胆红素致L-02细胞生长抑制与凋亡关系的研究
发布时间：2018-08-30 15:51
【摘要】：目的:探讨不同剂量胆红素致正常人肝细胞L-02生长及凋亡的变化。 方法:将不同浓度的胆红素(0 mg/L、100 mg/L、150 mg/L、200 mg/L、250mg/L、300 mg/L)作用于体外养的正常人肝细胞L-02,用倒置显微镜观察其形态变化;采用四甲基偶氮唑蓝(MTT)法观察不同浓度胆红素作用L-02细胞24h后的相对存活率的影响;不同浓度胆红素作用24h后,采用DNA琼脂糖凝胶电泳;不同浓度胆红素作用12h后流式细胞仪Annexin-V和PI双标等技术检测细胞凋亡率;将L-02接种于6孔板中进行细胞爬片后,用同浓度胆红素作用24h,多聚赖氨酸处理玻片做免疫组化染色检测Bcl-2与Bax蛋白的表达。 结果:在倒置相差显微镜下对正常人肝细胞L-02进行观察,对照组细胞24h形态无明显变化呈上皮型贴壁生长,细胞呈梭形或多边形,低浓度(100 mg/L、150 mg/L)胆红素作用的细胞12h内细胞细胞贴壁良好,可见黄染;高浓度(200 mg/L、250mg/L、300 mg/L)胆红素作用的细胞2h内形态无明显改变,4h后可见细胞被黄染,较多细胞突起、变圆,但细胞轮廓清晰,贴壁良好;6h可见细胞轮廓不清,少数细胞漂浮于培养基中;12h可见较多细胞漂浮,并已开始崩解。MTT法显示胆红素作用后L-02细胞的生长受到抑制,各组的OD值分别是0.2251±0.0324、0.2745±0.0466、0.6412±0.0734、0.7295±0.0337、0.7629±0.0327、0.8322±0.0336。各浓度和对照组间、各浓度组见差异具有统计学意义(F=74.4644,p0.05),除对照组与100mg/L组间比较差异无统计学意义外(q=2.464,p0.05),其余各时间组间差异有统计学意义(p0.05)。Hoechst33258荧光染色在各实验组均可见细胞凋亡,其中高浓度(200、250、300 mg/L)胆红素组更为明显,大量L-02细胞呈致密浓染,或呈碎块状致密浓染。低浓度(100、150 mg/L)胆红素组和对照组也可见少量细胞凋亡。对照组细胞琼脂糖凝胶电泳可见,DNA双链完整,相对分子质量大,滞留在加样孔附近,未形成DNA条带。胆红素溶液高浓度组(200 mg/L、250mg/L、300 mg/L)出现典型的云梯状细胞凋亡的DNA ladder,随浓度增加图像更清晰、典型。流式细胞仪检测结果显示,从0mmol/L剂量组到300mmol/L剂量组,凋亡率从1.24%上升到57.3%,各组之间差异有统计学意义(p0.05)。Bcl-2在300mg/L组偶见棕黄色颗粒状,呈散在、弱阳性表达,200mg/L组与300mg/L组相比较阳性细胞数增多,染色加深,呈黄褐色表达。Bax主要在肝细胞胞浆呈棕黄色颗粒状;对照组未见阳性表达,200mg/L组可见肝细胞散在、弱阳性表达,较150mg/L组有所增强,阳性细胞数增多,250mg/L组阳性染色进一步加深,阳性细胞多,300mg/L组肝细胞弥漫阳性染色,染色加深,阳性细胞数增多。 结论:胆红素可抑制正常成人离体肝细胞L-02的生长并诱导其发生凋亡,呈现良好的剂量-效应关系。该凋亡的可能与Bcl-2与Bax表达发生改变有关。
[Abstract]:Objective: To investigate the changes of L-02 growth and apoptosis in normal human hepatocytes induced by different doses of bilirubin.
Methods: Different concentrations of bilirubin (0 mg/L, 100 mg/L, 150 mg/L, 200 mg/L, 250 mg/L, 300 mg/L) were applied to cultured normal human hepatocytes L-02. The morphological changes were observed by inverted microscope. The relative survival rate of L-02 cells treated with different concentrations of bilirubin for 24 hours was observed by MTT method. Cell apoptosis was detected by DNA agarose gel electrophoresis, flow cytometry with Annexin-V and PI double labeling after 12 hours of bilirubin treatment, L-02 was inoculated into 6-well plate for cell climbing, treated with bilirubin at the same concentration for 24 hours, and the Bcl-2 and Bax proteins were detected by immunohistochemical staining with polylysine-treated slide. Expression.
Results: The normal human hepatocytes L-02 were observed under inverted phase contrast microscope. The cells in the control group showed no obvious morphological changes and epithelial adherent growth after 24 hours. The cells were spindle-shaped or polygonal. The cells treated with low concentration of bilirubin (100 mg/L, 150 mg/L) adhered well to the wall within 12 hours, and yellow staining was observed in the cells at high concentration (200 mg/L, 250 mg/L, 300 mg/L). After 4 hours, the cells were yellow stained, more protuberances and rounded, but the cell contour was clear and adherent to the wall was good. At 6 hours, the cell contour was unclear and a few cells were floating in the medium. At 12 hours, more cells were floating and began to disintegrate. MTT assay showed that the growth of L-02 cells was affected by bilirubin. Inhibition, the OD values of each group were 0.2251 (+ 0.0324), 0.2745 (+ 0.0466), 0.6412 (+ 0.0734), 0.7295 (+ 0.0337), 0.7629 (+ 0.0327), 0.8322 (+ 0.0336) respectively. There were significant differences between the concentration groups and the control group (F = 74.4644, p0.05), except for the difference between the control group and the 100mg/L group (q = 2.464, p0.05). Hoechst 33258 fluorescence staining showed apoptosis in all experimental groups, especially in high concentration (200,250,300 mg/L) bilirubin group. A large number of L-02 cells were densely stained or fragmented densely stained. Low concentration (100,150 mg/L) bilirubin group and control group also showed a small number of apoptosis. Agarose gel electrophoresis showed that the DNA double strands were intact, the relative molecular weight was large, and the DNA bands were not formed. DNA ladder of typical ladder cell apoptosis appeared in the high concentration group of bilirubin solution (200 mg/L, 250 mg/L, 300 mg/L), and the image became clearer and more typical with the increase of concentration. The apoptotic rate of the 300 mmol/L group increased from 1.24% to 57.3%. The difference was statistically significant (p0.05). In the 300 mg/L group, Bcl-2 showed occasional brown granular, scattered and weak positive expression. Compared with the 300 mg/L group, the number of positive cells in the 200 mg/L group increased and the staining deepened, showing yellowish-brown expression. No positive staining was found in the control group. The hepatocytes in the 200 mg/L group were scattered and weakly positive, and the number of positive cells was increased compared with the 150 mg/L group.
CONCLUSION: Bilirubin can inhibit the growth and induce apoptosis of normal adult hepatocytes L-02 in vitro in a dose-dependent manner, which may be related to the alteration of Bcl-2 and Bax expression.
【学位授予单位】：桂林医学院
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R363
本文编号：2213550
[Abstract]:Objective: To investigate the changes of L-02 growth and apoptosis in normal human hepatocytes induced by different doses of bilirubin.
Methods: Different concentrations of bilirubin (0 mg/L, 100 mg/L, 150 mg/L, 200 mg/L, 250 mg/L, 300 mg/L) were applied to cultured normal human hepatocytes L-02. The morphological changes were observed by inverted microscope. The relative survival rate of L-02 cells treated with different concentrations of bilirubin for 24 hours was observed by MTT method. Cell apoptosis was detected by DNA agarose gel electrophoresis, flow cytometry with Annexin-V and PI double labeling after 12 hours of bilirubin treatment, L-02 was inoculated into 6-well plate for cell climbing, treated with bilirubin at the same concentration for 24 hours, and the Bcl-2 and Bax proteins were detected by immunohistochemical staining with polylysine-treated slide. Expression.
Results: The normal human hepatocytes L-02 were observed under inverted phase contrast microscope. The cells in the control group showed no obvious morphological changes and epithelial adherent growth after 24 hours. The cells were spindle-shaped or polygonal. The cells treated with low concentration of bilirubin (100 mg/L, 150 mg/L) adhered well to the wall within 12 hours, and yellow staining was observed in the cells at high concentration (200 mg/L, 250 mg/L, 300 mg/L). After 4 hours, the cells were yellow stained, more protuberances and rounded, but the cell contour was clear and adherent to the wall was good. At 6 hours, the cell contour was unclear and a few cells were floating in the medium. At 12 hours, more cells were floating and began to disintegrate. MTT assay showed that the growth of L-02 cells was affected by bilirubin. Inhibition, the OD values of each group were 0.2251 (+ 0.0324), 0.2745 (+ 0.0466), 0.6412 (+ 0.0734), 0.7295 (+ 0.0337), 0.7629 (+ 0.0327), 0.8322 (+ 0.0336) respectively. There were significant differences between the concentration groups and the control group (F = 74.4644, p0.05), except for the difference between the control group and the 100mg/L group (q = 2.464, p0.05). Hoechst 33258 fluorescence staining showed apoptosis in all experimental groups, especially in high concentration (200,250,300 mg/L) bilirubin group. A large number of L-02 cells were densely stained or fragmented densely stained. Low concentration (100,150 mg/L) bilirubin group and control group also showed a small number of apoptosis. Agarose gel electrophoresis showed that the DNA double strands were intact, the relative molecular weight was large, and the DNA bands were not formed. DNA ladder of typical ladder cell apoptosis appeared in the high concentration group of bilirubin solution (200 mg/L, 250 mg/L, 300 mg/L), and the image became clearer and more typical with the increase of concentration. The apoptotic rate of the 300 mmol/L group increased from 1.24% to 57.3%. The difference was statistically significant (p0.05). In the 300 mg/L group, Bcl-2 showed occasional brown granular, scattered and weak positive expression. Compared with the 300 mg/L group, the number of positive cells in the 200 mg/L group increased and the staining deepened, showing yellowish-brown expression. No positive staining was found in the control group. The hepatocytes in the 200 mg/L group were scattered and weakly positive, and the number of positive cells was increased compared with the 150 mg/L group.
CONCLUSION: Bilirubin can inhibit the growth and induce apoptosis of normal adult hepatocytes L-02 in vitro in a dose-dependent manner, which may be related to the alteration of Bcl-2 and Bax expression.
【学位授予单位】：桂林医学院
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R363
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