Effects Of Alkaloid Sinomenine On ERK1/2 And TNF-α Expression Of CD4～+T Lymphocytes Of Human Peripheral Blood In Vitro

Objectives: To evaluate the immunosuppressive efficacy and outcome of alkaloid sinomenine(SIN) on the CD4~+T lymphocytes model of human peripheral blood in vitro. The chosen indicators are the expression p-ERK1/2 and TNF- α of CD4~+T cells. The aim is to study the synergistic effect of SIN combination with immunosuppressant Cyclosporin A (CsA) and provide theoretical foundation for applying the SIN as an immunosuppressive therapy in clinic.Methods: Establishment of human CD4~+T lymphocyte models of peripheral blood:(1), 50ml elbow vein blood in three healthy adult male volunteer was draw, adding 500U heprin anticoagulation;(2), PBMC were isolated from the vein blood with lymphocytes separation medium;(3), CD4~+T lymphocytes were isolated from PBMC suspension with immunomagnetic beads and cultured(37℃, 5%CO2) with 10%FCS RPMI-1640;(4), The CD4~+T lymphocytes were proliferated by PHA(final concentration: 5ug/ml) and IL-2 (final concentration: 100 U/ml) for 7 days. The proliferated CD4~+T lymphocytes suspension were divided 2 portions to treat as followings: 1) : The same concentration SIN effected on cell culture media for different time: (1) Group blank contrast: no medicine was added to cell culture medium;(2) Group SIN : SIN solutrope whose final concentration was 200 μ mol/L was added to cell culture media. Effecting on cell 15min, 30min, 1h, 1.5h, 24h. Every group was averagely divided into 3 sub-groups, which were tested through Western-blot to detect the expression of p-ERK1/2. 2): ELISA was used to test the levels of TNF- α quantitatively. (1) Group blank contrast: no medicine was added to cell culture medium; (2) Group CsA: CsA solutrope whose final concentration was...