【作者】 黄钢花；

【导师】 钟嘉熙；

【作者基本信息】 广州中医药大学 ， 中医临床基础， 2011， 博士

【摘要】 一、研究目的立足系统性红斑狼疮(SLE)免疫失衡角度,从临床研究和动物实验,观察Thl／Th2细胞群偏移及血管损伤因子与SLE发病机制关系,探讨SLE发病机理；分析清养透解法对该平衡的调节和干预血管损伤的作用,探讨其治疗SLE的主要作用环节。二、研究方法(一)临床试验采用前瞻性单盲随机对照的方法,纳入观察病例共42例,其中治疗组22例,对照组20例,每组各2例脱失,另设12人为正常组。治疗组给予清养透解合剂(由青蒿10g、鳖甲15g、水牛角20g、生地15g、丹皮10g、玄参15g、秦艽10组成)及常规西药(以口服强的松片为主,病情复发或合并狼疮肾炎、狼疮脑炎等用口服激素难以控制病情时,加用标准环磷酰胺冲击疗法)治疗；对照组给予安慰剂及常规西药(同前)治疗,治疗6个月,观察治疗前后病情活动性评分、主要症状评分、中医证候积分变化以及评估治疗期间出现的药物副作用,取静脉血采用酶联免疫法进行Thl类细胞因子(IFN-γ、IL-12)、Th2类细胞因子(IL-10、IL-4)及血管损伤因子(sTM、vWF)检测。(二)动物实验将24只8-10周雌性MRL/lpr小鼠按质量随机分为中药组、西药组、中西医结合组、模型对照组4组,每组6只,另取8-10周龄雌性BALB/C小鼠6只为正常对照组,中药组予清养透解合剂9.5g/kg生理盐水稀释按0.4ml/只,每日1次灌胃；西药组予强的松10mg/kg生理盐水稀释按0.4ml/只,每[211次灌胃；中西医结合组予强的松10mg/kg并清养透解合剂9.5g/kg生理盐水稀释0.4ml/只,每日1次灌胃；模型对照组予生理盐水按0.4ml/只,每日1次灌胃,连续给药6周,正常对照组则不给任何处理。6周后取血采用酶联免疫法进行Thl类细胞因子(IFN-γ、IL-12)、Th2类细胞因子(IL-10、IL-4)及血管损伤因子(sTM、vWF)检测,并取小鼠肾脏在光镜及透射电子显微镜下观察其病理变化。三、结果(一)临床实试验1、一般临床资料：本研究纳入观察病例共42例,38人完成了试验,其中治疗组完成试验20例,对照组完成试验18例,另设12人为正常组,三组在性别、年龄组成比较无显著性差异(p>0.05),治疗组和对照组在脱失率、病程、病情分布上比较无显著性差异(p>0.05)。2、疗效性临床资料分析：治疗组与对照组在治疗前病情活动性评分、主要症状评分、中医证候积分、强的松用量无显著性差异(p>0.05)；两组治疗前与治疗后6个月比较病情活动性评分、主要症状评分、中医证候积分、强的松用量有非常显著性差异,治疗6个月后明显低于治疗前(p<0.01)；治疗后6个月后两组间比较病情活动性评分、主要症状评分、中医证候积分、强的松用量有非常显著性差异,治疗组明显低于对照组(p<0.01)；2、对血清细胞因子的影响：治疗组和对照组治疗前与正常对照组比较血清Thl类细胞因子(IL-12、IFN-γ)、Th2类细胞因子(IL-10、IL-4)、Thl/Th2细胞因子比值(包括IFN-γ/IL-4、IL-12/IL-10的比值)、血管内皮损伤因子(包括sTM、vWF)均有非常显著性差异(p<0.01)；治疗6月与治疗前比较,治疗组能同时降低血清Thl类细胞因子(包括IL-12、IFN-γ)(p<0.05)、Th2类细胞因子(包括IL-10、IL-4)(p<0.05)、血管内皮损伤因子水平(包括sTM、vWF)(p<0.05),提高Thl/Th2细胞因子比值(包括IFN-γ/IL-4、IL-12/IL-10的比值)(p<0.05)；3、治疗6个月后出现的药物副作用：治疗组与对照组在柯兴氏征、高血压、消化性溃疡、脱发、继发白细胞减少、肝功能异常方面人数有非常显著性差异(p<0.01),治疗组发生这些副作用人数明显比对照组少。(二)动物实验：1、血清Thl类细胞因子：中药组、西药组、中西结合组、模型对照组、正常对照组两两比较血清IL-12水平无显著性差异(p>0.05)；中药组、西药组、中西结合组、模型对照组间两两比较血清IFN-γ水平无显著性差异(p>0.05),但此四组小鼠与正常对照组比较血清IFN-γ水平均有显著性差异(p<0.05)；2、血清Th2类细胞因子：与正常对照组比较,模型对照组小鼠血清IL-10、IL-4水平升高,有显著性差异(p<0.05)；与模型对照组小鼠,中药组、中西医结合组能同时降低血清Th2类细胞因子(包括IL-10、IL-4)水平,西药组可降低血清IL-4水平,有显著性差异(p<0.05)；3、对Thl/Th2平衡的影响：中药组、西药组、中西医结合组、模型对照组与正常对照组比较,IL-12/IL-10及IFN-γ/IL-4比值水平下降,有显著性差异(p<0.05)；与模型对照组比较,中药组、西药组、中西医结合组三组能提高MRL/lpr血清IFN-γ/IL-4比值(p<0.05),中西医结合组还可提高血清IL 12/IL 10的比值,有显著性差异(p<0.05)；4、对血管损伤因子的影响：模型对照组与正常对照组比较,血清vWF、sTM水平均上升,有非常显著性差异(p<0.01)；与模型对照组比较,中药组、中西结合能同时降低血清sTM、vWF水平,西药组能降低血清vWF水平,有显著性差异(p<0.05)；5、对肾脏病理学影响：MRL/lpr小鼠肾脏的光镜下检查可见肾小球增生或萎缩,大小不均匀,肾小球系膜区增宽,基底膜节断性增厚,间质细胞增生,小血管管壁纤维素样坏死,血管周围大量慢性炎细胞浸润；部分肾小管上皮细胞变性、坏死、脱落；电镜下可见肾小球基底膜增厚,足突细胞部分融合,内皮细胞肿胀,系膜基质较多,内皮下、基底膜、系膜区可见电子致密物。中药组、西药组、中西结合组能减轻肾脏病理改变。四、结论(一)临床试验提示,SLE患者体内存在Thl/Th2细胞平衡紊乱,Thl/Th2细胞向Th2漂移,血清vWF、sTM表达增高。以清养透解合剂为主的中西医结合治疗组在减轻SLE病情活动、改善SLE主要症状及阴虚证候方面明显优于单纯西药对照组；中西医结合治疗能改善Thl/Th2细胞群失衡,降低血清vWF、sTM的表达,减轻血管损伤；加速强的松的撤减,减少强的松用量,减轻强的松及环磷酰胺引起的毒副作用。(二)动物实验提示,MRL/lpr小鼠存在Th1/Th2细胞平衡紊乱,主要表现为Th2升高,Th1/Th2细胞向Th2漂移,血清vWF、sTM的表达增高,肾脏组织发生病变,清养透解合剂能降低Th2的表达,提高IFN-γ/IL-4比值,降低血清vWF、sTM的表达,改善肾脏病变。(三)动物实验与临床试验存在差异,MRL/Lpr小鼠血清Thl类细胞因子与正常对照组比较,表达降低或差异无显著性意义,而SLE患者血清Th1类细胞因子水平均明显增高。人和动物研究结果之所以存在差异性,可能是由于MRL/lpr小鼠与SLE患者发病机制不完全一致所致。(四)清养透解法可能通过改善Th1/Th2细胞失衡,从而维持T细胞功能正常,抑制B细胞的异常增殖,减轻因炎症因子的分泌和免疫复合物在组织沉积形成的炎性损伤,保护血管内皮而达到治疗SLE作用的。更多还原

【Abstract】 Objectives:Focusing on the imbalance of immunity in SLE, the study was to investigate the imbalance of Th1 and Th2 cell immunity as well as vascular damage factors in the pathological process of SLE. Clinical experiments were carried out to explore the effect of Qingyangtou jie therapy used to treat SLE.Methods:1、Clinical trial:Prospective a single blind Randomized Controlled trial was adopted, and 42 cases were divided into treatment group (22 cases) and control group (20 cases). We selected 12 persons as a normal group in Addition. The patients in treatment group were treated with a therapy with Qing-yang-tou-jie mixture and western medicine, while those in control group were treated with placebo and western medicine. The patients’ condition activity scores, main symptom scores, TCM syndrome scores and side effects of cortin were evaluated after 6 month of treatment. TH1 cell factors (IFN-γ, IL-12), TH2 cell factors (IL-10, IL-4) and vascular injury factors (sTM, vWF) in serum were tested by ELISA simultaneously.2、Animal experiment:Twenty-four female MRL/lpr mice of 8 to10 weeks were divided into therapentic group, western medicine treatment group, combined Chinese and western medicine treatment group and model control group. Six female BALB/C mice of 8 to 10 weeks were selected as a normal group. After continuous exposure therapy of six weeks, TH1 cell factors (IFN-γ, IL-12), TH2 cell factors (IL-10, IL-4) and vascular injury factors (sTM, vWF) in serum were tested; renal pathological changes and ultrastructure were observed by light microscopy and electronic microscopy. Result1、Clinical trial:Thirty-eight cases in the clinical trial were recorded completely, which involves 20 treatment cases,18 control cases and 12 normal control cases. There was no difference in sex and age of these cases. There was no difference in the case lost rate, disease progress between therapentic and control groups. (1)Clinical effects:After 6 month of treatment, the patients’condition activity score, main symptom score, TCM syndrome score and diversity of prednisone dosage were significantly ameliorated than those of pre-treatment. The improvement in the condition of treatment group was significantly superior to the control group (P<0.01).(2) Influence on cell factors:Before treatment, compared with normal group, the factors such as IL-12, IFN-γ, IL-10 and IL-4 in SLE patients increased obviously (p<0.05), and IL-12/IL-10, IFN-γ/IL-4 decreased significantly (P <0.01). Vascular injury factors also decreased significantly. After 6 month of treatment, the serous cell factors (both Thl and Th2 cell factors) decreased significantly in the treatment group, and vascular injury factor (sTM, vWF) decreased too. At the same time, Th1/Th2 ratio (both IFN-γ/IL-4 and IL-12/IL-10) in the treatment group increased, the difference was significant.(3) Compared with control group, the treatment group had reduced incidences of side effects, such as Cushing syndrome, hypertension, peptic ulcer, alopecia, secondary leucopenia and liver dysfunction, and the differences were extraordinary remarkable (P<0.01)2、Animal experiment:(1)Th1 cell factors:No significant differences in IL-12 level were found in these five groups, independently (p>0.05), but there was significant difference in IFN-γbetween MRL/lpr mice and BALB/C mice (p<0.05);(2)Th2 cell factors:Compared with normal group, the model mice’s serous level of IL-10 and IL-4 increased significantly (p<0.05). While the level of Th2 cell factors (IL-10, IL-4) decreased significantly in the therapentic group and the combined Chinese and western medicine treatment group. The serous level of IL-4 decreased in western medicine group.(3)Th1/Th2 balance:The ratio of Th1/Th2 (both IL-12/IL-10 and IFN-γ/IL-4) decreased in all groups except the normal group. But the ratio of IFN-γ/IL-4 was increased significantly in the therapic group, the western medicine group and the with group combined Chinese and western medicine. The serous level of IL-12/IL-10 was effectively increased in Chinese and western medicine group (p<0.05);(4)Vascular injury factor:The serous vWF and sTM increased greatly in model mice (MRL/lpr mice). The therapentic treatment and the combined Chinese and western medicine treatment lowed greatly both vWF and sTM in the model mice. Western medicine treatment lowered the level of serous vWF in model mice.(5)Renal Pathology:Under microscope, glomerulus proliferation or atrophy were found by observing renal pathological sections. There were also capillo fibrinoid necrosis and chronic inflammatory cells infiltrated around blood vessels. Under electron microscope, incrassation of basilar membrane, swollen endothelial cells, and dense deposits near basilar membrane were observed. All the treatments might relieve the micro pathology to some extent.Conclusion1. According to the result of clinical experiment, the balance of Th1/Th2 was disturbed in SLE patients, with the rise of Th2 and increase in the expression of vWF and STM in the serum. Qing-yang-tou-jie as the principal treatment in combination with western medicine was effective to reduce symptoms of SLE and Yin-deficiency, and the effect was superior to that of west medicine alone. The combined therapy was effective to improve the balance between Th1/Th2 cells, reduce the expression of vWF and sTM in the surum, relieve the vascular injury, and reduce the dosage of prednisone as well as its toxic side effects.2. Experiment on animal showed that MRL/lpr mice suffered disturbance in Th1/Th2 balance, which was reflected by increase of Th2, decrease in Th1/Th2, increased expression of vWF and sTM in the serum, and pathologic changes in the kidney tissue. Qing-yang-tou-jie therapy was able to decrease the expression of Th2, increase IFN-γ/IL-4 ratio, suppress the expression of vWF and sTM in the serum and relieve pathological changes of kidney.3. There was difference in the results of the animal experiment and clinical experiments. The Th1 cell factors in MRL/Lpr mice were lower or had no difference compared with the normal control mice, while in SLE patients, serum Th1 factors were significantly higher than in the control group. The difference in the results between the model mice and SLE patients might be due to difference in disease mechanisms.4 Qing-yang-tou-jie therapy effectively to ameliorate the imbalance of Th1/Th2 cell factors, and thus helped to maintain the normal function of T cells, suppress the abnormal reproduction of B cells, reduce the inflammatory injury caused by the deposition of inflammatory factors and immune complex, and protect vascular endothelium, achieving the effect to cure SLE.更多还原