實(shí)驗(yàn)性肝癌癌組織的發(fā)生和甲胎蛋白表達(dá)

1、    實(shí)驗(yàn)性肝癌癌組織的發(fā)生和甲胎蛋白表達(dá)        摘要目的：研究實(shí)驗(yàn)性肝癌的組織發(fā)生過(guò)程中甲胎蛋白的表達(dá)，旨在為肝癌診斷奠定形態(tài)學(xué)基礎(chǔ)。方法：Wistar大白鼠喂0.04% 3-Me-DAB不同日程處死，36周處死完，觀察動(dòng)力形態(tài)變化和免疫組化AFP表達(dá)。結(jié)果：實(shí)驗(yàn)早期卵圓細(xì)胞增生，該細(xì)胞是多向分化的干細(xì)胞。它進(jìn)一步分化成過(guò)渡細(xì)胞、胚胎性小肝細(xì)胞，后者可呈不典型增生，它們散在或群集，這些細(xì)胞AFP強(qiáng)陽(yáng)性，群集不典型增生的小肝細(xì)胞形態(tài)上不同于肝細(xì)胞增生結(jié)節(jié)，前者和肝細(xì)

2、胞癌形態(tài)相似。卵圓細(xì)胞向膽管分化形成纖維腺瘤樣增生，其上皮細(xì)胞也見(jiàn)不典型增生。58只實(shí)驗(yàn)動(dòng)物生成26例肝癌，其中肝細(xì)胞癌18例，混合癌8例，癌AFP強(qiáng)陽(yáng)性，其周圍宿主肝細(xì)胞和實(shí)驗(yàn)晚期無(wú)癌動(dòng)物的肝細(xì)胞也見(jiàn)程度不同的AFP陽(yáng)性表達(dá)。結(jié)論：小肝細(xì)胞和膽管上皮細(xì)胞的不典型增生是肝細(xì)胞癌和膽管癌的癌前病變。關(guān)鍵詞肝腫瘤，試驗(yàn)性；增生；甲胎蛋白類；大鼠分類號(hào)R735.7文獻(xiàn)標(biāo)識(shí)碼A文章編號(hào)1001-7399(1999)03-0224-03 Carcinohistogenesis and expression of alpha fetoprotein in experimental hepatocarcin

3、omaZhang Jizeng(Dept of Pathol, General Hospital of Lanzhou Military Area, Lanzhou730050)ABSTRACTPurposeTo investigate the carcinohistogenesis of hepatocarcinoma and AFP expression in oncogenesis and to lay a morpholoogical foundation for diagnosing and curing hepatocarcinoma. MethodsWistar rats wer

4、e fed on 0.04% 3-Me-DAB and killed according to different dates. All the animals were killed during 36 weeks. The morphologic changes of dynamics and expression of alpha fetoprotein (AFP) were observed with immunohistochemical method. ResultsHyperplasia of oval cells occurred in the early experimemt

5、al stage and the oval cells were multipotential stem cell. They further differentiated into transitional cell and embryoid small liver cell, and the latter may form atypical hyperplasia, scattered or crowded. AFP in these cells appeared to be strongly positive expression. The feature was different b

6、etween dysplastic small hepatocytes with crowd and proliferative nodules of hepatocytes. The former was similar to hepatocytic carcinoma morphologic ally. The oval cells differeatiated towards biliary duct to form fibroadenomatoid structure, and atypical hyperplasia was seen in their epithelial cell

7、s. 26 of 58 experimental animals were cases of hepatocarcinoma, among whom 18 cases of hepatocytic carcinoma and 8 mixed carcinoma were found. The hepatocellular carcinoma showed stuongly AFP positive expression. In the host hepatocytes around cancer and hepatocytes of non-neoplastic animals of late

8、r experimental period also appered to be various degrees of positive expression of the AFP. ConclusionThe precancerosis of hepatocellular carcinoma and bile duct caricnoma is atypical hyperplasia of small hepatocytes and epithelial cells of bile canaliculi.KEY WORDSliver neoplasms, experimental; hyp

9、erplasia; alpha-fetoproteins; rats1973年Anthony等1在烏干達(dá)研究人的肝硬化和肝癌時(shí)首次提出肝細(xì)胞不典型增生是癌前病變。Roncalli等2又把不典型增生的肝細(xì)胞分成大細(xì)胞和小細(xì)胞，認(rèn)為后者對(duì)肝細(xì)胞癌的生成關(guān)系密切。1988年徐元鼎等3認(rèn)為肝細(xì)胞癌來(lái)自肝細(xì)胞的嗜堿性和嗜酸性增生結(jié)節(jié)。誠(chéng)然，癌組織發(fā)生是很復(fù)雜的，特別是AFP在肝癌形成中的變化。AFP是確定細(xì)胞分化和腫瘤的標(biāo)記物4。此外，以往對(duì)膽管癌的組織發(fā)生以及小膽管上皮細(xì)胞不典型增生與癌發(fā)生的相關(guān)形態(tài)描述也較欠缺。為探討肝癌發(fā)生的動(dòng)力形態(tài)學(xué)，筆者試結(jié)合甲胎蛋白的變化對(duì)肝癌的組織發(fā)生進(jìn)行深入研究。1材料

10、與方法Wistar大白鼠喂0.04% 3-Me-DAB混合食物，其只數(shù)與誘癌時(shí)間見(jiàn)表1。在相應(yīng)時(shí)間里取2只正常動(dòng)物肝存檔蠟塊做切片對(duì)照。肝標(biāo)本系10%中性福爾馬林固定。AFP免疫組化用ABC法。一抗為兔抗大鼠AFP 1400稀釋；二抗是生物素標(biāo)記的馬抗兔IgG血清1200稀釋；三抗為ABC-HRP(Horseradish Peroxidase)1100稀釋，切片顯色用0.04-H2O2 DAB溶液，蘇木素輕淡襯染，常規(guī)制片。對(duì)照片用正常羊血清代一抗。AFP陽(yáng)性顆粒呈褐色，均勻地位于胞漿內(nèi)。在400倍光鏡下隨意轉(zhuǎn)動(dòng)10個(gè)視野在網(wǎng)格內(nèi)數(shù)陽(yáng)性細(xì)胞，以百分率計(jì)陽(yáng)性強(qiáng)度：“<"2 (10

11、1 bytes)" align=absMiddle src="/med/cano/201003/20100318190719463" 21 13>”示陽(yáng)性細(xì)胞41%，分布廣，深褐色強(qiáng)染；“+”示10%，稀落分布，淺褐弱染；“<"5 (140 bytes)" align=absMiddle src="/med/cano/201003/20100318190719247" 16 13>”示介于二者之間；“-”示陰性。為鑒別診斷而進(jìn)行了細(xì)胞角蛋白和上皮細(xì)胞膜抗原染色。表158只實(shí)驗(yàn)性大白鼠在不同日程內(nèi)誘發(fā)的肝臟病

12、變病變實(shí)驗(yàn)日程(周)<4512132021282936卵圓細(xì)胞增生441過(guò)渡細(xì)胞增生43胚胎性小肝細(xì)胞增生668肝細(xì)胞不典型增生5633肝細(xì)胞增生灶或結(jié)節(jié)5520肝假小葉4638小膽管纖維腺瘤樣增生4638肝癌2321處死動(dòng)物只數(shù)446638     表258例實(shí)驗(yàn)性大白鼠肝臟病變中AFP陽(yáng)性表達(dá)病變nAFP+<"3 (92 bytes)" src="/med/cano/201003/20100318190719247" 16 13><"2 (101 bytes)" sr

13、c="/med/cano/201003/20100318190719463" 21 13>-陽(yáng)性率(%)過(guò)渡細(xì)胞714271.4胚胎樣小肝細(xì)胞204610100.0不典型增生的肝細(xì)胞4471027100.0肝細(xì)胞增生灶或結(jié)節(jié)301119100.0假小葉內(nèi)的肝細(xì)胞48151016785.4小膽管纖維腺瘤樣增生的上皮細(xì)胞483423918.7癌細(xì)胞*261016100.0     *僅見(jiàn)肝細(xì)胞癌 2結(jié)果正常對(duì)照的肝切片無(wú)病變，AFP陰性。58只實(shí)驗(yàn)動(dòng)物主要肝病變見(jiàn)表1，AFP陽(yáng)性表達(dá)見(jiàn)表2。實(shí)驗(yàn)13周見(jiàn)肝細(xì)胞增生灶或結(jié)節(jié)。肝細(xì)胞壞死

14、增生破壞肝小葉形成假小葉。在假小葉和增生結(jié)節(jié)內(nèi)有不典型增生的肝細(xì)胞散在其中，該細(xì)胞體積有大有小。由過(guò)渡細(xì)胞而來(lái)的胚胎性小肝細(xì)胞偶見(jiàn)異型性。不典型增生的小肝細(xì)胞常群集，它們與增生結(jié)節(jié)不同，不向周圍組織壓迫性生長(zhǎng)，與周圍無(wú)明顯的界限(1)，AFP以<"2 (101 bytes)" align=absMiddle src="/med/cano/201003/20100318190719463" 21 13>反應(yīng)居多(2)。不典型增生肝細(xì)胞有大小之分。不典型增生的小肝細(xì)胞常和肝細(xì)胞癌毗鄰，有時(shí)和肝細(xì)胞癌不易分辨，但后者癌索或?qū)嵠g有血竇。肝細(xì)胞癌A

15、FP是<"3 (92 bytes)" align=absMiddle src="/med/cano/201003/20100318190719247" 16 13><"2 (101 bytes)" align=absMiddle src="/med/cano/201003/20100318190719463" 21 13>陽(yáng)性反應(yīng)(3)。此外也見(jiàn)癌細(xì)胞索與“正?！备渭?xì)胞索直接相連。肝小葉破壞后也能見(jiàn)到殘留的肝細(xì)胞不典型增生變成怪異狀核的小肝細(xì)胞。不典型增生的大肝細(xì)胞體積巨大，常為AFP陰性反

16、應(yīng)，它們位于不典型增生的小肝細(xì)胞群中或其附近(2)。<"t3501 (20302 bytes)" src="/med/cano/201003/20100318190719583" 250 188>1 不典型增生的小肝細(xì)胞(右中下方)和肝癌細(xì)胞(左下方).HE×100<"t3502 (16804 bytes)" src="/med/cano/201003/20100318190719853" 250 188>2 不典型增生的小肝細(xì)胞AFP(+),而大肝細(xì)胞AFP(-).ABC

17、5;200<"t3503 (20983 bytes)" src="/med/cano/201003/20100318190719910" 254 190>3 肝癌細(xì)胞AFP(+).ABC×400小膽管呈纖維腺瘤樣增生，該處卵圓細(xì)胞消失腺瘤樣增生的小膽管上皮細(xì)胞可見(jiàn)粘液分泌空泡；有的小管擴(kuò)張其上皮細(xì)胞扁平(4)。增生活躍的小膽管上皮細(xì)胞排成多層，擁擠，核深染。有6例膽管上皮細(xì)胞呈不典型增生，與膽管癌難鑒別，核變大，胞核淡染，含明顯的嗜酸性核仁，有較多的核分裂象，它們擁擠成多層，細(xì)胞AFP<"3 (92 bytes)&q

18、uot; align=absMiddle src="/med/cano/201003/20100318190719247" 16 13><"2 (101 bytes)" align=absMiddle src="/med/cano/201003/20100318190719463" 21 13>(5)，可見(jiàn)它們過(guò)渡到膽管癌的組織象，這種膽管大小不等和不規(guī)則的迂曲擴(kuò)張形成腺泡，基底膜模糊或消失。腺泡浸潤(rùn)于非常豐富的結(jié)締組織中，有時(shí)和腺樣肝細(xì)胞癌相間存在，膽管癌管腔內(nèi)含粘液，細(xì)胞膜抗原和細(xì)胞角蛋白染色呈陽(yáng)性反應(yīng)，AFP染

19、色呈陰性；腺樣結(jié)構(gòu)的肝細(xì)胞癌AFP陽(yáng)性反應(yīng)。<"t3504 (22003 bytes)" src="/med/cano/201003/20100318190719497" 254 190>4 小膽管的纖維腺瘤樣增生.HE×200<"t3505 (19427 bytes)" src="/med/cano/201003/20100318190719906" 254 190>5 小膽管的不典型增生,AFP(+).ABC×20026例肝癌中肝細(xì)胞癌18例；混合癌8例。癌周肝細(xì)胞有

20、不同程度的AFP陽(yáng)性反應(yīng)。實(shí)驗(yàn)晚期無(wú)癌動(dòng)物的肝細(xì)胞亦呈不同程度的AFP陽(yáng)性反應(yīng)(6)。<"t3506 (19063 bytes)" src="/med/cano/201003/20100318190720110" 254 190>6 無(wú)癌動(dòng)物和肝細(xì)胞AFP(+).ABC×4003討論不典型增生小肝細(xì)胞應(yīng)看作肝細(xì)胞癌的癌前細(xì)胞。它們散在于肝小葉，小葉破壞后的殘留肝細(xì)胞也可形成，因近似胚胎早期的肝細(xì)胞故AFP強(qiáng)陽(yáng)性反應(yīng)。不典型增生的小肝細(xì)胞群和肝細(xì)胞增生結(jié)節(jié)不同，周圍無(wú)壓迫性境界。它們常和肝細(xì)胞癌相鄰，有時(shí)兩者也難以辨認(rèn)，但后者瘤細(xì)胞索

21、形成，其間存有血竇。綜上所述，不典型增生在動(dòng)力形態(tài)的改變過(guò)程中有自己的演變過(guò)程、形態(tài)特點(diǎn)和存在形成。戴益民等5研究了人體肝癌癌周宿主肝細(xì)胞自己合成AFP。本研究中除肝細(xì)胞癌有AFP陽(yáng)性表達(dá)外，在實(shí)驗(yàn)晚期無(wú)癌動(dòng)物的肝細(xì)胞也有程度不同的AFP陽(yáng)性表達(dá)。顯然，這些細(xì)胞在致癌因子的作用下雖無(wú)形態(tài)上的異型性，但功能上已有返祖改變而處于胚胎狀態(tài)，出現(xiàn)了AFP陽(yáng)性表達(dá)。肝細(xì)胞癌相連的“正?！备渭?xì)胞索，可視為返祖功能改變的肝細(xì)胞被致癌因子啟動(dòng)失分化后形成的癌突變。實(shí)驗(yàn)晚期無(wú)癌動(dòng)物的肝細(xì)胞AFP陽(yáng)性為臨床非肝癌病人血清AFP水平的增高或提示癌突變的可能性提供了實(shí)驗(yàn)依據(jù)。眾所周知，肝細(xì)胞和膽管細(xì)胞來(lái)源于同一胚芽。

22、在實(shí)驗(yàn)性肝癌中Dempo6和Hixson等7指出卵圓細(xì)胞是胚胎性多潛能分化的干細(xì)胞，不言而喻，它既可生成肝細(xì)胞癌也可生成膽管癌和混合性肝癌。明顯的小膽管纖維腺瘤樣增生處卵圓細(xì)胞的消失與它向小膽管分化有關(guān)。不典型增生的小膽管上皮細(xì)胞在間變過(guò)程中返祖到與肝細(xì)胞同源的胚胎性質(zhì)時(shí)即可出現(xiàn)AFP陽(yáng)性表達(dá)，一旦這些細(xì)胞過(guò)渡到膽管癌時(shí)其本質(zhì)已不是肝細(xì)胞，故AFP呈陰性反應(yīng)，細(xì)胞角蛋白和上皮細(xì)胞膜抗原呈陽(yáng)性反應(yīng)，并以此同腺樣結(jié)構(gòu)的肝細(xì)胞癌鑒別。作者簡(jiǎn)介：張繼增，男，73歲，教授，主任醫(yī)師。主要從事癌前病變的病理形態(tài)學(xué)研究作者單位：張繼增（蘭州軍區(qū)總醫(yī)院病理科，蘭州730050）參考文獻(xiàn)1，Anthony PP, Vogel CL, Barker LF. Liver cell dysplasia. A premalignant condition. J Clin Pathology,1973;26(3):2172232，Roncalli M, Borzio M, Biagi GD et al. Liver cell dysplasia in cirrhosis serolog