中华皮肤科杂志
中華皮膚科雜誌
중화피부과잡지
Chinese Journal of Dermatology
2014年
3期
192-196
,共5页
CD8阳性T淋巴细胞%药物评价,临床前%中草药%细胞增殖
CD8暘性T淋巴細胞%藥物評價,臨床前%中草藥%細胞增殖
CD8양성T림파세포%약물평개,림상전%중초약%세포증식
CD8-positive T-lymphocytes%Drug evaluation,preclinical%Drugs,Chinese herbal%Cell proliferation
目的 建立CD8+细胞毒性T淋巴细胞(CTL)体外增殖模型,筛选免疫抑制性中药.方法 制备小鼠脾脏单细胞悬液,通过特异性抗体分离CD8+T淋巴细胞,CD3/CD28抗体诱导淋巴细胞活化增殖,分别加入23种中药提取物共培养,通过四唑盐MTS法检测各中药对淋巴细胞增殖的抑制效果,对抑制作用最强的4种中药作12.5～ 400 mg/L的浓度梯度分析.酶联免疫斑点法(ELISPOT)检测这4种中药对CD3/CD28抗体诱导CD8+T细胞分泌干扰素γ(IFN-γ)的作用.结果 23种中药提取物中,14味中药对淋巴细胞增殖有不同程度抑制,抑制作用最强的前4味分别为黄连、黄芩、木香和姜黄,其对CD8+T细胞增殖的50％抑制浓度(IC5o)分别约为25、35、50和60 mg/L,100％抑制的最低浓度分别为200、100、200、200 mg/L.黄芩、木香和姜黄在100 mg/L的浓度下对CD3/CD28抗体诱导CD8+T细胞分泌IFN-γ均有明显抑制作用,而黄连对CD8+T细胞分泌IFN-γ的抑制作用不明显.结论 成功建立CD8+ CTL体外增殖模型,并筛选出对小鼠脾脏CD8+T淋巴细胞增殖具有显著抑制作用的4味中药即黄连、黄芩、木香和姜黄.
目的 建立CD8+細胞毒性T淋巴細胞(CTL)體外增殖模型,篩選免疫抑製性中藥.方法 製備小鼠脾髒單細胞懸液,通過特異性抗體分離CD8+T淋巴細胞,CD3/CD28抗體誘導淋巴細胞活化增殖,分彆加入23種中藥提取物共培養,通過四唑鹽MTS法檢測各中藥對淋巴細胞增殖的抑製效果,對抑製作用最彊的4種中藥作12.5～ 400 mg/L的濃度梯度分析.酶聯免疫斑點法(ELISPOT)檢測這4種中藥對CD3/CD28抗體誘導CD8+T細胞分泌榦擾素γ(IFN-γ)的作用.結果 23種中藥提取物中,14味中藥對淋巴細胞增殖有不同程度抑製,抑製作用最彊的前4味分彆為黃連、黃芩、木香和薑黃,其對CD8+T細胞增殖的50％抑製濃度(IC5o)分彆約為25、35、50和60 mg/L,100％抑製的最低濃度分彆為200、100、200、200 mg/L.黃芩、木香和薑黃在100 mg/L的濃度下對CD3/CD28抗體誘導CD8+T細胞分泌IFN-γ均有明顯抑製作用,而黃連對CD8+T細胞分泌IFN-γ的抑製作用不明顯.結論 成功建立CD8+ CTL體外增殖模型,併篩選齣對小鼠脾髒CD8+T淋巴細胞增殖具有顯著抑製作用的4味中藥即黃連、黃芩、木香和薑黃.
목적 건립CD8+세포독성T림파세포(CTL)체외증식모형,사선면역억제성중약.방법 제비소서비장단세포현액,통과특이성항체분리CD8+T림파세포,CD3/CD28항체유도림파세포활화증식,분별가입23충중약제취물공배양,통과사서염MTS법검측각중약대림파세포증식적억제효과,대억제작용최강적4충중약작12.5～ 400 mg/L적농도제도분석.매련면역반점법(ELISPOT)검측저4충중약대CD3/CD28항체유도CD8+T세포분비간우소γ(IFN-γ)적작용.결과 23충중약제취물중,14미중약대림파세포증식유불동정도억제,억제작용최강적전4미분별위황련、황금、목향화강황,기대CD8+T세포증식적50％억제농도(IC5o)분별약위25、35、50화60 mg/L,100％억제적최저농도분별위200、100、200、200 mg/L.황금、목향화강황재100 mg/L적농도하대CD3/CD28항체유도CD8+T세포분비IFN-γ균유명현억제작용,이황련대CD8+T세포분비IFN-γ적억제작용불명현.결론 성공건립CD8+ CTL체외증식모형,병사선출대소서비장CD8+T림파세포증식구유현저억제작용적4미중약즉황련、황금、목향화강황.
Objective To establish a model for studying CD8+ cytotoxic T lymphocyte proliferation in vitro and to screen traditional Chinese drugs (TCDs) with immunosuppressive effects.Methods Spleen tissue was isolated from mice,and made into single cell suspensions followed by separation of CD8+ T lymphocytes with specific antibodies.Then,the CD8 + T lymphocytes were seeded into anti-CD3/CD28 antibody-coated 96-well plates and cocultured with the extracts of 23 TCDs (100 mg/L) separately for 96 hours.Those ceils cultured with and without the presence of anti-CD3/CD28 antibody alone served as the positive control and negative control respectively.The 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium,inner salt (MTS) assay was performed to evaluate the proliferation of cells and to select the top four TCDs with the strongest inhibitory effect.The relationship between the inhibitory effect and TCD concentrations was further assessed for the four selected TCDs.Enzyme-linked immunospot (ELISPOT) assay was carried out to estimate the influence of the four TCDs on the secretion of interferon (IFN)-γ by CD8+ T lymphocytes induced by anti-CD3/CD28 antibodies.Statistical analysis was done by nonparametric rank sum test.Results Of the 23 TCDs,14 significantly inhibited the proliferation of CD8+ T lymphocytes (all P ＜ 0.05),of which,Rhizoma Coptidis,Radix Scutellariae,Radix Aucklandiae and Rhizoma Curcumae Longae displayed the strongest inhibitory capacity with the 50％ inhibitory concentration being 25,35,50 and 60 mg/L respectively,and the 100％ inhibitory concentration being 200,100,200 and 200 mg/L respectively.The anti-CD3/CD28 antibody-induced secretion of IFN-γby CD8+ T lymphocytes was markedly suppressed by Radix Scutellariae,Radix Aucklandiae and Rhizoma Curcumae Longae at the concentration of 100 mg/L,but not by Rhizoma Coptidis at this concentration.Conclusions A model for studying the proliferation of CD8+ T lymphocytes is successfully developed in vitro,and four TCDs with strong inhibitory effects on the proliferation of CD8+ T lymphocytes have been screened out with this model.
