Relative quantification of mRNA levels in Jurkat T cells with RT-real time-PCR (RT-rt-PCR):: New possibilities for the screening of anti-inflammatory and cytotoxic compounds

被引：24

作者：

Gertsch, J ^{[1
]}

Güttinger, M ^{[1
]}

Sticher, O ^{[1
]}

Heilmann, J ^{[1
]}

机构：

[1] Univ Zurich, Inst Pharmaceut Sci, CH-8057 Zurich, Switzerland

来源：

PHARMACEUTICAL RESEARCH | 2002年 / 19卷 / 08期

关键词：

RT-real time-PCR; cytokines; Jurkat T cells; NF-kappa B; capsaicin; hypericin;

D O I：

10.1023/A:1019818814336

中图分类号：

O6 [化学];

学科分类号：

0703 ;

摘要：

Purpose. Quantification of the pro-inflammatory action of mitogens on mRNA levels of growth-related genes, transcription factors, and cytokines in T cells as markers for the screening of compounds with immunomodulatory, anti-inflammatory or cytotoxic potential. Method. A reverse transcription-real time-polymerase chain reaction assay with TaqMan probes was developed. Jurkat T cells were treated with cyclosporin A, hypericin, capsaicin, and catechin before phorbol 12-myristate 13-acetate stimulation, and their effects on the relative mRNA levels were determined. A cell viability assay was performed in parallel. Results. Cyclosporin A and capsaicin were potent inhibitors of PMA-induced cytokine transcription. Cyclosporin A further targeted cyclin D1 transcription. Capsaicin exhibited no effects on the cell viability at low concentrations, whereas cyclosporin A did. Hypericin down-regulated nearly all investigated mRNAs, resulting in a strong time-dependent cytotoxicity. Catechin showed no effects on mRNA levels and cell viability. Conclusions. The inhibition of the up-regulation of mRNA levels of cytokines points to a specific anti-inflammatory potential of capsaicin. Hypericin showed no specific effects on the mRNA expression. The overall decrease of mRNA levels is probably an early indication of the strong cytotoxic effect observed after 48 h. Therefore, quantification of mRNA levels by reverse transcription-real time-polymerase chain reaction is, in combination with the monitoring of cell viability, a valuable tool to distinguish between specific immunomodulatory and cytotoxic effects in vitro.

引用

页码：1236 / 1243

页数：8

共 32 条

[1]

AJCHENBAUM F, 1993, J BIOL CHEM, V268, P4113

[2] Green tea catechins partially protect DNA from (.)OH radical-induced strand breaks and base damage through fast chemical repair of DNA radicals [J].

Anderson, RF ;

Fisher, LJ ;

Hara, Y ;

Harris, T ;

Mak, WB ;

Melton, LD ;

Packer, JE .

CARCINOGENESIS, 2001, 22 (08) :1189-1193

[3]

Barnett J, 2000, IRISH VET J, V53, P583

[4] IL-2 MESSENGER-RNA LEVELS AND DEGRADATION RATES CHANGE WITH MODE OF STIMULATION AND PHORBOL ESTER TREATMENT OF LYMPHOCYTES [J].

BILL, O ;

GARLISI, CG ;

GROVE, DS ;

HOLT, GE ;

MASTRO, AM .

CYTOKINE, 1994, 6 (01) :102-110

[5] Hypericin as a non-antioxidant inhibitor of NF-κB [J].

Bork, PM ;

Bacher, S ;

Schmitz, ML ;

Kaspers, U ;

Heinrich, M .

PLANTA MEDICA, 1999, 65 (04) :297-300

[6]

Depraetere V, 1997, Semin Immunol, V9, P93, DOI 10.1006/smim.1997.0062

[7] Genetic approaches in mice to understand Rel/NF-κB and IκB function:: transgenics and knockouts [J].

Gerondakis, S ;

Grossmann, M ;

Nakamura, Y ;

Pohl, T ;

Grumont, R .

ONCOGENE, 1999, 18 (49) :6888-6895

[8] A novel method for real time quantitative RT PCR [J].

Gibson, UEM ;

Heid, CA ;

Williams, PM .

GENOME RESEARCH, 1996, 6 (10) :995-1001

[9]

HARDY KJ, 1987, J IMMUNOL, V138, P2353

[10] Real time quantitative PCR [J].

Heid, CA ;

Stevens, J ;

Livak, KJ ;

Williams, PM .

GENOME RESEARCH, 1996, 6 (10) :986-994

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