感毒清颗粒对免疫功能影响的实验和临床研究

【作者】 杨雯轩；

【导师】 王飞；

【作者基本信息】 成都中医药大学 ， 中医内科学， 2013， 博士

【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。更多还原

【Abstract】 Part one:Experimental Study of GanDuQing Granules on Immune System in Cyclophosphamide-induced immunesuppressive miceObjective: 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 patientsObjective: 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.更多还原