Key words primary liver oval cells in the physical characteristics of HCC

    Oval cells initially in 1944 by Opie etc. [1] found in rat chemical carcinogenesis process. 1956, Farber [2] for the first time its named oval cells. The occurrence of liver cancer, two different doctrines: One is that liver cancer is formed by the differentiation of mature hepatocytes dedifferentiated About HCC classic theory [3]; another theory that liver cancer is oval cells by the stem cells of the liver – abnormal differentiation due; representatives Sell S [4] According to the oval cell morphology, phenotypic characterization, oncogene expression of a large number of circumstantial evidence that hepatocellular carcinoma derived from eggs round cell. Oval cells are a heterogeneous cell populations, their biological characteristics have not accepted the conclusions, and thus lead to separation, purification oval cells also lack of a recognized standard. Possible sources of oval cells, oval cells with liver cancer the relationship between academia is still controversial issues. It is based on the above problems, we designed this experiment, tried by a dynamic method to observe the biological characteristics of oval cells and its possible sources to provide an experimental basis, and liver cancer stem cell theory of abnormal differentiation.

    1 Materials and methods

    The 1.1 animal model and specimen processing

    SD male rats were selected and standard feed provided by the Experimental Animal Center of Sun Yat-sen University, North Campus, added 0.06% 3 ‘ methyl  4  dimethylamino azobenzene carcinogenesis feed standard feed (3’  methyl  4  dimethylaminoazobenzene, 3 ‘ Me  DAB) (Tokyo Kasei Kogyo Co., Ltd.) is made. 29 SD rats were divided into two experimental groups: rats with induced cancer group 24, feeding induced cancer of fodder and normal drinking water; 5 control rats fed standard diet and normal drinking water. Rats in each group were in carcinogenesis sacrificed at 4,8,14,17 and 24 weeks (4 induced cancer group were sacrificed at 4 weeks, followed by 4 each group were sacrificed 5; each killed a control group ), liver tissue in 4% formalin-fixed, paraffin-embedded serial sections alternate.

    1.2 routine HE staining and immune staining (immunohistochemistry, IHC)

    HE staining in accordance with routine laboratory methods.

    1.2.1 Immunohistochemical choice of an anti-kit and method of operation.

    OV  6 monoclonal antibody, donated by the United States, Professor Stewart Sell benefits. Antibody dilution of 1:50, with two-step kit and DAB reagent were purchased from Fuzhou, the new company, with EDTA pressure repair. AFP (clone A0008) and C  kit the (cloning No. A4502) were purchased from DAKO Corporation, the former antibody dilution of 1:100, hot fix with citric acid; the latter antibody dilution of 1:50, with EDTA high pressure repair ; the SP method kit and DAB reagent were purchased from Fuzhou, the new company. The three kinds of immunohistochemistry in accordance with the instructions to operate.

    1.2.2 Immunohistochemistry the choice of the control group

    Each set of sections were set up a negative control, positive control, negative control. Negative blank on the photo instead of primary antibody with PBS, like the rest of the method steps; positive is known photo affirmative expression slices; the negative photo is certainly no expression slices.

    1.3 special staining

    Love the first blue (alcian blue, AB) (pH2.5) method [5], [5] References operation. Mucilage was blue positive reaction and negative reaction of the tissue, the cells were red.

    2 Results

    The 2.1 experimental hepatocarcinogenesis HE morphological characteristics

    With the time extension induced cancer agents stimulate oval cell proliferation is more and more obvious: starting from the first four weeks the portal area has a small amount of oval cell hyperplasia. The oval cells compared with the previous 8 weeks increased board hyperplasia along the hepatic lobule sector budding like an extension to the liver parenchyma. Also visible bile duct hyperplasia lesions and bile duct atypical hyperplastic lesions, liver cells transparent change, eosinophilic changes and small cell lesions, lesions above are collectively referred to as hepatic precancerous lesion [6,7]. Oval cells increased significantly at 14 weeks and split lobular hepatic lobule was false; hepatocellular carcinoma lesions than obvious the lesions formation of nodules, as shown in Figure 1; Some regions bile duct atypical hyperplasia foci of malignant transformation. Oval cell hyperplasia 17 weeks did not increase over the previous part of the region foci formation of significant intrahepatic bile duct cells and infiltration along the portal area growth, gradual integration into a larger piece of continuous foci, as shown in Figure 2. 24 weeks, the large distribution of foci surrounding visible relatively small amount of residual normal liver tissue, oval cells are rare; foci was the cable type, false adenoid or adenoid irregular tubular structures, as shown in Figure 3.

    Normal control group from 4 weeks to 24 weeks were no oval cell hyperplasia, to maintain the structure of the normal liver tissue.

    2.2 first love blue staining.

    24 weeks of carcinogenesis in the large areas of foci beam cable type, false adenoid type area love the first blue staining negative, confirmed HCC stove; without rules adenoid area of ​​tubular structures positive confirmed as intrahepatic bile duct the cell foci [8], as shown in Figure 4. There foci was rough, cable type, but there are a small number of cells of blue-stained cytoplasm, with negative staining cells and white mixed. Normal liver cells, bile ducts, oval cells love first blue staining were negative. The above characteristics description of the last occurrence of the carcinogenesis model is a combination of liver cancer.

    2.3 immunohistochemistry

    2.3.1 OV  6 expression characteristics

    OV-6 positive brownish yellow fine granular distribution in the cytoplasm. Oval cells, as shown in Figure 5, 6, biliary epithelial cells, and bile duct cell foci were positive, and liver cells, hepatocellular carcinoma foci negative. Normal bile duct cells OV-6-positive normal control group from 4 weeks to 24 weeks were no oval cell hyperplasia, visible in the portal area. Negative control background clean.

    2.3.2 AFP expression characteristics

    AFP weakly positive oval cells and bile duct cell foci in bile duct cells and hepatocytes negative. Carcinogenesis 24 weeks, in large tracts of foci, cable type, false adenoid hepatocellular carcinoma foci AFP positive expression strongly positive part of the region, as shown in Figure 7, adenoid irregular tubular structure of the bile duct cell foci was negative. The normal control group showed no expression of AFP-positive cells. Negative control background clean.

    2.3.3 c  kit expression characteristics

    c  kit weakly positive oval cells and bile duct cell foci; positive Kupffer cells in the normal liver tissue, liver cells and bile duct cells negative. Carcinogenesis 8 weeks, the portal area c  kit weakly positive oval cell hyperplasia obvious cell density area a few scattered strongly positive for c  kit (OV  6 negative) cells to 14 weeks when this The increase in cell number, as shown in Figure 8. 24 weeks of carcinogenesis, the expression of c  kit, large tracts of foci. The normal control group showed no oval cell hyperplasia, and the visible positive expression of Kupffer cells in the normal liver tissue. Negative control background clean.

    3 Discussion

    Experimental liver cancer animal model has an irreplaceable role in human specimens. It can simulate the the HCC natural process, with good controllability and repeatability. 3 ‘ methyl   dimethyl-aminoazobenzene (referred to as 3’  Me  DAB) and become the most widely used because of its effect induced cancer, stage of carcinogenesis induced cancer agent one [9] .

    The OV  6 University of Houston in 1989 [10] Preparation of monoclonal antibodies, its oval cell specificity as a tracer signs. 14 weeks ago in carcinogenesis the continuing role of the agent, the proliferation of oval cells with prolonged duration of carcinogenesis heavier. But after 14 weeks, part of the proliferation of oval cells differentiate to the bile duct and liver cells to repair damaged liver, the other part is in the malignant transformation induced cancer agent role, the formation of mixed hepatocellular carcinoma. Experimental liver cancer model good start from the oval cell hyperplasia, and then after the precancerous lesions of the liver cancer [6,7] process to mixed hepatocellular carcinoma. Evolution characteristics of oval cells observed in OV-6-positive cells in the process of HCC the distribution characteristics HE slice match from a new angle, the close relationship between oval cells and liver cancer.

    Cholangiocarcinoma induced in the animal models, according to its HE morphological characteristics to determine fine cholangiocarcinoma is a place in the the fine bile duct or Herring tube special type of bile duct cell carcinoma [11]. Because liver carcinogenesis agent sustained under the oval cell hyperplasia to the progress of cancer of the bile duct cells quickly start (in the first 14 weeks of the induced cancer cholangiocarcinoma formation to the first 24 weeks to be clearly hepatocellular carcinoma foci formation), cancer cells still retain the OV-6 of this molecular marker of oval cells, which also proved cholangiocarcinoma originated in oval cells.

    Oval cells by the portal area along the the liver limiting plate hyperplasia appears from the first eight weeks in our animal model, the organizational structure of the tube with Herring coincide confirmed oval Herring tubular epithelial cells may be derived from hyperplasia, consistent with most reported in the literature [12]. It is precisely because this oval cells and biliary epithelial cells co-expressed OV  6 is not surprising. Oval cells derived from the the Herring tubular epithelial fact, the continuing role of the agent of carcinogenesis is activated Herring tubular epithelial cells prone to genetic mutations, the formation of fine cholangiocarcinoma. While tubular epithelial stem cells derived from Herring oval cells to go through the various stages of differentiation, and then by small liver cells to differentiate into liver cells, differentiation of a longer route, so the time of the occurrence of hepatocellular carcinoma (HCC) has lagged far behind [1, 13]. The complexity of the mechanism of liver cancer carcinogenesis model hepatoma complex morphology.

    AFP, alpha-fetoprotein, a naive marker of liver cells, oval cells express AFP consistent with those reported [14], and oval cells have the characteristics of immature liver cells, has the potential to liver cells. Is generally believed that the very specificity of AFP is also hepatocellular carcinoma a less sensitive marker [15], the model mixed liver cancer, hepatocellular carcinoma foci positive expression, confirmed liver cell line-derived; However, bile duct cells foci also weak expression of AFP, which is soon due to the progress of cancer of the bile duct cells from oval cell hyperplasia, cancer still retains the oval cell molecular markers, even cholangiocarcinoma express weaker AFP .

    of c  kit also known as CD117, the receptor of stem cell factor (stem cell factor), is one of the signs of bone marrow stem cells [16]. The oval cell expression the weaker c  kit, between oval cells and bone marrow-derived stem cells have some sort of origins Contact [16]. In early fetal liver had a blood-forming organs; late in fetal hematopoietic stem cells migrate to the bone marrow by the liver, may still be some residue in the liver, and may be a precursor cells of oval cells, oval cells with The bone marrow-derived stem cells have the same molecular markers. Inherent sources exist in the liver, is likely to continue, serious damage to the liver to release some kind of signal molecules that stimulate and attract the migration of bone marrow stem cells to the liver, to reach the liver after liver damage local microenvironment (niche) can stimulate certain gene expression towards the oval cell differentiation, and differentiation into hepatocytes or bile duct epithelial cells, in order to mobilize the body’s potential to repair liver damage [17, 18]; With oval cells continue to divide, marker of bone marrow-derived stem cells gradually weakened to 24 balls are dropped mixed liver cancer, has been marked loss of bone marrow-derived stem cells, stem cell malignant transformation and thus c  kit no expression in foci.

    In summary, we have come to the following conclusions: oval cells intrahepatic stem cells, with two-way differentiation potential, in carcinogenesis the continuing role of the agent, to form a mixed type of liver cancer can be abnormal differentiation; characteristic expression OV  6 by AFP and c-kit, OV  6 oval cells specific molecular markers; c  kit expression in experimental liver cancer features indicate that there may be some kind of Original Relationship between oval cells and bone marrow stem cells, which little remains to be further research to find more direct evidence.