| Liver fibrosis is a necessarily stage in the development of chronic liver disease to cirrhosis, early diagnosis and real-time intervention is the key to the treatment of liver fibrosis. Although liver biopsy is the current gold standard for fibrosis assessment, it has some risks and limitations, including intra-observer and interobserver variation, sampling error and variability. The limitations of liver biopsy led to the search for non-invasive tests to assess liver fibrosis. At present, non-invasive diagnostic methods for liver fobrosis include non-invasive diagnostic models for liver fibrosis and biomarker molecular targeting diagnostic techniques, both of them has become a global research hotspot.The aim of our study is to evaluate the noninvasive diagnostic models for predicting liver fibrosis in patients with chronic hepatitis B and provide reference for clinical diagnosis and therapy; to investigate the expression ofαvβ3 integrin in human HSC and liver tissue of patients with chronic hepatitis B, and to demonstrate the relationship betweenαvβ3 integrin and liver fibrosis, to explore the possibilities of avP3 integrin as the biomarker of liver fibrosis progression.This study includes two parts:evaluation of noninvasive diagnostic models of liver fibrosis in patients with chronic hepatitis B; expression ofαvβ3 integrin in human HSC and expression ofαvβ3 integrin,α-smooth muscle actin (α-SMA) and CD31 in fibrotic liver tissue of patients with chronic hepatitis B.Part one Evaluation of noninvasive diagnostic models of liver fibrosis in patients with chronic hepatitis BBackground:In recent years, many studies and great interest have been dedicated to the development of non-invasive diagnostic models to substitute a liver biopsy for fibrosis assessment, but most of them are based on patients with chronic hepatitis C and have not been verified effective in patients with chronic hepatitis B in China.Objective:To evaluate the noninvasive diagnostic models for predicting liver fibrosis in patients with chronic hepatitis B and provide reference for clinic diagnosis and therapy.Methods:112 chronic hepatitis B patients were enrolled, liver biopsy were proceeded to assess the stage of fibrosis histologically, serum tests were measured simultaneously. Sensitivity, specificity, PPV, NPV and ROC curves were used to analyze the diagnostic value in models of APRI index,Forns index,S index,APAG index and Fibroindex.Results:Each model performed well in predicting fibrosis. APAG index and S index showed best among these models. The areas under ROC curve were 0.858, 0.891 and 0.930 for predicting significant fibrosis, advanced fibrosis and cirrhosis in APAG index models. Using a cutoff score of<0.27, the sensitivity, specificity and negative predictive value for exclusion of significant fibrosis was 98%,24% and 92%, meanwhile, applying a cutoff score of>0.8, the sensitivity, specificity and negative predictive value for diagnosis of significant fibrosis was 64%,91% and 90%.45.5% patients could be identified accurately. The areas under ROC curve were 0.837,0.847 and 0.866 for predicting significant fibrosis, advanced fibrosis and cirrhosis in S index models. Using a cutoff score of<0.1, the sensitivity, specificity and negative predictive value for exclusion of significant fibrosis was 94%,47% and 85%, similarly, applying a cutoff score of>0.5, the sensitivity, specificity and negative predictive value for diagnosis of significant fibrosis was 59%,91% and 90%.52.7% patients could be identified accurately.Conclusion:Noninvasive diagnostic models of liver fibrosis could predict significant fibrosis with high accuracy, APAG index and S index model was the most convenient and effective among them. The need for liver biopsy could be reduced in some patients with chronic hepatitis B.Part two Expression ofαvβ3 integrin in human HSC and expression ofαvβ3 integrin,α-SMA and CD31 in patients with chronic hepatitis BBackground:The activation and proliferation of hepatic stellate cell (HSC), the accumulation of extracellular matrix and angiogenesis is significant pathological characteristics of advance liver fibrosis. One family of adhesion molecules, integrins, mediate the structural and functional interactions of epithelial and mesenchymal cells with the extracellular matrix and transmit signals in both inside-out and outside-in directions. Among them, theαvβ3 subtype is an extensive extracellular matrix receptor that mediates cell events involved in proliferation, migration, activation, survival and apoptosis. It has been showed that expression ofαvβ3 integrin is weekly around the portal vein in a normal liver, with the liver parenchyma cells expressing even lessαvβ3 integrin. Its expression is up-regulated apparently in the activation of HSC and angiogenesis.αvβ3 integrin is up-regulated in rats liver fibrotic tissue. The overexpression involved in the activation of HSC and angiogenesis correspond with fibrotic remodeling. But the expression ofαvβ3 integrin and angiogenesis is not clear in patients with chronic hepatitis B liver fibrosis.Objective:To investigate the expression ofαvβ3 integrin and a-SMA in human HSC and the expression of avP3 integrin, a-SMA and CD31 in different stage of liver fibrosis in patients with chronic hepatitis B, to explore the possibilities of avP3 integrin as the biomarker of liver fibrosis progression.Methods:Human HSC line LX-2 were cultured and Western Bloting were detected for the expression ofαvβ3 integrin and a-SMA in human HSC. The expression ofαvβ3 integrin and a-SMA in human HSC were also identified by immunofluorescence staining.112 chronic hepatitis B patients were enrolled, liver biopsy were proceeded to assess the stage of fibrosis histologically. The expression ofαvβ3 integrin,α-SMA and CD31 in different stage of liver fibrosis were identified simultaneously by immunohistochemical staining.Results:Immunofluorescence staining showed that avP3 integrin expressed mostly on the membrane of activated HSC coinciding with the cell contour, while a-SMA expressed on the cytoplasma. Detection ofαvβ3 integrin andα-SMA in cell lysates by Western blotting showed that both of them were detectable in the activated human HSC. The expression ofαvβ3 integrin,α-SMA and CD31 were extended significantly along with the degree of liver fibrosis. The expression of CD31 was extended in the endothelial cells and portal area, and new blood vessels were seen in the fibrotic septum and hyperplastic bile ducts. Moreover, a close positive statistical correlation was observed between avP3 integrin,α-SMA, CD31 and the degree of liver fibrosis (P< 0.01). The expression ofαvβ3 integrin, a-SMA and CD31 were up-regulated in significant liver fibrosis (S2-4) (P<0.001). Immunofluorescence staining showed that there was an overlapping ofαvβ3 integrin andα-SMA expression.Conclusion:The close relationship betweenαvβ3 integrin andα-SMA on the cell structure suggested that the expression ofαvβ3 integrin was relevant to the HSC activation. Up-regulation ofαvβ3 integrin in the progression of liver fibrosis patients with chronic hepatitis B was closely related to HSC activation and angiogenesis,αvβ3 integrin can be used to identify whether there were significant liver fibrosis, which may be a biomarker of liver fibrosis progression.
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