雷公藤甲素SD大鼠睾丸毒性体外试验研究

In Vitro Study of Toxicity of Triptolide to the Testicles of SD Rats

探讨雷公藤甲素(Triptolide,TL)对体外原代培养SD大鼠睾丸支持细胞(sertolicell)和生精细胞(spermat-ogenic cells)增殖的影响及其广谱钙粘附蛋白(Pan-cadherin)的表达。MTT法检测雷公藤甲素8种不同浓度(0,0.1,1,10,1.0×102,1.0×103,1.0×104,1.0×105μg/L)对SD大鼠睾丸原代培养细胞增殖的影响;免疫细胞化学方法探讨在雷公藤甲素对单纯培养支持细胞的最小有毒浓度(0.1,1,10,1.0×102μg/L)水平,培养细胞pan-cadherin的表达。结果表明:雷公藤甲素浓度≥10μg/L时对混合培养细胞(主要为生精细胞),≥1.0×103μg/L时对单纯培养支持细胞的增殖有明显抑制作用,均呈剂量依赖性。细胞半数抑制浓度(IC50)分别为1.22mg/L和28.15mg/L。与溶剂对照组相比混合培养细胞在雷公藤甲素低浓度时(0.1和1μg/L)表达上调。雷公藤甲素低浓度即可抑制生精细胞增殖,而高浓度才能抑制支持细胞增殖,其细胞毒耐受性二者差异很大。睾丸毒性产生的原因可能与生精细胞的pan-cadherin表达上调有关。

To investigate the effect, in vitro, of triptolide on the proliferation and expression of the protein, pan-cadherin, in cultured primary sertoli and/or spermatogenic cells isolated from SD rat testis. METHODS ： Sertoli and spermatogenic cells from rats were isolated and cultured singly or mixture of two. The effect of triptolide at different concentrations （0, 0. 1, 1, 10, 1.0 × 10^2, 1.0 × 10^3, 1.0 × 10^4, 1.0 × 10^5 μg/L） on cell proliferation were studied by the MTT assay. Four doses which were below the level of least toxic dose to sertoli cells cultured singly, were selected and expression of the pan-cadherin protein in the cultured cells was examined useing immunocytochemistry. RESULTS： The cell proliferation was inhibited at the dose level of ≥ 10 μg/L in mixed cell culture （ most were spermatogenic cells ）, and ≥ 1.0 × 10^3 μg/L in pure sertoli cell culture, and that these inhibitions depended on doses. The two IC50 concentrations to cultured cells were 1.22 mg/L and 28. 15 mg/L, respectively. Increased expression of pan - cadherin was observed at lower TL concentrations（ 0. 1 and 1 μg/L ） in mixed culture compared with vehicle control. CONCLUSION： It is suggested that triptolide may inhibit proliferation of spermatogenic cells at lower concentrations, and that of sertoli cells at higher concentrations. The reasonof testicular toxicity of triptolide is probably the increased expression of pan-cadherin protein.