Effects Of Alkaloid Sinomenine On The Proliferation And Intracellular Ca～(2+) Concentration Of CD4～+ T Lymphocytes Of Human Peripheral Blood In Vitro

Object: To observe the effects of alkaloid sinomenine on the proliferation and intracellular Ca2+ concentration of CD4+T lymphocytes of human periphory blood in vitro for study of the efficacy and molecular biological mechanism of SIN immunosupressing human CD4+T lymphocytes .This research aims at providing theory foundation to clinical appliance of SIN in the field of transplantation, and providing a safe, effective and inexpensive administration scheme that can be offered to immunosupressiontherapy for transplantation recipients.Methods:Establishment of human CD4+ T lymphocyte models of peripheralblood: (1), 50ml elbow vein blood in one healthy adulted male volunteer was drawed, adding 500U heprin to protect the blood from coagulation; (2) , PBMCs were isolated from the vein blood with lymphocytes separation medium;(3K CD4+T lymphocytes were isolated from PBMC suspension with immunomagnetic beads and cultured(37℃, 5%C02) 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 averagely at random into 5 groups to treat as followings: (1) Group blank contrast:no medicine was added to cell culture medium; (2) Group CsA:CsA soluton whose final concentration was 50ng/ml was added to cell culture media; (3) Group low-concentration SIN: SIN soluton whose final concentration was 10 umol/L was added to cell culture media; (4) Group middle-concentration SIN:SIN soluton whose final concentration was 200 umol/L was added to cell culture media;(5) Group high-concentrationSIN :SIN soluton whose final concentration was 1000 umol/L was added to cell culture media. 6 hours later, anti-CD3 mAb soluton whose final concentration was lug/ml was added to cell culture media of above groups. And after 24 hours, every group was averagely divided into 3 sub-groups which were tested through following methods:(1) MTT chromatometry was used to test the proliferation of these models; (2) FCM was used to estimate the intracellular Ca2+ concentration of these models.Results: 1.The cell proliferation inhibition ratios(%) determined by MTT chromatometry: (1) Group blank contrast:0. 00±0. 00; (2) Group CsA:72. 62 ±0.85; (3) Group low-concentration SIN:2. 25±1. 58; (4) Group middle-concentration SIN: 27. 65 ± 1. 65; (5) Group high-concentration SIN:40. 12 ± 1. 08. The cell proliferation inhibition ratio of Group high-concentration SIN was significantly different from that of other groups (P<0. 05), so was that of Group middle-concentration SIN (P<0. 05), and that of Group low-concentration SIN was little changable in contrast to Group blank contrast (P >0. 05). Significant positive correlation between concentrations of SIN and proliferation inhibition ratios of CD4+T lymphocytes of human peripheral blood exist. The cell proliferation inhibition ratio of group CsA was the highest. 2. The data of intracellular Ca2+ fluorescent intensity estimated by FCM, which represented intracellular Ca2+ concentration: (1) Group blank contrast: 11. 00 ± 0. 20; (2) Group CsA: 11. 90±0. 27; (3) Group low-concentration SIN: 11.30± 0.26; (4) Group middle-concentration SIN: 7.57 ± 0. 15; (5) Group high-concentration SIN:6. 00 ± 0.10. The intracellular Ca2+ concentration of Group high-concentration SIN was significantly different from that of other groups (P<0. 05), so was that of Group middle-concentration SIN(P<0. 05), and the intracellular Ca2+con- centration of Group low-concentration SIN was little changable in contrast to that of Group blank contrast and Group CsA(P>0. 05). Significant nagitive correlation between the concentrations of SIN and intracellular Ca2+ concentrations of CD4+ T lymphocytes of human peripheral blood exist. 3% In the enviroment of treatment...