【作者】 陈修漾；

【导师】 陈达灿；

【作者基本信息】 广州中医药大学 ， 中西医结合临床， 2009， 博士

【摘要】 目的:观察中药益发口服液联合糖皮质激素(GC)治疗重型斑秃的临床疗效及药理机制。通过检测重型斑秃患者血皮质醇水平及外周血单个核细胞(PBMCs)中糖皮质激素受体(GR)mRNA的表达水平,探讨重型斑秃的发病机理,分析GR表达水平与病情程度及临床疗效的关系,进而深入探讨中药联合GC与糖皮质激素受体之间的关系。方法:参照《中药新药临床研究指导原则》第三辑内容,选择经临床确诊为重型斑秃并且中医辨证分型属肝肾不足证的患者32例,并设正常健康人对照组20例。将32例重型斑秃病人随机分为中西结合组(17例)和西药组(15例),中医结合组予益发口服液,每次20ml,每天3次;强的松0.25mg/kg,早8时一次顿服,每天1次,法莫替丁片20mg,口服,每天2次。西药组予强的松及法莫替丁片口服,用法用量同中西结合组。两组均治疗6个月后评定疗效,疗效性指标包括:治疗期间头发脱落的根数;治疗期间新发长出的面积;新生头发的密度、粗细、色泽;病人的整体状况。疗效判定标准参照《中药新药临床研究指导原则》第三辑有关内容。治疗前后均观察两组患者血、尿、便常规、心、肝、肾功能(EKG,ALT,AST,BUN,Cr)及不良反应情况。应用化学发光法检测正常对照组以及重型斑秃患者治疗前血清皮质醇含量并比较。应用逆转录-实时荧光定量聚合酶链反应(RT-qRealTime-PCR)检测正常对照组以及重型斑秃患者治疗前、后的GRmRNA表达水平并比较。采用SPSS13.0统计软件包建立数据库并进行统计分析。结果:(一)血清皮质醇方面:重型斑秃患者治疗前血清中皮质醇浓度与正常对照组相比无显著性差异(P＞0.05);中西结合组与西药组两组的血皮质醇浓度比较,差异也是无统计学意义(P＞0.05)。(二) GRmRNA表达水平方面:1.正常对照组与重型斑秃病例组比较:正常对照组与重型斑秃病例组PBMCs均有GRmRNA表达,重型斑秃患者治疗前、后GRmRNA表达水平均低于正常对照组,差异有统计学意义(P＜0.05);并且治疗后GRmRNA表达水平比治疗前更低,差异有统计学意义(P＜0.01)。2.中西结合组和西药组比较:(1)组内比较:两组治疗后GRmRNA表达水平均较治疗前明显降低,经配对样本比较,差异均有显著性意义,中医结合组(P＜0.05),西药组(P＜0.01)。但西药组比中西医组下降幅度更大。(2)组间比较:治疗前,中西结合组与西药组外周血GRmRNA表达水平无显著性差异(P＞0.05)。治疗后,中西结合组GRmRNA表达水平要明显高于西药组,差异有统计学意义(P＜0.05)。(三)疗效比较:采用符合方案集分析(PP)对依从性好且完成试验的受试者进行分析;采用ITT意图治疗分析(ITT)对全部的受试者进行统计分析。方案集分析:中西结合组治愈率58.82%,总有效率为94.1%;西药组治愈率为40%,总有效率为80%,两组间差异有统计学意义(P＜0.05)。两组的意图治疗数据集疗效比较结果与方案集分析结果相同。两组的不良反应比较,差异无显著性意义(P＞0.01)。(四)按脱发面积分为普秃、全秃、斑片组,结果斑片组GRmRNA表达水平较高。普秃、全秃组GRmRNA表达水平较低。(五)按疗效分成痊愈、显效、有效、无效四组,结果四组治疗前的GRmRNA表达水平组间比较无统计学差异(P＞0.05)。但四组治疗后的GRmRNA表达水平组间比较差异有统计学意义(P＜0.05)。组内比较:痊愈、显效、有效组治疗前后GRmRNA的差值比较差异均有统计学意义(P＜0.05);无效组治疗前后GRmRNA差值比较差异无统计学意义(P＞0.05)。结论:通过本次研究得到以下结论:1.国内外首次报道重型斑秃患者治疗前血皮质醇含量与正常对照相比无显著性差异,但外周血单一核细胞上的GR mRNA表达水平明显降低。表明重型斑秃患者存在GC-GR紊乱,GRmRNA表达水平降低可能参与了重型斑秃的发病机制。2.国内外首次报道肝肾不足证型的重型斑秃患者外周血单一核细胞上的GRmRNA表达水平显著低于正常对照组,这可能是重型斑秃肝肾不足证型在受体及基因水平上的病理变化。GRmRNA表达水平能否作为肝肾不足证型的现代病理学量化依据,还有待进一步深入的研究。3.国内外首次报道中西结合组治疗后GRmRNA表达水平要明显高于西药组,中西结合组治疗重型斑秃疗效要优于单纯西药组。表明益发口服液与GC合用,可提高患者血GRmRNA表达,或能减轻激素对自身受体的下调作用,从而增强GC敏感性,保证GC与GR的结合水平。从中也提示益发口服液联合GC治疗斑秃的作用与其对GR的调节作用有一定关联,这可能是益发口服液治疗斑秃的作用机制之一。4.国内外首次报道病情较轻的,GRmRNA表达水平较高。病情较重,GRmRNA表达水平较低。因此检测GRmRNA表达水平对判断重型斑秃的预后有一定作用。5.国内外首次报道疗效好的重型斑秃患者其PBMCs中GR mRNA表达水平相对高,疗效差者GR mRNA表达水平相对低。提示如果对反应不佳的患者盲目加大GC的用量只会增加不良反应,弊大于利。推测重型斑秃患者治疗前PBMCs中GRmRNA表达水平的检测可能有助于预测患者对GC的效果,也许能作为指导治疗与预后的参考指标之一。更多还原

【Abstract】 ObjectiveTo analyze the pharmacological mechanism of YIFA oral liquid combined whth glucocorticoid(GC) through the observation of its clinical treatment effect on severe alopecia areata patients.To investigate the pathogenesis of severe alopecia areata by detecting the level of glucocorticoid receptors(GR)mRNA in peripheral blood mononuclear cells(PBMCs) from patients with severe alopecia areata.Analyze both the relationship between the expression level of GR and pathogenetic condition,and between the the expression level of GR and clinical effect.Further more to explore the relationship between GR and Chinese medicine combined with GC.MethodsAccording to the third album《guide line of Chinese herbal medicine clinical research》,32 cases clinical diagnosed as severe alopecia areata and deficiency of liver and kidney as their TCM syndrome were selected.And 20 cases as normal control group.32 cases of severe alopecia areata patients were randomly divided into integrated Chinese and western medicine group(17 cases) and western medicine group(15 cases).Integrated Chinese and western medicine group was given YIFA oral liquid,20ml each time and three times a day;prednisone 0.25mg/kg,every 8 a.m.;famotidine 20mg each time,twice a day.Western medicine group was given prednisone and famotidine only,usage and dosage with integrated Chinese and western medicine group.Evaluting the treatment effect of two groups after six months treatment.The therapeutic indexes including:the root of hair loss during treatment,the new hair area during treatment,the color,density,thickness of new hair and the whole situation of the patients.The criteria for treatment effect also reffered to the third album《guide line of Chinese herbal medicine clinical research》. The patients of two groups were observed blood routine,urine routine,soil routine,cardiac function(EKG),liver function(ALT,AST),renal function(BUN, Cr) and adverse reactions before and after treatment.Before treament,serum cortisol levels was measured by Chemiluminescence from 32 cases of severe alopeeia areata patients and 20 normal controls.GR was measured by reverse transcription-real time fluorescence quantitative polymerase chain reaction (RT-FQRealTime-PCR) from severe alopecia areata patients(before and after treatment) and normal controls.The data were inputted into a computer and were analyzed by statistical package for the social science(SPSS) 13.0.Results(A).Comparison of serum cortisol level:Serum cortisol level from severe alopecia areata patients had no significant difference compared to the normal controls(P＞0.05);Serum cortisol level between integrated Chinese and western medicine group and western medicine group alos had no significant difference(P＞0.05).(B).Comparison of the GRmRNA expression level1.The normal control group compared with severe alopecia areata patients group:GRmRNA was detected in PBMCs from all severe alopecia areata patients and the normal controls.The expression of GRmRNA before and after treatment of severe alopecia areata patients was significantly lower than that of the normal controls(P＜0.05);And the expression of GRmRNA after treatment of alopecia areata patients was significantly lower than that before treatment(P＜0.01).2.Integrated Chinese and western medicine group compared with western medicine group1:(1) Comparison within the groupsGRmRNA expression level after treatment of two groups was significantly lower than that before treatment by two pairs sample comparison.And western medicine group dropped greater than integrated Chinese and western medicine group.Integrated Chinese and western medicine group(P＜0.05),western medicine group(P＜0.01).(2) Comparison between the groups Before treatment,GRmRNA expression level of integrated Chinese and western medicine group had no significant difference compared to western medicine group(P＞0.05).After treatment,GRmRNA expression of integrated Chinese and western medicine group was significantly higher than that of western medicine group(P＜0.05).(C).Comparison of treatment effectAnalyzed the testers with PP analysis who had good compliance and complete treatmeat test.Analyzed all the testers with ITT analysis.PP analysis:the cure rate of integrated Chinese and western medicine group was 58.82%,while the total effective rate was 94.1%.Tthe curative rate of western medicine group was 40%,while the total effective rate was 80%.The cure rate and the total effective rate of integrated Chinese and western medicine group was significantly higher than that of western medicine group(P＜0.05).The results of ITT analysis was the same to the results of PP analysis.The adverse reactions between integrated Chinese and western medicine group and western medicine group had no significant difference(P＞0.01).(D).The patients were divided into alopecia universalis group,alopecia totalis group and patiching group according to the bald area.As the result, the GRmRNA expression lever of patiching group was significantly higher than that of alopecia universalis group and alopecia capitis totalis group.(E).According to the treatment effect,the patients were divided into recovery group,excellence group,effective group and ineffective group.GRmRNA expression level before treatment of four groups had no statistically difference(P＞0.05).But after treatment,GRmRNA expression level of four groups had significant difference(P＜0.05).Comparison within the groups: Before and after treatment,the differential value of GRmRNA expression level in recovery group,excellence group and effective group had significant difference(P＜0.05);While the differential value of GRmRNA expression level in ineffective group had no significant difference(P＞0.05).ConclusionWe can draw a conclusion as the following:1.We firstly reported that serum cortisol level from severe alopecia areata patients had no significant difference compared to the normal controls. However,the expression of GRmRNA of severe alopecia areata patients was significantly lower than that of the normal controls.It suggested that the disorder of GC-GR exist in alopecia areata patients.And the decrease of GRmRNA in peripheral blood mononuclear cells may be invoved in the pathogenesis of severe alopecia areata2.We firstly reported that the expression of GRmRNA in peripheral blood mononuclear cells of severe alopecia areata patients and deficiency of liver and kidney as their TCM syndrome was significantly lower than that of the normal controls(P＜0.05).This maybe the pathological change on receptor and gene level in severe alopecia areata patients and deficiency of liver and kidney as their TCM syndrome.It depends on further research that whether GRmRNA expression level can be the mordern pathology quantitative basis or not.3.We firstly reported that GRmRNA expression of integrated Chinese and western medicine group after treatment was significantly higher than that of western medicine group.The treatment effect of integrated Chinese and western medicine group was better that that of western medicine group.It indicated that YIFA oral liquid combined with glucocorticoid can raise GRmRNA expression of severe alopecia areata patients,or decrease the down-regulation of glucocorticoid towards its receptor,which can increase the sensibility of glucocorticoid and guarantee the binding level between GC and GR.It pointed out that the treatment effect of YIFA oral liquid combined with glucocorticoid was related to its regulation towards GR.This maybe one of the action mechanism of YIFA oral liquid to alopecia areata.4.We firstly reported that the ligher pathogenetic condition was,the higher GRmRNA level expressed.On the contrary,the heavier pathogenetic condition was,the lower GRmRNA level expressed.For this reason,examining GRmRNA expression level is useful in judging the prognosis of severe alopecia areata.5.We firstly reported that the better the treatment effect was the higher GRmRNA expressed in PBMCs of severe alopecia areata.On the contrary,the worse the treatment effect was the lower GRmRNA expressed.It indicated it would increases adverse reactions if we blindly increase the dosage of GC to the patients who had bad response to GC.Disadvantage would exceed disadvantage. It is supposed that examining GRmRNA expression level of severe alopecia areata before treatment maybe contribute to the effect prediction which the patients towards GC.Moreover it maybe one of the reference index to direct treatment and prognosis.更多还原