Research On Detection Of Circulating Tumor Cells In Patients With Hepatocellular Carcinoma By Real-time PCR And The Clinical Study Of Circulating Tumor Cell Changes During Perioperative Period

Background: Hepatocellular carcinoma(HCC) is one of the most common malignant tumors in the world, its incidence rate ranks fifth in the malignant tumors,and its mortality rate ranks the third in the malignant tumors. In recent years, although the incidence of HCC in our country showed a downward trend, but the incidence of HCC is still grim. Data show that the global HCC new cases each year, the number of cases of death, of which, of which, the number of new cases and deaths accounted for about 50% of the world, is still in the world's first. Tumor resection or liver transplantation is the most effective treatment for the disease. However, the overall treatment effect of HCC is not ideal, the five year survival rate is still only 15%-40%,within two years of 62%-82% patients with recurrence, even the postoperative recurrence rate of small hepatocellular carcinoma can reach 43.5%. High metastasis and high recurrence of postoperative patients have become the main obstacles hindering the improvement of the prognosis of HCC, and it is the key problem to be solved in the study of hepatocellular carcinoma.HCC in transfer is different from other malignant tumor, lymph node transfer is relatively small, caused by hematogenous spread of liver metastasis and late is prone to metastasis to distant organs; HCC patients by affecting the liver is extremely easy to cause iatrogenic tumor cell diffusion, the reason may and liver has a sponge like structure related, affecting the liver organ extension and compression, so that part of the tumor cells into the blood. In some patients with hepatocellular carcinoma in tumor resection or liver transplantation, tumor cells shedding into the circulatorysystem, these tumor cells most after entering the circulatory system will by the immune system to recognize and be eliminated by the host, but there are still a small number of tumor cells because of the availability of new features and can survive in the blood, medical admiral cells in this part of the named as circulating tumor cells(CTCs). CTCs not only has the characteristics of epithelial cells, but also has some characteristics of tumor stem cells. A large number of studies have shown that CTCs is involved in the process of metastasis and recurrence of cancer, so there is a high correlation between the number of peripheral blood CTCs and the prognosis, survival rate and other parameters of the tumor patients. And studies have shown that the presence of CTCs is a prerequisite for distant metastasis, and the entry of CTCs is an important condition for the recurrence and metastasis of HCC after operation. The molecular biology study of CTCs is helpful to clarify the mechanism of tumor metastasis. This topic through establishing to detect patients with HCC CTCs in peripheral blood of real time PCR system and further detection of HCC patients undergoing operative CTCs specific target gene expression changes, find out the causes of the change trend of HCC hematogenous metastasis and CTCs spread, and seek to prevent HCC hematogenous metastasis.Objective: 1.Establishment of Real-time PCR system for detection of circulating tumor cells in peripheral blood of patients with hepatocellular carcinoma.2.Using PCR Real-time system to study the changes of circulating tumor cells in patients with hepatocellular carcinoma during perioperative period.Methods: 1.Literature research: using two databases of Pub Med and CNKI's literature retrieval, Pub Med to HCC; CTC "as keywords, CNKI with" Hepatocellular carcinoma; circulating tumor cells "as keywords; retrieval on liver cancer cells and circulating tumor cells in English journals, conference, graduation thesis literature,from the report to find the target genes associated with HCC.2.Target gene selection: through the tumor cell line Sprinking experiments,screening out one or more of the HCC CTCs specific target gene. Hep G2 positive cells and HL-60 cell negative as control(Chinese Academy of Sciences, Shanghai Institute of life science, cell resource center), in 1×106HL-60 cells were added to 0, 5,20, 80 and 320, 1280 Hep G2 cells, real time PCR was used to detect selected candidate target gene expression(AFP, GPC3, CK18, CK19, CD133) specificity and sensitivity. The experimental data were processed by SPSS17.0 computer medical statistical analysis software, and statistical charts were drawn using SPSS17.0 and Excel2003.3.Establish real time PCR for the detection of HCC CTCs technical system, the preliminary examination of the specificity and sensitivity of the system: selected in August 2011 to June 2012 period in Shandong Province Tumor Hospital from hepatocellular carcinoma patients blood samples and Shandong Province blood center of normal blood samples of all 8 cases, by real time PCR system and in comparison with the liver cell cancer patients and normal blood AFP, GPC3, CK19 three gene expression differences. The experimental data were processed by SPSS17.0 computer medical statistical analysis software, and statistical charts were drawn using SPSS17.0and Excel2003.4.Hepatocellular carcinoma patients with peri operative CTCs changes of clinical research: Department of Hepatobiliary Surgery; Shandong Provincial Tumor Hospital were collected from August 2011 to 2012 June were untreated HCC patients in 23 cases, respectively in 1 day before operation and after operation 1, 3, 5, 7, 10 days extraction outer peripheral venous blood by real time PCR system for the detection of peripheral blood in m RNA as a marker of the number of CTC. Using SPSS17.0 computer medical statistical analysis software for data processing,statistical charts are drawn using SPSS17.0 and Excel2003.Results: 1.Literature research results: AFP and DCP, GPC3, MAGE-1/MAGE-3,GGT H subtype, h TERT, E-cadherin, CK18, CK19, cadherin-17, CD90, CD133 gene in the aspects of clinical diagnosis and treatment of HCC have shown the great potential, this study selected AFP, GPC3, CK18, CK19, CD133 as a candidate target gene.2.In tumor cell lines Sprinking experiment: 1×106HL-60 cells with at least five Hep G2 cells, AFP, GPC3 and CK19 gene can detect differences. The first two genes more sensitive, as the target gene.3.The sensitivity experiment of establishing the technical system showed that there was no significant difference between the expression of AFP, GPC3 and CK19 in the patients with hepatocellular carcinoma and normal persons, which could not be distinguished.4.In 23 cases of hepatocellular carcinoma patients and the detection of peripheral blood showed: peripheral blood circulating tumor cells from surgical resection compared with surgical target gene expression decreased, but three screening of AFP, GPC3, CK19 gene after surgery in 1, 3, 5, 7, 10 days no detected statistically significant changes.Conclusions: 1.The CTCs in peripheral blood of patients with HCC was lower than that before operation, which indicated that the treatment of tumor by surgery may prevent the spread of HCC cancer cells in blood.2.In Sprinking experiment, AFP, GPC3, CK19 for the detection of target gene technology system with high specificity and sensitivity, can be used as a study of HCC patients with CTCs target target gene;3.At the present stage, the sensitivity of Real-time PCR system in the detection of peripheral blood CTCs in patients with HCC is not enough, and it needs further improvement and perfection.Background: Hyperspectral imaging technology is a new access to information technology, can get the object being detected two dimensional spatial information and one dimensional spectral information, it can in essence reflects the molecular regulation caused changes of organism physiological and molecular levels and overall function and reflect the biological group fabric changes of biochemical composition of specific pathological factors which causes. With the continuous development of science and technology, in recent ten years, the gradual emergence of the a can be used to obtain the microscopic sample information on the chemical composition of the new application technology, biomedical imaging technology, through the light to different cells produce spectra as the "fingerprint" of the cell and to distinguish between different types of cells. The application of hyperspectral imaging technology to the detection of hepatocellular carcinoma may improve the efficiency of the detection of hepatocellular carcinoma.Objective: Using hyperspectral imaging instrument detected the difference of tumor cells, normal cells and lymphocytes in the pathological sections of hepatocellular carcinoma, which laid a theoretical foundation for the establishment of an auxiliary pathological diagnosis system.Methods: Select 15 cases of hepatocellular carcinoma patients with hepatocellular carcinoma after surgery which were treated in Shandong provincial cancer hospital, from July 2012 to June 2013. Each slice selected 3 horizons, each of which included tumor cells, normal cells, and lymphocytes. Using hyperspectral imaging spectrometer and scanning each field, statistical hepatocellular carcinoma biopsy of hyperspectral image data, through the three band synthetic false color image,statistical spectral curve and multispectral fusion three ways for image processing, inthe form of image visual display of results.Results: The cell nuclear spectral curves of the three tumor cells, normal liver cells and lymphocytes were different, which were different in 620 nm, but the difference in 530 nm was small. But the difference was not universal.Conclusions: Detection of pathological sections of hepatocellular carcinoma by hyperspectral imaging technology, which can obtain some information about cancer.There were obvious differences in the cell nuclei of tumor cells, normal cells and lymphocytes in the three cell nuclei, but the difference was not universal.