| Hepatocellular carcinoma (HCC) is of the most common malignancies throughout the world, and there are about600,000new cases every year. The mortality caused by HCC is in the third of that caused by all carcinoma. HBV infection is one of the important risk factors. There are more than100,000,000carriers of HBV in China. So HCC is a multiple critical illness in our country. According to Chinese Ministry of Health statistics, in2007mortality caused by HCC in China urban residents is in the second of that caused by all malignancies(25.47/100000), and the mortality in rural residents is28.72/100000. Although the technology of hepatectomy is progressing, but many patients have poor prognosis because of delay in diagnosis and recurrence after operation. So prevention and early diagnosis of HCC is essential. Currently, because of lack of clinical diagnostic indicators, the rate of early diagnosis is low, many patients are diagnosed at an advanced stage. Early detection of precancerous lesions of HCC is key to early diagnosis and prognosis, currently the clinical diagnosis of precancerous lesions of HCC is still in lack of reliable indicators. As soon as possible to clarify the precancerous lesions of HCC and establish prevention strategies for hige-risk groups, improve prognosis, is an urgent task for the medical workers. Cirrhosis as a precancerous lesions of HCC, one of the pathological features in its organization change is the deposition of collagen fibers. We dynamic observed the characteristic changes of collagen fibers in rat HCC induced by DEN, in order to find the precancerous lesions of HCC and provide a pathological reference for clinical diagnosis and treatment.Objective1.Establish improved concurrency model of cirrhosis of HCC in rats, provide an ideal animal model for the research of characteristics of HCC and drug intervention.2. To observe the characteristic changes of collagen fibers in rat HCC induced by DEN dynamically, explore the pathological features of HCC precursor lesions, provide a reference for prevention and prognosis of HCC.Materials and methods1.Experimental protocol:50male Wistar rats of about100-120g were randomly divided into normal group(20rats) and model group(30rats). The model group was administered a dose of50mg DEN per kilogram intraperitoneal injection, twice a week, for4weeks, then once a week, for another10weeks. The normal group was given saline instead of DEN. Three rats of model and normal group were killd at2,4,8,12,14, weeks,respectively. Operation is under continuous ether inhalation anesthesia,5ml blood were taken for all animals before killed, blood serum stored at-20℃. Take foci of tumor and adjacent organization, which is lcm far from the foci edge, and every organization block size is of about0.5×0.5cm. Cut part of liver tissue of rats in normal group and in model group without tumor, randomly. Tissue were fixed in10%neutral formalin, then for pathological paraffin sections. The remaining liver stored at-80℃.2.Serum biochemistry:the serum gamma-glutamy transpeptidase, total bilirubin, alanine aminotransferase were tested by automatic-biochemical analyser.3. Western-blot was performed to evaluate the content of matrix metalloproteinase-2and matrix metalloproteinase-9in liver.4.PCR was performed to evaluate the content of collagen type Ⅰ and Ⅲ in liver.5. Histopathological analysis:paraffin sections w ere cut to stained by H.E., Masson’s Trichrome, argyrophilic fiber.The degree of necrosis, fibrosis, cirrhosis were analyzed by Knodell, Ishak, Metavir. 6.Fluorescent immunostaining is performed observe the change of CD31in liver.7.Statistical analysis:use spss13.0to analyse results.Results1.The rat model both with cirrhosis and HCC was established successfully after14weeks inducing. At14th week, there were3rats checked with cirrhosis and HCC in5rats, and3rats in3at18th week. The carcinogenesis rate was75%, and overall mortality was33%.2.Body weight of Model rats were lighter than the control group after2weeks of medicine inducing, and the result had a statistical significance.3.In model group, ALT in serum began to increase at2th week after inducing, reached a peak at14th week, and decreased from18th week. After2weeks of inducing, comparision between the two groups was statistically significant, P<0.05.4. In model group, TBIL in serum began to increase at4th week after inducing, reached a peak at14th week, and decreased from18th week. After4weeks of inducing, comparision between the two groups was statistically significant, except of12th week, P<0.01.5. In model group, y-GGT in serum began to increase at2th week after inducing, reached a peak at12th week, then at a steady level. Comparision between the two groups was statistically significant after12weeks of inducing, P<0.01.6.In the model group, when rats were cirrhosis, collagen deposition in liver tissue was significantly increased, in the time, perisinusoidal collagen deposition together. When rats geted HCC, collagen deposition was reduced in the foci.7. CD31expression in rat hepatoma tissue is stronger than in the cirrhotic liver tissue.8. Changes of typeⅠ and type Ⅲ collagen deposition in tissue was consistent with overall collagen.9.The result was similar in analysis of liver collagen, either using SHG and TPEF or traditional pathological techniques.conclusion 1.The improved model is better for HCC research than traditional methods.2.In the progress of HCC in this improved model, collagen fiber deposition in cirrhotic liver tissue was significantly increased, and decreased in HCC tissue.The result indicates that excessive collagen fiber deposition may occurrence before HCC formation, collagen fiber of HCC is negatively correlated with the progress of the foci.3.There was expression of CD31both in cirrhosis and HCC, and expression of CD31in HCC is higher than in cirrhotic liver tissue. This result indicates that CD31expression in cirrhosis may be related with hepatic sinusoidal capillarization, and related with neovascularization in HCC.4.In the progress of cirrhosis, type Ⅰ, type Ⅲ, and all collagen increased significantly, then decreased in HCC. It was negative related with MMP. This result indicates thatchanges of type Ⅰ, type Ⅲ are consistent with overall collagen in HCC formation, and may be related with prognosis.5. With preliminary exploration, we think the technology of TPEF united SHG is consistent with traditional pathology, and may be more effective, and reduce the human impact factors. It may be a better tool for liver collagen analysis.Application value1.Improved HCC model is an ideal tool for human desease research, because it is better reproducible,precise control of medicine intake, short time to carcinogenesis,lower mortality and similar to human HCC.2.This experiment confirm the characteristics of collagen deposition, provide a reference for clinical diagnosis and prognosis.3.Demonstrate a new technology of SHG united TPEF which can be used in the liver collagen fiber analysis. Hope to promote the development of analytical techniques in liver tissue collagen. |
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