祛银颗粒对KGF诱导的HaCaT细胞增殖及分泌TNF-α的影响

【作者】 鹿见香；

【导师】 白彦萍；

【作者基本信息】 北京中医药大学 ， 中医外科学， 2011， 硕士

【摘要】 本论文主要包括文献综述和研究报告两部分文献综述共两篇。第一篇综述了中药血清药理学的研究进展。中药血清药理学克服了用中药制剂直接进行体外实验时,排除了中药制剂本身的理化性质对实验的干扰。本文总结了中药药理学研究中动物模型的制备、动物给药方案、量效、含药血清的采集及处理等方面的经验和新的研究。第二篇综述了近十年来,中医药治疗寻常型银屑病实验研究进展,包括发病机制方面的实验研究和中医证型的实验研究两个方面,总结了最新的研究状况。研究报告部分主要是中药祛银颗粒含药血清对银屑病进行期角质形成细胞增殖及分泌TNF-α的影响,从这两个方面对中药治疗银屑病的作用机制进行研究。目的中药祛银颗粒是由清热凉血药物组成,在临床上治疗银屑病具有良好的效果,现应用中药血清药理学手段最大限度的模拟中药复方在体内吸收、代谢的过程。将其作用于由角质形成细胞生长因子(KGF)诱导HaCaT细胞增殖的体外银屑病进行期角质形成细胞的生长、增殖模型。观察中药含药血清对细胞的生长形态、增殖、凋亡及其分泌产生的TNF-α的影响,进而明确祛银颗粒治疗银屑病的作用机制。方法1.预实培养1000个/孔—10000个/孔不同数目的HaCaT细胞7天,并绘制细胞的生长曲线,获得符合实验的细胞数量为每孔3000。2.将HaCaT细胞每孔3000种植于96孔板,培养24小时,待细胞贴壁后,加入0ng/ml、2.5ng/ml、5ng/ml、10ng/ml、20ng/ml不同浓度的KGF体积2ul刺激HaCaT细胞的生长,测定药物刺激下的7天的细胞生长曲线,获得KGF促进HaCaT细胞生长的最佳浓度10ng/ml,并验证模型的稳定性。3.将实验SD雄性大鼠40只,随机分为5组(祛银颗粒低、中、高浓度组、生理盐水组、维A酸组),每组8只,根据血清药理学知识,对SD大鼠按照体重换算,将药物对大鼠每日灌胃一次,连续5天,禁食12小时,末次灌药2小时后,经水合氯醛麻醉,腹主动脉取血、离心、灭活保存,来制备含药血清。4.将HaCaT细胞以每孔3000个种植于96孔板,分为6组(NS含药血清组、中药低、中、高含药血清组、维A酸含药血清、空白对照组),一式三孔,24小时待细胞贴壁后以KGF刺激细胞使其快速增殖,从而模拟银屑病进行期的表皮角质形成细胞过度增殖的状态,同时予以不同浓度的祛银颗粒(低、中、高)药物血清、维A酸(阳性对照药物)含药血清、生理盐水(排除血清的干扰)含药血清对细胞模型进行刺激干预,倒置显微镜下观察药物作用后的细胞生长状态,用CCK-8测量药物血清对细胞增殖的影响。5.将将HaCaT细胞以每孔浓度1×105/ml的1ml种植于6孔板,培养24小时待细胞贴壁后,加入KGF40ul和含药血清共同培养48小时后,经过处理,上流式细胞仪检测细胞周期的影响；6.将HaCaT细胞以每孔3000个种植于96孔板,分为5组(祛银颗粒低、中、高浓度组、生理盐水组)按照ELISA法步骤,检测中药药物血清作用48小时后对细胞上清分泌TNF-α的影响。结果1.中药祛银颗粒药物血清可以影响KGF刺激的HaCaT细胞生长状态及其增殖,可以用于模拟银屑病进行期的表皮状态而且模型具有稳性定。2.药物血清对细胞的生长形态有着明显的作用：其中高剂量组和中剂量组的含药血清可以促进细胞的萎缩、变形、细胞核部分消失,低剂量组的含药血清作用不明显3.药物血清随着药物浓度的不同,抗增殖的能力不同,浓度越大,抑制作用就越明显,呈正相关性；药物血清的抑制作用随时间变化不同,其中48小时处得抑制作用最强,其中24小时、48小时及72小时的抑制率分别为：20.1363%、36.9866%,16.8975%、与阳性对照药物维A酸组在48小时的抑制率36.99%,无统计学差异(P>0.1),与阴性模型对照组相比有着明显的差异(P<0.05)。4.流式细胞仪检测细胞的周期及凋亡,结果可见,高浓度的药物血清组S期细胞数量较其他组较低,与模型组有着明显的差异性(P<0.05)。5.用ELISA法检测药物对细胞作用最强的时间点,48小时细胞培养上清液中的TNF-α,结果显示,高浓度的中药药物血清与模型组比较有着明显的差异性(P<0.05)。结论祛银颗粒药物血清具有抑制KG诱导的HaCaT细胞增殖的作用,并且可以影响细胞分泌的肿瘤坏死因子-α(TNF-α)的作用。从而提示以祛银颗粒为代表的清热凉血中药治疗银屑病的作用机制,并且能够从抑制表皮增殖和抑制细胞产生细胞炎症因子等方面验证中药祛银颗粒治疗银屑病的机制。更多还原

【Abstract】 This dissertation was composed of two parts,they were literature review and a experimental studyThere were two literature reviews.The first one reviews the progress of the serum pharmacology.Chinese medicine serum pharmacology (CMSP)was a new method of study the TCM.It eliminating the physical and chemical properties of traditional Chinese medicine itself,which may make the experiment not ture. This part summarizes the processing experience and new research of CMSP on the animal model, animal dosing regimen, dose, drug-containing serum collection and so on. The second literature review was on experimental research progress of vulgaris psoriasis in Chinese medicine treatment, including the pathogenesis and type of TCM.The secong part was on experimental study.It was about QuYin Granula Serum worked on the model of keratinocyte proliferation in the process of psoriasis,studying the mechanism of psoriasis two aspects of form keratinocyte proliferation and secretion of TNF-α.Objective QuYin Granula was formed by cleaning heat and cooling blood drugs,which is effective on treatment of psoriasis in clinical.Now the application of CMSP to simulate Chinese medicine compound in vivo absorption, metabolism process. The QuYin Granula Serum was worked on the model of acute period of psoriasis. The model was HaCaT cell which was induced by keratinocyte growth factor (KGF). Then the effects of serum was observed on proliferation, apoptosis and secretion of TNF-αproduced by the impact, and then studied the mechanism of psoriasis.Methods1.Planting 1000/hole-10000/well with different numbers of HaCaT cells for 7 days, then drew the cell growth curve, the number of cells obtained with the experiment for 3000 each hole in pre-implementation.2.The HaCaT cells were grown in 3000 for each hole for 24 hours until the cells adherent to the hole, add Ong/ml,2.5ng/ml,5ng/ml,10ng/ml,20ng/ml of different concentrations of KGF to stimulate HaCaT cell volume 2ul growth to stimulate the cells for 7 days,then drew the growth curve obtained KGF optimal concentration lOng/ml, and verify the stability of the model. 3.40 SD male rats were randomly divided into 5 groups (low, medium and high concentration of QuYin Granula Serum, saline group, retinoic acidA group), n=8. According to the knowledge of serum pharmacology, the drugs was fed on rats according to body weight conversion once a day for 5 days, 12 hours of fasting, after chloral hydrate anesthesia, getting the abdominal aorta blood, centrifugation, inactivated preservation, to prepare drug-containing serum.4. The HaCaT cells was grown to 3,000/ well in 96-well plates, divided into 6 groups (NS containing serum group, herbal low, medium and high serum group, retinoic acid A group serum, control group), using KGF to stimulate rapid proliferationafter 24 hours until cells adhered to the cells. observed under inverted microscope after the cell growth state, with the CCK-8 measuring serum on cell proliferation.5. HaCaT cells to the concentration of 1×105/ml per well in 6-well plates for 24 hours,add KGF40ul and QuYin Granula Serum for 48 hoursafter treatment, then flow Cytometry cell cycle6.T he HaCaT cells grown to 3,000 per well in 96-well plates, divided into 5 groups (herb serm of low, medium and high, saline group,KGF group)tested their secretion of TNF-a by ELISA method steps, when QuYin Granula Serum worked on the cells after 48 hours.Conclusion1. QuYin Granula Serum can affect the proliferation of HaCaT cell growth state and,KGF can be used to simulate the HaCaT cell rapidly proliferation simulation psoriasis condition, and the model was stable.2. QuYin Granula Serum has a significant role on cell morphology:in the high dose group and dose group containing serum to promote cell atrophy, deformation, partial disappearance of the nucleus, low-dose group containing serum is not obvious.3. Different QuYin Granula Serum concentration with the different ability of resistance to the proliferation:With different concentrations of serum concentrations of different drugs, anti-proliferative capacity is different,the higher concentrations of serm,the inhibition is more obvious. Inhibition of serum is different with time, which was strongest inhibition at 48 hours,24 hours,48 hours and 72 hours of inhibition rate:20.1363%,16.8975%,36.9866%, and the positive control drug-dimensional A Acid inhibition rate of 36.99% of group no significant difference, compared with the negative model group has significant difference (P<0.05).4. ELISA assay in cell culture supernatant TNF-α, results showed that high serum concentrations of Chinese medicines in 48 hours and the model group had significantly different (P<0.05).ConclusionQuYin Granula has the ability of inhibit KGF induced proliferation of HaCaT cells, and can affect cells in the tumor necrosis factor-α(TNF-α). QuYin Granula can treat disease of epidermal hyperplasia,such as psoriasis, QuYin Granula can inhibit the epidermal proliferation and cytokine productiondisease verifying the mechanism of QuYin Granula treatment of psoriasis更多还原