Experimental Study on Establishment of Cell Proliferation Model and Isolated Method in Vitro of Hepatic Oval Cells in Adult Rat_CHINESE JOURNAL OF BASES AND CLINICS IN GENERAL SURGERY

Authors：

HUANG Qinxian ,  LI Haiyang , HU Yi , SONG Jianning , DONG Biao , CAO Kun

Department of Hepatobiliary Surgery， Affiliated Hospital of Guiyang Medical University， Guiyang 550004， Guizhou Province， China;

Corresponding author：

LI Haiyang, Email: haiyang_li@sina.com

Keywords：

Hepatic oval cell; 2-acetaminofluorene; Proliferation model; Isolation; In vitro; Rat

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Objective 　To explore the proper dosage of establishment of stable hepatic oval cells （HOC） prolif-eration 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 experi-mental 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 every group， 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-6 （OV-6）. The number of OV-6 positive cells were increased significantly with the increase of dosage of 2-AAF between 4 days and 12 days after operation， and proliferation levels were related with dosages of 2-AAF （P＜0.05）. In all cultured cells， 80% of cells were OV-6 positive cells after isolation and culture by using collagenase perfusion method and percoll gradient centrifugation.
Conclusions 　The methods of gavage of 2-AAF at 15 mg/（kg ? d） combined with 2/3 PH surgery can establish the HOC proliferation model on the 12th day， as well as the rats have lower mortality and better tolerance， especially. The collagenase perfusion method and percoll gradient centrifugation can be used to isolate HOC effectively.

Citation： HUANG Qinxian,LI Haiyang,HU Yi,SONG Jianning,DONG Biao,CAO Kun,.. Experimental Study on Establishment of Cell Proliferation Model and Isolated Method in Vitro of Hepatic Oval Cells in Adult Rat. CHINESE JOURNAL OF BASES AND CLINICS IN GENERAL SURGERY, 2013, 20(10): 1100-1105. doi: Copy

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11.	Pham Van T， Couchie D， Martin-Garcia N， et al. Expression of matrix metalloproteinase-2 and -9 and of tissue inhibitor of matrix metalloproteinase-1 in liver regeneration from oval cells in rat[J]. Matrix Biol， 2008， 27(8)：674-681.
12.	Zhang W， Chen XP， Zhang WG， et al. Hepatic non-parenchymalcells and extracellular matrix participate in oval cell-mediated liverregeneration[J]. World J Gastroenterol， 2009， 15(5)：552-560.
13.	罗茜，王军，陈东风. 肝脏微环境对干细胞的分化调控作用[J]. 肝脏， 2012， 17(5)：347-349.
14.	陈琳，陈孝平，张伟，等. 肝星状细胞诱导卵圆细胞向成熟肝细胞分化的体外研究[J]. 中华肝脏病杂志， 2009， 17(10)：765-770.
15.	Xiong Q， Feng J， Xang J， et al. Comparison among curativeeffects of three different transplantation approaches of mesenchymalstem cells on rat model of cirrhosis[J]. J Third Mil Med Univ， 2011， 33(8)：804-808.
16.	Petersen BE， Zajac VF， Michalopoulos GK. Hepatic oval cell activation in response to injury following chemically induced periportal or pericentral damage in rats[J]. Hepatology， 1998， 27(4)：1030-1038.
17.	Suzuki A， Zheng Y， Kondo R， et al. Flow-cytometric separationand enrichment of hepatic progenitor cells in the developing mouseliver[J]. Hepatology， 2000， 32(6)：1230-1239.
18.	Suskind DL， Muench MO. Searching for common stem cells of the hepatic and hematopoietic systems in the human fetal liver：CD34+ cytokeratin 7/8+ cells express markers for stellate cells[J]. J Hepatol， 2004， 40(2)：261-268.
19.	崔潇. 肝癌干细胞研究进展[J]. 现代医药卫生， 2013， 29(4)：544-546.
20.	Yasui O， Miura N， Terada K， et al. Isolation of oval cells from Long-Evans Cinnamon rats and their transformation into hepatocytes in vivo in the rat liver[J]. Hepatology， 1997， 25(2)：329-334.

1. Jemal A， Siegel R， Xu J， et al. Cancer statistics， 2010[J]. CA Cancer J Clin， 2010， 60(5)：277-300.
2. 宋凯，杨硕菲，吴俊华，等. 肝癌干细胞的研究进展[J]. 世界华人消化杂志， 2009， 17(36)：3704-3710.
3. Sun CY， Zuo S. Stem cell research in hepatocellular carcinoma[J]. Front Med China， 2008， 2(1)：1-4.
4. 王楠，邹伟，刘鹏，等. 肝脏干细胞研究及应用前景展望[J]. 中国组织工程研究， 2012， 16(6)：1119-1124.
5. 王宇明，陈耀凯，郎松，等. 大鼠肝卵圆细胞的分离培养及脾内移植研究[J]. 中华肝脏病杂志， 2003， 11(6)：328-330.
6. Opie EL. The pathogenesis of tumors of the liver produced by butter yellow[J]. J Exp Med， 1944， 80(3)：231-246.
7. Farber E. Similarities in the sequence of early histological changes induced in the liver of the rat by ethionine， 2-acetylamino-fluorene， and 3’-methyl-4-dimethylaminoazobenzene[J]. CancerRes， 1956， 16(2)：142-148.
8. Crosby HA， Hubscher S， Fabris L， et al. Immunolocalization of putative human liver progenitor cells in livers from patients with end-stage primary biliary cirrhosis and sclerosing cholangitis using the monoclonal antibody OV-6[J]. Am J Pathol， 1998， 152(3)：771-779.
9. 邢秀伟，李建生. 干细胞移植治疗肝脏疾病的研究进展[J]. 中国组织工程研究， 2012， 16(6)：1115-1118.
10. Pi L， Ding X， Jorgensen M， et al. Connective tissue growth factorwith a novel fibronectin binding site promotes cell adhesion and migration during rat oval cell activation[J]. Hepatology， 2008， 47(3)：996-1004.
11. Pham Van T， Couchie D， Martin-Garcia N， et al. Expression of matrix metalloproteinase-2 and -9 and of tissue inhibitor of matrix metalloproteinase-1 in liver regeneration from oval cells in rat[J]. Matrix Biol， 2008， 27(8)：674-681.
12. Zhang W， Chen XP， Zhang WG， et al. Hepatic non-parenchymalcells and extracellular matrix participate in oval cell-mediated liverregeneration[J]. World J Gastroenterol， 2009， 15(5)：552-560.
13. 罗茜，王军，陈东风. 肝脏微环境对干细胞的分化调控作用[J]. 肝脏， 2012， 17(5)：347-349.
14. 陈琳，陈孝平，张伟，等. 肝星状细胞诱导卵圆细胞向成熟肝细胞分化的体外研究[J]. 中华肝脏病杂志， 2009， 17(10)：765-770.
15. Xiong Q， Feng J， Xang J， et al. Comparison among curativeeffects of three different transplantation approaches of mesenchymalstem cells on rat model of cirrhosis[J]. J Third Mil Med Univ， 2011， 33(8)：804-808.
16. Petersen BE， Zajac VF， Michalopoulos GK. Hepatic oval cell activation in response to injury following chemically induced periportal or pericentral damage in rats[J]. Hepatology， 1998， 27(4)：1030-1038.
17. Suzuki A， Zheng Y， Kondo R， et al. Flow-cytometric separationand enrichment of hepatic progenitor cells in the developing mouseliver[J]. Hepatology， 2000， 32(6)：1230-1239.
18. Suskind DL， Muench MO. Searching for common stem cells of the hepatic and hematopoietic systems in the human fetal liver：CD34+ cytokeratin 7/8+ cells express markers for stellate cells[J]. J Hepatol， 2004， 40(2)：261-268.
19. 崔潇. 肝癌干细胞研究进展[J]. 现代医药卫生， 2013， 29(4)：544-546.
20. Yasui O， Miura N， Terada K， et al. Isolation of oval cells from Long-Evans Cinnamon rats and their transformation into hepatocytes in vivo in the rat liver[J]. Hepatology， 1997， 25(2)：329-334.

CHINESE JOURNAL OF BASES AND CLINICS IN GENERAL SURGERY

Experimental Study on Establishment of Cell Proliferation Model and Isolated Method in Vitro of Hepatic Oval Cells in Adult Rat

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