小鼠体外T淋巴细胞模型的优化及在中药筛选中的应用

Optimization of the mouse T lymphocyte model in vitro and its applicationon TCM screening

目的 :优化MTT法测定小鼠体外T淋巴细胞的实验条件 ,并对 6种种子类中药的活性进行筛选。方法 :无菌分离小鼠脾脏 ,制备脾淋巴细胞 ,与待测药物预孵 1h后 ,加入刀豆蛋白A刺激剂继续培养 ,总计培养 72h后收获细胞 ,进行MTT染色 ,以酶标仪进行测定。结果 :应用MTT法 ,在 (2～ 3 0 )× 10 9/L范围内细胞密度与A值呈线性正相关 ,优化后的测定条件为细胞悬液中加入 10 μl的ConA ,孵育 72h后再加入 2 0 μl的MTT ,继续孵育 4h ,测定波长为 5 12nm。结果表明 ,这 6种种子类中药对小鼠T淋巴细胞均有一定的抑制作用。结论 :用于进行中药的抑制T淋巴细胞活性的体外筛选。

Objective:To develop a optimal method for assessing T lymphocytic proliferation effect by MTT colorimetric assays and determine the IC 50 of six TCMs;Methods:To break away the spleen from the mouse body with asepsis and incubate with treated drug for 1h.Adding the Con A,72 hours later to gain the cells.Dyeing the cells and determine with instrument.Results:There was a good correlativity between optical absorbance value and cell densities ranged 2～30×106/mL when assaying murine splenic lymphocyte proliferation by MTT colorimetric assays.The optimal incubating time after adding Con A and adding MTT are 72 hours and 4 hours separately.The best quantity of MTT is 20μL.The detected wavelength is 512nm.Conclusion:T lymphocytic proliferation effect can be used to screen the TCMs.