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目的探讨以2-乙酰氨基芴(2-AFF)灌胃与2/3肝切除法联合运用建立大鼠肝卵圆细胞增殖模型的适宜剂量,并探讨肝卵圆细胞的体外分离培养方法。方法将174只Wistar大鼠随机分为4个实验组(各30只)、未处理组(24只)和生理盐水组(30只)。4个实验组大鼠分别给予5、10、15及20 mg(/kg d)2-AAF灌胃(分别对应第1、2、3及4实验组),于第5天行2/3肝切除,术后继续灌胃至处死大鼠;生理盐水组大鼠以生理盐水灌胃,其余操作同实验组;未处理组大鼠不做任何处理。6组分别于肝切除术后第4、8、12及16天各随机处死6只大鼠,取肝组织行HE染色和免疫组化染色,观察肝卵圆细胞的增殖情况〔第4天时同时检测血清丙氨酸氨基转移酶(ALT)和天冬氨酸氨基转移酶(AST)水平〕;并采用二步胶原酶灌注结合Percoll密度梯度离心法对大鼠肝卵圆细胞进行分离和培养。结果肝切除术后第4天,未处理组、生理盐水组、第1、2、3及4实验组大鼠的存活率分别为100%(24/24)、93%(28/30)、93%(28/30)、90%(27/30)、90%(27/30)及80%(24/30)。肝切除后第4天,与未处理组及生理盐水组比较,第2、3和4实验组大鼠的血清ALT及AST水平均升高(P<0.05)。HE染色结果显示,第2、3及4实验组大鼠的肝组织均出现不同程度的肝损伤,其中第3和第4实验组均可见明显的肝卵圆细胞增殖反应;免疫组化染色结果显示,第2、3及4实验组卵圆细胞标志6(OV-6)表达阳性细胞数在术后第4、8、及12天,随2-AAF剂量的增加逐渐升高,与2-AAF间呈剂量-效应关系(P<0.05)。大鼠肝卵圆细胞进行体外分离和培养后,经OV-6免疫组化染色鉴定,有80%的细胞表达呈阳性。结论联合运用15 mg(/kg d)2-AFF灌胃加2/3肝切除,于术后第12天,有效诱导了肝卵圆细胞的增殖,且造模大鼠的耐受性较好、死亡率低。采用二步胶原酶灌注结合Percoll密度梯度离心法较成功地分离出了大鼠肝卵圆细胞。
Objective To investigate the appropriate dosage of 2-acetylaminofluorene (2-AFF) in combination with 2/3 hepatectomy to establish rat hepatic oval cell proliferation model and investigate the method of isolation and culture of hepatic oval cells in vitro. Methods 174 Wistar rats were randomly divided into 4 experimental groups (30 rats each), untreated group (24 rats) and normal saline group (30 rats). 4 experimental rats were given 5,10,15 and 20 mg (/ kg d) of 2-AAF intragastrically (corresponding to the first 1,2,3 and 4 experimental group), on the 5th day of 2/3 liver The rats in the saline group were given gavage with saline and the rest were the same as the experimental group. The rats in the untreated group were not treated. Six rats were randomly sacrificed on the 4th, 8th, 12th and 16th day after hepatectomy respectively. The liver tissues were harvested for HE staining and immunohistochemistry to observe the proliferation of hepatic oval cells [at day 4 Serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured. Two-step collagenase perfusion and Percoll density gradient centrifugation were used to isolate and culture rat hepatic oval cells. Results On the fourth day after hepatectomy, the survival rates of the untreated group, the saline group, the first, second, third and fourth experimental groups were 100% (24/24), 93% (28/30) 93% (28/30), 90% (27/30), 90% (27/30), and 80% (24/30). On day 4 after hepatectomy, serum ALT and AST levels of rats in groups 2, 3 and 4 were significantly higher than those in untreated group and saline group (P <0.05). The results of HE staining showed that the hepatic tissues of rats in the second, third and fourth experimental groups all had different degrees of hepatic injury, and the third and fourth experimental groups showed obvious hepatic oval cell proliferation reaction; the results of immunohistochemical staining The numbers of positive cells of oval cell marker 6 (OV-6) in experimental groups 2, 3 and 4 increased gradually with the increase of 2-AAF at the 4th, 8th, and 12th day after operation, AAF showed dose-effect relationship (P <0.05). After isolation and culture of rat hepatic oval cells in vitro, 80% of the cells were positive by immunohistochemical staining with OV-6. Conclusions Combined use of 15 mg / kg d-2 AFS plus 2/3 hepatectomy effectively induced the proliferation of hepatic oval cells on the 12th day after operation, and the tolerance of the model rats was better , The mortality rate is low. The rat hepatic oval cells were successfully isolated by two-step collagenase perfusion combined with Percoll density gradient centrifugation.