利用大鼠Y染色体特异性PCR技术检测卵圆细胞源性肝癌细胞

Detection of liver cancer cells originated from hepatic oval cells using rats' Y chromosome specific polymerase chain reaction technique

目的:利用Y染色体体细胞的特异性来寻找卵圆细胞分化为肝癌细胞的直接证据,从而为卵圆细胞源性的研究及临床肝癌治疗方法提供理论依据．方法:选择健康♂ Wistar大鼠30只饲喂每千克含0．6 g 3-甲基-4-二甲基偶氮苯(DAB)的饲料 4 wk,建立卵圆细胞增生的♂ Wistar大鼠动物模型．卵圆细胞的分离、提取和鉴定(光镜、电镜、免疫组化)．将实验用♀Wistar大鼠60只随机平分成对照组和实验组．对照组不接种细胞悬液,连续喂含DAB饲料14 wk．实验组按 25 mg／kg体重戊巴比妥腹腔麻醉,消毒开腹, 用吸取♂ Wistar大鼠卵圆细胞悬液,接种至肝脏包膜下,每点106个,之后连续喂含DAB饲料 14 wk,促进肝肿瘤的生成．从GenBank调出雄性大鼠的SRY基因,利用引物设计软件设计引物片断．14 wk后提取两组大鼠肝肿瘤组织的基因组DNA,利用所设计的引物进行PCR扩增,对PCR产物进行电泳分析．结果:光镜下可找到卵圆细胞,核仁小而清晰,胞质少,细胞体积较小,约为正常肝细胞的 1／3,直径约9-12 μm不等．电镜下观察可见细胞表面少量短而小的微绒毛突起,呈现未分化细胞的形态．免疫组化观察肝卵圆细胞胞质 c-kit染色呈棕黄色阳性信号．14 wk后对照组和实验组共计60只大鼠均见肝脏肿瘤生成,肿瘤组织在外观上和性状上无显著性差异．实验组电泳后见与设计片断长度相符的电泳阳性条带．结论:大鼠的卵圆细胞可以分化为肝癌细胞．为卵圆细胞源性肝癌细胞的假说提供实验依据．

AIM： To find out the evidence that hepatic oval cells could differentiated into liver cancer cells, and to provide the theoretical basis for clinical therapy of liver cancer. METHODS： Thirty male Wistar rats were fed with the animal food containing 0.6 g 3′3-diaminobenzidine （3＇-Me-DAB） per kilogram for 4 weeks to establish the model of hepatic oval cell proliferation. Then the hepatic oval ceils were extracted and identified by immunohistochemistry under light and electron microscope. Sixty female Wistar rats were randomly and averagely divided into experiment group and control group. The rats in the control group were fed with the DAB-containing food for 14 weeks without transplantation. While the rats in experiment group were narcotized with 25 mg/kg nembutal via abdominal cavity, and the male hepatic oval cells （in suspension） were inoculated （10^6 each point） under the liver envelope after the abdominal part was cut open. The rats received transplantation were also fed with DAB-containing food for 14 weeks to promote the formation of liver cancer. At the end of 14 wk, the DNA of cancer tissues was extracted from both groups. According to the GenBank, the specific primer of rat SRY gene was designed. Then the extracted DNA was amplified using the specific primer by polymerase chain reaction （PCR）. The PCR product was analyzed by electrophoresis. RESULTS： Under light microscope, hepatic oval cells were observed, showing the characteristics of small and clear nucleolus, small sizes （9-12 lun in diameter）, which were one third of normal liver cells. By electron microscopy, hepatic oval cells manifested typical morphological features of undifferentiation, with some short and tiny microvillus on their surfaces. By immunohistochemistry, c-kit was positively expressed in the oval cells. At the end of 14 wk, liver tumor formed in both groups of rats, and the tumor characters and appearances had no significant difference between experiment and control group. The PCR product showed the same length of positive bands after electrophoresis at the designed ones. CONCLUSION： Hepatic oval cells can differentiate into liver cancer cells.