Diagnostic Value of Nanomagicbeabs Sorting-time Resolved Fluoroimmuno Assay for Serum Calgizzar in Pancreatic Carcinoma / 现代检验医学杂志

Objective To detect the serum levels of calpainin (S100A11) using nanomagicbeabs sorting-time resolved fluoroimmuno assay (NMBS-TRFIA) and evaluate its diagnostic value in pancreatic carcinoma.Methods 88 patients with pancreatic carcinoma,50 patients with acute pancreatitis,10 patients with pancreatic cyst and 20 healthy controls were selected as the study subjects.The human peripheral serum blood was sorted with S100A11 antibody coupled nanomagicbeabs,and the concentration of S100A11 was detected by TRFIA method.The area under the receiver operating characteristic (ROC) curve (AUC) was used to determine the cut-off level for diagnosis of pancreatic carcinoma,in order to evaluate the sensitivity and specificity for diagnosis of pancreatic carcinoma.Results S100A11 showed a linear relationship within the range of 6.08～ 500 ng/ml using NMBS-TRFIA method,intraassay CV≤6.35％,inter-assay CV≤7.12％,and the average recovery rate was 104.7％.The serum levels of S100A11 in patients with pancreatic carcinoma,patients with acute pancreatitis and patients with pancreatic cyst were 185.53 ± 161.19,106.06±113.83 and 68.99± 47.83 ng/ml respectively.Compared with the normal control group (37.98±25.14 ng/ml),the differences were statistically significant (t=-8.065,-3.375,-2.266,all P ＜0.01).The serum levels of S100A11 in patients with pancreatic carcinoma was significantly higher than those in patients with acute pancreatitis and patients with pancreatic cyst (all P＜0.05).According to the ROC curve,ROCAUC=0.985 (95％ CI:0.972 ～ 0.997),the best cut-off level for the diagnosis of pancreatic carcinoma was 89.5 ng/ml (sensitivity 81.8 ％,specificity 67.5 ％).Conclusion NMBS-TRFIA can enrich S100A11 in serum and improve the detection sensitivity of serum S100A11,and the method is simple and easy to be popularized.Serum S100A11 has a high sensitivity and specificity in the diagnosis of pancreatic carcinoma,and is a new serum marker for the diagnosis of early pancreatic carcinoma.

Serum and tissue expressions of galectin-3 in hepatocellular carcinoma and the clinical significances / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To study the expression of Galectin-3 in human hepatocellular carcinoma (HCC) tissues and the clinical value of serum Galectin-3 in the diagnosis of hepatocellular carcinoma.</p><p><b>METHODS</b>Immunohistochemistry method was used to detect the expression of Galectin-3 in the 46 pairs of HCC tissues and their para cancerous tissues. The relationship between expression levels of Galectin-3 and clinical parameters was analyzed. Serum Galectin-3 in different liver diseases were measured with ELISA. The sensitivity and specificity of galectin-3, alpha fetoprotein (AFP) and gamma-glutamyltranspeptidase II (GGT-II) for diagnosis of HCC were compared and the complementary diagnostic values of Galectin-3 and AFP and GGT-II for HCC were studied.</p><p><b>RESULTS</b>(1) The positive rate of Galectin-3 in the tissue of HCC was 78.2%, dramatically higher than that in para cancerous tissues (15.2%) (P is less than 0.01). The expression levels were correlated with differentiation and with the high expression in poor differentiation tissues; (2) Based on ROC curve, the cut-off of serum Galectin-3 for HCC diagnosis was set as 0.62mug/L, the serum galectin-3 positive rate was 64.5% in HCC cases, which was apparently higher than that in liver cirrhosis, chronic hepatitis and healthy persons (P is less than 0.05); (3) Serum Galectin-3 was not correlated with AFP and GGT-II. Combined determination of the three markers had the complementary diagnostic value for HCC and might increase the diagnostic sensitivity to 94.7%.</p><p><b>CONCLUSION</b>Galectin-3 is overexpressed in HCC tissues and is correlated with the tumor differentiation, suggesting that Galectin-3 may be associated with the carcinogenesis and development of HCC. Serum galectin-3 increases in the HCC cases and combined determination of serum Galectin-3, AFP and GGT-II can increase the diagnostic efficiency for HCC. Galectin-3 could be a novel serum tumor marker for HCC.</p>

Effects of decreased leptin expression on liver fibrosis / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To study the effects of decreased leptin expression on liver fibrosis.</p><p><b>METHODS</b>The small interfering RNA, targeting leptin gene, was designed according to the secondary structure of leptin gene. The recombinant plasmids were encapsulated with lipofectamine and then injected into carbon tetrachloride (CCl4) induced rat liver fibrosis models. Leptin and I, III collage were detected by immunohistochemistry and reverse transcription polymerase chain reaction (RT-PCR).</p><p><b>RESULTS</b>The mRNA and protein levels of leptin in the fibrotic liver transfected with leptin shRNA were significantly decreased compared with those in controls (P less than 0.01). The depositions of type I and type III collagens were also decreased (P less than 0.01).</p><p><b>CONCLUSION</b>Decreased leptin expression prevents liver fibrosis.</p>

Effects of antisense RNA of connective tissue growth factor expressing plasmid on rat liver fibrosis / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To observe the effects of antisense RNA of connective tissue growth factor (CTGF) on rat liver fibrosis.</p><p><b>METHODS</b>Gene recombinant techniques were used to construct a rat antisense RNA of CTGF recombinant plasmid which could be expressed in eukaryotic cells. The recombinant plasmids were encapsulated with lipofectamine and then transducted into a carbon tetrachloride (CCl4) induced rat liver fibrosis model. Expression of CTGF was assessed by RT-PCR, Western blot and immunohistochemistry. Immunohistochemistry was used to identify type I and III collagens. HE stained liver slides were used for pathological study.</p><p><b>RESULTS</b>The mRNA and protein expression of CTGF in the fibrotic liver transfected with antisense-CTGF were significantly decreased compared with those of the controls (P<0.01). The depositions of type I and type III collagens were also decreased (P<0.05). Antisense-CTGF also minimized the pathological fibrosis in the rat livers (P<0.01).</p><p><b>CONCLUSION</b>The results demonstrate that the antisense RNA of CTGF recombinant plasmid has certain effects in preventing liver fibrosis and makes it a possible candidate for use in future gene therapy.</p>

Effects of ribozyme targeting platelet-derived growth factor receptor beta subunit gene on the proliferation and apoptosis of hepatic stellate cells in vitro / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Activation and proliferation of hepatic stellate cells (HSC) is essentially involved in the development and progression of hepatic fibrosis. The most potent growth factor for HSC is platelet-derived growth factor receptor (PDGF) and PDGF receptor beta subunit (PDGFR-beta) is the predominant signal transduction pathway of PDGF which is overexpressed in activated HSC. This study investigated the cleavage activity of hammerhead ribozyme targeting PDGFR-beta mRNA in HSC and the effect on biological characteristics of HSC.</p><p><b>METHODS</b>Expression vector of anti-PDGFR-beta ribozyme was constructed and transfected into rat activated HSC with lipofectamin. The positive cell clones were gained by G418 selection. The expression of PDGFR-beta, alpha-smooth muscle actin, and typeI and type III collagen were detected by using Northern blot, Western blot and immunocytochemical staining, respectively. The cell proliferation was determined with MTT colorimetric assay. The cell apoptosis was analyzed by using flow cytometry, acridine orange fluorescence vital staining and transmission electron microscopy.</p><p><b>RESULTS</b>The expression of PDGFR-beta at mRNA and protein level was markedly reduced in ribozyme-transfected HSC by 49% - 57% (P < 0.05 - 0.01). The proliferation and alpha-smooth muscle actin expression of ribozyme-transfected HSC were significantly decreased (P < 0.05 - 0.01), and the type I and type III collagen synthesis were also reduced (P < 0.01). In addition, the proliferative response of ribozyme-transfected HSC to PDGF BB was significantly inhibited. Otherwise, the apoptotic cells were significantly increased in ribozyme-transfected HSC (P < 0.01), and typical apoptotic cells could be found under transmission electron microscopy.</p><p><b>CONCLUSIONS</b>The anti-PDGFR-beta ribozyme effectively cleaved the target RNA and significantly inhibited its expression, which blocked the signal transduction of PDGF at receptor level, inhibited HSC proliferation and collagen synthesis, and induced HSC apoptosis. These results suggest that inhibiting PDGFR-beta expression of HSC may be a new target for the therapy of liver fibrogenesis, and ribozyme may be a useful tool for inhibiting PDGFR-beta expression.</p>

The expression of platelet-derived growth factor (PDGF) receptor-beta and its correlation with extracellular matrix in hepatic tissue in hepatic fibrosis rats / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To investigate the expression of PDGF receptor-beta and its correlation with extracellular matrix in hepatic tissue during hepatic fibrosis.</p><p><b>METHODS</b>The model of hepatic fibrosis in rats was induced by carbon tetrachloride. PDGF receptor-beta subunit, collagen I, collagen III and a-SMA in hepatic tissues of these rats were examined using immunohistochemistry. The correlation between PDGF receptor-beta subunit and collagen I, III was analyzed using SAS software after the results of immunohistochemistry were semi-quantified.</p><p><b>RESULTS</b>PDGF receptor-beta subunit and a-SMA were not detected in normal controls. Collagen I and III were distributed in the portal tracts and beneath the endothelia of the central veins and of the Disse spaces. Two weeks after CCl4 injection, the PDGF receptor-beta and a-SMA were detected, and the expression of collagen I and III increased. At the end of 4 and 6 weeks, the above four proteins were further increased. Two weeks after CCl4 injection, PDGF receptor-beta had no apparent correlation with collagen I and III. However, PDGF receptor-beta had a significant correlation with collagen I and III 2 weeks later, and the correlation coefficient was 0.74 and 0.60 respectively at 4 weeks, and 0.83 and 0.67 respectively at 6 weeks. PDGF receptor-beta had a significant correlation with a-SMA during the whole process of hepatic fibrosis and the correlation coefficient was 0.62, 0.69 and 0.81, respectively at the time of 2, 4 and 6 weeks after CCl4 injection.</p><p><b>CONCLUSION</b>The PDGF receptor-beta was overexpressed during the process of hepatic fibrosis development, and it significantly correlated with collagen I and collagen III.</p>

Effect of ribozyme against platelet-derived growth factor receptor beta subunit mRNA on the biological characters of hepatic stellate cells / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To study the cleavage activity of hammerhead ribozyme targeting at platelet-derived growth factor receptor beta subunit (PDGFR- beta) mRNA in hepatic stellate cells (HSCs) and its effect on the biological characters of HSCs.</p><p><b>METHODS</b>Expression vector of anti-PDGFR- beta ribozyme was constructed and transfected into rat-derived HSC-T6 cells with lipofectin. The positive cell clones were gained by G418 selection. The expression of PDGFR- beta, alpha-smooth muscle actin (alpha-SMA), and type I and type III collagen was detected by means of northern blot, Western blot and immunocytochemical staining respectively. The cell proliferation was determined with MTT colorimetric assay. The cell apoptosis was demonstrated with flow cytometry, acridine orange fluorescence vital staining and transmission electron microscopy.</p><p><b>RESULTS</b>The expression of PDGFR- beta at mRNA and protein level was markedly reduced in ribozyme-transfected HSCs only 43% to 51% of that in control cells (t > or = 3.957, P < 0.05), and alpha-SMA expression level, type I and type III collagen synthesis ability were also reduced (t > or = 6.790, P < 0.01). The proliferation of ribozyme-transfected HSCs was significantly decreased (t > or = 3.858, P < 0.05), and the proliferation response to PDGF BB was markedly inhibited. However the apoptotic rate was significantly increased in ribozyme-transfected HSCs (chi2 > or = 14.157, P < 0.01), and typical apoptotic cells could be found under transmission electron microscopy.</p><p><b>CONCLUSIONS</b>The anti-PDGFR- beta ribozyme can be expressed stably in HSCs, cleave the target RNA effectively, inhibit HSCs proliferation and collagen synthesis, and induce HSC apoptosis. The results suggest that inhibiting PDGFR- beta expression in HSCs may be a new therapy for liver fibrosis.</p>