【作者】 孙雯；

【导师】 俞建； 汪永红； 张皓；

【作者基本信息】 复旦大学 ， 儿科学， 2009， 硕士

【摘要】 目的通过临床观察,客观评价槐耳对哮喘缓解期儿童的干预效果;并通过体外实验,探讨槐耳改善哮喘缓解期儿童T淋巴细胞调控失衡的机理,为槐耳的进一步应用提供理论依据。方法第一部分:槐耳对哮喘缓解期儿童临床干预作用的评估将60例哮喘缓解期儿童分为2组,即对照组和槐耳组,分别给予中药调理方及中药调理方+槐杞黄颗粒,通过门诊随访的方式,观察并记录以下情况:①用药前后患儿中医证候群的累计例数;②应用ACT/C-ACT量表,每隔4周对病人进行一次哮喘控制水平评估,其时间点为首诊、第4周、第8周和第12周时。第二部分:槐耳对哮喘缓解期儿童T淋巴细胞体外干预作用的观察应用肝素抗凝管采集哮喘缓解期儿童外周血3ml,用Ficoll法分离PBMCs,均分为3组,即对照组、中剂量组和大剂量组,分别与不同浓度的槐耳菌质配液(0、3、6μg/ml)及培养液作用48小时;然后利用Real-Time PCR的方法测定细胞培养沉淀中IFN-γ,、IL-4、IL-10 mRNA的表达及T-bet、GATA-3、Foxp3 mRNA的表达,利用ELISA的方法测定细胞培养上清中IFN-γ、IL-4及IL-10的表达。统计学分析:第一部分数据为计数资料,第二部分数据以均数±标准差((?)±s)表示。采用SPSS16.0统计软件处理。P<0.05表示有统计学意义。结果第一部分:1、中医证候群的差异表达1.1在槐耳组和对照组中,治疗后患儿面色淡白、自汗、倦怠乏力、舌苔白、舌苔花剥及脉细无力的中医证候较治疗前有所改善(23vs14/21vs12,21vs11/20vs11,18vs9/15vs6等等,P值<0.05);其中,治疗后槐耳组中的自汗症状、两组中的舌苔花剥和脉细无力症状较治疗前有明显的改善(21vs11,10vs1,12vs0等等,P值<0.01)。但是,患儿纳差的中医证候在治疗前后改善不明显(12vs6,10vs4,P值>0.05)。1.2在对照组中,患儿大便干结与盗汗的中医证候在治疗前后改善不明显(13vs8,22vs15,P值>0.05);在槐耳组中,以上两个证候治疗前后有明显的改善(12vs2,19vs7,P值<0.01)。1.3在治疗前,中医证候大便干结与盗汗在槐耳组与对照组间无差异表达(12vs13,19vs22,P值>0.05);但在治疗后,大便干结与盗汗在槐耳组与对照组中的组间差异具有统计学意义(2vs8,7vs15,P值<0.05)。2、ACT/C-ACT的差异表达2.1在槐耳组,将第4周、第8周和第12周的哮喘控制测试(Asthma ControlTest,ACT)水平随访结果,分别与首诊时的ACT水平相比较,其差异表达无统计学意义P值>0.05);而在对照组,第4周与第8周的ACT水平随访结果,与首诊的ACT水平相比较,其差异表达存在统计学意义(P值<0.05)。2.2在治疗后第4周和第12周时,槐耳组与对照组的ACT水平间的差异表达无统计学意义(P值>0.05);而在治疗后第8周时,槐耳组与对照组ACT水平之间存在统计学差异(P值<0.05)。第二部分:1、T淋巴细胞相关细胞因子mRNA水平的表达:细胞沉淀中IFN-γmRNA在三组中两两组间的差异表达无统计学意义(2.21±0.26vs2.17±0.33vs2.10±0.31,P值>0.05)。IL-4 mRNA在大剂量组的表达明显低于对照组(0.86±0.13vs1.28±0.14,P值<0.01);同时,亦低于中剂量组(0.86±0.13vs1.21±0.17,P值<0.05)。IL-10 mRNA在大剂量组的表达高于对照组(2.99±0.33vs2.63±0.36,P值<0.05)。2、T淋巴细胞相关转录因子mRNA水平的表达:细胞沉淀中T-bet、Foxp3mRNA在大剂量组的表达均低于对照组(2.40±0.30vs2.63±0.25,2.31±0.31vs2.60±0.39,P值<0.05);GATA-3 mRNA在大剂量组的表达明显低于与对照组(1.56±0.22vs1.87±0.29,P值<0.01);同时,办低于中剂量组(1.56±0.22vs1.71±0.24,P值<0.05)。3、T淋巴细胞相关细胞因子蛋白水平的表达:细胞培养上清中IFN-γ、IL-10在大剂量组的表达均高于对照组(672.78±184.72vs503.74±189.78,586.47±191.83vs431.67±135.25,P值<0.05);IL-4在三组中两两组间差异表达无统计学意义(22.76±7.08vs21.17±6.84vs18.73±4.55,P值>0.05)。4、Th1/Th2细胞相关细胞因子及转录因子比值的表达:细胞沉淀中IFN-γ/IL-4 mRNA在大剂量组的表达明显高于对照组和中剂量组(2.61±0.59vs1.91±0.61,2.61±0.59vs2.00±0.62,P值<0.01);T-bet/GATA-3 mRNA在三组中两两组间差异的表达没有统计学意义(1.43±0.22vs1.52±0.22vs1.55±0.24,P值>0.05)。细胞培养上清中IFN-γ/IL-4在大剂量组的表达高于对照组(37.90±13.59vs25.62±15.28,P值<0.05)。结论1、与对照组相比较,槐耳组可有效改善中医证候群中的大便干结、盗汗两项症状;此外,在治疗后第8周的随访中发现,槐耳组在控制哮喘发作时优于对照组,以上结果提示槐耳可以对哮喘缓解期儿童进行有效的干预,从而改善患儿体质,调整肺脾肾不足,以此达到维持病情稳定的目的。2、体外实验结果显示,槐耳可上调培养上清中IFN-γ的表达,同时下调细胞沉淀中IL-4 mRNA的表达。以上结果提示槐耳可以通过上调Th1类细胞因子及下调Th2类细胞因子的途径,纠正Th1/Th2活性细胞所分泌细胞因子的偏移,从而进一步平衡Th1/Th2细胞比值的失衡。3、体外实验结果显示,槐耳可下调细胞沉淀中GATA-3 mRNA的表达,同时伴有T-bet mRNA的消耗。提示槐耳亦可通过下调Th2相关转录因子的途径,抑制Th0向Th2的分化,达到平衡Th1/Th2细胞比值的目的。4、体外实验结果显示,槐耳可上调细胞沉淀及培养上清中IL-10的表达,同时伴有Foxp3 mRNA的消耗。以上结果提示,槐耳可通过IL-10途径减轻患儿体内的慢性变应性炎症,同时也可作用于转录因子Foxp3,促进了未分化T细胞向CD4~+CD25~+Treg细胞的转化,抑制了Th2细胞的产生,调节了Th1/Th2细胞比值的失衡。更多还原

【Abstract】 Objective:To evaluate the effect of Trametes Robinioplila on asthmatic children during remission stage and the mechanism of Trametes Robinioplila,we also explored its regulating effects on the T lymphocyte imbalance through the experience in vitro, in order to find the theoretical basis of the fungus for treating asthma in remission stage.Methods:PartⅠ:The effect evaluation of Trametes Robinioplila on asthmatic children during remission stage.In clinical study,60 asthmatic children were divided into two groups,which were the Huaier group and the control group.All the children were followed up clinically being observed and the information was recorded as shown below:①the sample number of one of the TCM symptoms before and after the drug administering;②the asthma control levels on patients evaluated by the questionnaire of ACT/C-ACT every 4 weeks at the beginnng of the first-care.PartⅡ:The investigation of Trametes Robinioplila on the T lymphocyte in vitro in asthmatic children during remission stage.Peripheral venous blood（3ml） was collected from each of these children. PBMCs were isolated by density gradient centrifugation and seperated into three groups（the control group,the middle-dose group and the large-dose group）.The samples were then incubated in vitro for 48 hours in the absence（the control group） or presence（the middle-dose group and the large-dose group） of Trametes Robinioplila in different concentration（0,3,6μg/ml）.The expression of IFN-γ,IL-4,IL-10,T-bet, GATA-3 and Foxp3 in cellular precipitation was measured at mRNA levelby real-time fluorescence quantitative polymerase chain reaction technology（Real-Time PCR）, and that of IFN-γ,IL-4 and IL-10 in supernatant was measured by ELISA. Statistical analysis:All Data were analyzed with SPSS 16.0.The data were numeration data in the partⅠ,and those were measurement data presented as Means±SD in partⅡ.The P values are two-tailed and a P value of less than 0.05 is considered to be statistically significant. Results:PartⅠ:1.The expression of TCM symptoms1.1 In the both groups,after the drug administering the pale complexion, spontaneous sweat,fatigue,white coating tongue,cersicolor exfoliative tongue and abnormal pulse presentation（thready and weak） got better than those before that （23vs14/21vs12,21vs11/20vs11,18vs9/15vs6,et al,P value＜0.05）;especially,the spontaneous sweat in the Huaier group,cersicolor exfoliative tongue and abnormal pulse presentation in both groups improved markedly after the drug administering （21vs11,10vs1,12vs0,et al,p value＜0.01）.But the differences of poor appetite showed no statistical significance in the same group（12vs6,10vs4,P value＞0.05）.1.2 In the control group,after the drug administering the constipation and night sweat got no better than those before that（13vs8,22vs15,P value＞0.05）,but in Huaier group,the symptoms improved markedly（12vs2,19vs7,P value＜0.01）.1.3 Before the drug administering,the constipation and night sweat presented no significant differences between the Huaier group and the control group（12vs13, 19vs22,P value＞0.05）;but after that,the two symptoms got better in the Huaier group than those in the control group（2vs8,7vs15,P value＜0.05）.2.The expression of ACT/C-ACT2.1 Between before and after the drug administering,the differences of the asthma control levels presented no statistical significance（Pvalue＞0.05） in Huaier group.However at the 4^th and 8^th week in the control group,the differences of the asthma control levels were no better than the levels at the first（P value＜0.05）.2.2 At the 4^th and 12^th week after the drug administering,the expression of the asthma control levels were insignificantly different between the Huaier group and the control group（P value＞0.05）;but at the 8^th they were significantly different in the both groups（P value＜0.05）.PartⅡ:1.The expression of T LC cytokines at mRNA levelThe expression of the IFN-γmRNA was insignificant statistically among any two groups（2.21±0.26vs2.17±0.33vs2.10±0.31,P value＞0.05）.The levels of the IL-4 mRNA in the large-dosel group were lower than those of the control group and the middle-dose group（0.86±0.13vs 1.28±0.14,0.86±0.13vs 1.21±0.17,p value＜0.01, P value＜0.05）.The expression of the IL-10 mRNA in the large-dose group was higher than those in the control group（2.99±0.33vs2.63±0.36,P value＜0.05）.2.The expression of T LC transcription factors at mRNA levelThe expression of the T-bet、Foxp3 mRNA in the large-dose group was lower than those in the control group（2.40±0.30vs2.63±0.25,2.31±0.31vs2.60±0.39,P value＜0.05）.The levels of GATA-3 mRNA were markedly statistical significance between the large-dose group and the control group（1.56±0.22vs1.87±0.29,P value＜0.01）,and there were statistical significance between the large-dose group and the middle-dose group（1.56±0.22vs1.71±0.24,P value＜0.05）.3.The expression of T LC cytokines at protein levelThe levels of the IFN-γ,IL-10 in large-dose group were higher than those in the control group（672.78±184.72vs503.74±189.78,586.47±191.83vs431.67±135.25,P value＜0.05）,but the differences of the IL-4 presented no statistic significance among any two groups（22.76±7.08vs21.17±6.84 vs18.73±4.55,Pvalue＞0.05）.4.The ratio expression of the TLC cytokines＆transcription factorsThe levels of the IFN-γ/IL-4 mRNA in large-dose group were higher markedly than those in the control group,and there were significant differences between large-dose group and middle-dose group（2.61±0.59vs 1.91±0.61,2.61±0.59 vs2.00±0.62,P value＜0.01）.But the differences of the T-bet /GATA-3 mRNA presented no statistic significance among any two groups（1.43±0.22vs1.52±0.22vs 1.55±0.24,P value＞0.05）.The expression of the IFN-γ/IL-4 showed the statistic significance between large-dose group and control group（37.90±13.59vs 25.62±15.28, Pvalue＜0.05）.Conclusions:1.Compared with the control group,the constipation and night sweat improved in the Huaier group;and at the 8^th week after the drug administering,the asthma control levels in Huaier group were better than those in the control group.It’s demonstrated that Trametes Robinioplila could be benefit for the asthmatic children, improving the kids’ body-constitution and adjusting the deficiency of that,in order to maintain the patient’s condition.2.The experiences in vitro showed that Trametes Robinioplila could increase the levels of IFN-γin supernatant,and decrease the levels of IL-4 mRNA in cellular precipitation.The results indicated that Trametes Robinioplila could correct the drift of Th1/Th2 cytokines and regulate the imbalance of Th1/Th2 by up-regulating the Th1 cytokines in supernatant and down-regulating the Th2 cytokines. 3.The experiences in vitro presented that Trametes Robinioplila could down-regulate the expressions of GATA-3 mRNA in cellular precipitation,and the levels of T-bet mRNA were consumed.It’s supposed that Trametes Robinioplila could inhibit the differentiation of Th0 into Th2 and adjust the ratio of Th1/Th2 cells by down-regulating the Th2 transcription factor.4.The experiences in vitro showed that Trametes Robinioplila could increase the expressions of IL-10 mRNA in cellular precipitation and IL-10 in supernatant,and the levels of Foxp3 mRNA were consumed.The results demonstrated that Trametes Robinioplila could alleviate the chronic allergic inflammation by up-regulating anti-inflammatory cytokine;meanwhile the drug could induce the differentiation of ThO into CD4⁺CD25⁺ Treg,in order to inhibit the Th2 and adjust the imbalance of Th1/Th2 cells.更多还原