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目的:研究胆固醇(CL)对兔胆管成纤维细胞增生的影响,并对其机制进行探讨。 方法:分离、纯化、培养兔胆管成纤维细胞,完全随机分为两组:对照组、中等浓度胆固醇组(0.6g/L)和高浓度胆固醇组(1.2g/L)。以MTT法测定各组细胞的增生情况、并以流式细胞仪(FCM)测定细胞周期。进而采用免疫组化法定量分析各组细胞内增生细胞核抗原(PCNA)的表达情况。 结果:中等浓度CL(0.4～0.8g/L)可以使培养的兔胆管成纤维细胞MTT A_(490nm)值显著升高,CL(0.4g/L)A_(490nm值为0.422±0.015,较对照组增加51.4％(P<0.05);CL(0.6g/L)A_(490nm)值达到最大为0.486±0.019,较对照组增加95.2％(P<0.01);0.8g/L CL组A_(490nm)值为0.472±0.017较对照组增加89.6％(P<0.01);高浓度CL(1.2g/L)明显抑制细胞生长,A_(490nm)值为0.163±0.018,较对照组下降34.5％(P<0.05)。流式细胞仪分析显示,与正常组相比,中等浓度CL(0.6g/L)显著增加G_2/M期细胞比例,从9.8％上升为13.0％,S期细胞比例从11.7％上升为33.4％,而显著减少进入G_0/G_1期的细胞比例,从78.5％下降为53.6％。高浓度CL(1.2g/L)显著减少G_2/M期细胞比例,从9.8％降低为6.7％,S期细胞比例从11.7％下降为5.9％,而显著增加进入G_0/G_1期的细胞比例,从78.5％上升为87.4％,使更多的胆管成纤维
Objective: To study the effect of cholesterol (CL) on proliferation of bile duct fibroblasts in rabbits and to explore its mechanism. Methods: Rabbit bile duct fibroblasts were isolated, purified, and cultured. They were randomly divided into two groups: control group, medium concentration cholesterol group (0.6g / L) and high concentration cholesterol group (1.2g / L). Cell proliferation was measured by MTT assay and cell cycle was determined by flow cytometry (FCM). Then immunohistochemistry was used to quantitatively analyze the expression of proliferating cell nuclear antigen (PCNA) in each group. Results: The MTT A 490nm value of rabbit bile duct fibroblasts cultured in medium concentration of CL (0.4-0.8g / L) was significantly higher than that of control (0.4g / L) (P <0.01). The average value of A (490nm) of CL (0.6g / L) reached to 0.486 ± 0.019, which was 95.2% (P <0.01) ) Value was 0.472 ± 0.017, increased by 89.6% (P <0.01). High concentration CL (1.2g / L) significantly inhibited the cell growth. The value of 490 nm was 0.163 ± 0.018, which was 34.5% lower than that of the control group <0.05) .Flow cytometry analysis showed that the medium concentration of CL (0.6g / L) significantly increased the proportion of G 2 / M phase cells from 9.8% to 13.0% compared with the normal group, and the percentage of S phase cells increased from 11.7% Increased from 33.5% to 33.4%, while decreased significantly from 78.5% to 53.6% in the G_0 / G_1 phase.High concentration of CL (1.2g / L) significantly decreased the proportion of cells in the G_2 / M phase from 9.8% to 6.7% , The proportion of S phase cells decreased from 11.7% to 5.9%, while the percentage of cells entering G_0 / G_1 phase increased significantly from 78.5% to 87.4%, leading to more bile duct fibrogenesis