消银解毒饮及拆方对角质形成细胞COLO-16增殖的调控作用

探讨消银解毒饮是否对角质形成细胞的增殖具有直接作用;以及消银解毒饮中凉血、解毒、祛风除湿等三组主要成分在治疗银屑病中所起的作用.本研究以角质形成细胞株COLO-16为研究对象;用四唑盐比色法观察了消银解毒饮及拆方后各组药物血清对COLO-16细胞增殖的影响.结果表明消银解毒组和凉血方组体外培养COLO-16细胞的存活率明显降低,而且存活率与含药血清浓度呈负相关.     

白消一号方对黑素瘤细胞与角质形成细胞混合培养模型中黑素合成的影响

目的 体外构建黑素瘤细胞与角质形成细胞直接接触的混合培养模型,观察白消一号方对此模型中黑素瘤细胞的影响,探讨其治疗白癜风的药理作用.方法 分别培养角质形成细胞、黑素瘤细胞;体外构建黑素瘤细胞与角质形成细胞直接接触的混合培养模型,大鼠中药灌胃后不同时间取血清加入此模型中,用MTT法、多巴氧化法、及NaOH法.检测含药血清对黑素瘤细胞增殖、酪氧酸酶活性以及黑素合成的影响.结果 含药血清可促进黑素瘤细胞增殖,上调酪氨酸酶活性.增加黑素合成.结论 成功构建了黑素瘤细胞与角质形成细胞直接接触的混合培养模型,白消一号方作用于此模型对黑素瘤细胞增殖、酪氨酸酶活性及黑素的合成均呈促进作用.     

白消一号方作用于黑素瘤细胞与角质形成细胞共培养模型中黑素小体转运的观察

目的体外建立黑素瘤细胞与角质形成细胞直接接触的培养模型,观察白消一号方对此模型中黑素小体转运的影响,探讨其治疗白癜风的药理作用。方法体外培养黑素瘤细胞和角质形成细胞,用二乙酰羧基荧光素-琥珀酰亚胺酯(CFDA-SE)标记黑素瘤细胞内的黑素小体,然后将标记的黑素瘤细胞与角质形成细胞共同培养。大鼠中药灌胃后取血清加入此模型中,在倒置显微镜下观察混合细胞模型生长及共聚焦显微镜下观察共培养模型中黑素小体由黑素瘤细胞向角质形成细胞的转运情况。结果含药血清可使黑素小体由黑素瘤细胞向角质形成细胞的转运增加。结论在共聚焦显微镜下观察到白消一号方可以促进黑素小体转运,此途径可能为白消一号方治疗白癜风的作用机制之一。     

不同培养条件对皮肤角质形成细胞和成纤维细胞的活性增殖影响

目的 观察含不同血清浓度培养基对皮肤角质形成细胞和成纤维细胞体外活性的影响. 方法 体外培养、扩增皮肤角质形成细胞和成纤维细胞,按3×105/ ml的密度接种于96孔板,加入含不同血清浓度的K-SFM培养基培养.并用MTT比色法观察细胞活性. 结果 应用K-SFM+10%FBS组培养基培养成纤维细胞与DMEM+10%FBS为对照组培养细胞的活性影响相同(P＞0.05);应用K-SFM+1%FBS培养基培养角质形成细胞与K-SFM为对照组培养细胞的活性影响相同(P＞0.05); 结论 在构建人工皮肤体外模型时,角质形成细胞和成纤维细胞可分别加用含1%FBS、10%FBS的K-SFM培养基进行培养.     

不同培养条件对皮肤角质形成细胞和成纤维细胞增殖的影响

目的观察含不同血清浓度培养基对皮肤角质形成细胞和成纤维细胞体外活性的影响。方法体外培养、扩增皮肤角质形成细胞和成纤维细胞,按3×10^5／ml的密度接种于96孔板,加入含不同血清浓度的K-SFM培养基培养。并用MTY比色法观察细胞活性。结果应用K—SFM＋10％FBS组培养基培养成纤维细胞与DMEM＋10％FBS为对照组培养细胞的活性影响相同（P〉0．05）;应用K-SFM＋1％FBS培养基培养角质形成细胞与K—SFM为对照组培养细胞的活性影响相同（P〉0．05）;结论在构建人工皮肤体外模型时,角质形成细胞和成纤维细胞可分别加用含1％FBS、10％FBS的K—SFM培养基进行培养。     

复方青黛饮含药血清对体外HaCaT细胞增殖和凋亡的影响

目的体外观察复方青黛饮大鼠含药血清对人永生化角质形成细胞株HaCaT细胞增殖、凋亡和细胞周期的影响,并探讨其作用机制。方法根据中药血清药理学方法,用SD大鼠制备实验血清,采用MTT法、流式细胞术、免疫荧光Hoechst染色,观察含药血清对细胞生长抑制率、凋亡率、细胞周期分布及细胞形态学的影响。结果体外培养HaCaT细胞24h和48h后,复方青黛饮高、中、低三个剂量不同浓度的含药血清组,对HaCaT细胞的生长均有不同程度的抑制,与空白血清组相比,差异有统计学意义(P<0.05),其中高剂量组抑制作用最明显。复方青黛饮含药血清组细胞凋亡率和G0/G1比例明显增加,与空白血清组相比,差异有统计学意义(P<0.05),在镜下呈现凋亡的特征性形态改变。结论复方青黛饮大鼠含药血清对体外培养的角质形成细胞株HaCaT细胞有生长抑制和诱导凋亡作用,并对细胞周期有一定的影响。     

UVB与钙离子对体外培养人角质形成细胞表达天疱疮抗原的影响

目的 研究中波紫外线(UVB)照射以及不同钙离子浓度对角质形成细胞表达天疱疮抗原的影响.方法 通过改变培养基中的钙离子浓度以及采用不同剂量UVB照射体外培养的人角质形成细胞,在不同时间段以寻常型天疱疮(PV)或落叶型天疱疮(PF)血清作为一抗进行免疫荧光检测,观察寻常型天疱疮抗原(PVA)和落叶型天疱疮抗原(PFA)的表达情况;提取细胞或皮肤表皮组织蛋白质,用PV和PF血清进行免疫印迹检测.结果 无论是否增加钙离子浓度,体外培养人角质形成细胞间隙均可以检测到PV血清特异性着色.只有增加培养基中钙离子浓度后,形成的复层化角质形成细胞间隙才可以检测到PF血清特异性荧光着色.不同剂量UVB照射后的角质形成细胞均不产生PF血清的特异性着色.PF血清与160000条带反应,PV血清可与130000和160000两条带反应.结论 体外培养的单层或复层角质形成细胞均可以表达PVA,提高培养基中钙离子浓度可以诱导培养人角质形成细胞复层化并表达PFA,而UVB照射不能促使人角质形成细胞在体外培养条件下表达PFA.     

凉血活血胶囊对皮肤角质形成细胞凋亡的研究

目的 观察凉血活血胶囊对体外角质形成细胞株凋亡的影响,初步探讨其治疗银屑病的机制。方法 以TUNEL法检测银屑病患者皮损细胞凋亡、PCNA检测增殖细胞核抗原,并以碘化丙啶法和膜联蛋白V法在流式细胞仪上检测凉血活血胶囊对体外角质形成细胞凋亡作用的影响。结果 银屑病皮损中基底层和棘细胞中下层角质形成细胞增殖和凋亡较正常皮肤均有所增加。凉血活血胶囊可诱导培养的角质形成细胞凋亡,在1、5、10 mg/mL时分别为24.67±5.07、50.33±10.04、66.20±6.91,随着浓度增加,细胞凋亡率增加,与正常对照组比较差异有显著性,与试验对照复方青黛胶囊组比较差异无显著性。结论 银屑病皮损中角质形成细胞增殖和凋亡发生紊乱,两者在较高水平保持相对平衡。凉血活血胶囊可以诱导细胞凋亡,从而达到治疗银屑病的目的。     

甲氧沙林对培养人表皮黑素细胞黑素合成的影响及信号转导的研究

目的：观察甲氧沙林(8-甲氧补骨脂素，8-MOP)对体外培养的人表皮黑素细胞黑素合成和酪氨酸酶活性的影响，并初步探讨8-MOP诱导表皮黑素细胞分化的信号转导途径。方法：采用4种浓度(10～500μmol／L）的8-MOP作用于体外培养的人表皮黑素细胞，观察不同浓度、不同时间8-MOP对黑素细胞的形态、增殖、酪氨酸酶活性、黑素含量的影响，并用放射免疫法测定8-MOP对细胞内环磷腺苷酸(cAMP)含量的影响。结果：100μmol／L 8-MOP作用黑素细胞120h能显著促进酪氨酸酶的活性，提高黑素细胞的黑素含量，8-MOP抑制黑素细胞增殖和提高细胞内cAMP的水平呈浓度依赖性。结论：8-MOP在体外能直接刺激表皮黑素细胞的黑素合成，上述变化可能通过cAMP依赖的蛋白激酶(PK)A信号通路发挥作用。     

姜黄素对角质形成细胞凋亡的诱导及其机制

目的研究姜黄素在体外诱导角质形成细胞凋亡及其作用机制。方法采用MTT法测定不同浓度的姜黄素对角质形成细胞增殖的影响,流式细胞仪观察细胞凋亡情况,分光光度法检测细胞内半胱天冬蛋白酶3(Caspase-3)及半胱天冬蛋白酶9(Caspase-9)活性的改变情况。结果浓度>5μmol/L的姜黄素处理后细胞早期凋亡率显著升高(P<0.05),细胞内Caspase-3及Caspase-9的活性明显增加(P<0.05),均与姜黄素呈浓度依赖性。结论一定浓度范围的姜黄素可诱导角质形成细胞的凋亡,Caspase-3及Caspase-9活性增加可能为其诱导凋亡的重要机制之一。     

加味桃红四物汤对角质形成细胞分泌内皮素-1和碱性成纤维细胞生长因子的影响

目的 研究加味桃红四物汤对角质形成细胞(KC)分泌内皮素-1和碱性成纤维细胞生长因子的影响。方法 以体外培养的人角质形成细胞为对象,加入不同浓度加味桃红四物汤后,用MTT法反映细胞增殖的变化。ELISA方法测定细胞上清液中内皮素-1和碱性成纤维细胞生长因子的分泌量。结果 加味桃红四物汤浓度4.27、5.49、6.1mg/mL时可促进KC的增殖。加味桃红四物汤浓度1.83mg/mL和5.49mg/mL可刺激KC分泌内皮素-1,而对KC分泌碱性成纤维细胞生长因子无影响。结论 高浓度的加味桃红四物汤可促进KC的增殖。不同浓度的加味桃红四物汤均能刺激KC分泌内皮素-1,提示加味桃红四物汤在体外可刺激KC分泌内皮素-1。     

益肾活血方改善肾虚血瘀型不孕症患者子宫内膜容受性临床研究

目的:观察益肾活血方改善肾虚血瘀型不孕症子宫内膜容受性的临床疗效。方法:选择2014年6月至2016年6月于我院不孕不育科门诊就诊的患者60例,采用随机数字表法分为两组各30例,观察组服用益肾活血方,对照组服用戊酸雌二醇。通过B超监测排卵,于排卵后6~8d B超监测子宫内膜情况,计算Salle评分值等进行非劣效研究,评价临床疗效。结果:治疗后分析发现两组患者治疗前后,Salle评分、PI评分与内膜厚度具有统计学差异(P0.05),观察组非劣效于对照组。观察组与对照组受孕率分别为40%(12/30)和20%(6/30),观察组的受孕率明显优于对照组,差异具有统计学意义(P0.05)。结论:益肾活血方可以改善肾虚血瘀型不孕症患者子宫内膜容受性,增加受孕率。     

中药消银汤药物血清对EGF诱导的HaCaT细胞生长增殖的影响

目的探讨中药消银汤药物血清对表皮生长因子诱导的HaCaT细胞生长增殖的影响。方法HaCaT细胞株为靶细胞.观察中药消银汤药物血清对10ng/mL EGF信号刺激下HaCaT细胞生长曲线的影响;用MTT法检测细胞增殖情况:用流式细胞术测定细胞周期及凋亡率变化。结果中药消银汤药物血清影响EGF信号刺激下HaCaT细胞的生长状态并抑制其生长速度;不同浓度消银汤处理的HaCaT细胞,48h时表现出对细胞增殖的抑制作用,随着时间延长和药物剂量加大,抗增殖作用愈明显;高浓度组,48h和72h时的抑制率分别为59.47%和73.76%。与对照组相比,经消银汤作用48h后,细胞G_1期比例显著增加,S期与G_2期比例则显著下降,并可抑制G_1/G_2期转换。结论消银汤具有抑制表皮生长因子诱导的HaCaT细胞增殖及诱导其分化的作用     

凉血活血复方对体外培养HaCaT细胞增殖和凋亡的影响

目的观察中药凉血活血复方水醇粗提液对人永生化表皮细胞(HaCaT)的增殖抑制效应和诱导细胞凋亡作用的影响,探讨该复方治疗银屑病的可能机制。方法将不同浓度的凉血活血复方水醇粗提液分别作用于体外培养的HaCaT细胞,采用MTT法检测凉血活血复方水醇粗提液对细胞的增殖抑制作用以及药物的有效浓度;采用倒置显微镜、透射电子显微镜观察细胞处理前后的形态学改变:流式细胞术检测细胞周期的变化及凋亡比率。结果凉血活血复方以时间和浓度依赖性方式抑制HaCaT细胞增殖,作用24、48、72h其IC50分别为155.83 mg/mL、71.57mg/mL,41.27 mg/mL。细胞形态学和流式细胞仪检测发现药物以浓度依赖性的方式干扰细胞周期、诱导细胞凋亡,细胞分裂周期阻皆滞于G2/M期。结论凉血活血复方可能通过抑制角质形成细胞的增殖、诱导其凋亡而治疗银屑病。     

The proteinase-activated receptor-2 mediates phagocytosis in a Rho-dependent manner in human keratinocytes

Recent work shows that the G-protein-coupled receptor proteinase activated receptor-2 activates signals that stimulate melanosome uptake in keratinocytes in vivo and in vitro. The Rho family of GTP-binding proteins is involved in cytoskeletal remodeling during phagocytosis. We show that proteinase-activated receptor-2 mediated phagocytosis in human keratinocytes is Rho dependent and that proteinase-activated receptor-2 signals to activate Rho. In contrast, Rho activity did not affect either proteinase-activated receptor-2 activity or mRNA and protein levels. We explored the signaling mechanisms of proteinase-activated receptor-2 mediated Rho activation in human keratinocytes and show that activation of proteinase-activated receptor-2, either through specific proteinase-activated receptor-2 activating peptides or through trypsinization, elevates cAMP in keratinocytes. Proteinase-activated receptor-2 mediated Rho activation was pertussis toxin insensitive and independent of the protein kinase A signaling pathway. These data are the first to show that proteinase-activated receptor-2 mediated phagocytosis is Rho dependent and that proteinase-activated receptor-2 signals to Rho and cAMP in keratinocytes. Because phagocytosis of melanosomes is recognized as an important mechanism for melanosome transfer to keratinocytes, these results suggest that Rho is a critical signaling intermediate in melanosome uptake in keratinocytes.     

Levels of cyclosporin in epidermis of treated psoriasis patients differentially inhibit growth of keratinocytes cultured in serum free versus serum containing media

Cyclosporin A, which is a specific immunosuppressive compound, has recently been demonstrated to be of significant benefit in the treatment of psoriasis. Because hyperplasia is a major feature of psoriasis, we have investigated whether this drug acts directly to inhibit keratinocyte growth. We have determined the concentration range of cyclosporin in the epidermis of psoriatic patients undergoing cyclosporin therapy and the effect of this concentration range on the growth of cultured keratinocytes. After 7 days of treatment, psoriatic involved epidermis contained 1.1 +/- 0.3 ng cyclosporin/micrograms DNA. Based on tissue wet weight this is approximately 2.8 micrograms cyclosporin/ml. This value was 10 times that of trough blood samples. On day 3 of treatment, involved epidermis contained 7 times more cyclosporin than scale, while on day 7 this ratio was reduced to 2. This suggests that cyclosporin was initially associated with the lower layers of the epidermis and distributed upward with time. Exposure of adult human keratinocytes, cultured on collagen in the presence of human serum, to cyclosporin (0.1-30 micrograms/ml, 0.4-13-fold higher than lesional cyclosporin) for 2-6 d had no effect on the rate of incorporation of thymidine into DNA. Cyclosporin (1-30 micrograms/ml) also had not effect on the reinitiation of DNA synthesis of quiescent cells subsequent to the reintroduction of serum. In contrast, cyclosporin (1-10 micrograms/ml) inhibited growth of keratinocytes cultured on plastic culture dishes in serum free media (MCDB 153). For a given dose of cyclosporin, cell associated drug was 2-3 times greater in serum free compared to serum containing media. This may contribute in part to the sensitivity of keratinocytes in serum free media to growth inhibition by cyclosporin. These data demonstrate that human epidermis contains a high concentration of cyclosporin after oral administration, and that, under certain conditions, concentrations of cyclosporin within the range found in vivo can inhibit growth of cultured keratinocytes. Because cyclosporin regulates lymphocyte function in vivo and in vitro, the demonstrated antiproliferative effects of cyclosporin on psoriatic keratinocytes in vivo may be due to inhibition of a mononuclear leukocyte-derived keratinocyte growth factor(s) in combination with direct inhibition of keratinocyte growth.     

17beta-estradiol stimulates the growth of human keratinocytes by inducing cyclin D2 expression

Estrogen is reported to prevent age-associated epidermal thinning in the skin. We examined if 17beta-estradiol (E2) may enhance the growth of human keratinocytes, focusing on its effects on the expression of cell cycle-regulatory proteins. E2 enhanced proliferation, bromodeoxyuridine incorporation of keratinocytes, and increased the proportion of cells in the S phase. The E2-induced stimulation of proliferation and bromodeoxyuridine incorporation was suppressed by antisense oligonucleotide against cyclin D2, which induces G1 to S phase progression. E2 increased protein and mRNA levels of cyclin D2, and resultantly enhanced assembly and kinase activities of cyclin D2-cyclin-dependent kinases 4 or 6 complexes. E2 enhanced cyclin D2 promoter activity, and the element homologous to cAMP response element (CRE) on the promoter was responsible for the effect. Cyclin D2 expression was enhanced by antiestrogens, ICI 182,780 and 4-hydroxytamoxifen, and membrane-impermeable bovine serum albumin-conjugated E2, indicating the effects via membrane E2-binding sites. E2 increased the enhancer activity of CRE-like element and the amount of phosphorylated cAMP response element binding protein (CREB) binding this element, and the increases were suppressed by H-89, an inhibitor of cAMP-dependent protein kinase A. H-89 also suppressed E2-induced cyclin D2 expression, proliferation, and bromodeoxyuridine incorporation in keratinocytes. Antisense oligonucleotide against G-protein-coupled receptor GPR30 suppressed the E2-induced increases of phosphorylated CREB, cyclin D2 level, proliferation, and bromodeoxyuridine incorporation in keratinocytes. These results suggest that E2 may stimulate the growth of keratinocytes by inducing cyclin D2 expression via CREB phosphorylation by protein kinase A, dependent on cAMP. These effects of E2 may be mediated via cell surface GPR30.     

依巴斯汀联合中药养血活血止痒方治疗慢性湿疹70例疗效观察

目的观察依巴斯汀片联合中药养血活血止痒方治疗湿疹的临床疗效。方法将130例慢性湿疹患者随机分为治疗组70例与对照组60例,对照组予口服依巴斯汀10mg,1次/d,外用复方氟米松软膏,2次/d;治疗组在对照组治疗基础上加用中药养血活血止痒方汤剂(主要由当归、丹参、鸡血藤、白藓皮、荆芥、防风、蝉衣、生地、全虫和陈皮组成),水煎服,日一剂,早晚分服。两组疗程均为2周。结果治疗结束后两组EASI评分较治疗前均明显下降,组间比较差异有统计学意义(P<0.01),治疗后两组瘙痒评分较治疗前均明显下降,组间比较差异有统计学意义(P均<0.01),治疗组有效率为92.86%,对照组为63.33%,疗效优于对照组(P<0.01)。结论依巴斯汀片联合中药养血活血止痒方治疗慢性湿疹疗效明显,安全方便。