出版年:

2000

研究生:

陳怡安

研究生(英文姓名):

Yi-An Chen

論文名稱:

原位肝癌小鼠模式

英文論文名稱:

Establishment of a murine in situ hepatoma model

指導教授:

黎煥耀

指導教授(英文姓名):

Huan-Yao Lei

學位類別:

碩士

校院名稱:

國立成功大學　

系所名稱:

微生物暨免疫學研究所

學號:

s46871128

學年度:

88

語文別:

英文

論文頁數:

47

關鍵詞:

肝癌 ; B型肝炎表面抗原 ; BALB/c小鼠模式

英文關鍵詞:

hepatoma ; HBsAg ; hepatitis B virus surface antigen ;
murine ; BALB/c

被引用次數:

0

[ 摘要 ]

人類B型肝炎病毒（Human hepatitis B virus, HBV）的感染被認為與肝癌息息相關。然而，對其致癌機制及相關免疫反應的了解並不多。HBV具有三種表面抗原（HBV surface a
ntigen, HBsAg），分別為大、中及小。有些肝癌患者中，累積於肝癌細胞質中的B型肝炎表面抗原會以邊緣形式（marginal type）表現。我們希望建立一種原位肝癌小鼠模式以進行其在肝臟部位相
關的免疫學研究。首先，將三種質體，分別是大表面抗原野生型（large S wild type, w）、大表面抗原突變型（large S mutant, △2）及小表面抗原（small S, △pst）
，與帶有neo基因的質體進行共同轉植（cotransfection）至取自BALB/c小鼠的ML-14a肝癌細胞株內，再以G418為篩選標記。我們發現帶有大表面抗原突變型的基因轉植株（△2-ML-14a）與其他基因轉植株相比，其細胞質內累積了較多表面抗原。同時，△2-ML-14a在軟洋菜膠測定中，生成較其他基因轉植株為大的細胞群落。我們經由在BALB/c小鼠的脾臟注入3x106個ML-14a細胞，
建立一種原位肝癌小鼠模式，二週後可在小鼠肝臟以肉眼觀察到肝癌結節，且小鼠於3到4週死亡。接受△pst-ML-14a基因轉植株的小鼠，直到注射後六週才可以肉眼觀察到較明顯的結節生成。然而接受△2-ML-14a基因轉植株的小鼠，在注射後二週可在肝臟處被觀察到肝癌結節，結節數目在三週達到最大量。組織切片的結果顯示，相對於由ML-14a細胞株所生成的結節，由△2-ML-14a及△pst-ML-14a細胞株所生成的結節處，有較多淋巴球浸潤的現象。大部分由△pst-ML-14a細胞株所生成的結節處有細胞壞死（necrosis）的情形。相反地，由△2-ML-14a細胞株所生成的結節處卻沒有細胞壞死的情形，且此種結節在6到7週時，淋巴球浸潤的現象會消失。對照以抗B型肝炎病毒表面抗原的抗體進行免疫組織化學染色的結果，△2-ML-14a細胞株所生成的結節處在早期可以染到表面抗原的存在，但到了晚期有些細胞卻染不

[ 英文摘要 ]

Human hepatitis B virus (HBV) is closely associated with hepatocellular carcinoma (HCC). However, neither the mechanism of carcinogenesis nor the immune responses to HBV associated HCC is well understood. HBV has three types of surface antigens (HBsAg): large, middle, and small. In some HCC patients, the large HBsAg accumulation in cytoplasm of HCC was associated with marginal type expression of HBsAg. We are interested in establishing a murine in situ hepatoma model, and studying its immune responses in liver. First of all, ML-14a hepatoma cell line from BALB/c mice was transfected with plasmid including large S wild type（w）, large S mutant (△2), and major S (△pst) with neo gene. The transfectants were selected with G418. We found that ML-14a cells transfected with large S mutant (△2-ML-14a) showed higher accumulation of HBsAg in cytoplasm comparing with other transfectants. △2-ML-14a formed more foci than △pst-ML-14a or ML-14a on soft agar assay. An in situ HCC was established by injecting 3x106 ML-14a cells into spleen of BALB/c mice. Tumor nodules were found on liver at 2 weeks after injection and these mice died at 3 to 4 weeks. The small S transfectants （△pst-ML-14a） did not form such tumor nodules until 6 weeks after injection. But, △2-ML-14a formed tumor nodules at 2 weeks post injection and achieved peak at 3 weeks. Histologically, nodules formed by either △2-ML-14a or△pst-ML-14a had more lymphocytic infiltration than ML-14a one. Most nodules formed by △pst-ML-14a h
ad necrotic lesions. On the contrary, nodules of △2-ML-14a did not observed necrosis. The lymphocyti
c infiltration would disappear at 6-7 weeks. Interestingly, immunohistochemistry stain with anti-HBs
Ab on △2-ML-14a nodules showed HBsAg was stained in early stage, but in late stage, it was not stain
ed on some cells. Based on the above data, the large S mutant gene （△2-ML-14a） would affect the hepa