痤疮的流行病调查及中医分型和积雪甙对造模大鼠炎症结节作用机理探讨

【作者】 陈慧；

【导师】 王玮蓁；

【作者基本信息】 湖北中医学院 ， 中西医结合， 2008， 硕士

【摘要】 本课题主要包括两个方面的内容:1痤疮的流行病调查及中医分型;2积雪甙对造模大鼠局部炎症结节的作用机理。第一部分:痤疮的流行病调查及中医分型目的本课题主要通过流行病调查研究的方法,探讨痤疮的中医分型以及诱导或加重痤疮的危险因素,进一步了解中国痤疮流行病发展趋势,为指导临床治疗提供理论帮助。研究方法本课题采用流行病学方法,设计统一的流行病调查表,收集武汉市第一医院每周痤疮专科门诊的痤疮患者的资料,采用医学统计方法对调查对象的性别、年龄、皮肤类型、精神状态、睡眠时间、女性月经与痤疮的关系以及中医辨证分型进行统计学分析,最后得出结论。结果1、2558例痤疮患者中,女性1748例,男性810例,就诊患者中以女性多见,发病年龄以青春期为主,男性发病年龄集中在15～20岁的年龄段,女性患者的发病年龄集中在小于15岁的年龄段。2、油性皮肤的痤疮患者最为多见所占比例最大,其次为中性皮肤、混合性皮肤、干性皮肤。3、2558例痤疮患者中,精神紧张的痤疮患者1619例,占63.29%,认为与痤疮发病相关的占30.70%;无精神紧张的患者939例,占36.71%。4、经常熬夜的患者所占比例为21.85%,偶尔熬夜的患者占42.65%,认为与痤疮发病相关的占49.27%;不熬夜的患者所占比例为35.50%。5、2558例痤疮患者中,痰瘀结聚型最多,占37.57%,其后依次为肝气郁结型,占28.03%,湿热蕴结型占26.94%,肝肾阴虚型占6.53%,肺经风热型占0.67%。男性痤疮以痰瘀结聚型最为多见,青春期后女性患者以肝气郁结型最为多见,青春期女性患者证型分布无明显差别。6、女性痤疮患者中,痤疮加重与月经有关的为1103例,主要为月经前痤疮加重,占56.18%,其次为经中加重,经后加重。肝肾阴虚型与月经的关系最密切。结论1、2558例就诊痤疮患者中以青春期女性患者多见,发病年龄女性早于男性。2、油性皮肤、精神紧张、熬夜可能为诱发或加重痤疮的危险因素,女性患者多在月经前痤疮加重。3、我科痤疮门诊的患者中,男性以痰瘀结聚型最为多见,青春期后女性患者以肝气郁结型最为多见,青春期女性患者证型分布无明显差别。第二部分:积雪甙对造模大鼠局部炎症结节的作用机理的探讨目的本研究拟从动物模型中,探讨积雪甙对炎症后成纤维细胞作用机理,通过对光镜和电镜的观察,了解在炎症发展过程中成纤维细胞的超微结构的改变,为积雪甙在临床广泛应用提供理论基础。实验方法1.预实验:1.1分别用单纯的痤疮丙酸杆菌、不完全福氏佐剂、痤疮丙酸杆菌+不完全福氏佐剂造模。在造模后不给予药物干预,观察造模后各组大鼠局部组织的改变,并通过光镜观察不同时期的病理改变,从而确定炎症反应高峰期,成纤维细胞、胶原纤维在不同时期的改变情况以及三组模型中最佳的动物模型。1.2疮丙酸杆菌培养:冻干痤疮丙酸杆菌以脑心浸液复苏后,室温下放置数分钟,取少许溶液于哥伦比亚血平板,厌氧环境下35-37℃培养48-72小时,调整痤疮丙酸杆菌浓度为2×10¹⁰/ml备用。并经镜下检验观察为蓝色棒状痤疮丙酸杆菌。1.3造模:大鼠背部皮肤备皮,约1×1cm大小,每只大鼠四处。痤疮丙酸杆菌组将调整好浓度的痤疮丙酸杆菌接种到大鼠皮下,每处0.1ml,约2000万单位的细菌;不完全福氏佐剂组,将不完全福氏佐剂与生理盐水以3:7的比例乳化后接种到大鼠皮下,每处0.1ml;痤疮丙酸杆菌+不完全福氏佐剂,将不完全福氏佐剂与痤疮丙酸杆菌以3:7的比例乳化后接种到大鼠皮下,每处0.1ml,约2000万单位的细菌。2.实验2.1痤疮丙酸杆菌培养:同前2.2造模:大鼠背部皮肤备皮,约1×1cm大小,每只大鼠四处。将调整好浓度的痤疮丙酸杆菌与不完全福氏佐剂以7:3的比例乳化后接种到大鼠皮下,每处0.1ml,约2000万单位的细菌。2.3.分组:将四十只大鼠随机分为四组,积雪甙和阿奇霉素组、积雪甙组、阿奇霉素组、空白对照组,每组十只。2.4灌胃:于造模后第二天开始灌胃,每只大鼠灌2ml,药物浓度分别为:积雪甙和阿奇霉素组,积雪甙浓度为1.5mg/ml,阿奇霉素浓度为10.42mg/ml;积雪甙组,积雪甙浓度为0.75mg/ml;阿奇霉素组,阿奇霉素浓度为5.21mg/ml;空白对照组,0.9%生理盐水。（《实验动物学》湖北科学技术出版社）2.5光镜标本制作及观察:于造模后第20、30天切除大鼠造模组织,10%甲醛固定,按石蜡切片常规处理,用HE染色,在光镜下观察。2.6电镜标本制作及观察:在造模后第20、30天切除大鼠皮肤,2.5%戊二醛前固定,1%锇酸固定,梯度乙醇脱水,树脂包埋,半薄切片定位,LKB2V型超薄切片机切片,日立H2600型透射电镜观察,观察接种部位成纤维细胞内细胞器结构的变化。结果1.预实验结果:三组模型比较发现,以痤疮丙酸杆菌+不完全福氏佐剂的炎症反应、成纤维细胞、胶原纤维的改变最为明显。不同时期的病理改变显示,造模后第10天炎症反应最剧烈;第20天成纤维细胞增殖,胶原纤维增多,排列致密最明显;第30天各组炎症反应较轻甚至消失,但仍有成纤维细胞的增殖和胶原纤维增多增粗,较20天有减轻。2.实验结果2.1光镜结果:空白组胶原增生、增粗明显,排列致密,30天较20天的改变不明显。阿奇霉素组胶原改变较空白组稍轻,但同组前后比较改善不明显,有积雪甙干预的胶原改变较空白组明显要轻,同组前后对比,亦有明显改善。2.2电镜结果:空白对照组成纤维细胞增殖,凋亡,细胞体积变大,成纤维细胞体积增大,胞浆内线粒体数目较多,肿胀,粗面内质网扩张明显,部分细胞核内异染色质明显增多。阿奇霉素组成纤维细胞的胞浆改变较空白组轻。经积雪甙干预的成纤维细胞胞浆改变较空白组明显要轻,第30天时,成纤维细胞胞浆改变接近正常。结论积雪甙通过抑制成纤维细胞过于活跃的生物学活性从而使其分泌胶原的功能下降,进而起到抑制胶原增生的作用,减少了造模大鼠炎症结节愈后留下纤维性结节的可能性。更多还原

【Abstract】 This paper includes two parts:one is acne’s epidemic investigation and chinese medicine syndrome differentation.The other is to discuss the mechanism of asiaticoside to the fibroblast of the rat model.Part 1:acne’s epidemic investigation and chinese medicine syndrome differentation.ObjectiveThis subject is to research acne’s chinese medicine syndrome differntation and the risk factors of inducing or aggravating acne, further understand China acne vulgaris epidemiological trends,provide theoretical guidance to help clinical treatment.MenthodsUsing epidemiological methods,designinguniform epidemiological survey,collecting acne patients from acne specialist out-patient clinics of the first hospital of Wuhan.The investigation of sex,age,skin type, mental state,sleep time,the relationship between women acne and menstrual and the TCM differntation for statistical analysis,finally concluded.Result1.In the 2558 cases of acne patients,women 1,748 cases,male 810 cases,women is most of the treatment patients,age of Incidence is still mainly to puberty,male is to 15 to 20-year-old age group,wemon is to less than 15-year-old age group.2.Oily skin acne patients with the largest proportion,followed by neutral skin,mixed skin,dry skin.3.In the 2558 cases of acne patients,patients with spirit intension accounted for 63.29%.4.Patients with regular insufficient sleep accounted for 21.85%,patients with occasional insufficien sleep accounted for 42.65%,patients with no insufficient sleep accounted for 35.50%.5.In the 2558 cases of acne patients,phlegm and blood stasis of the guitar is most,accounted for 37.57%;Followed by liver-qi stagnation,accounted for 8.03%,damp heat guitar 26.94%,yin deficiency of liver and kidney6.53%,wind-heat of feijing0.67%.Male acne with phlegm and blood stasis of the guitar most common,wemon acne after puberty with liver-qi stagnation most common,there is no significant difference in the puberty6.In wemon acne patients there are 1103 cases having relation with menstrual, major increasing in pre-menstrual,for56.18%,followed by increasing in menstrualand increasing after menstrual,yin deficiency of liver and kidney most closely related to menstrual,followed by liver-qi stagnation.Conclusion1.In the 2558 cases of acne patients,adolescent female patients are most common. Age of Incidence is earlier in wemon than man.2.Oily skin,spirit tension,insufficient sleep can induce or exacerbate acne.3.The most common acne TCM syndrome differentation followed by phlegm and blood stasis of the guitar,liver-qi stagnation,damp heat guitar, yin deficiency of liver and kidney,wind-heat of feijing.Male acne with phlegm and blood stasis of the guitar most common,wemon acne after puberty with liver-qi stagnation most common,there is no significant difference in the puberty.Part 2:To discuss the mechanism of asiaticoside to the fibroblast of rat modelObjectiveThe experiment is to discuss the mechanism of asiaticoside to fibroblast after inflammation from the rat model.To understanding the change of ultramicrostructure of fibroblast in the inflammation developing process through observating the light mirror and the electricity mirror,providing the theoretical basis for the asiaticoside in the clinical application.Methods1.Pre-experiment1.1 With a simple Propionibacterium acnes bacteria,not complete Freund’s adjuvant,Propionibacterium acnes Bacillus+incomplete Freund’s adjuvant model.After the model does not give drug intervention.After the model,observe the pathological changes in different periods,to determine the inflammatory response at its peak,fibroblasts and collagen fibers at different times of change,as well as the three models in the best animal model1.2Propionibacterium acnes culture:The freezing and drying Propionibacterium acnes dissolutes in the brain heart infusion,then layes aside several minutes under the room temperature,takes the little solution culture in 5%blood agar,tires of the oxygen environment in the middle of 35-37℃to culture for 5-7 day,washes 3 times with the physiological saline,adjusts Propionibacterium acnes concertration for 2×10¹⁰/ml spare,and through light mirror confirms that it’s rod-shaped Propionibacterium acnes.1.3Animal model:Shearing fur of the rat back,approximately 1×1 centimeter size,each rat four sides;then respectively injecting cultured propionibacterium acnes,incompleteFreund’sadjuvant,propionibacterium acnes and incomplete Freund’s adjuvant injected into rat subcutaneous.2Experiment2.1 Propionibacterium acnes culture:With the former2.2Batching:Dividing the experiment mouse into four groups:the asiaticoside and azithromycin group、azithromycin group、asiaticoside group、azithromycin group;blank contrast group. 2.3Intragastral:2 ml of every rat.The asiaticoside+azithromycin group: asiaticoside density is 1.5mg/ml,azithromycin density is10.42 mg/ml;The asiaticoside group:asiaticoside density is 0.75mg/ml;azithromycin group:azithromycin density is5.21 mg/ml;blank Contrast group: physiological saline.According to stipulation dosage gives the medicine.in the morning 8-10 of every day.《Experimental Zoolog》Hubei science and technology publishing house)2.4.Light microscopy specimens manufacture and observation:Excising the rat tissue on 10,20,30days.Fixed with 20%formaldehyde.According to paraffin section convention processing,dyed with HE,Observes the vaccination spot to know the structural change of fibroblast under the light mirror.2.5Electron microscope specimen manufacture and observation:On 20, 30days excisions rat tissue,firstly fixes specimen with 2.5%glutaric dialdehydes,then fixes with 1%perosmic.Hands over the ethyl alcohol dehydration,Resin embedding,half thin slice localization;Slices with LKB2V ultra thin slicer,Hitachi H2600 passes the radio mirror observation. observes the vaccination spot fibroblast organ structure changing.Results1 Pre-results:Comparing three group s of ainimal models,the changes of of the inflammatory response,the fibroblasts and collagen fibers are the most obvious in the group of Propionibacterium acnes+Incomplete Freund’s adjuvant.Pathological changes in different periods show that the inflammatory response on the tenth day is the most intense;on the twentith day,fibroblast prolifered mostly,collagen Increased mostly and ordered tightly.On the thirtith day,the inflammatory response of every group is weak even disappear,but there is still the proliferation of fibroblasts and collagen fibers increased thickening,better than the twentith day’s.2Formal experiment results 2.1 OM results:The collagen of the blank contrast group increasing and thicking obviously,ordered tightly,the change on the thietith day’s was not more obvious than the twentith day’s.The change of the collagen of the aithromycin group is slightly Improved than the blank contrast group,but compared with the group before and after the improvement was not obvious.With the asiaticoside’s intervention,The change of the collagen of the asiaticoside group and the asiaticoside+azithromycin group is obviosely impoved than the blank contrast group,the change on the thietith day was more obvious than the twentith day’s.2.2 Electron microscope results:The fibroblasts of the blank contrast group prolifered,vlume increased,Larger number of mitochondria, swelling,RER expanded obviousely.And that fibroblasts apoptosed,the nucleus of heterochromatin increased obviousely.Changes in the cytoplasm of the aithromycin group is better than the blank contrast group’s,but the change on the thietith day was more obvious than the twentith day’ s.With the asiaticoside’s intervention,changes in the cytoplasm of the fibroblasts are improved obviousely than the blank contrast group’s, changes fibroblast closed to normal on the thirtith day.ConclusionAsiaticoside plays a role in inhibiting the proliferation of collagen, by inhibiting fibroblasts’ too active biological activity,so that the function of secretion collagen decline,reduce the possibility of lefting the fibrous nodules after the inflammatory nodules of the model of the rat更多还原