摘要
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 explore the proper dosage of establishment of stable hepatic oval cells （HOC） proliferation model by using 2-acetaminofluorene （2-AAF） combined with two-third partial hepatectomy （2/3 PH） surgery, and to explore isolated and cultured method of HOC in vitro. Methods The 174 Wistar rats were randomly divided into 4 experimental groups （each group enrolled 30 rats）, saline group （n=30）, and untreated group （n=24）. Rats of 4 experimental groups were underwent gavage of 5, 10, 15, and 20 mg/（kg·d） 2-AAF, corresponding to the groups from No. 1 to No.4 group. Rats of saline group received saline gavage and rats of untreated group didn＇t received any treatment. A standard 2/3 PH surgery was performed on the 5th day after gavage, then the same gavage method was still administrated as preoperation untill rats were sacrificed. The liver tissues of 6 selected rats were adopted and identified by HE staining and immunohistochemical staining on 4, 8, 12, and 16 days after PH for observation of the proliferation of HOC in everygroup, on 4 days, levels of alanine aminotransferase （ALT） and aspartate aminotransferase （AST） were tested in addition. HOC were isolated and purified by collagenase perfusion method and percoll gradient centrifugation. Results The surv- ival rates of untreated group, saline group, No.1 group, No.2 group, No.3 group, and No.4 group were 100% （24/24）, 93% （28/30）, 93% （28/30）, 90% （27/30）, 90% （27/30）, and 80% （24/30） respectively. Compared with the saline group and untreated group, the levels of serum ALT and AST increased significantly in No.2, No.3, and No.4 group on the 4th day after PH （P〈 0.05）. The results of HE staining showed that No.2, No.3, and No.4 group were observed visibly different level of damage at liver tissue, and the proliferation level of HOC were most obviously in No.3 and No.4 group. The results of immunohistochemical staining revealed that proliferation cells were positively expressed oval cell marker
出处
《中国普外基础与临床杂志》
CAS
2013年第10期1100-1105,共6页
Chinese Journal of Bases and Clinics In General Surgery
基金
贵州省优秀教育人才省长基金资助(项目编号:2007-74)
关键词
肝卵圆细胞
2-乙酰氨基芴
增殖模型
分离
体外
大鼠
Hepatic oval cell
2-acetaminofluorene
Proliferation model
Isolation
In vitro
Rat
