【作者】 江志超；

【导师】 唐发清； 田道法；

【作者基本信息】 湖南中医药大学 ， 中西医结合临床， 2013， 博士

【摘要】 目的：通过检测鼻咽癌患者CD4+CD25+T调节细胞比例、转录因子Foxp3mRNA表达水平、细胞因子IFN-γ、IL-2、TGF-β、IL-10、IL-6、 IL-17含量、益气解毒方的干预作用及其对BALB/c小鼠的影响,分析CD4+CD25+T调节细胞在鼻咽癌肿瘤微环境免疫耐受现象中的免疫病理意义,探讨益气解毒方对鼻咽癌肿瘤微环境中CD4+CD25+T调节细胞主导的免疫耐受现象的调节作用,为建立鼻咽癌中医药免疫调节治疗提供新的思路。方法：1、收集健康人群、中晚期鼻咽癌患者外周血,流式细胞术检测外周血CD4+CD25+T调节细胞、CD4+CD25+Foxp3+T调节细胞和Th17细胞比例,比较分析其组间差异。2、将中晚期鼻咽癌患者分为常规治疗组和益气解毒方+常规治疗组,进行临床研究。流式细胞术检测外周血CD4+CD25+T调节细胞、CD4+CD25+Foxp3+T调节细胞和Th17细胞比例,对比分析常规治疗组、益气解毒方+常规治疗组的组间差异。3、随机将BALB/c小鼠分成三个组,空白组、生理盐水组和益气解毒方组,进行相应处理。采集小鼠外周血,流式细胞术检测血标本CD4+CD25+T调节细胞、CD4+CD25+Foxp3+T调节细胞比例,比较其组间差异。4、采集中晚期鼻咽癌患者和健康人群外周血,分离血清,ELISA检测血清细胞因子IFN-γ、IL-2、TGF-β、IL-10、IL-6、IL-17含量,比较分析组间差异。5、采集鼻咽癌患者常规治疗组和益气解毒方+常规治疗组外周血,分离血清。ELISA法检测血清细胞因子IFN-γ、IL-2、TGF-β、 IL-10、IL-6、IL-17含量,比较分析其组间差异。6、采集空白组、生理盐水组和益气解毒方组BALB/c小鼠静脉血,ELISA法检血清细胞因子IFN-γ、IL-2、TGF-β、IL-10、IL-6、 IL-17含量,比较分析其组间差异。7、采集中晚期鼻咽癌患者和健康人群静脉血,RT-PCR法检测外周血Foxp3mRNA、ROR-γt mRNA转录水平,比较其组间差异,分析其与CD4+CD25+T调节细胞比例的关系。8、分别采集常规治疗组、益气解毒方+常规治疗组鼻咽癌患者外周血,分离单个核细胞。提取总RNA, RT-PCR法检测外周血Foxp3mRNA、ROR-γt mRNA转录水平,比较分析其组间差异。结果：1、流式细胞术检测结果表明,健康人群、中晚期鼻咽癌患者外周血CD4+CD25+T调节细胞比例分别为(2.65±0.31)%、(4.23±0.53)%, CD4+CD25+Foxp3+T调节细胞比例分别为(0.48±0.05)%、(0.98±0.15)%,Th17细胞比例分别为(1.80±0.30)%、(0.50±0.21)%。中晚期鼻咽癌患者外周血CD4+CD25+、CD4+CD25+Foxp3+T细胞比例显著高于健康人群(P<0.05),而Th17细胞比例显著降低(P<0.05)。2、临床治疗观察结果显示,常规治疗组、益气解毒方+常规治疗组外周血CD4+CD25+T细胞比例分别为(4.75±0.39)%、(3.09±0.49)%, CD4+CD25+Foxp3’T调节细胞比例分别为(0.93±0.15)%、(0.53±0.04)%,Th17细胞比例分别为(0.63±0.20)%、(2.09±0.34)%。益气解毒方+常规治疗组患者外周血CD4+CD25+T调节细胞、CD4+CD25+Foxp3+T调节细胞比例显著降低(P<0.05),而Th17细胞比例则显著升高(P<0.05)。3、动物实验结果显示,空白对照组、生理盐水组、益气解毒方组BALB/c小鼠外周血CD4+CD25+T细胞比例分别为(6.53±1.09)%.(6.79±1.22)%.(4.52±0.61)%,CD4+CD25+Foxp3+T调节细胞比例分别为(12.16±2.51)%、(11.01±2.00)%、(8.25±1.43)%。益气解毒方组小鼠外周血CD4+CD25+T调节细胞、CD4+CD25+Foxp3+T调节细胞显著降低(P<0.01)；而空白对照组与生理盐水组相比较,则其无明显差异(P>0.05)。4、外周血细胞因子测定表明,健康人群IFN-γ、IL-2、IL-17、 TGF-β、IL-10、IL-6分别为(220.36±18.76)pg/ml.(174.79±7.55) pg/ml、(7.71±0.34)pg/ml、(488.82±36.91)pg/ml、(0.68±0.08)pg/ml、(1.37±0.03)pg/ml,中晚期鼻咽癌患者IFN-γ、IL-2、IL-17、TGF-β、 IL-10、IL-6分别为(124.95±4.22)pg/ml、(81.88±10.84)pg/ml、(4.61±0.09)pg/ml、(645.56±39.61)pg/ml、(1.27±0.21)pg/ml、(1.88±0.13)pg/ml、中晚期鼻咽癌患者IFN-γ、IL-2、IL-17含量低于健康人群(P<0.05),而TGF-β、IL-10、IL-6含量则高于健康人群(P<0.05)。5、中晚期鼻咽癌患者临床治疗实验结果表明,常规治疗组患者血清IFN-γ、IL-2、IL-17、TGF-β、IL-10、IL-6分别为(155.26±4.99) pg/ml、(128.89±9.27) pg/ml、(2.97±0.14) pg/ml、(544.47±21.96) pg/ml、(0.99±0.04) pg/ml、(2.86±0.19) pg/ml。益气解毒方+常规治疗组IFN-γ、IL-2、IL-17、TGF-β、IL-10、IL-6分别为(321.60±41.19) pg/ml、(231.91±24.24) pg/ml、(5.54±0.14) pg/ml、(421.62±25.42) pg/ml、(0.67±0.11) pg/ml、(1.47±0.05) pg/ml。益气解毒方+常规治疗组患者IFN-γ、IL-2、IL-17含量显著升高(P<0.05),而TGF-β、 IL-10、IL-6含量则显著降低(P<0.05)。6、在益气解毒方干预实验中,益气解毒方组IFN-γ、IL-2、IL-17、 TGF-β、IL-10、IL-6分别为(229.72±5.72) pg/ml、(70.24±3.34) pg/ml、(11.22±0.35) pg/ml、(126.31±16.50) pg/ml、(128.43±9.86) pg/ml、(20.55±0.92) pg/ml。与空白组、生理盐水组相比,益气解毒方组BABL/c小鼠TGF-β、IL-10含量显著降低(P<0.05),而IFN-γ、IL-2、 IL-17、IL-6含量明显升高(P<0.05)；空白组与对照组相比较则差异无统计学意义(P>0.05)。7、Foxp3mRNA检测结果发现,中晚期鼻咽癌患者外周血Foxp3mRNA转录水平(3.699±0.309)显著高于健康人群(1.019±0.146)(P<0.05),而ROR-γt mRNA水平(0.303±0.115)则低于健康人群(1.007±0.039)(P<0.05), CD4+CD25+Treg细胞与CD4+CD25Toxp3-细胞比例呈正相关(r2=0.163,P<0.05),而Foxp3mRNA与ROR-yt mRNA转录水平并不相关(r2=-0.127,P>0.05)。加用益气解毒方干预后,患者外周血Foxp3mRNA转录水平明显降低(P<0.05),而ROR-yt mRNA水平则明显升高(P<0.05)。结论：1. CD4+CD25+T调节细胞比例及功能活性是中晚期鼻咽癌患者肿瘤微环境免疫耐受形成的重要因素之一,有效干预CD4+CD25+T调节细胞和Th17细胞免疫活性及分化影响因素,可改善鼻咽癌肿瘤微环境中的免疫耐受现象。2、细胞因子IFN-γ、IL-2、IL-17及转录因子ROR-yt mRNA是中晚期鼻咽癌患者CD4+CD25+T调节细胞的负性调节因子,而细胞因子TGF-β、IL-6、IL-10及转录因子Foxp3mRNA是其正性因子。3、中药复方益气解毒方的有效干预可以降低中晚期鼻咽癌患者免疫抑制细胞CD4+CD25+T调节细胞比例和活性,提高免疫效应细胞Th17比例和功能活性,有助于实现鼻咽癌肿瘤微环境中免疫耐受现象的逆转,进而增强鼻咽癌患者的抗瘤能力。4、益气解毒方对CD4+CD25+T调节细胞的抑制效应,可能是通过对其负性与正性双调节而实现的,体现了中药复方多靶点微效应的药理作用模式特点。5、益气解毒方对BALB/c小鼠CD4+CD25+T调节细胞比例及功能活性、负性调节因子TGF-β、IL-10表达水平、正性调节因子IFN-γ、IL-2、IL-17、JL-6分泌水平的作用模式,佐证了益气解毒方对中晚期鼻咽癌患者CD4+CD25+T调节细胞的干预作用模式特点。6、由CD4+CD25+T调节细胞主导的肿瘤微环境免疫耐受现象的逆转,可能成为中药抗肿瘤疗法的重要途径,藉此体现并充分发挥中医药抗肿瘤的独特优势。更多还原

【Abstract】 OBJECTIVETo detect the percentage of CD4+CD25+Tregs cells,the levels of Foxp3mRNA, the content of its relative cytokines (IFN-γ、IL-2、TGF-β、 IL-10、IL-6、IL-17), intervention reaction of QBTRF in the patients with middle to late staged NPC and QBTRF’s influence on BALB/c animals, the pathological significance of CD4+CD25+Tregs cells and the reversing effect of Qi-Boosting Toxin-Resolving Formula(QBTRF) were investigated in the immune tolerance of tumor microenvironment among patients with middle to late staged nasopharyngeal carcinoma (NPC), which it is provided a new clue for establishing immune regulation therapy of traditional Chinese medicine in NPC.METHODS1.The peripheral blood samples were taken form healthy population, patients with middle to late staged NPC. Flow-cytometry was performed to detect the percentage of CD4+CD25+Tregs cells, CD4+CD25+Foxp3Tregs cells and Th17cells of peripheral blood cells, which the differences was compared between these groups.2.The percentage of CD4+CD25+Tregs cells, CD4+CD25+Foxp3Tregs cells and Th17cells were detected by Flow-cytometry in the peripheral blood among middle to late staged NPC patients treated by conventional therapy and conventional therapy add to QBTRF, which the differences was compared between these groups.3.The percentage of CD4+CD25+Tregs cells, CD4+CD25+Foxp3Tregs cells and Th17cells were detected by Flow-cytometry in peripheral blood of the blank group, saline group and QBTRF group of BALB/c mice, which the differences was compared between these groups.4.The serum levels of IFN-γ, IL-2, TGF-p, IL-10, EL-6and IL-17were determined by ELISA in the healthy population and patients with middle to late staged NPC, which the differences of every cytokine was compared between these groups.5.The serum levels of IFN-γ, IL-2, TGF-β, IL-10, IL-6and IL-17were determined by ELISA in the peripheral blood among middle to late staged NPC patients treated by conventional therapy and conventional therapy add to QBTRF, which the differences of every cytokine was compared between these groups.6.To further confirm the regulatory effect of QBTRF on immune system, the serum levels of IFN-γ, IL-2, TGF-β, IL-10, IL-6and IL-17were determined by ELISA in peripheral blood of the blank group, saline group and QBTRF group of BALB/c mice, which the differences of every cytokine was compared between these groups.7.The transcriptional levels of Foxp3mRNA and ROR-yt mRNA were detected by real-time PCR in the peripheral blood among the healthy population and patients with middle to late staged NPC, which the differences the relation of CD4+CD25+Tregs cells, Foxp3mRNA and ROR-yt mRNA was compared between these groups.8.The transcriptional levels of Foxp3mRNA and ROR-yt mRNA were detected by real-time PCR in the peripheral blood among middle to late staged NPC patients treated by conventional therapy and conventional therapy add to QBTRF, which the differences was compared between these groups.RESULTS1. The results of flow cytometry show that the percentage of CD4+CD25+Tregs cells were respectively (2.65±0.31)%、(4.23±0.53)%, the percentage of CD4+CD25+Foxp3Tregs cells were respectively (0.48±0.05)%、(0.98±0.15)%, and the percentage of Th17cell were respectively (1.80±0.30)%、(0.50±0.21)%in peripheral blood among healthy control and middle to late staged NPC patients. Compared with the healthy control, the percentage of CD4+CD25+Tregs cells and CD4+CD25TFoxp3Tregs cells was significantly higher in peripheral blood among middle to late staged NPC patients (P<0.05), while the percentage of Th17cell is significantly decreased (P<0.05).2. The results of clinic treatment show that the percentage of CD4+CD25+Tregs cells were respectively (4.75±0.39)%、(3.09±0.49)%, the percentage of CD4+CD25+Foxp3Tregs cells were respectively (0.93±0.15)%、(0.53±0.04)%, and the percentage of Th17cell were respectively (0.63±0.20)%、(2.09±0.34)%in peripheral blood among NPC patients treated by conventional therapy and conventional therapy add to QBTRF. Compared with NPC patients treated by conventional therapy, the percentage of CD4+CD25+Tregs cells and CD4+CD25+Foxp3Tregs cells was significantly lower in peripheral blood among NPC patients treated by conventional therapy add to QBTRF (P<0.05), while the percentage of Th17cell is significantly increased (P<0.05).3. The results of animal experiment show that the percentage of CD4+CD25+Tregs cells were respectively (6.53±1.09)%、(6.79±1.22)%、(4.52±0.61)%, and the percentage of CD4+CD25+Foxp3Tregs cells were respectively (12.16±2.51)%、(11.01±2.00)%、(8.25±1.43)%in peripheral blood among the blank group, the NS group and QBTRF of BALB/c mouse. Compared with the blank group and the NS group of BALB/c mouse, the percentage of CD4+CD25+Tregs cells and CD4+CD25+Foxp3Tregs cells was significantly lower in peripheral blood among BABL/c mice intervened by QBTRF (P<0.05), while the percentage of CD4+CD25+Tregs cells and CD4+CD25+Foxp3Tregs cells had no significant difference between the blank group and the NS group (P>0.05).4. The detection of cytokines show that the serum levels of IFN-γ、IL-2、 IL-17、TGF-β、IL-10、IL-6were respectively (220.36±18.76) pg/ml、(174.79±7.55) pg/ml、(7.71±0.34) pg/ml、(488.82±36.91) pg/ml、(0.68±0.08)pg/ml、(1.37±0.03)pg/ml in peripheral blood among among the healthy control, the serum levels of IFN-γ、IL-2、IL-17、TGF-β、 IL-10、IL-6were respectively (124.95±4.22) pg/ml、(81.88±10.84) pg/ml、(4.61±0.09) pg/ml、(645.56±39.61) pg/ml、(1.27±0.21) pg/ml、(1.88±0.13) pg/ml in peripheral blood among middle to late staged NPC patients. Compared with the healthy control, the serum levels of TGF-β、IL-10、IL-6was significantly higher in peripheral blood among middle to late staged NPC patients (P<0.05), while the serum levels of IFN-γ、IL-2、IL-17is significantly decreased (P<0.05).5.The experimental result of clinical treatment in middle to late staged NPC show that the serum levels of IFN-γ、IL-2、IL-17、TGF-β、IL-10、 IL-6were respectively (155.26±4.99) pg/ml、(128.89±9.27) pg/ml、(2.97±0.14) pg/ml、(544.47±21.96) pg/ml、(0.99±0.04) pg/ml、(2.86±0.19) pg/ml in the NPC group treated by conventional therapy. The serum levels of IFN-γ、IL-2、IL-17、TGF-β、IL-10、IL-6were respectively (321.60±41.19)pg/ml、(231.91±24.24)pg/ml、(5.54±0.14) pg/ml、(421.62±25.42) pg/ml、(0.67±0.11) pg/ml、(1.47±0.05) pg/ml in the NPC group treated by conventional therapy add to QBTRF. Compared with the NPC group treated by conventional therapy, the serum levels of IFN-γ、IL-2、IL-17was significantly higher in the NPC group treated by conventional therapy add to QBTRF (P<0.05), while the serum levels of TGF-P, IL-10, IL-6is significantly decreased (P<0.05).6. The serum levels of IFN-γ、IL-2、IL-17、TGF-β、IL-10、IL-6were respectively (229.72±5.72) pg/ml.(70.24±3.34) pg/ml、(11.22±0.35) pg/ml、(126.31±16.50)pg/ml、(128.43±9.86)pg/ml、(20.55±0.92) pg/ml in peripheral blood among BABL/c mice intervened by QBTRF. Compared with the blank group and NS group of BALB/c mouse, the serum levels of TGF-β、IL-10、IL-6was significantly decreased (P<0.05), but IFN-γ、IL-2、IL-17was significantly higher in peripheral blood among BABL/c mice intervened by QBTRF (P<0.05), while there was no significant difference between the blank group and the NS group (P>0.05).7.Compared with the healthy controls, the transcriptional levels(3.699±0.309) of Foxp3mRNA was significantly higher in peripheral blood among patients with middle to late staged NPC (P<0.05), while ROR-yt mRNA(O.303±0.115) was significantly decreased (P <0.05). The ratio of CD4+CD25+Tregs cells was positively related with CD4+CD25+Foxp3Tregs cells in peripheral blood among patients with middle to late staged NPC (r2=0.163, P<0.05),but there was no relation between the transcriptional levels of Foxp3mRNA and ROR-yt mRNA(r2=-0.127, P>0.05). The transcriptional levels of ROR-yt mRNA is significantly higher in peripheral blood among the NPC treated by QBTRF (P<0.05), while the transcriptional levels of Foxp3mRNA is significantly decreased (P<0.05).CONCLUSION1.The advantaged percentage and functional activity of CD4+CD25+Tregs cells is one of important reasons produced the phenomenon of immune tolerance in the tumor microenvironment in patients with middle to late staged NPC, which was maintained by influencing the differentiatial factors of CD4+CD25+Tregs cells and Th17cells and its immune activity.2.The activity regulation and effect factors of CD4+CD25+Tregs cells is negatively related with the cytokines of IFN-y, IL-2, IL-17and transcriptional factor ROR-yt mRNA in patients with middle to late staged NPC, but positively with the level of TGF-β、IL-6、IL-10and transcriptional factor Foxp3mRNA3. Intervention effect of compound Chinese medicine QBTRF can reduce the percentage and functional activity of suppressor cell CD4+CD25+Tregs cells in patients with middle to late staged NPC, improve the percentage and functional activity of effect cell Th17cells, which be help to reverse the phenomenon of immune tolerance in tumor microenvironment and enhance the resistance of tumor in patients with NPC.4. Inhibition effect of QBTRF for CD4+CD25+Tregs cells may be realized on the extensively intervention of negative and positive regulating factors, which embodies pharmacological pattern characteristics of more target micro effect in traditional Chinese medicine compound.5. The percentage and functional change of CD4+CD25+Tregs cells, the expression levels of negative regulation factors(TGF-β、IL-10) and the secretion levels of positive regulation factors (IFN-γ、IL-2、IL-17、IL-6) in BALB/c mice intervened by QBTRF further supported the role characteristics of QBTRF for CD4+CD25+Tregs cells in patients with middle to late staged NPC.6.The reversal of immune tolerance dominated by CD4+CD25+Tregs cells in the tumor microenvironment should be the important way of antitumor therapy of traditional Chinese medicine, which embody and give full play to the unique advantages of traditional Chinese medicine anticancer.更多还原