自拟凉血消疕汤治疗寻常型银屑病临床观察及其作用机制研究

【作者】 黄琪；

【导师】 周飞红；

【作者基本信息】 湖北中医学院 ， 中西医结合临床， 2008， 硕士

【摘要】 目的:1.观察凉血消疕汤治疗寻常型银屑病的临床疗效及安全性。2.通过体外传代培养人永生化角质形成细胞株（HaCaT）,观察不同浓度凉血消疙汤含药血清对HaCaT细胞增殖、血管内皮生长因子（VEGF）表达的影响并探讨其治疗银屑病的作用机制。为凉血消疕汤在临床的广泛应用提供理论基础。方法:一.观察凉血消疕汤治疗寻常型银屑病的临床疗效1病例来源2005年8月-2008年3月武汉市第一医院皮肤科银屑病专科门诊患者,临床诊断寻常型银屑病,皮损面积小于30%体表面积,就诊前一月未经口服中药治疗未接受免疫抑制剂治疗的患者46例。2患者按随机数字表法分为2组,两组在年龄、性别、病程、严重程度上无显著性差异（P＞0.05）,具有可比性。治疗组26例服用凉血消疕汤,每日一剂,分两次口服。对照组20例服用郁金银屑片,每日3次,每次4片。局部治疗二组均采用10%煤焦油软膏。每2周为1疗程,连续观察8周后根据银屑病皮损面积和严重度指数PASI评分系统,来评价临床疗效。同时监测患者血尿常规,肝肾功能,记录不良反应。3观察结束后采用SPSS14.0软件系统对结果进行分析。二.观察不同浓度凉血消疕汤含药血清对HaCaT细胞增殖及血管内皮生长因子（VEGF）表达的影响1体外培养HaCaT细胞传至第三代备用。当细胞融合80%以上时,加入0.25%胰酶消化,37℃、5%CO₂培养箱中孵育5min,倒置显微镜下观察细胞形态,细胞变圆,细胞间连接中断,加入适量培养基终止消化,轻轻吹打瓶壁使细胞呈单细胞悬液,添加适量培养基吹打混匀,接种至96孔板备用。2大鼠灌胃制备盐水,低、中、高剂量凉血消疙汤血清分别作用于HaCaT细胞。将24只大鼠随机分为4组,空白对照组（生理盐水）,凉血消疕汤低、中、高剂量组（0.5g/ml、1g/ml、2g/ml）,每组6只,雌雄各半。给药组,每天灌胃给药一次,连续7天;对照组,每天灌服等量生理盐水。具体灌胃剂量按照《中医科研设计与统计学》（湖南科学技术出版社48实验动物给药方法52中药血清药理学方法）计算。末次灌胃1h后取血,分离血清,-20℃冰箱保存备用。3采用MTT法观察凉血消疙汤含药血清对细胞增殖影响。调整细胞数至2×10⁴的密度接种于96孔板,细胞70%-80%融合后弃去原培养液,加入无血清的DMEM培养基培养4小时后去上清,加入各组受试品,在酶标仪上测OD值。4运用ELIsA法检测上清液中VEGF的表达,调整细胞数至1×10⁵接种于24孔板,细胞70%-80%融合后弃去原培养液加入各组受试品,培养24小时后取上清按照人VEGF ELISA试剂盒（深圳晶美）步骤操作。结果:一.临床观察根据PASI评分下降比值,治疗组痊愈率7.69%,显效率57.69%,总显效率65.38%;对照组痊愈率5%,显效率30%,总显效率35%;治疗组总显效率高于对照组,经统计学分析差异有统计学意义（P＜0.05）。治疗前,治疗组患者PASI评分9.64±3.86,对照组PASI评分9.58±2.48。治疗后,治疗组患者PSAI评分4.23±3.55,对照组患者PASI评分5.48±3.83,两组患者治疗后总评分比较差异有显著统计学意义（P＜0.01）,治疗组优于对照组。二.实验研究1.抑制细胞增殖:各实验组分别作用HaCaT细胞24小时后,HaCaT细胞组OD₄₉₂为0.961±0.028,TGF-α刺激HaCaT细胞组OD₄₉₂为1.163±0.073,生理盐水大鼠血清组OD₄₉₂值为1.098±0.067,低、中、高含药血清组OD₄₉₂值分别为0.908±0.041、0.914±0.096、0.553±0.159,TGF-α刺激空白组OD₄₉₂值明显高于空白组OD₄₉₂（P＜0.05）随药物浓度梯度的升高,各含药血清组细胞增殖活性逐渐降低,与盐水组比较差异有统计学意义（P＜0.05）,各加药组组间比较,低剂量组与中剂量组比较差异无统计学意义（P＞0.05）,高剂量组与盐水血清组,低、中剂量组比较差异均有显著统计学意义（P＜0.01）。2.抑制VEGF分泌:各实验组分别作用HaCaT细胞24小时后,VEGF表达量HaCaT细胞组OD₄₅₀值为0.47 3±0.142,TGF-α刺激HaCaT细胞组OD₄₅₀值为1.007±0.261,生理盐水大鼠血清组OD₄₅₀值为1.500±0.041,低、中、高含药血清组OD₄₅₀值分别为1.185±0.133、0.964±0.403、0.33±0.067,TGF-α刺激HaCaT细胞组OD₄₅₀值与HaCaT细胞组OD₄₅₀值比较差异有显著统计学意义（P＜0.01）,并且随着药物浓度梯度的升高,各含药血清组VEGF分泌量逐渐降低,与盐水组比较差异有显著统计学意义（P＜0.01）,各加药组组间比较差异亦有显著统计学意义（P＜0.01）。经相关回归分析显示,VEGF表达量与呈高度负相关,r=-0.998,P＜0.01。提示以上各凉血消疕汤含药血清对HaCaT细胞VEGF分泌有明显抑制作用,且具有浓度效应关系。结论:1.凉血消疕汤对寻常型银屑病临床治疗作用显著,明显优于对照组。2.凉血消疙汤含药血清能够抑制HaCaT细胞增殖,有效下调HaCaT细胞VEGF的分泌量,抑制VEGF表达,从而抑制血管新生,这可能是其临床治疗银屑病的药理作用机制之一。更多还原

【Abstract】 Objective1.To observe the clinic curative effect and safety of Liangxuexiaobi Decoction（LXXB） for psoriasis vulgaris.2.To observe the cell proliferation and VEGF expression of HaCaT cells which are cultured in vitro and intervened by different concentration of Liangxuexiaobi Decoction. Discuss the effective therapy mechanism of traditional Chinese medicine in psoriasis vulgaris and provide scientific basis for asiaticoside application in clinic.MethodsⅠclinical observation1.A total of 46 psoriasis vulgaris sample patients who came from the psoriasis clinic in The NO.1 Hospital of Wuhan from August,2005 to March,2008 were observed according to the standards of psoriasis area below 30%of human’s body surface area,and never accepted the therapy of traditional Chinese medicine or immunosuppressive one month before observed.2.The patients were divided into 2 groups randomly.Two groups have no obvious differrences in age、sex and PASI（P＞0.05）has comparability.The treatment group includes 26 cases which are given Liangxuexiaobi Decoction,one dose for one day,taken at two times,while the comparison group includes 20 cases which are given Yujinyinxie PIece, three times one day,with 4 pieces each time.Take 10%coal tar ointment as the local treatment both groups.We set every two weeks as a treatment course.Then the clinic curative effects of the two groups are compared according to the PASI,after observing four treatment courses consecutively.Also blood analysis、urine analysis、Hepatic function and renal function is monitored and reactions are recorded.3.Statistics:After the observation,we analyzed all the data by SPSS14.0.ⅡExperiment1.HaCaT cells were cultured in vitro in 10%DMEM culture medium.Digest HaCaT cell by 0.25%pancreas enzyme,37℃、5%CO₂,5min,after the cells were pasted with the wall of culture bottle 70%-80%.Observed the morphological change of HaCaT through the convert microscope,add appropriate culture medium to stop the digestion when the cells turn round and the cells junctions get interrupted,blow the wall of culture bottle until it becomes unicellular suspension,then inoculated to 96 hole board.2.Rats are divided into groups to make serum contained Liangxuexiaopi Decoction by intragastral.Divided 24 rats into 4 groups:blank comparison group(physiological Saline group,liangxuexiaobi decoction groups（the end concentration is 0.5g/ml、1g/ml、2g/ml）,every group contain 6,half is female and the other is male.The rats of the LXXBD groups were medicated once a day,continuing for 7 days.The rats of blank compareis- on groups（physiological saline group）were medicated equal physiological saline ever- yday.（《TCM Research Design & Statistics》Hunan science and technology publicshing house 48 the method of medication about experimental animals）.Collect blood 1 hour after the last intragastral,distill the serum,store at -20℃refrigerator.3.Obtain the proliferation level of the cell which is intervened by LXXBD using MTT. Inoculated cells to 96 holes board when density is 2×10⁴.Discard the old culture medium when the cells were pasted with the wall of culture bottle 70%-80%,then keep the culture medium without FBS for 4 hours,check the OD₄₉₂by the enzyme marks board after adding different concentration medicine.4.Inoculated the cells to 96 holes board when density is 1×10⁵.Discard the old culture medium when the cells were pasted with the wall of culture bottle 70%-80%,add different concentration medicine,collect supernatant after 24 hours,Check the expression of VEGF by ELISA according to the approach of Human VEGF ELISA KIT from Jingmei.Result:一.clinical observationAccording to the decline ratio of PASI,as for the treatment group,the convalesceng ate is 7.69%,the showing results rate is 57.69%,and the total showing results rate is 65.38%.While for the comparison group,the convalescing rate is 5%,showing results rate is 30%,and the total showing results rate is 35%.The treatment group total showing results rate is higher than the comparison group.The score of the treatment group is 9.64±3.86 while the score of the comparison group is 9.58±2.48 before accept treatment,we get the the score of the treatment group is 4.23±3.55 while the score of the comparison group is 5.48±3.83 at the end of the observation,there is obvious differences between the scores below（P＜0.01）.二.experiment1.In the experiment of MTT,the OD₄₉₂value of blank control group is 0.961±0.028, the OD₄₉₂value of blank control group which is intervened by TGF-a is 1.163±0.073, the OD₄₉₂value of blind comparison group is 1.098±0.067,the OD492 value of different LXXBD groups sequentially are 0.908±0.041、0.914±0.096、0.553±0.159.We can see that the OD₄₉₂value of blank control group which is intervened by TGF-a is higher than the OD₄₉₂value of blank control group（P＜0.05）.Following the rising of medicine concentration gradient,the cell proliferation activation gradually reduced,the cell proliferation levels of LXXBD groups are obviously lower than which is in HaCaT cell group（P＜0.05）,and among LXXBD groups,there is no significance difference between the low concentration group and medium concentration group （P＞0.05）.But it shows an obvious difference between high concentration group and blind comparison group or low concentration group or medium concentration group （P＜0.01）.2.From the result of ELISA,we can find that the VEGF expression of blank control group is 0.473±0.142,the value of blank control group which is intervened by TGF-a is 1.001±0.261,and the value of different concentration LXXBD sequentially are 1.185±0.133、0.964±0.403、0.33±0.067.There is significant difference between blank control group which is intervened by TGF-a and blank control group（P＜0.01）.the VEGF expression of LXXBD groups are obviously lower than which is in physiological saline group（P＜0.01）.It shows that following the rising of medicine concentration gradient,the VEGF expression gradually reduced,and among the LXXBD groups,the values of VEGF expression also has difference（P＜0.01）.By Correlation analysis,the VEGF expression is highly negatively related to medicine concentration gradient,r=-0.998,P＜0.01.It shows that LXXBD can evidently suppress the VEGF expression of HaCaT cell. Conclusion:1.The LXXBD has evident clinic curative effect for psoriasis vulgaris,it is better than the comparing group obviously.2.The serum with LXXBD could restrain the proliferation of HaCaT cell and has a good effect of reducing the VEGF secretion,bating the expression of VEGF,and controlling the angiogenesis,which might be an effective therapy mechanism for psoriasis vulgaris curing.更多还原