猪十二指肠类肝素对动脉平滑肌细胞增殖的影响

目的　研究类肝素对平滑肌细胞增殖的影响及其机制。方法　分别以结晶紫和MTT染色、透射电镜及流式细胞仪等方法，观察类肝素对平滑肌细胞增殖，细胞形态，细胞内α-肌动蛋白含量及原癌基因c-myc，c-fos表达的影响。结果　类肝素0.2，0.4及0.8 mg.mL^-1可以抑制10%小牛血清所致平滑肌细胞增殖，类肝素0.8 mg.mL^-1可以使细胞维持类似收缩型的形态学表现，使细胞内α-actin含量增加，c-myc, c-fos原癌基因表达减少。结论　类肝素可以抑制平滑肌细胞增殖，其机制可能与抑制细胞表型转化，抑制细胞内原癌基因表达有关。

EFFECT OF HEPARINOID DERIVED FROM PORCINE DUODENUM ON THE PROLIFERATION OF CULTURED SMOOTH MUSCLE CELLS

AIM: To study the antiproliferative effect of haparinoid derived from porcine duodenum (heparinoid) on cultured vascular smooth muscle cells. METHODS: Cultured bovine aortic smooth muscle cells (BASMCs) of 5-10 passages were seeded into 24 and 72-well cluster culture plates and were synchronized by 48 h serum deprivation. Then, the cells were re-stimulated by serum repletion with or without heparinoid. The antiproliferative effect of heparinoid was evaluated by crystal violet staining and MTT assay 72 h after serum repletion. To study the drug action on cytomorphological changes, three kinds of cells [quiescent cells, cells treated with 10% fetal calf serum (FCS) with or without heparinoid] were observed by transmission electron microscopy. After synchronized and re-stimulated as above, BASMCs were treated with heparinoid 0.8 mg.mL-1 at selected points during serum repletion. The cells were harvested at specified times after serum repletion, then cellular DNA contents (to estimate the proportions of cells in different phases of the cell cycle) and the contents of alpha-actin, c-myc and c-fos proteins were measured by flow cytometry. RESULTS: Heparinoid was shown to inhibit the proliferation of BASMCs induced by 10% FCS. The inhibitory effect was weakened when heparinoid was added 2 h after serum repletion, and there was no antiproliferative effect when heparinoid was added 12 h after serum repletion. Electron micrographs showed that cells treated with 10% FCS and heparinoid expressed a contractile phenotype, while cells treated with 10% FCS only expressed a synthetic phenotype. Flow cytometry study showed remarkable increase of alpha-actin, and decrease of c-myc and c-fos proteins in the cells treated with heparinoid. CONCLUSION: Heparinoid was found to inhibit the proliferation of BASMCs. The antiproliferative effect occurred at the early phase of the cell cycle. It might be due to the drug's influence on cell phenotype modulation and the down regulation of c-myc and c-fos proto-oncogenes expression.