【作者】 张波；

【导师】 王家祥；

【作者基本信息】 郑州大学 ， 小儿外科， 2005， 硕士

【摘要】 研究背景:长期以来,我国肝癌的死亡率在恶性肿瘤中占居第二位,仅次于肺癌。原发性肝癌的诊断对于具有典型临床表现者并不困难,但这时往往已届中晚期,疗效不佳。AFP一直是肝癌诊断的最佳指标,AFP是胚胎期肝细胞和卵黄囊产生的一种蛋白,出生一周后即消失,当肝细胞癌变后又获得合成此蛋白的能力。从病理类型看,高分化和分化很差的肝癌细胞均不产生AFP,只有中度分化的肝癌细胞能合成,因此,AFP对肝癌诊断的阳性率一般为60%～70%。肝硬化患者中AFP,也有很高的阳性率,说明AFP在原发性肝癌诊断中存在假阳性问题。因此,探索和建立一种简单、快速、敏感性高和特异性强的早期诊断技术已经成了临床医学上的迫切需要。 任何恶性肿瘤在出现病理变化之前,细胞内的蛋白质在成分和数量上都会有相应的改变,所以从理论上说,通过对蛋白质的动态观察可以筛选出疾病早期的指标和征兆。当然实现这种诊断的前提就是要找出各种疾病的特殊标志的分子,这种筛选是高通量的,用以前的常规方法很难做到,蛋白质组学的开展和研究及其新兴技术的出现是这种大规模筛选成为可能。同时也给临床疾病的诊断,尤其是肿瘤诊断开辟了一个全新的分子诊断技术时期。更多还原

【Abstract】 Backgroud: For a long time, the liver cancer comes to the second place in the mortality rate in the malignancies in China, just behind the lung cancer. It is not difficult in the diagnosis of the primary liver cancer with the typical signs and symptoms. Hence, liver cancer tends to present at a late clinical stage with poor prognosis. The AFP (alpha fetoprotein) has been a gold standard for diagnosis of the liver cancer in early stage. The AFP is a kind of protein produced by liver cells and yolk sac of the fetus which will disappear one week after the birth. After canceration, the liver cell is capable of producing AFP again. According to the pathological grading of liver cancer, well-differentiated and poor-differentiated liver cancer cells do not produce the AFP. Only the moderate-differentiated liver cancer cells can produce the AFP. So the positive rate of the AFP in diagnosis of liver cancer ranges from 60% to 70%. And there are also high positive rate of AFP among the patients with liver cirrhosis . It is indicated that there is a problem of false positive when the AFP is applied in the diagnosis of primary liver cancer. So it is urgent to find out a simple technique of early diagnosis with high sensitivity, specificity and quickness, particularity at the clinical medical ascend.The component and quantity of proteins in the cells will change prior to theoccurrence of the pathological changes of malignant tumor. So in theory, it is possible to screen the index and signs of diseases in early stage by dynamic observation of the protein. The precondition is the specific molecule for certain disease for early diagnosis. The screening method is high-throughput and cannot be performed with the traditional techniques. The research and application of proteomics along with its related high technology make this large scale screening come to truth. It also opens a fresh new stage of the molecular diagnosis technology for the clinical application, especially for the tumor diagnosis.This research adoption the United States ciphergen SELDI- TOF- MS to analysis contrast the serum protein of liver cancer, normal person and cirrhosis, this method sensitive and specific, intend to proceed examination to test this method whether can used to choose the specific serum protein marker of the earlier period of liver cancer.Purpose: Establish a technique to examinate the serum protein mass spectometry, and study the value of serum protein mass spectrometry according to artificial nerve the network model in the diagnosis of the liver cancer.Material and method: 74 serum samples obtained, 52 were from patients with liver cancer, 22 from patients with liver cirrhosis in the Department of Oncology of the Second Affiliated Hospital of Zhejiang University, China. The diagnosis of the liver cancer was confirmed postoperatively by pathological examination in all 52 patients including 32 males and 20 females with an average age of 60.6 years old (39-81 years old). All the 22 liver cirrhosis samples were age and gender matched, which were obtained in the early morning under the condition of no eating and drinking and stored at -80 ℃ in a cryogenic refrigerator after separation. And the serum samples from the patients with liver cancer were collected prior to the start of treatment. This research used SELDI- TOF- MS to analysis contrast the serum protein of liver cancer, normal person and cirrhosis. At the same time, 74 example specimens is divided into two set in radiam: train set(49) and test set(25), firstly train49 example specimens proceeds artificial nerve network, get a model,then examinate this model. Secondly,use the same technique and m/zta collect method to estimate 25 unknown serum specimen( include the liver cancer, the cirrhosis and the health person). Use same method to collect the serum protein mass spectometry of 25 examples , get the spectra m/zta equally to the train set,proceeding matrix converting after output the primitive m/zta, then input the establishing artificial nerve network, output the estimate m/zta.The m/zta handles: The raw intensity m/zta were normalized with the ProteinChip Software version 3.1 (homogenizing of the total ion current and molecular weight) in the groups compared. The mass/charge (m/z) peak intensities of the samples with the molecular weight more than 2kDa were normalized with biomaker wizard of ProteinChip Software version 3.0 for noise filtering. The first threshold for noise filtering was set at 5, and the second was set at 2. The 10% was set for the minimum threshold for clustering. The statistics analysis of the m/zta of serum protein mass spectrum was performed on the groups (normal vs. liver cancer and liver cirrhosis vs. liver cancer) with the t test.Result: Analysis 2 training sets of 49 sample and get 21 peaks, among them two peaks(7759 m/z,13134 m/z)show the difference obviously.At 7759 m/z,Its protein of opposite contain respectively for the liver cancer sets is 5.916 ± 0.149, for cirrhosis set is 3.194 ± 0.063(P<0.001); at 13134 m/z, Its protein of opposite contain respectively for the liver cancer sets is 3.242±0.082,for cirrhosis set is 1.837±0.042(P<0.001); Make use of the protein’s m/zta of these two different molecular mass, we can get a model, expectated output towards the liver cancer and cirrhosis establish 1 or 0 respectively. Matrix converting the m/zta of the train set, then put the statistics into the artificial nerve network model, output the estimate m/zta. Through the test, the model of the artificial nerve network can be used to comparise the liver cancer set and the cinhosis set. Use the method which already be tested correctly above to measure the 25 unknow serum specimen , put the serum protein mass spectometry of each example into the established artificial nerve network to estimate the result, 15 example liver cancerpatient in 17 sample,8 example cirrhosis in 8 sample is accurately be predicted, Sensitivity of this method is 88.2%(15/17), specificity is 100%.(8/8).Conclusion: The particularity marker that select by the SELDI technique can used fordiagnosis of the liver cancer and liver cirrhosis.更多还原