大鼠肝癌形成过程中癌基因表达变化的意义

摘要

目的

探讨癌基因在实验性大鼠肝癌形成过程中的表达变化特点及其生物学意义.

方法

应用化学致癌剂3'-Me-DAB建立大鼠肝癌模型, 取其肝脏用核酸原位杂交和RNA Slot blot杂交方法, 动态检测c-myc、Ha-ras和Ki-ras在实验性大鼠肝癌形成过程中的表达变化.

结果

在诱癌全过程中, c-myc和Ha-ras均为同步表达; 在诱癌的早期可检测到较多c-myc和Ha-ras的阳性表达细胞, 而Ki-ras阳性的细胞数则很少, 且反应强度弱; 而诱癌后期, 各癌基因mRNA阳性表达细胞均减少; 至17 wk, 所有癌组织内均显示3种癌基因表达阴性, 或仅见个别小癌巢内少数癌细胞呈弱阳性. 而癌旁肝组织内却可见三种癌基因的大量表达.

结论

c-myc与Ha-ras被激活是肝癌发生过程中的早期事件, 且二者之间具有协同作用, 而这种协同作用在肝癌的启动上可能具有重要意义; Ki-ras的激活则发生较晚, 可能与促进细胞恶性转化有关.

关键词: N/A

Expressive characteristic and biological significance of oncogenes during rat hepatocarcinogenesis

Ling Xue, Bing Liao, Guo-Qiang Zhao, Rui-De Hu, Li-Hong Che, Jun Dong

Ling Xue, Bing Liao, Guo-Qiang Zhao, Rui-De Hu, Li-Hong Che, Jun Dong, Department of Pathology, Zhongshan Medical College, Zhongshan University, Guangzhou 510080, Guangdong Province China

Supported by: the founation of National Education Committee, No. 2000479.

Correspondence to: Dr. Ling Xue, Department of Pathology, Zhongshan Medical College, 74 Zhongshan 2th Road, Guangzhou 510080, Guangdong Province, China. pathol@gzsums.edu.cn

Received: November 6, 2002
Revised: November 10, 2002
Accepted: November 14, 2002
Published online: July 15, 2003

Abstract

AIM

To explore the expressive characteristic and biological significance of oncogenes during experimental hepatocarcinogenesis in rat.

METHODS

A rat model of liver carcinoma was induced with carcinogen 3-Me-DAB. The hepatic expressive characteristic of Ha-ras, c-myc and Ki-ras rats in varied stages in induction of cancer were examined with in situ hybridization and RNA slot blot hybridization.

RESULTS

The hepatic expression and distribution of Ha-ras and c-myc were similar in different periods of cancer induction. In early stage of carcinoma induction, there were more positive cells of Ha-ras and c-myc but little Ki-ras positive cells. In late stage of carcinoma induced, the number of positive cells of three oncogenes was decreased; On the 17th week of induction of cancer, the signals of three oncogenes in all the cancer nodules were negative or weak but stronger in hepatic peri-cancer tissues.

CONCLUSION

Oncogenes Ha-ras and c-myc were activated and had synergistic effect on the malignant transformation of cells in early stage of hepatocarcinogenesis. Ki-ras might be activated in late stage of hepatocarcinogenesis and associated with malignant transformation of hepatocytes.

Key Words: N/A

0 引言

肝癌的发生涉及多种癌基因的激活和抑癌基因的失活[1-11], 但对于不同癌基因的选择性表达以及他们在时间上的顺序激活对肿瘤的发生发展有何意义仍不完全清楚. 我们应用核酸原位杂交和RNA Slot blot杂交, 动态检测c-myc、Ha-ras和Ki-ras在实验性大鼠肝癌形成过程中的表达变化, 旨在探讨上述癌基因在肝癌发生中的作用及其生物学意义.

1 材料和方法

1.1 材料

3'-Me-DAB诱发大鼠肝癌模型: 由本校实验动物中心提供标准动物, 选择健康♂SD品系大白鼠50只, 体质量100-120 g, 稳定饲养1 wk后, 40只作为实验组, 用含0.6 g/L 3'-Me-DAB的饲料喂养14 wk 后停药, 之后继续以正常饲料喂养至17 wk; 另外10只作为对照组, 以标准饲料喂养. 自投以致癌剂之日起, 分别在4, 6, 8, 14和17 wk处死5只大鼠, 取其肝脏, 部分置液氮中冻存, 用于提取组织总RNA; 部分用40 g/L中性甲醛固定, 石蜡包埋. 所有蜡块均作5 μm厚的连续切片, 用于HE染色及核酸原位杂交.

1.2 方法

癌基因核酸原位杂交: 所用癌基因探针质粒(c-Ha-ras PBR322 6.6 kb BamH I、Ki-ras PBR322 1.0 kb ECoRI、c-myc PBR322 8.5 kb Hind I/ECoRIII)由北京中国医学科学院肿瘤研究所提供, DIG标记(标记试剂盒为德国宝灵曼公司产品), 标记方法为随机引物法. 石蜡包埋切片5 μm, 蛋白酶K 40 mg/L 37 °C, 30 min; 预杂交, 42 °C, 1 h; 杂交过夜, 42 °C; 1/500辣根过氧化酶标记的抗Dig抗体1 h, DAB显色, TE终止显色, 中性树胶封片, 镜检. 常规提取不同时期大鼠肝组织标本的总RNA, 紫外分光光度计(pharmacia, ultrospec 2000)测其A值, 260/280在1.7以上者用于杂交实验. RNA-DNA狭缝杂交(slot blot hybridazation)按《分子克隆》一书介绍的方法加以改进进行. 用狭缝点样器将变性之RNA点于尼龙膜(德国宝灵曼公司)后进行杂交. 所用癌基因探针标记方法同上. 常规洗膜. 将膜封入杂交袋内, 加入50 g/LCSPD(德国宝灵曼公司), 10 min后倾出CSPD液, 将膜压入X光暗盒曝光于X光胶片, 常规显影, 定影. 胶片用Kontron IBASS 2.0图像分析处理仪(German)扫描分析结果.

2 结果

诱癌4 wk的大鼠肝体积明显缩小, 质地柔软, 镜下除见大量肝细胞坏死外, 显著的变化为门管区及坏死的肝细胞区内均见大量增生的卵圆细胞. 诱癌6 wk, 大鼠肝内仍有大量卵圆细胞, 部分卵圆细胞变为立方形, 核呈圆形, 似胆管上皮细胞, 并可围成管腔样结构. 与此同时, 在大片的卵圆细胞中还可看到一些嗜碱性细胞、小肝细胞及过渡细胞. 诱癌8 wk, 大鼠肝镜下可见典型假小叶形成, 假小叶周围为增生的纤维组织及较多的卵圆细胞, 其间仍可见过渡细胞和胆管上皮细胞. 诱癌14 wk, 肝内已出现不典型增生的肝细胞灶. 诱癌17 wk, 大鼠肝脏除肝硬化改变外, 还可见癌结节形成. 所形成的5例肝癌中, 1例为胆管上皮癌, 其余4例为混合性肝癌.

2.1 癌基因c-myc、Ha-ras、Ki-ras mRNA原位杂交

凡细胞胞质内见有均匀一致分布的棕黄色颗粒者即为阳性反应细胞. 在化学诱癌不同时期的大鼠肝脏标本中均可检测到3种癌基因的表达, 其中以Ha-ras检出率最高且反应强度最强, Ki-ras的反应最弱. 3种癌基因的表达分布范围基本一致. 在诱癌的4-8wk, 癌基因c-myc和Ha-ras的阳性表达细胞呈逐渐增多趋势, 而Ki-ras阳性的细胞数很少, 且反应强度弱. 阳性表达细胞多为肝细胞坏死区周围或假小叶外围的小肝细胞; 而门管区卵圆细胞和过渡细胞亦可见少量阳性反应细胞. 诱癌14 wk时, 各癌基因阳性表达细胞数量均减少, 但仍可见部分小肝细胞、嗜碱性肝细胞呈阳性反应. 至17 wk, 所有癌组织内均显示三种癌基因表达阴性, 或仅见个别小癌巢内少数癌细胞呈弱阳性. 而癌旁肝组织内无论是HE切片中显示形态完全正常的肝细胞, 亦或是具有一定异型性, 甚至不典型增生之肝细胞均可见三种癌基因的大量表达(图1-3).

图1 诱癌8 wk时c-Ha-ras原位杂交示大量阳性细胞.

图2 诱癌17 wk时癌旁肝组织内见大量c-Ha-ras阳性细胞.

图3 诱癌17 wk时癌旁肝组织内见较多c-myc阳性细胞.

2.2 c-myc, Ha-ras, Ki-ras mRNA表达的Slot blot杂交半定量

在大鼠诱癌的过程中, c-myc、Ha-ras和Ki-ras的表达程度如何, 仅靠原位杂交来反映是不够的, 故我们提取了诱癌不同时期大鼠肝的总RNA进行Slot blot杂交, 然后通过图像分析仪将杂交信号的强弱转换为平均灰度值, 以求客观反应上述三种癌基因在大鼠肝癌发生、发展过程中相对量的表达变化情况, 可见, c-myc与Ha-ras的表达是同步的, 二者均从诱癌4 wk开始升高, 8 wk升高更为显著, 之后下降; 至肝癌形成时(17 wk), 癌组织内二者的表达均很低, 而癌旁组织则有高表达. Ki-ras仅在癌旁组织中检出. (表1).

表1 癌基因Slot blot杂交信号的平均灰度值.

t/wk	c-myc	Ha-ras	Ki-ras
0	7.0	1.2	-
4	7.5	6.4	-
6	12.1	7.7	-
8	18.6	10.9	-
14	5.4	4.1	-
17/癌组织	4.3	1.8	-
癌旁组织	28.3	18.9	5.2

3 讨论

在肝癌发生发展的过程中, 各阶段有其特异的基因表达[12]. N-ras在人原发性肝癌中是一个转化基因, 他可能在癌形成的早期或癌前期即被激活[13,14]. 而在动物(包括大鼠和小鼠), 不少化学致癌剂首先激活的是Ha-ras和Ki-ras, 从而导致肝癌的形成, 这已为许多实验所证实[15-20]. 由此看来, 无论在人或动物, ras基因都可能是肝的致瘤因子作用的靶基因, 从而也提示他可能是在肿瘤发生的启动阶段被激活的肿瘤基因. c-myc的过量表达普遍见于各种肝癌细胞系和肝癌组织中, 表明c-myc的激活亦是肝癌发生中的常见事件, 可能参与肝癌发生的机制[21-29]. 但对于他在肝癌发生过程中检出的时间, 各家结果不尽一致. Yaswen et al[30]在诱癌早期增生的卵圆细胞内即发现有c-myc的表达升高; 而Beer et al[31]则仅在诱癌后期检测到c-myc的表达. 以往的研究发现, 绝大多数人肝癌组织中同时有c-myc和ras的过表达. 动物实验也发现, 在化学诱癌早期可出现c-myc或Ha-ras的过表达[32]. c-myc和ras的表达对细胞的增生发挥作用, 可能参与肝癌的形成或维持. Tashiro et al[33]用黄曲霉素B1诱发大鼠肝癌, 分析癌细胞内的RNA和DNA, 发现c-myc与Ha-ras之间具有协同作用; Corcos et al [34]在DENA诱发的大鼠肝癌及癌旁肝细胞内检测到Ha-ras、Ki-ras和c-myc, 认为这可能是化学致肝癌过程中较早出现的事件, 与肝癌的形成始动有关, 由此认为二者可能是一类互补基因, 之间具有一定特异性的协同作用, 而这种特异的协同作用在肝癌的启动上可能有重要意义.

我们的结果显示, 在诱癌的整个过程中, Ha-ras的表达不仅总是与c-myc的表达相伴出现, 呈现出同步升高或同步降低, 而且二者在核酸原位杂交中的显示阳性表达细胞的类型及分布范围基本一致, 表明二者之间确实存在某种联系, 这与以往许多学者的研究结果相吻合. 在诱癌的早期和中期(诱癌的4-8 wk), 在增生的卵圆细胞、过渡细胞和小肝细胞中都可以检测到c-myc和Ha-ras的表达, 且表达强度不断升高. 而在诱癌晚期(诱癌的14 wk), 上述两种癌基因的表达强度降低. 在肝癌形成之后, 我们仅在癌旁组织内检出c-myc、Ha-ras的表达升高, 而癌组织内则呈低水平表达. 这提示二者可能是在肝细胞癌变的早期(启动阶段)被激活, 而在肝癌形成的后期, 不需要上述二种癌基因的过表达来维持细胞的恶性表型, 而可能有其他癌基因或抑癌基因参与其中. Ki-ras的表达仅在癌旁组织中检测到, 表明他可能是本模型中较晚出现的事件, 其作用可能与促进细胞恶性转化有关.

备注

$[Footnotes]

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