【摘要】：目的 通過改良型間斷飲用二乙基亞硝胺（diethylnitrosamine,DEN）誘導(dǎo)大鼠原發(fā)性肝癌模型，觀察大鼠肝臟在不同誘癌階段的病理變化，同時(shí)檢測(cè)Survivin和Apollon在大鼠肝臟不同誘癌階段的表達(dá)情況，旨在研究Survivin和Apollon在誘導(dǎo)的大鼠肝細(xì)胞癌發(fā)生、發(fā)展過程中的變化，探討Survivin和Apollon作為肝癌早期診斷標(biāo)志物的可行性；收集部分臨床人肝癌標(biāo)本及其癌旁組織、肝良性病變組織，檢測(cè)Survivin和Apollon表達(dá)情況，并結(jié)合臨床病理參數(shù)，探討Survivin和Apollon在人肝癌形成過程中的意義。 方法 4周齡的雌雄性近交系wista大鼠共80只，隨機(jī)分為實(shí)驗(yàn)組（A組，n=68）和對(duì)照組（B組，n=12）。A組動(dòng)物間斷飲用0.01%二乙基亞硝胺(DEN)溶液，B組自由飲用滅菌自來水。實(shí)驗(yàn)中觀察大鼠的一般情況，于實(shí)驗(yàn)第4周、8周、12周、16周、18周分別取A組大鼠12只，B組大鼠2-3只，稱重后麻醉，開腹摘取大鼠肝臟，觀察肝臟大體情況及組織病理學(xué)變化；分別采用免疫組織化學(xué)法檢測(cè)Survivin和Apollon在大鼠肝癌形成不同階段的表達(dá)情況，并將兩者在各階段表達(dá)情況進(jìn)行比較分析；用免疫組織化學(xué)方法檢測(cè)Survivin和Apollon蛋白在42例原發(fā)性肝癌組織及其癌旁組織、15例肝臟良性病變組織的表達(dá)情況，，實(shí)驗(yàn)數(shù)據(jù)結(jié)合臨床病理參數(shù)進(jìn)行統(tǒng)計(jì)學(xué)分析。 結(jié)果 1.改良型間斷飲用DEN成功誘導(dǎo)了大鼠肝癌模型，大鼠肝臟癌變大致經(jīng)過肝細(xì)胞損傷期、肝硬化期和肝細(xì)胞癌變期三個(gè)階段。至誘癌結(jié)束時(shí)實(shí)驗(yàn)組大鼠自然死亡4只，死亡率5.88%（4/68）。實(shí)驗(yàn)組第18周誘癌結(jié)束時(shí)存活的16只大鼠15只形成肝癌，誘癌率93.75%（15/16）。 2.Survivin和Apollon在正常大鼠肝臟組織不表達(dá)，伴隨著肝癌的發(fā)展，Survivin和Apollon蛋白陽性表達(dá)率和表達(dá)強(qiáng)度依次升高，各組間陽性率比較差異有統(tǒng)計(jì)學(xué)意義（p0.05）。與對(duì)照組相比，Survivin和Apollon在肝硬化期、肝癌變期陽性率均上升（P0.05/3），肝癌變組與肝硬化組比較，Survivin和Apollon的陽性表達(dá)率亦出現(xiàn)上升(P0.05/3)。 3.Survivin蛋白在臨床肝癌組、癌旁組及良性組的陽性率分別為71.43%（30/42）、40.48%(17/42)、0.00%（0/15），各組間差異有統(tǒng)計(jì)學(xué)意義（p0.05）。Survivin蛋白在HCC中的表達(dá)與患者性別、年齡、HBsAg、AFP、腫瘤大小無明顯相關(guān)（p0.05），而與病理分級(jí)、門靜脈癌栓相關(guān)（p0.05）。 4.Apollon蛋白在臨床肝癌組、癌旁組及良性組的陽性率分別為66.67%（28/42)、38.09%(16/42)、0.00%（0/15），各組間差異有統(tǒng)計(jì)學(xué)意義（p0.05）。Apollon蛋白在HCC中的表達(dá)與患者性別、年齡、HBsAg、AFP、腫瘤大小無明顯相關(guān)（p0.05），而與病理分級(jí)、門靜脈癌栓相關(guān)（p0.05）。 5.兩者相關(guān)性：臨床人肝癌組織中Survivin和Apollon蛋白的表達(dá)呈正相關(guān)關(guān)系（r=0.409,p=0.005）。 結(jié)論 1.本研究通過間斷飲用DEN成功誘導(dǎo)了大鼠肝癌模型，該模型簡(jiǎn)單、安全有效，誘癌大鼠具有低死亡率和高誘癌率的特點(diǎn)。 2.Survivin和Apollon參與了大鼠肝癌的發(fā)生、發(fā)展，對(duì)于大鼠肝癌發(fā)生各個(gè)階段均有提示作用，提示其可作為肝癌早期診斷的潛在標(biāo)志物。 3.Survivin和Apollon在人肝癌組表達(dá)明顯高于良性對(duì)照組，說明其表達(dá)異常和失衡與肝癌的發(fā)生、發(fā)展密切相關(guān)。Survivin和Apollon與門脈癌栓的形成及HCC的病理分級(jí)有一定的關(guān)系，提示Survivin和Apollon可作為判斷肝癌侵襲和轉(zhuǎn)移的指標(biāo)。 4.Survivin和Apollon在人肝癌旁組織中表達(dá)與良性對(duì)照組有統(tǒng)計(jì)學(xué)意義，提示陽性表達(dá)的癌旁肝組織具有癌變危險(xiǎn)性，癌旁肝組織可能是一群異常過度增生的肝細(xì)胞群，其中部分細(xì)胞有可能發(fā)展成為HCC。 5.大鼠肝癌的Survivin和Apollon蛋白表達(dá)情況和人肝癌的表達(dá)情況基本一致，該模型能較好地從分子層面模擬人類肝癌發(fā)生，為今后肝癌的研究提供了有效的手段。
[Abstract]:objective
To study the effect of diethylnitrosamine (DEN) on hepatocellular carcinoma (HCC) induced by diethylnitrosamine (DEN) in rats, the pathological changes of rat liver in different stages of carcinogenesis were observed, and the expressions of Survivin and Apollon in different stages of hepatocarcinogenesis were detected. To explore the feasibility of Survivin and Apollon as early diagnostic markers of hepatocellular carcinoma, to collect some clinical specimens of human hepatocellular carcinoma and its adjacent tissues, benign liver lesions, to detect the expression of Survivin and Apollon, and to explore the role of Survivin and Apollon in the formation of human hepatocellular carcinoma in combination with clinicopathological parameters. Significance.
Method
Eighty four-week-old male and female WISTA rats were randomly divided into experimental group (group A, n=68) and control group (group B, n=12). The rats in group A were given 0.01% diethylnitrosamine (DEN) solution intermittently and the rats in group B were given tap water freely. Two to three rats were anesthetized after weighing and abdominal liver was taken out to observe the general situation and histopathological changes of the liver. The expression of Survivin and Apollon in different stages of hepatocarcinogenesis was detected by immunohistochemical method, and the expression of Survivin and Apollon in different stages were compared and analyzed. Survivin and Apollon proteins were detected in 42 cases of primary hepatocellular carcinoma and its adjacent tissues, and 15 cases of benign liver lesions. The experimental data were analyzed statistically with clinicopathological parameters.
Result
1. Modified intermittent drinking of DEN successfully induced hepatocarcinogenesis in rats. The hepatocarcinogenesis of the rats went through three stages: hepatocyte injury stage, liver cirrhosis stage and hepatocyte carcinogenesis stage. The cancer rate was 93.75% (15/16).
2. Survivin and Apollon were not expressed in normal rat liver tissues. With the development of hepatocellular carcinoma, the positive expression rate and intensity of Survivin and Apollon protein increased in turn, and the difference was statistically significant (p0.05). The positive expression rates of Survivin and Apollon in HCC group were higher than those in cirrhosis group (P0.05/3).
3. The positive rate of Survivin protein was 71.43% (30/42), 40.48% (17/42) and 0.00% (0/15) in clinical hepatocellular carcinoma group, adjacent cancer group and benign group, respectively. The difference was statistically significant (p0.05). The expression of Survivin protein in HCC was not significantly correlated with gender, age, HBsAg, AFP, tumor size (p0.05), but with pathological grade and portal vein tumor thrombus (p0.05). P0.05).
4. The positive rate of Apollon protein was 66.67% (28/42), 38.09% (16/42) and 0.00% (0/15) in clinical hepatocellular carcinoma group, adjacent cancer group and benign group, respectively. The difference was statistically significant (p0.05). The expression of Apollon protein in HCC was not significantly correlated with gender, age, HBsAg, AFP, tumor size (p0.05), but with pathological grade and portal vein tumor thrombus (p0.05). .05).
5. The correlation between the expression of Survivin and Apollon protein was positive (r = 0.409, P = 0.005).
conclusion
1. In this study, a rat model of hepatocellular carcinoma was successfully induced by intermittent drinking of DEN. The model was simple, safe and effective, and the induced cancer rats had the characteristics of low mortality and high induced cancer rate.
2. Survivin and Apollon are involved in the occurrence and development of hepatocellular carcinoma in rats, which may be a potential marker for early diagnosis of hepatocellular carcinoma.
3. The expression of Survivin and Apollon in HCC group was significantly higher than that in benign control group, indicating that the abnormal and unbalanced expression of Survivin and Apollon was closely related to the occurrence and development of HCC.
4. The expression of Survivin and Apollon in the tissues adjacent to HCC was statistically significant with that in the benign control group, suggesting that the positive expression of Survivin and Apollon in the tissues adjacent to HCC is at risk of carcinogenesis.
5. The expression of Survivin and Apollon protein in rat hepatocellular carcinoma is basically the same as that in human hepatocellular carcinoma. The model can simulate the occurrence of human hepatocellular carcinoma from the molecular level and provide an effective means for the future study of hepatocellular carcinoma.
【學(xué)位授予單位】：廣州醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R735.7

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