感毒清颗粒对免疫功能影响的实验和临床研究
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摘要
第一部分感毒清颗粒对环磷酰胺所致免疫抑制小鼠免疫功能影响的实验研究
目的：为了探讨感毒清颗粒对免疫抑制小鼠的免疫调节作用,本研究通过建立免疫抑制小鼠模型,观察感毒清颗粒对免疫抑制小鼠免疫器官脏器指数和免疫细胞、免疫分子含量等方面的影响,并进行作用机制研究,为其临床应用提供理论依据。
方法：1.SPF级昆明种小鼠60只,体重18／22g,随机分为六组,分别为：正常对照组(生理盐水灌胃0.2mL/10g,连续7天)；环磷酰胺模型组(生理盐水灌胃0.2mL/10g,连续7天,同时连续3天腹腔注射环磷酰胺50mg/kg)；阳性对照组(连续3天腹腔注射环磷酰胺50mg/kg造模后,每天灌胃玉屏风颗粒混悬液2.25g/kg,连续7天)；感毒清颗粒高、中、低剂量组(连续3天腹腔注射环磷酰胺50mg/kg造模后,每天分别灌胃感毒清颗粒8. lg/kg,4.05g/kg,2.025g/kg,连续7天),建立免疫功能低下小鼠模型。末次给药后小鼠禁食12h,摘除眼球采血,采用流式细胞术检测血液中NKT细胞百分比含量。
2.造模及给药方法同1,眼球采血后,处死小鼠,分别取胸腺、脾脏称重,计算胸腺、脾脏指数。将胸腺制成组织匀浆,离心,取上清,进行IFN-γ、IL-2、IL-4检测。
3.造模及给药方法同1,小鼠处死后,取脾,立即置液氮中,随后转移置-80℃低温冰箱保存,采用免疫组织化染色法检测脾脏T-Bet、GATA-3蛋白表达。
结果：1.与正常对照组相比,环磷酰胺模型组胸腺指数、脾脏指数显著降低(P<0.05),给药后,感毒清颗粒各剂量组、玉屏风阳性对照组、模型组之间比较,无显著性差异(P>0.05)。
2.与正常对照组相比,环磷酰胺模型组NKT数量明显高于正常对照组,差异有统计学意义(P<0.05)；给药后,感毒清颗粒高剂量组外周血NKT细胞数量明显高于环磷酰胺模型组,差异有统计学意义(P<0.05)。感毒清颗粒中剂量组、玉屏风阳性对照组外周血NKT细胞数量亦有所提高,但与环磷酰胺模型组比较,无显著性差异(P>0.05)
3.与正常对照组相比,环磷酰胺模型组1FN-γ、1L-2数量明显低于正常对照组,差异有统计学意义(P<0.05)；给药后,感毒清颗粒高、中、低剂量组胸腺组织匀浆液IFN-γ表达明显高于环磷酰胺模型组,有显著性差异(P<0.05)；玉屏风阳性对照组胸腺组织匀浆液IFN-γ表达与环磷酰胺模型组比较,无显著性差异(P>0.05)。
给药后,感毒清颗粒高剂量组胸腺组织匀浆液IL-2表达明显高于环磷酰胺模型组,差异有统计学意义(P<0.05)。感毒清颗粒中、低剂量组、玉屏风阳性对照组胸腺组织匀浆液IL-2表达与环磷酰胺模型组相比,差异无统计学意义(P>0.05)。
与正常对照组相比,环磷酰胺模型组IL-4表达明显高于正常对照组,差异有统计学意义(P<0.05)；给药后,感毒清颗粒高、中剂量组、玉屏风阳性对照组胸腺组织匀浆液IL-4表达明显低于环磷酰胺模型组,有显著性差异(P<0.05)；感毒清颗粒高、中剂量组胸腺组织匀浆液IL-4表达与玉屏风阳性对照组相比,差异无统计学意义(P>0.05)。
4.与正常对照组相比,环磷酰胺模型组脾脏T-bet表达明显低于正常对照组,差异有统计学意义(P<0.05)；给药后,感毒清颗粒高、中剂量组、玉屏风阳性对照组脾脏T-bet表达明显高于环磷酰胺模型组,有显著性差异(P<0.05),说明感毒清颗粒、玉屏风颗粒可以提高免疫抑制小鼠脾脏组织T-bet的表达。感毒清颗粒高剂量组脾脏组织T-bet表达与玉屏风阳性对照组相比,差异无统计学意义(P>0.05),说明感毒清颗粒提高免疫抑制小鼠脾脏组织T-bet表达的作用与玉屏风颗粒相当。
与正常对照组相比,环磷酰胺模型组脾脏GATA-3表达明显高于正常对照组,差异有统计学意义(P<0.05)；给药后,感毒清颗粒高、中、低剂量组、玉屏风阳性对照组脾脏GATA-3表达明显低于环磷酰胺模型组,有显著性差异(P<0.05),说明感毒清颗粒、玉屏风颗粒可以降低免疫抑制小鼠脾脏组织GATA-3的表达。感毒清颗粒高剂量组脾脏组织GATA-3表达与玉屏风阳性对照组相比,差异无统计学意义(P>0.05),说明感毒清颗粒降低免疫抑制小鼠脾脏组织GATA-3表达的作用与玉屏风颗粒相当。
5.感毒清颗粒的上述作用皆呈现一定的量效依赖关系,高、中剂量组作用优于低剂量组。
结论：1.采用免疫抑制剂环磷酰胺制造免疫抑制小鼠模型,造模后小鼠胸腺指数、脾脏指数下降,说明使用环磷酰胺造模成功。
2.感毒清颗粒可以提高免疫抑制小鼠外周血NKT细胞数量,通过影响外周血NKT细胞数量,使机体的固有免疫向正常方向恢复。
3.感毒清颗粒可以提高免疫抑制小鼠胸腺组织IFN-γ、IL-2含量,降低IL-4含量,平衡TH1/TH2亚群细胞因子的比例,调节免疫细胞含量,其效果与玉屏风颗粒相当。
4.感毒清颗粒调节TH1/TH2亚群细胞因子比例的作用,可能部分源于对TH1/TH2亚群主要转录因子T-bet和GATA-3表达的影响,其效果与玉屏风颗粒相当。
第二部分感毒清颗粒治疗急性上呼吸道感染(气虚邪犯证)的临床观察及对患者免疫功能影响的临床研究
目的：通过临床观察,分析感毒清颗粒治疗急性上呼吸道感染(气虚邪犯证)的临床疗效及其对患者外周血T细胞亚群、B细胞、NK细胞含量的影响。论证感毒清颗粒治疗急性上呼吸道感染气虚邪犯证的可行性和有效性,为临床用药提供依据,为开发新型治疗急性上呼吸道感染制剂奠定基础。
方法：将2012年12月至2013年3月期间,就诊于成都中医药大学附属医院、成都市西安路羊西线社区卫生服务站的急性上呼吸道感染患者39例纳入研究范围,随机分为治疗组26例,对照组13例。治疗组给予感毒清颗粒,对照组给予抗病毒颗粒,进行疗程为3天的临床观察。从治疗组随机选取11例,对照组随机选取11例,采用流式细胞术分别检测两组患者治疗前后全血中T细胞亚群、B细胞、NK细胞百分比含量,并进行比较。
结果：1、治疗组和对照组疗效比较,愈显率比较有显著性差异(P<0.05)。感毒清治疗组愈显率为87.5%,总有效率为100%；抗病毒对照组愈显率为27.3%,总有效率为100%；治疗组愈显率明显高于对照组,差异有统计学意义(P<0.05)。
2、两组治疗后主症发热、咽痛、倦怠乏力、气短懒言均有明显缓解。感毒清组各主症治疗前后比较均有显著性差异(P<0.05)；抗病毒组发热、咽痛治疗前后比较有显著性差异(P<0.05),倦怠乏力、气短懒言治疗前后比较无显著性差异(P>0.05)。感毒清颗粒缓解主症发热、咽痛、倦怠乏力、气短懒言的疗效优于抗病毒颗粒。
3、两组治疗后次要症状恶风寒、汗出不畅、头痛、鼻塞、流涕、咳嗽治疗3天后也有缓解。感毒清组治疗前后各次要症状比较有显著性差异(P<0.05)；抗病毒组汗出不畅、头痛、鼻塞、咳嗽治疗前后比较有显著性差异(P<0.05),恶风寒、流涕治疗前后比较无显著性差异(P>0.05)。感毒清颗粒缓解次要症状恶风寒、汗出不畅、头痛、鼻塞、流涕、咳嗽的疗效优于抗病毒颗粒。
4、治疗后,感毒清组全血中T细胞亚群、B细胞、NK细胞百分比含量较治疗前均有明显的上升趋势,其中CD4/CD8的比值治疗前后比较有显著性差异(P<0.05)；抗病毒组全血中T细胞亚群治疗前后未有明显的变化,B细胞、NK细胞百分比有上升的趋势,但与治疗前比较均无显著性差异(P>0.05)。两组组间全血中T细胞亚群、B细胞、NK细胞百分比含量水平比较,差异无统计学意义(P>0.05)。
结论：感毒清颗粒在改善急性上呼吸道感染主要症状发热、咽痛、倦怠乏力、气短懒言和次要症状恶风寒、汗出不畅、头痛、鼻塞、流涕、咳嗽等方面有显著的疗效,其作用优于抗病毒颗粒。感毒清颗粒能使上呼吸道感染患者的免疫细胞T细胞亚群、B细胞、NK细胞上调,调节免疫力。感毒清颗粒对急性上呼吸道感染(气虚邪犯证)有较好的治疗效果,对各种症状有明显的缓解作用,并可以提高上呼吸道感染患者的免疫功能。
Part one:Experimental Study of GanDuQing Granules on Immune System in Cyclophosphamide-induced immunesuppressive mice
Objective: To investigate the immune regulation effects of GanDuQingGranules in immunosuppressive mice, the immunosuppressive mice model was established to observe the impact on Immune System of GanDuQing Granules in Immune Organ Index, immune cell function, immune molecules content of immunosuppressive mice, and to explore its mechanisms.This study provide a theoretical basis for its clinical application.
Methods:1. SPF grade Kunming mouse60, weight18-22g, were randomly divided into six groups, as follows:Normal control group(lavaged Physiological saline,7consecutive days); Cyclophosphamide model group (Lavaged saline0.2mL/10g, seven days, at the same time abdominal subcutaneous injected cyclophosphamide50mg/kg for3days); Positive control group (intraperitoneal subcutaneous injected cyclophosphamide50mg/kg modeling3consecutive days, then lavaged YuPingFeng Granules suspension2.25g/kg7consecutive days);GanDuQing Granules high group、middle grope、low dose group (intraperitoneal injected cyclophosphamide50mg/kg modeling daily3consecutive days, administered GanDuQing Granules8.1g/kg,4.05g/kg,2.025g/kg,7consecutive days). After the last administration, mouse were fasted12h, the eye was removed to collect blood, using flow cytometry to detect the proportion of NKT cells in the blood.
2. To make model was the same as the first one. After blood collection from eyes, all mouse were killed to take thymus and spleen, which weighed to calculate the thymus and spleen index. Thymus was made to thymic tissue homogenate after centrifugation, and to detect the supernatant of IFN-y、IL-2、IL-4.
3. To make model was the same as the first one. The mouse was killed to take its spleen, and the spleen was set into liquid nitrogen immediately, and subsequently transfered to-80℃cryogenic refrigerator, using immunohistochemical methods to detect the protein content of T-Bet and GATA-3in the spleen.
Results:1.Compared with the normal control group, the thymus index and spleen index of cyclophosphamide model group was decreased significant(P<0.05), after the administration,the comparison between GanDuQing Granules dose group, YuPingFeng Granules control group, model group has no significant difference (P>0.05).
2.Compared with normal control group, the content of NKT in the cyclophosphamide model group was obviously higher than normal control group, which has significant difference (P<0.05); after administration, compared with model group, NKT cells of Peripheral blood in GanDuQing high dose group was obviously higher than cyclophosphamide model group, which has significant difference(P<0.05); Compared with cyclophosphamide model group, the middle dose group of GanDuQing and YuPingFeng Positive control group was also increased, but the difference has no statistical significance(P>0.05).
3.Compared with the normal control group, the content of IFN-y, IL-2in the cyclophosphamide model group was significantly lower than the control group, which has significant difference (P<0.05); after administration,the content of BFN-y in every dose group thymus tissue homogenate was significantly higher than the cyclophosphamide model group, the difference has statistical significance (P<0.05).The content of IL-2in the high dose group thymus tissue homogenate was significantly higher than the cyclophosphamide model group, the difference has statistical significance(P<0.05).
Compared with the normal control group, the content of IL-4in the cyclophosphamide model group was significantly higher than the normal control group, the difference has statistical significance (P<0.05); after administration, the content of IL-4in the high and middle dose group thymus tissue homogenate was significantly lower than the cyclophospamide model group, the difference has statistical significance (P<0.05).
4. Compared with the normal control group, the content of T-bet in the spleen of the cyclophosphamide model group was significantly lower than the control group, the difference has statistical significance (P<0.05); after administration, the content of T-bet in the high and middle dose group of GanDuQing Granules, YuPingFeng Positive control group was ignificantly higher than the cyclophosphamide model group, the difference has statistical significance(P<0.05). Compared with YuPingFeng Positive control group, the difference of the content of T-bet in the high dose group of GanDuQing has no statistical significance (P>0.05). It proved that GanDuQing Granules can improve the content of T-bet in the spleen tissue in immunosuppressed mice, and the effect was the same as YuPingFeng Granules.
Compared with normal control group, the content of GATA-3in the spleen of the cyclophosphamide model groups was significantly higher than the normal control group, the difference has statistical significance (P<0.05); after administration, the content of GATA-3in the spleen of all GanDuQing dose group and YuPingFeng Positive control group was significantly lower than cyclophosphamide model group, and the difference was significant (P<0.05). Compared with YuPingFeng Positive control group, the difference of the content of GATA-3in the high dose group of GanDuQing has no statistical significance (P>0.05). It indicted that GanDuQing Granules can reduce the content of GATA-3in the spleen tissue of immunosuppressed mice, and the effect was the same as YuPingFeng Granules.
5. Effects of GanDuQing Granules on Immune System were relative with dose, and the high and middle dose group was better than the low dose group.
Conclusion:1.Using cyclophosphamide to make immunosuppressed mice model, which leaded to the thymus index, spleen index decreased, indicate that using cyclophosphamide to make immunosuppressive model was successful.
2. GanDuQing Granules can improve the content of NKT cells in the peripheral blood of immunosuppressed mice, so that the body's inherent immune can recover to the normal standard.
3. GanDuQing Granules can improve the content of IFN-γand IL-2, decrease the content of IL-4in immunosuppressed mice; and balance cytokines of TH1/TH2subsets, regulate the content of immune cells; its effect was the same as YuPinFeng Granules.
4. GanDuQing Granules can regulate proportion of Thl and Th2, which may be partly due to the effect of transcription factor T-bet and GATA-3.
Part two: Clinical observation of GanDuQing Granules to cure acute upper respiratory tract infection (Pattern of Qi Deficiency and Evil Invaded) and Clinical research on immune function in patients
Objective: Through clinical observation, to explore the clinical effect of GanDuQing Granules on treating acute upper respiratory tract infection and its impact on the content of T cells, B cells, NK cells from peripheral blood in patients. The study would demonstrate effectiveness and feasibility of GanDuQing Granules on treating acute upper respiratory tract infection (Pattern of Qi Deficiency and Evil Invaded), and provide a theoretical basic for clinical medicine, develop a new treatment of acute upper respiratory tract infection.
Methods:In December2012to March2013, to collect39clinical patients with acute upper respiratory tract infection, which see a doctor in attachment hospital of ChengDu University of TCM, ChengDu Xi'an Road QingYang Community health service station. All the patients were divided into the treatment group (26patients) and the control group (13patients).The treatment group had been treated with GanDuQing Granules, the control group had been treated with anti-viral Granules, then made clinical observation of treatment for three days. To select11cases from the treatment group randomly and11cases from the control group randomly, each of them adopted flow cytometry to detect the percentage of T cells, B cells, NK cells in the peripheral blood of patients at the same day and3day latter, then compared two group.
Results:1. Compared with control group, the cure and markedly cure rate of GanDuQing treatment group have significant difference(p<0.05).The cure rate of GanDuQing group was87.5%, the total effective rate was100%; the cure rate of the anti-viral control group was27.3%, the total effective rate was100%. The cure rate of GanDuQing Granules treatment group was significantly higher than the anti-viral control group, the difference was statistically significant (p<0.05).
2. After treatment with GanDuQing Granules and anti-viral Granules, the main symptoms of two groups such as fever, sore throat, malaise and fatigue, shortness of breath were all significantly alleviated. Compared with3days ago, the curative effect of GanDuQing treatmen group have significant difference (P<0.05). Compared with3days ago, the curative effect of Anti-viral treatment group, such as the fever, sore throat have significant difference (P<0.05), but malaise, fatigue, shortness of breath was no significant difference (P>0.05). It proved that GanDuQing Granules can alleviate the main symptoms of cold such as fever, sore throat, fatigue, shortness of breath, and GanDuQing Granules were more effective than anti-viral Granules.
3. After3days treatment, the secondary symptoms of cold of two groups such as fear of cold, poor sweating, headache, nasal congestion, runny nose, cough were also alleviate. Compare with treatment ago, the secondary symptoms of cold were significant difference after GanDuQing treatment (P<0.05). And Compared with treatment ago, the secondary symptoms of cold such as poor sweating, headache, nasal congestion, cough were more significant difference (P<0.05) after Anti-viral drugs treatment, but the symptoms of bad cold, runny nose were no significant difference (P>0.05). The effective effect of GanDuQing Granules to alleviate the secondary symptoms of a bad cold such as poor sweating, headache, nasal congestion, runny nose, cough were more effective than anti-viral Granules.
4. Compared with GanDuQing treatment ago, the percentages of T cells, B cells, NK cells from patient blood had a clear upward trend after treatment, and the ratio of CD4/CD8had a more significant difference(P<0.05). Compare with anti-viral treatment ago, the content of T cells did not change significantly, and there was an upward trend in the percentage of B cells, NK cells after anti-viral treatment, but there was no significant difference (P>0.05). The percentage of T cell subsets, B cells, NK cells between two groups was not significant difference (P>0.05).
Conclusion:GanDuQing Granules had a significant effect on curing acute upper respiratory tract infection symptoms such as fever, sore throat, fatigue, shortness of breath, and relieve secondary symptoms such as fear of cold, poor sweating, headache, nasal congestion, runny nose, cough, etc. The therapeutic effect of GanDuQing Granules was better than anti-virus Granules. GanDuQing Granules can increase the content of immune cells such as T cell subsets, B cells, NK cells and other cells in upper respiratory tract infection patient's blood, enhance their immunity. GanDuQing Granules had a better therapeutic effect on curing acute upper respiratory tract infection (Pattern of Qi Deficiency and Evil Invaded), which can relieve a variety of cold symptoms significantly. At the same time, GanDuQing Granules can improve the immune function of patient with the upper respiratory tract infection.
目的：为了探讨感毒清颗粒对免疫抑制小鼠的免疫调节作用,本研究通过建立免疫抑制小鼠模型,观察感毒清颗粒对免疫抑制小鼠免疫器官脏器指数和免疫细胞、免疫分子含量等方面的影响,并进行作用机制研究,为其临床应用提供理论依据。
方法：1.SPF级昆明种小鼠60只,体重18／22g,随机分为六组,分别为：正常对照组(生理盐水灌胃0.2mL/10g,连续7天)；环磷酰胺模型组(生理盐水灌胃0.2mL/10g,连续7天,同时连续3天腹腔注射环磷酰胺50mg/kg)；阳性对照组(连续3天腹腔注射环磷酰胺50mg/kg造模后,每天灌胃玉屏风颗粒混悬液2.25g/kg,连续7天)；感毒清颗粒高、中、低剂量组(连续3天腹腔注射环磷酰胺50mg/kg造模后,每天分别灌胃感毒清颗粒8. lg/kg,4.05g/kg,2.025g/kg,连续7天),建立免疫功能低下小鼠模型。末次给药后小鼠禁食12h,摘除眼球采血,采用流式细胞术检测血液中NKT细胞百分比含量。
2.造模及给药方法同1,眼球采血后,处死小鼠,分别取胸腺、脾脏称重,计算胸腺、脾脏指数。将胸腺制成组织匀浆,离心,取上清,进行IFN-γ、IL-2、IL-4检测。
3.造模及给药方法同1,小鼠处死后,取脾,立即置液氮中,随后转移置-80℃低温冰箱保存,采用免疫组织化染色法检测脾脏T-Bet、GATA-3蛋白表达。
结果：1.与正常对照组相比,环磷酰胺模型组胸腺指数、脾脏指数显著降低(P<0.05),给药后,感毒清颗粒各剂量组、玉屏风阳性对照组、模型组之间比较,无显著性差异(P>0.05)。
2.与正常对照组相比,环磷酰胺模型组NKT数量明显高于正常对照组,差异有统计学意义(P<0.05)；给药后,感毒清颗粒高剂量组外周血NKT细胞数量明显高于环磷酰胺模型组,差异有统计学意义(P<0.05)。感毒清颗粒中剂量组、玉屏风阳性对照组外周血NKT细胞数量亦有所提高,但与环磷酰胺模型组比较,无显著性差异(P>0.05)
3.与正常对照组相比,环磷酰胺模型组1FN-γ、1L-2数量明显低于正常对照组,差异有统计学意义(P<0.05)；给药后,感毒清颗粒高、中、低剂量组胸腺组织匀浆液IFN-γ表达明显高于环磷酰胺模型组,有显著性差异(P<0.05)；玉屏风阳性对照组胸腺组织匀浆液IFN-γ表达与环磷酰胺模型组比较,无显著性差异(P>0.05)。
给药后,感毒清颗粒高剂量组胸腺组织匀浆液IL-2表达明显高于环磷酰胺模型组,差异有统计学意义(P<0.05)。感毒清颗粒中、低剂量组、玉屏风阳性对照组胸腺组织匀浆液IL-2表达与环磷酰胺模型组相比,差异无统计学意义(P>0.05)。
与正常对照组相比,环磷酰胺模型组IL-4表达明显高于正常对照组,差异有统计学意义(P<0.05)；给药后,感毒清颗粒高、中剂量组、玉屏风阳性对照组胸腺组织匀浆液IL-4表达明显低于环磷酰胺模型组,有显著性差异(P<0.05)；感毒清颗粒高、中剂量组胸腺组织匀浆液IL-4表达与玉屏风阳性对照组相比,差异无统计学意义(P>0.05)。
4.与正常对照组相比,环磷酰胺模型组脾脏T-bet表达明显低于正常对照组,差异有统计学意义(P<0.05)；给药后,感毒清颗粒高、中剂量组、玉屏风阳性对照组脾脏T-bet表达明显高于环磷酰胺模型组,有显著性差异(P<0.05),说明感毒清颗粒、玉屏风颗粒可以提高免疫抑制小鼠脾脏组织T-bet的表达。感毒清颗粒高剂量组脾脏组织T-bet表达与玉屏风阳性对照组相比,差异无统计学意义(P>0.05),说明感毒清颗粒提高免疫抑制小鼠脾脏组织T-bet表达的作用与玉屏风颗粒相当。
与正常对照组相比,环磷酰胺模型组脾脏GATA-3表达明显高于正常对照组,差异有统计学意义(P<0.05)；给药后,感毒清颗粒高、中、低剂量组、玉屏风阳性对照组脾脏GATA-3表达明显低于环磷酰胺模型组,有显著性差异(P<0.05),说明感毒清颗粒、玉屏风颗粒可以降低免疫抑制小鼠脾脏组织GATA-3的表达。感毒清颗粒高剂量组脾脏组织GATA-3表达与玉屏风阳性对照组相比,差异无统计学意义(P>0.05),说明感毒清颗粒降低免疫抑制小鼠脾脏组织GATA-3表达的作用与玉屏风颗粒相当。
5.感毒清颗粒的上述作用皆呈现一定的量效依赖关系,高、中剂量组作用优于低剂量组。
结论：1.采用免疫抑制剂环磷酰胺制造免疫抑制小鼠模型,造模后小鼠胸腺指数、脾脏指数下降,说明使用环磷酰胺造模成功。
2.感毒清颗粒可以提高免疫抑制小鼠外周血NKT细胞数量,通过影响外周血NKT细胞数量,使机体的固有免疫向正常方向恢复。
3.感毒清颗粒可以提高免疫抑制小鼠胸腺组织IFN-γ、IL-2含量,降低IL-4含量,平衡TH1/TH2亚群细胞因子的比例,调节免疫细胞含量,其效果与玉屏风颗粒相当。
4.感毒清颗粒调节TH1/TH2亚群细胞因子比例的作用,可能部分源于对TH1/TH2亚群主要转录因子T-bet和GATA-3表达的影响,其效果与玉屏风颗粒相当。
第二部分感毒清颗粒治疗急性上呼吸道感染(气虚邪犯证)的临床观察及对患者免疫功能影响的临床研究
目的：通过临床观察,分析感毒清颗粒治疗急性上呼吸道感染(气虚邪犯证)的临床疗效及其对患者外周血T细胞亚群、B细胞、NK细胞含量的影响。论证感毒清颗粒治疗急性上呼吸道感染气虚邪犯证的可行性和有效性,为临床用药提供依据,为开发新型治疗急性上呼吸道感染制剂奠定基础。
方法：将2012年12月至2013年3月期间,就诊于成都中医药大学附属医院、成都市西安路羊西线社区卫生服务站的急性上呼吸道感染患者39例纳入研究范围,随机分为治疗组26例,对照组13例。治疗组给予感毒清颗粒,对照组给予抗病毒颗粒,进行疗程为3天的临床观察。从治疗组随机选取11例,对照组随机选取11例,采用流式细胞术分别检测两组患者治疗前后全血中T细胞亚群、B细胞、NK细胞百分比含量,并进行比较。
结果：1、治疗组和对照组疗效比较,愈显率比较有显著性差异(P<0.05)。感毒清治疗组愈显率为87.5%,总有效率为100%；抗病毒对照组愈显率为27.3%,总有效率为100%；治疗组愈显率明显高于对照组,差异有统计学意义(P<0.05)。
2、两组治疗后主症发热、咽痛、倦怠乏力、气短懒言均有明显缓解。感毒清组各主症治疗前后比较均有显著性差异(P<0.05)；抗病毒组发热、咽痛治疗前后比较有显著性差异(P<0.05),倦怠乏力、气短懒言治疗前后比较无显著性差异(P>0.05)。感毒清颗粒缓解主症发热、咽痛、倦怠乏力、气短懒言的疗效优于抗病毒颗粒。
3、两组治疗后次要症状恶风寒、汗出不畅、头痛、鼻塞、流涕、咳嗽治疗3天后也有缓解。感毒清组治疗前后各次要症状比较有显著性差异(P<0.05)；抗病毒组汗出不畅、头痛、鼻塞、咳嗽治疗前后比较有显著性差异(P<0.05),恶风寒、流涕治疗前后比较无显著性差异(P>0.05)。感毒清颗粒缓解次要症状恶风寒、汗出不畅、头痛、鼻塞、流涕、咳嗽的疗效优于抗病毒颗粒。
4、治疗后,感毒清组全血中T细胞亚群、B细胞、NK细胞百分比含量较治疗前均有明显的上升趋势,其中CD4/CD8的比值治疗前后比较有显著性差异(P<0.05)；抗病毒组全血中T细胞亚群治疗前后未有明显的变化,B细胞、NK细胞百分比有上升的趋势,但与治疗前比较均无显著性差异(P>0.05)。两组组间全血中T细胞亚群、B细胞、NK细胞百分比含量水平比较,差异无统计学意义(P>0.05)。
结论：感毒清颗粒在改善急性上呼吸道感染主要症状发热、咽痛、倦怠乏力、气短懒言和次要症状恶风寒、汗出不畅、头痛、鼻塞、流涕、咳嗽等方面有显著的疗效,其作用优于抗病毒颗粒。感毒清颗粒能使上呼吸道感染患者的免疫细胞T细胞亚群、B细胞、NK细胞上调,调节免疫力。感毒清颗粒对急性上呼吸道感染(气虚邪犯证)有较好的治疗效果,对各种症状有明显的缓解作用,并可以提高上呼吸道感染患者的免疫功能。
Part one:Experimental Study of GanDuQing Granules on Immune System in Cyclophosphamide-induced immunesuppressive mice
Objective: To investigate the immune regulation effects of GanDuQingGranules in immunosuppressive mice, the immunosuppressive mice model was established to observe the impact on Immune System of GanDuQing Granules in Immune Organ Index, immune cell function, immune molecules content of immunosuppressive mice, and to explore its mechanisms.This study provide a theoretical basis for its clinical application.
Methods:1. SPF grade Kunming mouse60, weight18-22g, were randomly divided into six groups, as follows:Normal control group(lavaged Physiological saline,7consecutive days); Cyclophosphamide model group (Lavaged saline0.2mL/10g, seven days, at the same time abdominal subcutaneous injected cyclophosphamide50mg/kg for3days); Positive control group (intraperitoneal subcutaneous injected cyclophosphamide50mg/kg modeling3consecutive days, then lavaged YuPingFeng Granules suspension2.25g/kg7consecutive days);GanDuQing Granules high group、middle grope、low dose group (intraperitoneal injected cyclophosphamide50mg/kg modeling daily3consecutive days, administered GanDuQing Granules8.1g/kg,4.05g/kg,2.025g/kg,7consecutive days). After the last administration, mouse were fasted12h, the eye was removed to collect blood, using flow cytometry to detect the proportion of NKT cells in the blood.
2. To make model was the same as the first one. After blood collection from eyes, all mouse were killed to take thymus and spleen, which weighed to calculate the thymus and spleen index. Thymus was made to thymic tissue homogenate after centrifugation, and to detect the supernatant of IFN-y、IL-2、IL-4.
3. To make model was the same as the first one. The mouse was killed to take its spleen, and the spleen was set into liquid nitrogen immediately, and subsequently transfered to-80℃cryogenic refrigerator, using immunohistochemical methods to detect the protein content of T-Bet and GATA-3in the spleen.
Results:1.Compared with the normal control group, the thymus index and spleen index of cyclophosphamide model group was decreased significant(P<0.05), after the administration,the comparison between GanDuQing Granules dose group, YuPingFeng Granules control group, model group has no significant difference (P>0.05).
2.Compared with normal control group, the content of NKT in the cyclophosphamide model group was obviously higher than normal control group, which has significant difference (P<0.05); after administration, compared with model group, NKT cells of Peripheral blood in GanDuQing high dose group was obviously higher than cyclophosphamide model group, which has significant difference(P<0.05); Compared with cyclophosphamide model group, the middle dose group of GanDuQing and YuPingFeng Positive control group was also increased, but the difference has no statistical significance(P>0.05).
3.Compared with the normal control group, the content of IFN-y, IL-2in the cyclophosphamide model group was significantly lower than the control group, which has significant difference (P<0.05); after administration,the content of BFN-y in every dose group thymus tissue homogenate was significantly higher than the cyclophosphamide model group, the difference has statistical significance (P<0.05).The content of IL-2in the high dose group thymus tissue homogenate was significantly higher than the cyclophosphamide model group, the difference has statistical significance(P<0.05).
Compared with the normal control group, the content of IL-4in the cyclophosphamide model group was significantly higher than the normal control group, the difference has statistical significance (P<0.05); after administration, the content of IL-4in the high and middle dose group thymus tissue homogenate was significantly lower than the cyclophospamide model group, the difference has statistical significance (P<0.05).
4. Compared with the normal control group, the content of T-bet in the spleen of the cyclophosphamide model group was significantly lower than the control group, the difference has statistical significance (P<0.05); after administration, the content of T-bet in the high and middle dose group of GanDuQing Granules, YuPingFeng Positive control group was ignificantly higher than the cyclophosphamide model group, the difference has statistical significance(P<0.05). Compared with YuPingFeng Positive control group, the difference of the content of T-bet in the high dose group of GanDuQing has no statistical significance (P>0.05). It proved that GanDuQing Granules can improve the content of T-bet in the spleen tissue in immunosuppressed mice, and the effect was the same as YuPingFeng Granules.
Compared with normal control group, the content of GATA-3in the spleen of the cyclophosphamide model groups was significantly higher than the normal control group, the difference has statistical significance (P<0.05); after administration, the content of GATA-3in the spleen of all GanDuQing dose group and YuPingFeng Positive control group was significantly lower than cyclophosphamide model group, and the difference was significant (P<0.05). Compared with YuPingFeng Positive control group, the difference of the content of GATA-3in the high dose group of GanDuQing has no statistical significance (P>0.05). It indicted that GanDuQing Granules can reduce the content of GATA-3in the spleen tissue of immunosuppressed mice, and the effect was the same as YuPingFeng Granules.
5. Effects of GanDuQing Granules on Immune System were relative with dose, and the high and middle dose group was better than the low dose group.
Conclusion:1.Using cyclophosphamide to make immunosuppressed mice model, which leaded to the thymus index, spleen index decreased, indicate that using cyclophosphamide to make immunosuppressive model was successful.
2. GanDuQing Granules can improve the content of NKT cells in the peripheral blood of immunosuppressed mice, so that the body's inherent immune can recover to the normal standard.
3. GanDuQing Granules can improve the content of IFN-γand IL-2, decrease the content of IL-4in immunosuppressed mice; and balance cytokines of TH1/TH2subsets, regulate the content of immune cells; its effect was the same as YuPinFeng Granules.
4. GanDuQing Granules can regulate proportion of Thl and Th2, which may be partly due to the effect of transcription factor T-bet and GATA-3.
Part two: Clinical observation of GanDuQing Granules to cure acute upper respiratory tract infection (Pattern of Qi Deficiency and Evil Invaded) and Clinical research on immune function in patients
Objective: Through clinical observation, to explore the clinical effect of GanDuQing Granules on treating acute upper respiratory tract infection and its impact on the content of T cells, B cells, NK cells from peripheral blood in patients. The study would demonstrate effectiveness and feasibility of GanDuQing Granules on treating acute upper respiratory tract infection (Pattern of Qi Deficiency and Evil Invaded), and provide a theoretical basic for clinical medicine, develop a new treatment of acute upper respiratory tract infection.
Methods:In December2012to March2013, to collect39clinical patients with acute upper respiratory tract infection, which see a doctor in attachment hospital of ChengDu University of TCM, ChengDu Xi'an Road QingYang Community health service station. All the patients were divided into the treatment group (26patients) and the control group (13patients).The treatment group had been treated with GanDuQing Granules, the control group had been treated with anti-viral Granules, then made clinical observation of treatment for three days. To select11cases from the treatment group randomly and11cases from the control group randomly, each of them adopted flow cytometry to detect the percentage of T cells, B cells, NK cells in the peripheral blood of patients at the same day and3day latter, then compared two group.
Results:1. Compared with control group, the cure and markedly cure rate of GanDuQing treatment group have significant difference(p<0.05).The cure rate of GanDuQing group was87.5%, the total effective rate was100%; the cure rate of the anti-viral control group was27.3%, the total effective rate was100%. The cure rate of GanDuQing Granules treatment group was significantly higher than the anti-viral control group, the difference was statistically significant (p<0.05).
2. After treatment with GanDuQing Granules and anti-viral Granules, the main symptoms of two groups such as fever, sore throat, malaise and fatigue, shortness of breath were all significantly alleviated. Compared with3days ago, the curative effect of GanDuQing treatmen group have significant difference (P<0.05). Compared with3days ago, the curative effect of Anti-viral treatment group, such as the fever, sore throat have significant difference (P<0.05), but malaise, fatigue, shortness of breath was no significant difference (P>0.05). It proved that GanDuQing Granules can alleviate the main symptoms of cold such as fever, sore throat, fatigue, shortness of breath, and GanDuQing Granules were more effective than anti-viral Granules.
3. After3days treatment, the secondary symptoms of cold of two groups such as fear of cold, poor sweating, headache, nasal congestion, runny nose, cough were also alleviate. Compare with treatment ago, the secondary symptoms of cold were significant difference after GanDuQing treatment (P<0.05). And Compared with treatment ago, the secondary symptoms of cold such as poor sweating, headache, nasal congestion, cough were more significant difference (P<0.05) after Anti-viral drugs treatment, but the symptoms of bad cold, runny nose were no significant difference (P>0.05). The effective effect of GanDuQing Granules to alleviate the secondary symptoms of a bad cold such as poor sweating, headache, nasal congestion, runny nose, cough were more effective than anti-viral Granules.
4. Compared with GanDuQing treatment ago, the percentages of T cells, B cells, NK cells from patient blood had a clear upward trend after treatment, and the ratio of CD4/CD8had a more significant difference(P<0.05). Compare with anti-viral treatment ago, the content of T cells did not change significantly, and there was an upward trend in the percentage of B cells, NK cells after anti-viral treatment, but there was no significant difference (P>0.05). The percentage of T cell subsets, B cells, NK cells between two groups was not significant difference (P>0.05).
Conclusion:GanDuQing Granules had a significant effect on curing acute upper respiratory tract infection symptoms such as fever, sore throat, fatigue, shortness of breath, and relieve secondary symptoms such as fear of cold, poor sweating, headache, nasal congestion, runny nose, cough, etc. The therapeutic effect of GanDuQing Granules was better than anti-virus Granules. GanDuQing Granules can increase the content of immune cells such as T cell subsets, B cells, NK cells and other cells in upper respiratory tract infection patient's blood, enhance their immunity. GanDuQing Granules had a better therapeutic effect on curing acute upper respiratory tract infection (Pattern of Qi Deficiency and Evil Invaded), which can relieve a variety of cold symptoms significantly. At the same time, GanDuQing Granules can improve the immune function of patient with the upper respiratory tract infection.
引文
[1]Nestler G. Traditional Chinese medicine. Med Clin North Am,2002,86:63-73.
[2]朱丹.金黄扶正散对免疫抑制小鼠的免疫调节作用及机制研究.博士学位论文,2012,06：14-17.
[3]陈敏.感毒清口崩片抗病毒作用及免疫机理的实验研究.硕士学位论文,2007,04.
[4]王飞.感毒清口崩片防治呼吸道病毒感染药效及免疫机理研究.博士学位论文,2006,04.
[5]刘维庆.免疫与中医正气探微.陕西中医,2003,24(7)：629-632.
[6]张熙.关于中医免疫学与现代免疫学相关性的思考.湖南中医杂志,2005,21(3)：45-46.
[7]Rodriguez Lopez-Vazquez A, Lopez-Larrea C. Immune systems evolution[J]. Adv Exp Med Biol,2012,739:237-251.
[8]McGargill MA, Derbinski JM, Hogquist KA, et al. Receptor editing in developing T cells [J].NatRev Immunol,2000,1(4):336-341.
[9]Tousseyn T, De Wolf-Peeters C. T cell/histiocyte-rich large B-cell lymphoma: an update on its biology and classification [J]. Virchows Arch,2011,459 (6):557-563.
[10]郭志辉.射干的化学成分药理和临床研究进展.天津药学,2009,21(4)：63-66.
[11]钟明,关旭俊,黄炳生,等.中药射干现代研究进展.中药材,2001,24(12)：904-907.
[12]韩杨,孔红,李宜平.射干的抗病毒实验研究.中草药,2004,3(3)：306-307.
[13]陈云珠,钟鸣.射干药理作用的现代研究进展.中国中医药现代远程教育,2006,4(10)27-29.
[14]林久茂,王瑞国,郑良朴.射干对小鼠免疫功能的影响.福建中医学院学报,2005,15(3)：39-40.
[15]何雯,李檬,张英.中西医结合治疗小儿急性化脓性扁桃体炎38例.中国儿科杂志,2009,5(1)：29.
[16]邱勇波,刘锦,武飞.黄芪化学成分及药理作用研究进展.中国疗养医学,2011,20(5)：435-436.
[17]段亚丽,谢梅冬.黄芪化学成分及其有效成分黄茂甲苷含量测定的研究现状.中国兽药医学,2005,39(3)：35-38.
[18]郑秀琴.黄芪的免疫调节作用及其临床应用.云南中医中药,2007,28(7)：30-31.
[19]Shao BM, Xu W, Dai H. A study on the immune receptors for polysaccharides from the roots of Astragalus membranaceus, A Chinese medicinal herb. Biochem Biophys Res Commun,2004,20(4):1103-1111.
[20]吴宝明,李俊.黄芪多糖在免疫调节中的作用[J].安徽医药,2008,12(7)：577-579.
[21]李燕玉,徐丽梅,刘又宁.黄芪的药理作用及其在呼吸系统疾病中的应用.空军总医院学报,2007,23(2)：99-104.
[22]张靖飞,李小丽,李蜜蜡,等.复方黄芪多糖防治禽流感临床试验[J].动物医学进展,2003,24(3)：124-125.
[23]Smale ST. Transcriptional regulation in the innate immune system [J].Curr Opin Immunol,2012,24(1):51-57.
[24]Artym J, Zimecki M, Kruzel ML. Reconstitution of the cellular immune response by lactoferrin in cyclophosphamide-treated mice is correlated with renewal of T cell compartment [J]. Immunobiology,2003,207(3):197-205.
[25]祝绚,鲍依稀.云芝糖肽、丹参酮IIA对荷瘤小鼠的抗肿瘤及免疫调节作用[J].中医中药与免疫,2008,24(6)：526-529.
[26]国家药典委员会.中华人民共和国药典[M].北京：化学工业出版社,2005：409.
[27]王亮,卞慧敏.玉屏风散的免疫效应研究[J].甘肃中医学院学报,2008,25(5)：48-51.
[28]王秋菊.玉屏风散的免疫药理学研究进展[J].兽医导刊,2010(1)：45-47.
[29]Dardenne M. Role of thymic peptides as transmitters between the neuroendocrine and immune systems [J].Ann Med,1999,2:34-39.
[30]章晓联.医学免疫学[M].武汉：武汉大学出版社,2008：12-19.
[31]陈慰峰.医学免疫学(第4版)[M].北京：人民卫生出版社,2004：148-152.
[32]刘源,陆平成.中药复方顽疣净对小鼠免疫器官重量和淋巴细胞转化的影响[J].湖北中医学院学报,2006,8(1)：25-26.
[33]Iho S, Shau H, Sidney H, et al. Characteristics of interleukin-6 enhanced lymphokin-Activated Killer cell function[J]. Cell Immunol,1991,135:66.
[34]Taniguchi M. A novel lymphocyte-NKT[J]. JSI Newsletter,2000,8(1)':15-16.
[35]Godfrey DI, MacDonald HR, Kronenberg M, et al. NKT cells:what's in a name?[J].Nat Rev Immunol,2004,4(3):231-237.
[36]Godfrey DI, Hammond KJ, Poulton LK, et al. NKT cells:Facts, functions and fallacies [J]. Immunol Today,2000,21(11):573-583.
[37]Eberl G, Lees R, Smiley ST, et al. Tissue-specific segregation of CDld-dependent and CDld-independent NK T cells [J].J Immunol,1999,162:6410.
[38]Kawano T, Cui JQ, Koezuka Y, et al. Analysis of human Va24+ CD4+ NKT cells activated by a-galactosy lceramide pulsed monocytederived dendritic cells [J].J Immunol,2000,164:4458.
[39]邵慧,孙德明.免疫调节细胞的研究现状：NKT细胞[J].上海免疫学杂志,2003,23(3)：145-147.
[40]李子涛,杨滨燕,吴长有.人外周血NKT细胞亚群表型及生物学特征[J].中国免疫学杂志,2011,27(2)：110-114.
[41]陈杰,陈勇.NKT细胞及其免疫功能[J].山西医科大学学报,2006,37(5)：552-554.
[42]Oki S, Chiba A, Yamamura T, et al. The clinical implication and molecular mechanism of preferential 1L-4 production by modified glycolipid-stimulated NKT cells[J].J Clin Invest,2004,113(11):1631-1640.
[43]Yu KOA, Im JS, Molano A, et al. Modulation of CDld restricted NKT cell responses by using N-acyl variants of alpha-galactosylceramides [J]. Proc Natl A cad Sci USA. 2005,102(9):3333-3338.
[44]Hansen DS, Siomos MA, Buckingham L, et al. Regulation of murine cerebral malaria pathogenesisby CDld-restricted NKT cell, and the natural killer complex [J]. Immunity, 2003,18(3):391-402.
[45]Lang GA, Devera TS, Lang ML. Requirement for CDld expression by B cells to stimulate NKT cell-enhanced antibody product ion [J]. Blood,2008,111(4):2158-2162.
[46]章晓红,王曾礼.呼吸道病毒感染局部细胞免疫研究进展[J].国外医学·呼吸系统分册,2005,25(1)：20-22.
[47]张香斋,贾青辉,李蕴玉,等.中药制剂对热应激下雏鸡T淋巴细胞转化率和免疫器官指数的影响[J].河北科技师范学院学报,2009,23(3)：31-32.
[48]刘廷,鞠玉琳,李杨,等.中药复方连黄对小鼠T淋巴细胞亚群CD4+,CD8+的影响[J].四川动物,2009,28(1)：115-117.
[49]章晓联.医学免疫学[M].武汉：武汉大学出版社,2008：139-140.
[50]何荣.益气清瘟片对免疫低下小鼠T淋巴细胞亚群和IL-2、IFN-γ影响的实验研究.硕士学位论文,2005,06：21.
[51]王月飞,赵红晔,王玉阁,等.玉屏风散的生物转化液对免疫抑制小鼠血清IL-2的影响[J].齐齐哈尔医学院学报,2010,31(8)：1178-1179.
[52]章晓联.医学免疫学(第1版)[M].武汉：武汉大学出版社,2008：68-70.
[53]JKim JW, Ferris RL, Whiteside TL. Chemokine C Receptor Expression and Protection of Circulating CD8+ TLymphocytes from Apoptosis[J]. Clin CancerRes,2005, 11(21):7901-7910.
[54]高允海.自拟健脾清疲汤对术后肠功能障碍的治疗作用研究[J].吉林中医药,2007,27(5)：16-17.
[55]卢健,于彩娜,马骥,等.四逆散对实验性溃疡性结肠炎大鼠IL-4的影响[J].中华中医药学刊,2011,29(4)：813-814.
[56]赵向中,董群.不同剂量黄柏组方大补阴丸(汤)对CJ小鼠IL-2/IL-4免疫调节作用的初步研究[J]皖南医学院学报,2005,24(3)：164-166.
[57]Rengarajan J, Szabo S J, Glim cher LH, et al Transc Riptional regulation of Thl/Th2 polarization [J]. Immunol Today,2002,21(10):479-483.
[58]S zabo SJ, Kim ST, Cos ta GL, et al A novel transcription factor, T-bet, directs Thl lineage commitment. Cell,2000,100:655-669.
[59]Faedo A, Ficara F, Ghian iM, et al. Development express -ion of the T-box transcription factor T-bet/Tbx21 during mouse embryogenesis [J]. Mechanisms of Development,2002,116(1-2):157-160.
[60]王锁英T-bet的研究进展[J].国外医学：免疫学分册,2005,28(3)：136-140.
[61]王强,冀朝晖,李若瑜T-bet/GATA3对Thl/Th2分化的调节及其与疾病的关系[J].国外医学：皮肤性病学分册,2005,31(3)：189-191.
[62]S zabo S, J Sullivan BM, S temm ann C, et al. Distinct effects of T-bet in Thl lineage commitment and IFN-gamma produ-ction in CD4 and CD8 T cell[J]. Science, 2002,295(553):338-342.
[63]Sek iN, M iyazak iM, Su zuk iW, et. al IL- 4- induced GATA-3 expression is a time-restricted in struction switch for Th2 cell differentiation. J Immunol 2004, 172:6158-6166.
[64]Chak irH, Wang H, Lefebvre DE, et al. T-bet/GATA-3 ration as a measure of the Thl/Th2 cytokine profile in mixed cell popu -lations:predominant role of GATA-3 [J].J Immunol Methods,2003,278:157-169.
[65]Sun J, Pearce E J. Suppression of early IL-4 production underlies the failure of CD4 T cells activated by TLR-stimu-lated dendritic cells to differentiate into Th2 cells [J].J Immunol 2007,178(3):1635-1644.
[66]陆再英,钟南山.内科学[M].第7版.北京：人民卫生出版社,2008,1114.
[67]胡敏.银翘散治疗老年人病毒性上呼吸道感染的临床观察[J].新中医,2012,44(5)：29-30.
[68]张莉莉.抗病毒药物及作用机制[J].科技信息,2007,20：42-43.
[69]刘颂.抗流感病毒药物的研究进展[J].国外医学药学分册,2005,32(2)：111-115.
[70]Hay den FG, de Jong MD. Emerging influenza antiviral resistance threats [J]. J Infect Dis,2011,203(1):6-10.
[71]Nabel GJ, Fauci AS. Induction of unnatural immunity:prospects for a broadly protective universal influenza vaccine [J]. Nat Med,2010,16(12):1389-1391.
[72]张进陶,王飞,陈勇.中医对呼吸道病毒感染的病因病机探析.四川中医,2008,26(1)：15-16.
[73]周妍.柴板汤治疗流行性感冒表寒里热证的临床观察及对患者血清IL-2和IFN-r含量的影响,硕士学位论文,2010,6.
[74]吴澄.不居集[M].北京：中国中医药出版社,2002：176.
[75]赵梅英,李莉.抗病毒中药在临床中的应用及优势[J].实用医技杂志,2007,14(8)：3218-3219.
[76]孙平华.中药抗流感病毒的最新进展[J].中医药刊,2006,24(4)：733-735.
[77]曹羽.中药抗流感病毒实验研究进展[J].浙江临床医学,2008,10(12)：1604-1605.
[78]窦肇华,张远强,郭顺根.免疫细胞学与疾病[M].北京：中国医药科技出版社,2004：30-31.
[79]王自正.现代标记免疫学[M].北京：人民军医出版社,2000：79-81.
[80]Davids S J, Ikemizu S, Evans E J, et al. The nature of molecular recognition by T cells[J].Nat Immunol,2003,4:217-224.
[81]杨锡强.T细胞亚群的临床意义[J].中国实用儿科杂志,2000,15(4)：250-260.
[82]韩瑞珠,郝艳艳,侯安存.反复呼吸道感染儿童细胞免疫及体液免疫状况[J].实用儿科临床杂志,2008,23(10)：736-737.
[83]Patricia D B, Susan G, Megan R R, et al Effect of CVTE002(TM)(COLD-fX) Versus a ginsenoside extract on systan ic and gut-associated inmune function [J]. Int lnmu-nophaim acol,2008,8(8):1134-1142.
[84]冀湧,盖建芳,姚建宏.甲型H1N1流感病毒重症感染8例临床分析[J].中国小儿急救医学,2010,17(1)：83-84.
[85]Sawaki J, Tsut sui H, Hayashi N, et al.Type lcytokine/chemokine production by mouse N K cells following activation of their TLR/MyD882mediated pathways [J]. Int Immunol,2007,19(3):311-320.
[86]Newman KC, Riley E M. Whatever turns you on:Accessory-cell-dependent activation of NK cells by pathogens [J]. Nat Rev Immunol,2007,7(4):279-291.
[87]Moretta L, Bottino C, Cantoni C, et al. Human natural killer cell function and receptors[J]. Current Opinion in Pharmcology,2001,1(4):387.
[88]于建中.临床流式分析[M].上海：上海科技出版社,2006：61-62.
[89]Lissioni P, Brovio F, Ferrante R, et al. Circulating immature and mature dendritic cell in relation to lymphocyte subsets in patients w ith gastro intestinal tract cancer [J]. Int J Boil Markers,2000,15(1):22-25.
[90]沈玉光,许金良,杨宗林,等.流式细胞术测肺癌患者外周血T细胞亚群及其临床病理的关系[J].实用诊断与治疗杂志,2006,2(2)：101-104.
[1)张静等.小儿反复呼吸道感染发病机制中西医研究进展[J].云南中医学院学报,2002,(9)：34
[2]叶任高,陆再英.内科学[M].第6版.北京：人民卫生出版社,2004：26.
[3]卫生部.传染性非典型性肺炎(SARS)诊疗方案,2003：13-22.
[4]胡敏.银翘散治疗老年人病毒性上呼吸道感染的临床观察[J].新中医,2012,44(5)29-30.
[5]张莉莉.抗病毒药物及作用机制[J].科技信息,2007,20：42-43.
[6]刘颂.抗流感病毒药物的研究进展[J].国外医学药学分册,2005,32(2)：111-115.
[7]陈本川.抗流感病毒药物[J].医药导报,2009,28(7)：964-967.
[8]刘岩,李建华.浅述抗病毒中药的研究及临床疗效优势[J].时珍国医国药,2007,18(12)：3122-3123.
[9]李莹.中药抗呼吸道病毒感染药理作用研究进展[J].山西中医,2007,23(4)：71-72.
[10]胡克杰,王跃红,王栋.金银花中氯原酸在体外抗病毒作用的实验研究[J].中医药信息,2010,27(3)：27-28.
[11]郑虎占,董泽宏,余靖.中药现代研究与应用(4)[M].北京：学苑出版社,1998：3957.
[12]杨洁,刘萍.黄芩提取物及含药血清体外2抑制呼吸道合胞病毒作用的实验研究[J].科学技术与工程,2007,7(12)：2784-2786.
[13]王志杰.鱼腥草抗病毒实验研究[J].预防医学文献,1999,5(4)：43-47.
[14]李京鹤,李志成,陈雯.鱼腥草注射液治疗急性上呼吸道感染196例疗效观察[J].时珍国医国药,2006,17(3)：404.
[15]胡克俭,徐凯建,王跃红,等.连翘酯甙体外抗病毒作用的实验研究[J].2001,8(2)：89.
[16]陈莉莉,张洪泉.中药抗病毒性呼吸道感染的研究进展[J].中国野生植物资源,2006,25(6)：16-18.
[17]李珊珊,李洪源,朴英爱,等.败酱草抗病毒有效部位体外抑制呼吸道合胞病毒作用研究[J].中华流行病学杂志,2004,25(2)：150-153.
[18]韩杨,孔红,李宜平.射干的抗病毒实验研究.中草药,2004,3(3)：306-307.
[19]陈云珠,钟鸣.射干药理作用的现代研究进展.中国中医药现代远程教育,2006, 4(10)27-29.
[20]张振亚,方学平,刁志花,等.野菊花提取物抑制呼吸道合胞病毒作用的体外实验研究[J].解放军药学学报,2002,22(4)：273-274.
[21]左丽,陈丽,唐小敏,等.黄芪A6组分对流感病毒感染小鼠的防治作用[J].中国病毒学,1997,12(4)：342-345.
[22]赵文,任永风,樊建航,等.新疆黄芪抗病毒作用研究[J].中国药学杂志,2001,36(1)：23-25.
[23]张建军,李洪源,王吉锡,等.甘草活性成分抗呼吸道合胞病毒的作用[J].中国公共卫生,2007,23(6)：723-724.
[24]陈连剑,李婷,李成.抗病毒复方制剂的药效学研究[J].中药材,2003,26(9)：659-662.
[25]白明学.柴连辛口服液抗病毒及抑菌的实验研究[J].中国中医药现代运程教育,2008,6(8)：896.
[26]高莉莉,王和平,王亚贤,等.蓝草空气消毒乳剂的研制及其抗菌抗病毒实验[J].时珍国医国药,2009,20(3)：561-562.
[27]张国刚,宋少江,徐绥绪,等.抗病毒无菌粉针剂抗病毒作用的研究[J].沈阳药科大学学报,2002,19(6)：433-434.
[28]石钺,石任岳,刘斌等.银翘散抗流感病毒有效部位群中黄酮类成分研究[J].中国中药杂志,2001,26(5)：320-323.
[29]刘培民.升降散抗流感病毒实验研究[J].山东中医药大学学报,2001,25(1)：43-45.
[30]高益民.战胜“非典”中医药应当登上主战场[J].首都医药,2003,10(12)：25.
[2]朱丹.金黄扶正散对免疫抑制小鼠的免疫调节作用及机制研究.博士学位论文,2012,06：14-17.
[3]陈敏.感毒清口崩片抗病毒作用及免疫机理的实验研究.硕士学位论文,2007,04.
[4]王飞.感毒清口崩片防治呼吸道病毒感染药效及免疫机理研究.博士学位论文,2006,04.
[5]刘维庆.免疫与中医正气探微.陕西中医,2003,24(7)：629-632.
[6]张熙.关于中医免疫学与现代免疫学相关性的思考.湖南中医杂志,2005,21(3)：45-46.
[7]Rodriguez Lopez-Vazquez A, Lopez-Larrea C. Immune systems evolution[J]. Adv Exp Med Biol,2012,739:237-251.
[8]McGargill MA, Derbinski JM, Hogquist KA, et al. Receptor editing in developing T cells [J].NatRev Immunol,2000,1(4):336-341.
[9]Tousseyn T, De Wolf-Peeters C. T cell/histiocyte-rich large B-cell lymphoma: an update on its biology and classification [J]. Virchows Arch,2011,459 (6):557-563.
[10]郭志辉.射干的化学成分药理和临床研究进展.天津药学,2009,21(4)：63-66.
[11]钟明,关旭俊,黄炳生,等.中药射干现代研究进展.中药材,2001,24(12)：904-907.
[12]韩杨,孔红,李宜平.射干的抗病毒实验研究.中草药,2004,3(3)：306-307.
[13]陈云珠,钟鸣.射干药理作用的现代研究进展.中国中医药现代远程教育,2006,4(10)27-29.
[14]林久茂,王瑞国,郑良朴.射干对小鼠免疫功能的影响.福建中医学院学报,2005,15(3)：39-40.
[15]何雯,李檬,张英.中西医结合治疗小儿急性化脓性扁桃体炎38例.中国儿科杂志,2009,5(1)：29.
[16]邱勇波,刘锦,武飞.黄芪化学成分及药理作用研究进展.中国疗养医学,2011,20(5)：435-436.
[17]段亚丽,谢梅冬.黄芪化学成分及其有效成分黄茂甲苷含量测定的研究现状.中国兽药医学,2005,39(3)：35-38.
[18]郑秀琴.黄芪的免疫调节作用及其临床应用.云南中医中药,2007,28(7)：30-31.
[19]Shao BM, Xu W, Dai H. A study on the immune receptors for polysaccharides from the roots of Astragalus membranaceus, A Chinese medicinal herb. Biochem Biophys Res Commun,2004,20(4):1103-1111.
[20]吴宝明,李俊.黄芪多糖在免疫调节中的作用[J].安徽医药,2008,12(7)：577-579.
[21]李燕玉,徐丽梅,刘又宁.黄芪的药理作用及其在呼吸系统疾病中的应用.空军总医院学报,2007,23(2)：99-104.
[22]张靖飞,李小丽,李蜜蜡,等.复方黄芪多糖防治禽流感临床试验[J].动物医学进展,2003,24(3)：124-125.
[23]Smale ST. Transcriptional regulation in the innate immune system [J].Curr Opin Immunol,2012,24(1):51-57.
[24]Artym J, Zimecki M, Kruzel ML. Reconstitution of the cellular immune response by lactoferrin in cyclophosphamide-treated mice is correlated with renewal of T cell compartment [J]. Immunobiology,2003,207(3):197-205.
[25]祝绚,鲍依稀.云芝糖肽、丹参酮IIA对荷瘤小鼠的抗肿瘤及免疫调节作用[J].中医中药与免疫,2008,24(6)：526-529.
[26]国家药典委员会.中华人民共和国药典[M].北京：化学工业出版社,2005：409.
[27]王亮,卞慧敏.玉屏风散的免疫效应研究[J].甘肃中医学院学报,2008,25(5)：48-51.
[28]王秋菊.玉屏风散的免疫药理学研究进展[J].兽医导刊,2010(1)：45-47.
[29]Dardenne M. Role of thymic peptides as transmitters between the neuroendocrine and immune systems [J].Ann Med,1999,2:34-39.
[30]章晓联.医学免疫学[M].武汉：武汉大学出版社,2008：12-19.
[31]陈慰峰.医学免疫学(第4版)[M].北京：人民卫生出版社,2004：148-152.
[32]刘源,陆平成.中药复方顽疣净对小鼠免疫器官重量和淋巴细胞转化的影响[J].湖北中医学院学报,2006,8(1)：25-26.
[33]Iho S, Shau H, Sidney H, et al. Characteristics of interleukin-6 enhanced lymphokin-Activated Killer cell function[J]. Cell Immunol,1991,135:66.
[34]Taniguchi M. A novel lymphocyte-NKT[J]. JSI Newsletter,2000,8(1)':15-16.
[35]Godfrey DI, MacDonald HR, Kronenberg M, et al. NKT cells:what's in a name?[J].Nat Rev Immunol,2004,4(3):231-237.
[36]Godfrey DI, Hammond KJ, Poulton LK, et al. NKT cells:Facts, functions and fallacies [J]. Immunol Today,2000,21(11):573-583.
[37]Eberl G, Lees R, Smiley ST, et al. Tissue-specific segregation of CDld-dependent and CDld-independent NK T cells [J].J Immunol,1999,162:6410.
[38]Kawano T, Cui JQ, Koezuka Y, et al. Analysis of human Va24+ CD4+ NKT cells activated by a-galactosy lceramide pulsed monocytederived dendritic cells [J].J Immunol,2000,164:4458.
[39]邵慧,孙德明.免疫调节细胞的研究现状：NKT细胞[J].上海免疫学杂志,2003,23(3)：145-147.
[40]李子涛,杨滨燕,吴长有.人外周血NKT细胞亚群表型及生物学特征[J].中国免疫学杂志,2011,27(2)：110-114.
[41]陈杰,陈勇.NKT细胞及其免疫功能[J].山西医科大学学报,2006,37(5)：552-554.
[42]Oki S, Chiba A, Yamamura T, et al. The clinical implication and molecular mechanism of preferential 1L-4 production by modified glycolipid-stimulated NKT cells[J].J Clin Invest,2004,113(11):1631-1640.
[43]Yu KOA, Im JS, Molano A, et al. Modulation of CDld restricted NKT cell responses by using N-acyl variants of alpha-galactosylceramides [J]. Proc Natl A cad Sci USA. 2005,102(9):3333-3338.
[44]Hansen DS, Siomos MA, Buckingham L, et al. Regulation of murine cerebral malaria pathogenesisby CDld-restricted NKT cell, and the natural killer complex [J]. Immunity, 2003,18(3):391-402.
[45]Lang GA, Devera TS, Lang ML. Requirement for CDld expression by B cells to stimulate NKT cell-enhanced antibody product ion [J]. Blood,2008,111(4):2158-2162.
[46]章晓红,王曾礼.呼吸道病毒感染局部细胞免疫研究进展[J].国外医学·呼吸系统分册,2005,25(1)：20-22.
[47]张香斋,贾青辉,李蕴玉,等.中药制剂对热应激下雏鸡T淋巴细胞转化率和免疫器官指数的影响[J].河北科技师范学院学报,2009,23(3)：31-32.
[48]刘廷,鞠玉琳,李杨,等.中药复方连黄对小鼠T淋巴细胞亚群CD4+,CD8+的影响[J].四川动物,2009,28(1)：115-117.
[49]章晓联.医学免疫学[M].武汉：武汉大学出版社,2008：139-140.
[50]何荣.益气清瘟片对免疫低下小鼠T淋巴细胞亚群和IL-2、IFN-γ影响的实验研究.硕士学位论文,2005,06：21.
[51]王月飞,赵红晔,王玉阁,等.玉屏风散的生物转化液对免疫抑制小鼠血清IL-2的影响[J].齐齐哈尔医学院学报,2010,31(8)：1178-1179.
[52]章晓联.医学免疫学(第1版)[M].武汉：武汉大学出版社,2008：68-70.
[53]JKim JW, Ferris RL, Whiteside TL. Chemokine C Receptor Expression and Protection of Circulating CD8+ TLymphocytes from Apoptosis[J]. Clin CancerRes,2005, 11(21):7901-7910.
[54]高允海.自拟健脾清疲汤对术后肠功能障碍的治疗作用研究[J].吉林中医药,2007,27(5)：16-17.
[55]卢健,于彩娜,马骥,等.四逆散对实验性溃疡性结肠炎大鼠IL-4的影响[J].中华中医药学刊,2011,29(4)：813-814.
[56]赵向中,董群.不同剂量黄柏组方大补阴丸(汤)对CJ小鼠IL-2/IL-4免疫调节作用的初步研究[J]皖南医学院学报,2005,24(3)：164-166.
[57]Rengarajan J, Szabo S J, Glim cher LH, et al Transc Riptional regulation of Thl/Th2 polarization [J]. Immunol Today,2002,21(10):479-483.
[58]S zabo SJ, Kim ST, Cos ta GL, et al A novel transcription factor, T-bet, directs Thl lineage commitment. Cell,2000,100:655-669.
[59]Faedo A, Ficara F, Ghian iM, et al. Development express -ion of the T-box transcription factor T-bet/Tbx21 during mouse embryogenesis [J]. Mechanisms of Development,2002,116(1-2):157-160.
[60]王锁英T-bet的研究进展[J].国外医学：免疫学分册,2005,28(3)：136-140.
[61]王强,冀朝晖,李若瑜T-bet/GATA3对Thl/Th2分化的调节及其与疾病的关系[J].国外医学：皮肤性病学分册,2005,31(3)：189-191.
[62]S zabo S, J Sullivan BM, S temm ann C, et al. Distinct effects of T-bet in Thl lineage commitment and IFN-gamma produ-ction in CD4 and CD8 T cell[J]. Science, 2002,295(553):338-342.
[63]Sek iN, M iyazak iM, Su zuk iW, et. al IL- 4- induced GATA-3 expression is a time-restricted in struction switch for Th2 cell differentiation. J Immunol 2004, 172:6158-6166.
[64]Chak irH, Wang H, Lefebvre DE, et al. T-bet/GATA-3 ration as a measure of the Thl/Th2 cytokine profile in mixed cell popu -lations:predominant role of GATA-3 [J].J Immunol Methods,2003,278:157-169.
[65]Sun J, Pearce E J. Suppression of early IL-4 production underlies the failure of CD4 T cells activated by TLR-stimu-lated dendritic cells to differentiate into Th2 cells [J].J Immunol 2007,178(3):1635-1644.
[66]陆再英,钟南山.内科学[M].第7版.北京：人民卫生出版社,2008,1114.
[67]胡敏.银翘散治疗老年人病毒性上呼吸道感染的临床观察[J].新中医,2012,44(5)：29-30.
[68]张莉莉.抗病毒药物及作用机制[J].科技信息,2007,20：42-43.
[69]刘颂.抗流感病毒药物的研究进展[J].国外医学药学分册,2005,32(2)：111-115.
[70]Hay den FG, de Jong MD. Emerging influenza antiviral resistance threats [J]. J Infect Dis,2011,203(1):6-10.
[71]Nabel GJ, Fauci AS. Induction of unnatural immunity:prospects for a broadly protective universal influenza vaccine [J]. Nat Med,2010,16(12):1389-1391.
[72]张进陶,王飞,陈勇.中医对呼吸道病毒感染的病因病机探析.四川中医,2008,26(1)：15-16.
[73]周妍.柴板汤治疗流行性感冒表寒里热证的临床观察及对患者血清IL-2和IFN-r含量的影响,硕士学位论文,2010,6.
[74]吴澄.不居集[M].北京：中国中医药出版社,2002：176.
[75]赵梅英,李莉.抗病毒中药在临床中的应用及优势[J].实用医技杂志,2007,14(8)：3218-3219.
[76]孙平华.中药抗流感病毒的最新进展[J].中医药刊,2006,24(4)：733-735.
[77]曹羽.中药抗流感病毒实验研究进展[J].浙江临床医学,2008,10(12)：1604-1605.
[78]窦肇华,张远强,郭顺根.免疫细胞学与疾病[M].北京：中国医药科技出版社,2004：30-31.
[79]王自正.现代标记免疫学[M].北京：人民军医出版社,2000：79-81.
[80]Davids S J, Ikemizu S, Evans E J, et al. The nature of molecular recognition by T cells[J].Nat Immunol,2003,4:217-224.
[81]杨锡强.T细胞亚群的临床意义[J].中国实用儿科杂志,2000,15(4)：250-260.
[82]韩瑞珠,郝艳艳,侯安存.反复呼吸道感染儿童细胞免疫及体液免疫状况[J].实用儿科临床杂志,2008,23(10)：736-737.
[83]Patricia D B, Susan G, Megan R R, et al Effect of CVTE002(TM)(COLD-fX) Versus a ginsenoside extract on systan ic and gut-associated inmune function [J]. Int lnmu-nophaim acol,2008,8(8):1134-1142.
[84]冀湧,盖建芳,姚建宏.甲型H1N1流感病毒重症感染8例临床分析[J].中国小儿急救医学,2010,17(1)：83-84.
[85]Sawaki J, Tsut sui H, Hayashi N, et al.Type lcytokine/chemokine production by mouse N K cells following activation of their TLR/MyD882mediated pathways [J]. Int Immunol,2007,19(3):311-320.
[86]Newman KC, Riley E M. Whatever turns you on:Accessory-cell-dependent activation of NK cells by pathogens [J]. Nat Rev Immunol,2007,7(4):279-291.
[87]Moretta L, Bottino C, Cantoni C, et al. Human natural killer cell function and receptors[J]. Current Opinion in Pharmcology,2001,1(4):387.
[88]于建中.临床流式分析[M].上海：上海科技出版社,2006：61-62.
[89]Lissioni P, Brovio F, Ferrante R, et al. Circulating immature and mature dendritic cell in relation to lymphocyte subsets in patients w ith gastro intestinal tract cancer [J]. Int J Boil Markers,2000,15(1):22-25.
[90]沈玉光,许金良,杨宗林,等.流式细胞术测肺癌患者外周血T细胞亚群及其临床病理的关系[J].实用诊断与治疗杂志,2006,2(2)：101-104.
[1)张静等.小儿反复呼吸道感染发病机制中西医研究进展[J].云南中医学院学报,2002,(9)：34
[2]叶任高,陆再英.内科学[M].第6版.北京：人民卫生出版社,2004：26.
[3]卫生部.传染性非典型性肺炎(SARS)诊疗方案,2003：13-22.
[4]胡敏.银翘散治疗老年人病毒性上呼吸道感染的临床观察[J].新中医,2012,44(5)29-30.
[5]张莉莉.抗病毒药物及作用机制[J].科技信息,2007,20：42-43.
[6]刘颂.抗流感病毒药物的研究进展[J].国外医学药学分册,2005,32(2)：111-115.
[7]陈本川.抗流感病毒药物[J].医药导报,2009,28(7)：964-967.
[8]刘岩,李建华.浅述抗病毒中药的研究及临床疗效优势[J].时珍国医国药,2007,18(12)：3122-3123.
[9]李莹.中药抗呼吸道病毒感染药理作用研究进展[J].山西中医,2007,23(4)：71-72.
[10]胡克杰,王跃红,王栋.金银花中氯原酸在体外抗病毒作用的实验研究[J].中医药信息,2010,27(3)：27-28.
[11]郑虎占,董泽宏,余靖.中药现代研究与应用(4)[M].北京：学苑出版社,1998：3957.
[12]杨洁,刘萍.黄芩提取物及含药血清体外2抑制呼吸道合胞病毒作用的实验研究[J].科学技术与工程,2007,7(12)：2784-2786.
[13]王志杰.鱼腥草抗病毒实验研究[J].预防医学文献,1999,5(4)：43-47.
[14]李京鹤,李志成,陈雯.鱼腥草注射液治疗急性上呼吸道感染196例疗效观察[J].时珍国医国药,2006,17(3)：404.
[15]胡克俭,徐凯建,王跃红,等.连翘酯甙体外抗病毒作用的实验研究[J].2001,8(2)：89.
[16]陈莉莉,张洪泉.中药抗病毒性呼吸道感染的研究进展[J].中国野生植物资源,2006,25(6)：16-18.
[17]李珊珊,李洪源,朴英爱,等.败酱草抗病毒有效部位体外抑制呼吸道合胞病毒作用研究[J].中华流行病学杂志,2004,25(2)：150-153.
[18]韩杨,孔红,李宜平.射干的抗病毒实验研究.中草药,2004,3(3)：306-307.
[19]陈云珠,钟鸣.射干药理作用的现代研究进展.中国中医药现代远程教育,2006, 4(10)27-29.
[20]张振亚,方学平,刁志花,等.野菊花提取物抑制呼吸道合胞病毒作用的体外实验研究[J].解放军药学学报,2002,22(4)：273-274.
[21]左丽,陈丽,唐小敏,等.黄芪A6组分对流感病毒感染小鼠的防治作用[J].中国病毒学,1997,12(4)：342-345.
[22]赵文,任永风,樊建航,等.新疆黄芪抗病毒作用研究[J].中国药学杂志,2001,36(1)：23-25.
[23]张建军,李洪源,王吉锡,等.甘草活性成分抗呼吸道合胞病毒的作用[J].中国公共卫生,2007,23(6)：723-724.
[24]陈连剑,李婷,李成.抗病毒复方制剂的药效学研究[J].中药材,2003,26(9)：659-662.
[25]白明学.柴连辛口服液抗病毒及抑菌的实验研究[J].中国中医药现代运程教育,2008,6(8)：896.
[26]高莉莉,王和平,王亚贤,等.蓝草空气消毒乳剂的研制及其抗菌抗病毒实验[J].时珍国医国药,2009,20(3)：561-562.
[27]张国刚,宋少江,徐绥绪,等.抗病毒无菌粉针剂抗病毒作用的研究[J].沈阳药科大学学报,2002,19(6)：433-434.
[28]石钺,石任岳,刘斌等.银翘散抗流感病毒有效部位群中黄酮类成分研究[J].中国中药杂志,2001,26(5)：320-323.
[29]刘培民.升降散抗流感病毒实验研究[J].山东中医药大学学报,2001,25(1)：43-45.
[30]高益民.战胜“非典”中医药应当登上主战场[J].首都医药,2003,10(12)：25.