DC in vitro intervened by culture supernatant of PG
In this study,PBMC was isolated from human peripheral blood,differentiation into different mature degrees of DC after induction in vitro.Then culture supernatant of PG was added into the culture system to simulate tumor microenvironment.By flow cytometry,we detected the expressions of phenotypes of DC in the two culture systems.We discovered that:The culture supernatant of PG could decrease the mature degree of DC in vitro.These results had provided premise conditions and experimental method for further study.
1.9 The effects on the mixed culture system of DCs,CD4~+T cells and culture supernatant of PG intervened by Caesalpinina sappan(SM) and Caesalpinina sappan+Radix Astragali(SMHQ) medicated serum
In this study,we established the mixed culture system of DCs,CD4~+T cells and culture supernatant of PG.By detecting the expression of CD4~+CD25~+Foxp3~+Treg, the level of IL-12 and the expression of Jagged1,Notch1 mRNA in the mixed culture system,we found that:①In the mixed culture system intervened by culture supernatant of PG,the expression of CD4~+CD25~+Foxp3~+Treg and Jagged1,Notch1 mRNA was higher compared with the control group while the level of IL-12 was decreased.②Caesalpinina sappan(SM) and Caesalpinina sappan+Radix Astragali (SMHQ) medicated serum had no significant effect on the differentiation of T cells and the level of IL-12 in the mixed culture system.③Caesalpinina sappan+Radix Astragali(SMHQ) medicated serum had inhibitory effect on CD4~+CD25-T cells differentiating into CD4~+CD25~+T cells.④Caesalpinina sappan+Radix Astragali (SMHQ) medicated serum could increase the level of IL-12 in the mixed culture system.⑤Caesalpinina sappan(SM) and Caesalpinina sappan+Radix Astragali (SMHQ) medicated serum had no significant effect on the expression of Notch1, Jagged1 mRNA in the mixed culture system.These results showed that the CD4~+CD25T cells differentiating into CD4~+CD25~+T cells in the mixed culture system intervened by culture supematant of PG related to many factors,including inhibitory cytokines secreted by tumor cells,the mature degree of DC and stimulating amount of tumor antigen.
In this research,patients with NSCLC,Lewis lung cancer-bearing mice and PG cells were taken as the research objects.We discussed on the relationship between CD4~+CD25~+Foxp3~+Treg and the tumor growth and metastasis from three different levels of clinical experiments,animal experiments and in vitro experiments.The expressions of CD4~+CD25~+Treg and its related activated molecules,regulatory molecules,effector molecules,main genes in Jaggedl-Notch signal transduction pathway and cell surface molecules of DC intervened by Caesalpinina sappan(SM) and Caesalpinina sappan+Radix Astragali(SMHQ) were observed in both tumor tissue and T cells.The study explored the molecular mechanisms and key links of the different effects of blood activating drugs and supplementing qi and activating blood circulation drugs on tumor growth and metastasis from the levels of whole body,cell mechanism and molecular mechanism.The results had provided a scientific basis for more reasonable and effective clinical application of blood activating drugs and supplementing qi and activating blood circulation drugs.
本研究是在导师前期工作的基础上,以国家自然科学基金课题为依托开展的。研究对原发性非小细胞肺癌患者外周血CD4~+CD25~+Foxp3~+调节性T细胞与健康志愿者的差异、与肺癌主要临床特征及主要单证的关系进行了小样本的研究;从丹参、苏木、水蛭三种活血药及其与益气药黄芪配伍后的三种益气活血药中选择出一组对小鼠Lewis肺癌肺转移干预作用差异最大的药物,即苏木和苏木+黄芪作为活血药与益气活血药的代表进行更深入观察;动物实验研究中主要通过免疫磁珠细胞分选、流式细胞术、实时定量PCR、荧光显微镜图像分析、ELISA、CBA flexset等技术,从Lewis肺癌荷瘤小鼠肿瘤组织和T淋巴细胞两方面观察苏木、苏木+黄芪对CD4~+CD25~+调节性T细胞、相关活化和调控分子、效应因子、Jagged1-Notch信号转导通路中主要基因表达及树突状细胞表面分子的干预作用;体外实验研究中主要通过MTT、transwell小室法检测苏木、苏木+黄芪含药血清对PG细胞增殖、运动、侵袭能力的干预作用,并运用共培养技术,建立人外周血DC、外周血CD4~+细胞、PG细胞培养上清混合培养体系,观察苏木、苏木+黄芪含药血清对T细胞分化的影响。研究从影响肿瘤免疫逃逸角度探讨活血药、益气活血药对肿瘤生长转移干预作用差异的分子机制及关键环节,取得了具有一定价值的研究结果:
1主要研究结论
1.1原发性非小细胞肺癌患者外周血CD4~+CD25~+Foxp3~+调节性T细胞检测及临床意义
通过对30例原发性非小细胞肺癌患者和12例健康志愿者外周血CD4~+CD25~+Foxp3~+Treg流式细胞仪检测和对肺癌患者的临床资料分析证实,肺癌患者外周血CD4~+CD25~+Foxp3~+细胞百分比高于健康志愿者,与文献报道相一致;肺癌患者CD4~+CD25~+Foxp3~+细胞百分比升高与病期进展和肿瘤负荷增加有关,在不同性别、年龄、病理、KPS及肿瘤标志物正常与否之间比较无差异;肺癌患者CD4~+CD25~+Foxp3~+细胞百分比升高与患者出现主要单证中的气虚证和血瘀证关系最为密切。这一研究结果在CD4~+CD25~+Foxp3~+Treg与肺癌关系方面为动物和体外实验研究提供了临床数据依据,并对人外周血CD4~+CD25~+Foxp3~+细胞百分比流式检测作为一项新的免疫功能监测指标的临床意义和方法可行性作了初步探讨。
1.2不同类型活血药、益气活血药对小鼠Lewis肺癌生长转移的干预作用观察
选取肿瘤科常用的和血、活血、破血代表药丹参、苏木、水蛭及其与益气药代表黄芪配伍的组方用于抑瘤实验,结果表明:①丹参有促进Lewis肺癌生长及转移的趋势,丹参与黄芪配伍干预后不能逆转这种趋势;苏木、苏木+黄芪、水蛭、水蛭+黄芪在抑制小鼠Lewis肺癌生长转移方面有良好的疗效,提示不同类型活血药、益气活血药对肿瘤生长转移的干预作用不尽相同;②丹参、苏木、水蛭三种不同类型活血药配伍益气药后在增强活血药抑瘤、抗转移作用方面有共同的趋势。通过以上实验结果,选取对小鼠Lewis肺癌肺转移干预作用差异最大的一组即苏木和苏木+黄芪作为活血药和益气活血药的代表进行后续研究。
1.3活血药、益气活血药对荷瘤小鼠脾CD4~+CD25~+Foxp3~+Treg表达干预作用动态观察
通过对苏木、苏木+黄芪作用后Lewis肺癌荷瘤小鼠体重、瘤重、小鼠脾CD4~+CD25~+Foxp3~+细胞表达的动态观察及小鼠肺转移病理观察、生存期的分析,结果显示:①苏木、苏木+黄芪的抑瘤作用随荷瘤天数的增加总体呈降低趋势,提示中药在肿瘤早期,瘤负荷较小时抑瘤作用更明显;②苏木在肿瘤早期抑瘤作用较强,但苏木+黄芪在提高肺转移抑制率、延长生存期方面作用均优于苏木;③荷瘤对照组小鼠脾CD4~+CD25~+Foxp3~+细胞表达随荷瘤天数的增加呈上升趋势;④苏木、苏木+黄芪对荷瘤小鼠脾CD4~+CD25~+Foxp3~+细胞表达的变化趋势有不同影响,20d苏木+黄芪组小鼠脾CD4~+CD25~+Foxp3~+细胞百分比显著低于荷瘤对照组和苏木组。以上结果显示,中药可通过调控荷瘤小鼠脾CD4~+CD25~+Treg表达改善其体内存在的免疫耐受状态,益气活血药苏木+黄芪作用优于其单纯活血药成分,这可能是益气活血中药抑制小鼠Lewis肺癌生长转移、延长生存的作用靶点之一。
1.4活血药、益气活血药对荷瘤小鼠脾CD4~+细胞培养上清中细胞因子及Jagged1-Notch1信号转导通路主要基因的影响
通过对苏木、苏木+黄芪作用后Lewis肺癌荷瘤小鼠脾CD4~+细胞培养上清中细胞因子及Jagged1-Notch1信号转导通路主要基因检测,发现:①苏木+黄芪组小鼠脾CD4~+细胞培养上清IL-2、IFN-γ水平显著高于荷瘤对照组,而TGF-β1水平低于荷瘤对照组;②苏木、苏木+黄芪对荷瘤小鼠脾CD4~+细胞Notch1、Jagged1mRNA表达无明显干预作用。说明中药可通过调控荷瘤小鼠体内细胞因子水平改善体内存在的免疫耐受状态,苏木+黄芪组优于苏木组,而二种中药对Jagged1-Notch1信号转导通路的影响有待进一步研究。
1.5活血药、益气活血药对荷瘤小鼠免疫逃逸相关基因表达的影响
通过对苏木、苏木+黄芪作用后Lewis肺癌荷瘤小鼠脾CD4~+CD25~+细胞和小鼠肿瘤组织Foxp3、CTLA-4基因的表达的同步检测,发现:苏木+黄芪能减低脾CD4~+CD25~+分选后细胞Foxp3及荷瘤小鼠瘤组织CTLA-4、Foxp3 mRNA表达,作用优于苏木。说明苏木+黄芪可通过降低CTLA-4、Foxp3 mRNA的表达而减低CD4~+CD25~+Treg的活性,并且在淋巴组织和肿瘤微环境都表现出作用,为中药的全身调节特点提供了佐证。
1.6活血药、益气活血药对荷瘤小鼠脾树突状细胞表型的影响
通过对正常小鼠和苏木、苏木+黄芪作用后Lewis肺癌荷瘤小鼠脾树突状细胞表型CD11c、CD80、CD86百分比检测发现:①荷瘤机体存在明显的成熟DC数量减少;②苏木+黄芪干预能够显著增加荷瘤小鼠脾成熟DC表型,作用优于苏木。说明苏木+黄芪具有改善荷瘤机体中成熟DC数量减少的作用,同时为体外实验中研究DC成熟程度与T细胞分化的关系提供动物实验的基础。
1.7活血药、益气活血药含药血清对PG细胞增殖及运动、侵袭能力干预作用观察
为体外实验研究需要,制备了苏木、苏木+黄芪大鼠含药血清,并检测其对PG细胞增殖及运动、侵袭能力的干预作用,结果显示:①苏木、苏木+黄芪含药血清对PG细胞增殖无明显抑制作用;②苏木+黄芪含药血清对PG细胞趋化运动、侵袭能力的抑制作用显著高于对照大鼠血清和苏木含药血清。说明中药能够在多个靶点、从多种途径发挥抗肿瘤生长转移作用,对肿瘤细胞的直接抑制杀灭作用只是其中的一个方面。
1.8 PG细胞培养上清对体外培养树突状细胞成熟的影响
本实验从人外周血分离单个核细胞,并采用不同细胞因子体外诱导其分化为不同成熟状态的树突状细胞,以PG细胞培养上清加入DC培养体系,在体外模拟体内肿瘤微环境,并检测PG细胞培养上清干预组和无干预组中DC表型CD11c、CD86百分比,结果证实:PG细胞培养上清干预能显著抑制体外培养DC的成熟。本实验为后续体外实验提供肿瘤细胞培养上清干预抑制体外培养DC成熟的前提结论和方法学铺垫。
1.9 PG细胞培养上清对DC、T细胞混合培养体系的影响及活血药、益气活血药对其干预作用
本实验建立了人外周血DC、外周血CD4~+细胞、PG细胞培养上清混合培养体系,并观察混合培养体系中DC、T细胞分布,检测混合培养体系中CD4~+CD25~+Foxp3~+细胞百分比,细胞培养上清IL-12水平,细胞Jagged1、Notch1 mRNA表达,结果显示:①PG细胞培养上清干预后人外周血DC、外周血CD4~+细胞混合培养体系与无PG干预组相比具有细胞CD4~+CD25~+Foxp3~+表达显著增高、细胞Jagged1、Notch1 mRNA表达明显增高、细胞培养上清IL-12水平明显下降的特点;②苏木、苏木+黄芪含药血清对无PG细胞培养上清干预的人外周血DC、外周血CD4~+细胞混合培养体系中CD4~+T细胞向Treg的分化无干预作用,对细胞培养上清IL-12水平也无影响;③苏木+黄芪含药血清在体外人外周血DC、外周血CD4~+细胞混合培养体系中有抑制CD4~+CD25~-T细胞向CD4~+CD25~+ Treg转化的作用;④苏木+黄芪含药血清在体外有改善肿瘤细胞培养上清干预体系中IL-12水平降低作用;⑤苏木、苏木+黄芪含药血清对人外周血DC、外周血CD4~+细胞混合培养体系中细胞Jagged1、Notch1 mRNA表达无明显干预作用。说明PG细胞培养上清干预体系中Treg的产生与肿瘤细胞分泌大量免疫抑制性细胞因子、DC成熟状态和肿瘤抗原刺激量有关,是多方因素共同作用的结果,而苏木+黄芪含药血清对Treg产生的抑制及对T细胞活化的促进可能与上述因素有关。
本研究分别以非小细胞肺癌患者、Lewis肺癌荷瘤小鼠、高转移性人巨细胞肺癌细胞PG干预的DC、T细胞共培养体系为研究对象,从临床实验、动物实验、体外实验三个不同层次探讨了CD4~+CD25~+Foxp3~+Treg与肺癌生长转移关系,从肿瘤组织和T淋巴细胞两方面观察苏木、苏木+黄芪对CD4~+CD25~+Treg、相关活化和调控分子、效应因子、Jagged1-Notch信号传导通路中主要基因表达及树突状细胞表面分子的干预作用,从整体-细胞-分子基因水平较系统地探讨了活血药、益气活血药对肿瘤生长转移干预作用差异的分子机制及关键环节,为临床更加合理有效地应用活血药和益气活血药提供了科学依据。
2主要创新点
本研究的创新之处主要有以下几点:
2.1本研究分别以非小细胞肺癌患者、Lewis肺癌荷瘤小鼠、高转移性人巨细胞肺癌细胞PG干预的DC、T细胞共培养体系为研究对象,从整体-细胞-分子基因水平探讨了活血药苏木、益气活血药苏木+黄芪对肺癌生长转移过程中CD4~+CD25~+Treg介导的肿瘤免疫逃逸影响差异的分子机制和关键作用环节,为肿瘤临床治疗中更加合理有效地应用活血药、益气活血药提供了科学依据。
2.2在既往研究基础上,通过动态观察Lewis肺癌荷瘤小鼠在肿瘤自然病程中不同时间点脾CD4~+CD25~+Foxp3~+细胞表达,并分析其与小鼠体重、生存期等的关系以及活血药苏木、益气活血药苏木+黄芪的影响作用,提出干预CD4~+CD25~+Treg及相关活化和调控分子、效应因子表达是活血药、益气活血药在肿瘤生长转移过程中作用差异产生的重要机制,首次结合肿瘤免疫编辑学说中免疫监视和免疫逃逸理论,从影响T淋巴细胞和肿瘤微环境两方面评价不同中医治法各自特点和治疗结果差异的原因。
2.3本研究从小样本临床研究切入,证实肺癌患者CD4~+CD25~+Treg检测的重要临床意义;继而通过动物抑瘤实验从三组不同活血药、益气活血药中筛选出一组对Lewis肺癌转移影响差异最大的药物苏木、苏木+黄芪作为代表进行深入研究;然后采用细胞分选、流式细胞术、实时定量PCR、荧光显微镜图像分析、ELISA、CBAflexset等技术从动物实验角度研究活血药、益气活血药对CD4~+CD25~+Treg及相关活化和调控分子、效应因子表达量的影响;最后采用MTT、transwell小室、细胞共培养等技术从体外实验角度观察活血药、益气活血药对Treg的产生的干预作用。围绕CD4~+CD25~+Treg作用机制的主线,通过不同层次研究,较为全面地观察了中药对肺癌生长转移过程中免疫逃逸相关分子表达和功能的干预作用。
2.4临床实验研究中将免疫学新指标与中医基础理论相结合,首次探讨了原发性非小细胞肺癌患者外周血CD4~+CD25~+Foxp3~+Treg表达与肺癌中医证候的关系。
The role that blood activating drugs play in tumor therapy is always in dispute. The clinical experience showed that we can enhance the effect of blood activating drugs on improving the high blood coagulation state of tumor-bearing body and reverse(or decrease) the possible trend of promoting tumor metastasis by reasonable compatibility.The earlier studies of my mentor have observed influence of different types of blood activating drugs compatibility with qi-deficiency drug(Radix Astragali) in different doses on tumor metastasis.The results showed that high dosage qi-deficiency drug compatibility with blood activating drugs could improve the tumor inhibiting rate and reverse the trend of promoting tumor metastasis of simple blood activating drugs.The results of mechanism study showed that the supplementing qi and activating blood circulation drugs could down-regulate the expression of CD4~+CD25~+ Treg.The study needs further research.
The study was carried out on the base of the earlier studies,and was supported by NSFC.We carried out a small sample clinical research on the difference of the expression of CD4~+CD25~+Foxp3~+ Treg in peripheral blood between patients with NSCLC and healthy volunteers and its relationship with main features and TCM syndrome of lung cancer.After the difference comparison studying on the inhibiting rate of lung metastasis in Lewis lung cancer model in mice,we chose a pair of representative drugs(Caesalpinina sappan(SM) and Caesalpinina sappan+Radix Astragali(SMHQ)) from three types of blood activating drugs(Salvia Miltiorrhiza Bunge,Caesalpinina sappan,Hirudo) and three types of supplementing qi and activating blood circulation drugs(blood activating drugs compatibility with Radix Astragali).In animal experiments we observed the expressions of CD4~+CD25~+Treg and its related activated molecules,regulatory molecules,effector molecules,main genes in Jagged1-Notch signal transduction pathway and cell surface molecules of DC intervened by Caesalpinina sappan(SM) and Caesalpinina sappan+Radix Astragali(SMHQ),using the main techniques of MACS,FCM,real-time PCR, fluorescence microscope,ELISA and CBA flexset.In vitro experiments we observed the ability of cell proliferation,movement,invasion in PG intervened by Caesalpinina sappan(SM) and Caesalpinina sappan+Radix Astragali(SMHQ) medicated serum, using the main techniques of MTT and transwell.We also established the mixed culture system of DCs,CD4~+T cells and culture supernatant of PG to study the T cell differentiation intervened by Caesalpinina sappan(SM) and Caesalpinina sappan+ Radix Astragali(SMHQ) medicated serum.The study explored the molecular mechanisms and key links of the different effects of blood activating drugs and supplementing qi and activating blood circulation drugs on tumor growth and metastasis from the angle of tumor immune escape.We have obtained high academic valuable data.
1 The primary research conclusions
1.1 Detection of CD4~+CD25~+Foxp3~+ regulatory T cells in peripheral blood of patients with NSCLC and its clinical significance
By detecting CD4~+CD25~+Foxp3~+ regulatory T cells in peripheral blood of 30 patients with NSCLC and 12 healthy volunteers and analyzing their clinical information,we found that:The population of CD4~+CD25~+Foxp3~+ Treg in peripheral blood from patients with NSCLC was higher in comparison with that in healthy volunteers,which conformed the documental reports.The proportion of CD4~+CD25~+Foxp3~+ Treg in the patients with stageⅣlung cancer was higher than those in stageⅠ-Ⅲ.The proportion of CD4~+CD25~+Foxp3~+ Treg in the patients with tumor was higher than those in the patients without tumor.Treg expression was not obviously correlated with different genders,ages,pathological types,KPS and level of tumor markers.The increase of the proportion of CD4~+CD25~+Foxp3~+ Treg in the patients most closely correlated with Qi deficiency syndrome and blood stasis syndrome.These results had provided data basis for animal and vitro experiments on the relationship between CD4~+CD25~+Foxp3~+Treg and lung cancer.
1.2 The effects on Lewis lung cancer growth and metastasis intervened by different types of blood activating drugs and supplementing qi and activating blood circulation drugs
By studying on the inhibiting rate of lung metastasis in Lewis lung cancer model in mice,we observed the intervention effect of three types of blood activating drugs (Salvia Miltiorrhiza Bunge,Caesalpinina sappan,Hirudo) and three types of supplementing qi and activating blood circulation drugs(blood activating drugs compatibility with Radix Astragali).We found that:①Salvia Miltiorrhiza Bunge had showed the trend of promoting tumor metastasis while Salvia Miltiorrhiza Bunge+Radix Astragali could not reverse it.Caesalpinina sappan,Caesalpinina sappan+Radix Astragali,Hirudo,Hirudo+Radix Astragali had showed good curative effect on decrease tumor metastasis,which suggested that different types of blood activating drugs and supplementing qi and activating blood circulation drugs had different effects.②Supplementing qi and activating blood circulation drugs had better effect on decrease tumor growth and metastasis than blood activating drugs.So we chose a pair of representative drugs(Caesalpinina sappan(SM) and Caesalpinina sappan+Radix Astragali(SMHQ)) for further research.
1.3 Continuous observation on the expression of CD4~+CD25~+Foxp3~+ regulatory T cells in spleen of tumor-bearing mice intervened by blood activating drugs and supplementing qi and activating blood circulation drugs
By Continuous observing on the body weight,tumor weight,expression of CD4~+CD25~+Foxp3~+ regulatory T cells in spleen of tumor-beating mice intervened by Caesalpinina sappan(SM) and Caesalpinina sappan+Radix Astragali(SMHQ) and their survival time,we found that:①Traditional Chinese medicine had showed a more obvious tumor inhibiting effect in the early stage.②Caesalpinina sappan(SM) had showed a better tumor inhibiting effect in the early stage,while the advantages of Caesalpinina sappan+Radix Astragali(SMHQ) was in the inhibiting rate of lung metastasis and survival time.③The expression of CD4~+CD25~+Foxp3~+ Treg in spleen of tumor-beating mice in control group was increased followed with the tumor progression.④Caesalpinina sappan(SM) and Caesalpinina sappan+Radix Astragali (SMHQ) had different effect on the changing tendency of the expression of CD4~+CD25~+Foxp3~+ Treg.The decreased of CD4~+CD25~+Foxp3~+ Treg(20d) was obvious in SMHQ group.These results showed that the supplementing qi and activating blood circulation drugs could improve the tumor-bearing mouse immune tolerance condition by Treg down-regulation and this effect was better than that of blood activating drugs.
1.4 The effects on the levels of cytokines in CD4~+ cell culture supernatant in spleen of tumor-bearing mice and the expression of main genes in Jagged1-Notch signal transduction pathway intervened by blood activating drugs and supplementing qi and activating blood circulation drugs
By detecting the levels of cytokines in CD4~+ cell culture supernatant in spleen of tumor-bearing mice and the expression of main genes in Jagged1-Notch signal transduction pathway intervened by Caesalpinina sappan(SM) and Caesalpinina sappan+Radix Astragali(SMHQ),we found that:①The levels of IL-2,IFN-γin CD4~+ cell culture supernatant in spleen of tumor-bearing mice in SMHQ group was obviously higher than that in control group,while the levels of TGF-β1 was lower than that in control group.②Caesalpinina sappan(SM) and Caesalpinina sappan+Radix Astragali (SMHQ) had no significant effect on the expression of Notch1,Jagged1 mRNA.These results showed that the supplementing qi and activating blood circulation drugs could improve the tumor-bearing mouse immune tolerance condition by regulating the levels of cytokines in tumor-bearing mice and this effect was better than that of blood activating drugs.
1.5 The effects on the expressions of genes related with immune escape in tumor-bearing mice intervened by blood activating drugs and supplementing qi and activating blood circulation drugs
By real-time RT-PCR,we detected the expressions of Foxp3 and CTLA-4 mRNA in tumor tissues and in CD4~+CD25~+cells selected from spleen cells of tumor-bearing mice by magnetic activated cell sorting system.We discovered that:The expressions of Foxp3 and CTLA-4 mRNA in tumor tissues and the expression of Foxp3 mRNA in CD4~+CD25~+cells selected from spleen cells of tumor-bearing mice in SMHQ group were lower than that in control group.These results showed that the supplementing qi and activating blood circulation drugs could improve the tumor-bearing mouse immune tolerance condition by down-regulating the expressions of genes related with immune escape and this effect was showed in both lymphoid tissue and tumor micro-environment.
1.6 The effects on the expressions of phenotypes of DC in spleen of tumor-bearing mice intervened by blood activating drugs and supplementing qi and activating blood circulation drugs
By flow cytometry,we detected the expressions of phenotypes of DC in spleen of tumor-bearing mice.We discovered that:①There was a significantly decrease of the expressions ofphenotypes of DC in tumor-bearing bodies.②Radix Astragali(SMHQ) could increase the expressions of mature phenotypes of DC in spleen of tumor-bearing mice and this effect was better than that of blood activating drugs.These results had provided data basis for vitro experiments on the relationship between the mature degree of DC and differentiation of T cells.
1.7 The effects on the ability of cell proliferation,movement,invasion in PG intervened by Caesalpinina sappan(SM) and Caesalpinina sappan+Radix Astragali(SMHQ) medicated serum
By detecting the effects on the ability of cell proliferation,movement,invasion in PG intervened by Caesalpinina sappan(SM) and Caesalpinina sappan+Radix Astragali (SMHQ) medicated serum,we found that:①Caesalpinina sappan(SM) and Caesalpinina sappan+Radix Astragali(SMHQ) had no significant effect on the ability of cell proliferation in PG.②Caesalpinina sappan+ Radix Astragali(SMHQ) medicated serum had significant inhibitory effect on the ability of cell movement,invasion in PG.These results showed that traditional Chinese medicine could inhibit tumor growth and metastasis in many different targets and many different ways.Directly killing the tumor cells was only one side of these effects.
1.8 The effects on the expressions of phenotypes of DC in vitro intervened by culture supernatant of PG
In this study,PBMC was isolated from human peripheral blood,differentiation into different mature degrees of DC after induction in vitro.Then culture supernatant of PG was added into the culture system to simulate tumor microenvironment.By flow cytometry,we detected the expressions of phenotypes of DC in the two culture systems.We discovered that:The culture supernatant of PG could decrease the mature degree of DC in vitro.These results had provided premise conditions and experimental method for further study.
1.9 The effects on the mixed culture system of DCs,CD4~+T cells and culture supernatant of PG intervened by Caesalpinina sappan(SM) and Caesalpinina sappan+Radix Astragali(SMHQ) medicated serum
In this study,we established the mixed culture system of DCs,CD4~+T cells and culture supernatant of PG.By detecting the expression of CD4~+CD25~+Foxp3~+Treg, the level of IL-12 and the expression of Jagged1,Notch1 mRNA in the mixed culture system,we found that:①In the mixed culture system intervened by culture supernatant of PG,the expression of CD4~+CD25~+Foxp3~+Treg and Jagged1,Notch1 mRNA was higher compared with the control group while the level of IL-12 was decreased.②Caesalpinina sappan(SM) and Caesalpinina sappan+Radix Astragali (SMHQ) medicated serum had no significant effect on the differentiation of T cells and the level of IL-12 in the mixed culture system.③Caesalpinina sappan+Radix Astragali(SMHQ) medicated serum had inhibitory effect on CD4~+CD25-T cells differentiating into CD4~+CD25~+T cells.④Caesalpinina sappan+Radix Astragali (SMHQ) medicated serum could increase the level of IL-12 in the mixed culture system.⑤Caesalpinina sappan(SM) and Caesalpinina sappan+Radix Astragali (SMHQ) medicated serum had no significant effect on the expression of Notch1, Jagged1 mRNA in the mixed culture system.These results showed that the CD4~+CD25T cells differentiating into CD4~+CD25~+T cells in the mixed culture system intervened by culture supematant of PG related to many factors,including inhibitory cytokines secreted by tumor cells,the mature degree of DC and stimulating amount of tumor antigen.
In this research,patients with NSCLC,Lewis lung cancer-bearing mice and PG cells were taken as the research objects.We discussed on the relationship between CD4~+CD25~+Foxp3~+Treg and the tumor growth and metastasis from three different levels of clinical experiments,animal experiments and in vitro experiments.The expressions of CD4~+CD25~+Treg and its related activated molecules,regulatory molecules,effector molecules,main genes in Jaggedl-Notch signal transduction pathway and cell surface molecules of DC intervened by Caesalpinina sappan(SM) and Caesalpinina sappan+Radix Astragali(SMHQ) were observed in both tumor tissue and T cells.The study explored the molecular mechanisms and key links of the different effects of blood activating drugs and supplementing qi and activating blood circulation drugs on tumor growth and metastasis from the levels of whole body,cell mechanism and molecular mechanism.The results had provided a scientific basis for more reasonable and effective clinical application of blood activating drugs and supplementing qi and activating blood circulation drugs.
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