为探寻PI3K/AKT抑制剂渥曼青霉素(Wortmannin, WM)对猪前体脂肪细胞增殖和凋亡均无影响的适宜浓度, 文章首先分离并验证了猪原代前体脂肪细胞的分化潜能, 然后对不同浓度渥曼青霉素处理11 d的细胞采用Annexin V-FITC/PI双标法检测细胞凋亡, 并通过凋亡相关基因的表达以及DNA损伤程度进行验证, 同时利用甲烷硫代磺酸盐(Methanethiosulfonate , MTS)检测了细胞的增殖活性。结果表明, 100 nmol/L渥曼青霉素对猪前体脂肪细胞的增殖和凋亡均无显著影响, 而200 nmol/L的渥曼青霉素对猪原代脂肪细胞的增殖活性虽没有显著影响, 但对细胞凋亡有显著促进作用。研究发现, 处理后促凋亡因子caspase8和TNFR1表达显著上调, 非caspase依赖促凋亡因子GZMA表达无显著性差异, 而GZMB表达则显著上调, 抗凋亡因子Bcl-x1表达显著上调, cFLIP表达则无显著性差异。100 nmol/L的渥曼青霉素对细胞DNA的损伤不显著。因此, 100 nmol/L的渥曼青霉素对猪前体脂肪细胞的增殖和凋亡均无显著影响, 是在不影响细胞生长的情况下研究PI3K通路对脂肪细胞分化的较为理想的浓度。
The purpose of this study was to determine the proper concentration of wortmannin that effectively inhibits PI3K/AKT but does not affect the proliferation and apoptosis of primary porcine preadipocytes. Firstly, primary porcine preadipocytes were isolated and their abilities to be induced to differentiation into mature adipocytes were evaluated. The preadipocytes were then treated with different concentrations of wortmannin, and the proliferation of the cells was de-tected with methanethiosulfonate (MTS). Annexin V- FITC/PI double-staining was used to detect the level of cell apoptosis. The apoptosis-related gene expressions were also quantified by qRT-PCR. At the same time, single cell electrophoresis was used to examine the extent of cellular DNA damage. Our data demonstrated that the primary porcine preadipocytes could differentiate into mature adipocytes. Up to 200 nmol/L of wortmannin had no effect on the proliferation ability of primary porcine preadipocytes (P>0.05). Results from the flow cytometry Annexin V- FITC/PI double-staining showed that 200 nmol/L wortmannin significantly induced apoptosis of the primary porcine preadipocytes (P<0.05). QRT-PCR results also showed that the expressions of caspase8, TNFR1, GZMB, and Bcl-x1 were significantly upregu-lated, while the expression of GZMA and cFLIP were not significantly affected when treated with 200 nmol/L wortmannin. In addition, results from the single cell gel electrophoresis indicated that 100 nmol/L wortmannin did not induce DNA damage. In conclusion, our results collectively showed that 100 nmol/L wortmanin can be used to study the role of PI3k pathway on the preadipocytes differention without affecting the cell proliferation and apoptosis.
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