膽紅素致L-02細(xì)胞生長抑制與凋亡關(guān)系的研究
發(fā)布時(shí)間：2018-08-30 15:51
【摘要】：目的:探討不同劑量膽紅素致正常人肝細(xì)胞L-02生長及凋亡的變化。 方法:將不同濃度的膽紅素(0 mg/L、100 mg/L、150 mg/L、200 mg/L、250mg/L、300 mg/L)作用于體外養(yǎng)的正常人肝細(xì)胞L-02,用倒置顯微鏡觀察其形態(tài)變化;采用四甲基偶氮唑藍(lán)(MTT)法觀察不同濃度膽紅素作用L-02細(xì)胞24h后的相對(duì)存活率的影響;不同濃度膽紅素作用24h后,采用DNA瓊脂糖凝膠電泳;不同濃度膽紅素作用12h后流式細(xì)胞儀Annexin-V和PI雙標(biāo)等技術(shù)檢測(cè)細(xì)胞凋亡率;將L-02接種于6孔板中進(jìn)行細(xì)胞爬片后,用同濃度膽紅素作用24h,多聚賴氨酸處理玻片做免疫組化染色檢測(cè)Bcl-2與Bax蛋白的表達(dá)。 結(jié)果:在倒置相差顯微鏡下對(duì)正常人肝細(xì)胞L-02進(jìn)行觀察,對(duì)照組細(xì)胞24h形態(tài)無明顯變化呈上皮型貼壁生長,細(xì)胞呈梭形或多邊形,低濃度(100 mg/L、150 mg/L)膽紅素作用的細(xì)胞12h內(nèi)細(xì)胞細(xì)胞貼壁良好,可見黃染;高濃度(200 mg/L、250mg/L、300 mg/L)膽紅素作用的細(xì)胞2h內(nèi)形態(tài)無明顯改變,4h后可見細(xì)胞被黃染,較多細(xì)胞突起、變圓,但細(xì)胞輪廓清晰,貼壁良好;6h可見細(xì)胞輪廓不清,少數(shù)細(xì)胞漂浮于培養(yǎng)基中;12h可見較多細(xì)胞漂浮,并已開始崩解。MTT法顯示膽紅素作用后L-02細(xì)胞的生長受到抑制,各組的OD值分別是0.2251±0.0324、0.2745±0.0466、0.6412±0.0734、0.7295±0.0337、0.7629±0.0327、0.8322±0.0336。各濃度和對(duì)照組間、各濃度組見差異具有統(tǒng)計(jì)學(xué)意義(F=74.4644,p0.05),除對(duì)照組與100mg/L組間比較差異無統(tǒng)計(jì)學(xué)意義外(q=2.464,p0.05),其余各時(shí)間組間差異有統(tǒng)計(jì)學(xué)意義(p0.05)。Hoechst33258熒光染色在各實(shí)驗(yàn)組均可見細(xì)胞凋亡,其中高濃度(200、250、300 mg/L)膽紅素組更為明顯,大量L-02細(xì)胞呈致密濃染,或呈碎塊狀致密濃染。低濃度(100、150 mg/L)膽紅素組和對(duì)照組也可見少量細(xì)胞凋亡。對(duì)照組細(xì)胞瓊脂糖凝膠電泳可見,DNA雙鏈完整,相對(duì)分子質(zhì)量大,滯留在加樣孔附近,未形成DNA條帶。膽紅素溶液高濃度組(200 mg/L、250mg/L、300 mg/L)出現(xiàn)典型的云梯狀細(xì)胞凋亡的DNA ladder,隨濃度增加圖像更清晰、典型。流式細(xì)胞儀檢測(cè)結(jié)果顯示,從0mmol/L劑量組到300mmol/L劑量組,凋亡率從1.24%上升到57.3%,各組之間差異有統(tǒng)計(jì)學(xué)意義(p0.05)。Bcl-2在300mg/L組偶見棕黃色顆粒狀,呈散在、弱陽性表達(dá),200mg/L組與300mg/L組相比較陽性細(xì)胞數(shù)增多,染色加深,呈黃褐色表達(dá)。Bax主要在肝細(xì)胞胞漿呈棕黃色顆粒狀;對(duì)照組未見陽性表達(dá),200mg/L組可見肝細(xì)胞散在、弱陽性表達(dá),較150mg/L組有所增強(qiáng),陽性細(xì)胞數(shù)增多,250mg/L組陽性染色進(jìn)一步加深,陽性細(xì)胞多,300mg/L組肝細(xì)胞彌漫陽性染色,染色加深,陽性細(xì)胞數(shù)增多。 結(jié)論:膽紅素可抑制正常成人離體肝細(xì)胞L-02的生長并誘導(dǎo)其發(fā)生凋亡,呈現(xiàn)良好的劑量-效應(yīng)關(guān)系。該凋亡的可能與Bcl-2與Bax表達(dá)發(fā)生改變有關(guān)。
[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.
【學(xué)位授予單位】：桂林醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類號(hào)】：R363
本文編號(hào)：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.
【學(xué)位授予單位】：桂林醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類號(hào)】：R363
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