Objective To investigate the prognostic value of epithelial-mesenchymal transition （EMT） related proteins （Snail， E-cadherin， and N-cadherin） in gastric cancer and its relationship with tumor initiating cells （TICs） marker （CD133）. Methods The expressions of EMT-related proteins and CD133 protein in the gastric cancer tissues and normal gastric mucosa tissues adjacent to gastric cancer were detected by Western blot method. The relations between the expressions of EMT-related factors proteins and CD133 protein and the clinicopathologic characters were analyzed. The correlations between EMT-related factors and CD133 were analyzed by Spearman. The correlations between EMT-related factors expressions and CD133 expression and survival were analyzed by Kaplan-Meier method and Log-rank test. Results ① The protein expression levels of Snail， N-cadherin， and CD133 in the gastric cancer tissues were significantly higher than those in the normal gastric mucosa tissues adjacent to gastric cancer （Snail：0.599±0.114 versus 0.259±0.108， P=0.020；N-cadherin：0.754±0.154 versus 0.329±0.134， P=0.001；CD133：0.635±0.119 versus 0.485±0.116， P=0.029）， while the protein expression level of E-cadherin was lower than that in the normal gastric mucosa tissues adjacent to gastric cancer （0.378±0.123 versus 0.752±0.156， P=0.003）.② The expression levels of Snail and N-cadherin in the gastric cancer patients with vascular invasion， lymphatic vessel invasion，N3 lymph node metastasis， diameter more than 5 cm， and Ⅲ＋Ⅳ staging were significantly higher than those in the patients without vascular invasion， lymphatic vessel invasion， N0-N2 lymph node metastasis， diameter less than 5 cm， andⅠ＋Ⅱ staging（P＜0.05）， while E-cadherin protein expression was lower than that in the patients without vascular invasion， lymphatic vessel invasion， N0-N2 lymph nodes metastasis， andⅠ＋Ⅱstaging （P＜0.05）. The expression levels of CD133 in the gastric cancer patients with lymphatic vessel invasion， diameter more than 5 cm， and Ⅲ＋Ⅳ staging were significantly higher than those in the patients without lymphatic vessel invasion， diameter less than 5 cm， andⅠ＋Ⅱ staging （P＜0.05）. ③The Snail and N-cadherin protein expressions were significantly positive correlated with CD133 protein expression， respectively （rs=0.278， P=0.048；rs=0.406， P=0.003）， while E-cadherin protein expression was significantly negative correlated with CD133 protein expression （rs=-0.504， P=0.000）.④ The survival time in the patients with lower expressions of Snail， N-cadherin， and CD133 were significantly longer than those in the patients with higher expressions of Snail， N-cadherin， and CD133 （P＜0.05）. The combination of Snail， N-cadherin， E-cadherin， and CD133 could effectively predict survival. Conclusions There is a significant correlation between EMT and gastric cancer TICs， and which are correlated with aggressive clinicopathologic features of gastric cancer. The combination of Snail， E-cadherin， N-cadherin， and CD133 may be effectively predict the prognosis of gastric cancer patients.
ObjectiveTo investigate the early diagnostic value of transforming growth factor-β1(TGF-β1) on acute rejection after liver transplantation in rhesus by detecting the expression of TGF-β1 in the liver tissue. MethodsLiver transplantation models in rhesus were constructed by the improved vascular dual cuff, supporting tube of biliary tract, and artery anastomosis method.The successful models were randomly divided into experimental group (no immunosuppressant treatment in perioperative period) and control group (treated by immunosuppressant in perioperative period).Then the blood samples and liver tissues were collected at 6, 12, 24, and 72 hours after surgery.Allograft rejections of liver tissue after liver transplantation were monitored by liver function test, hematoxylin-eosin staining and Banff score.Finally, the expression level of TGF-β1 was detected by Western blot analysis or immunohistochemistry technique. Results①The acute rejection happened in all the rhesus at 12 h, 24 h and 72 h after liver transplantation, especially at 72 h after liver transplantation in the experimental group, the Banff grade levels of acute rejection in the liver tissue was more severe than that in the control group (P < 0.05).②The levels of ALT, AST, and TBIL after liver transplantation was gradually increased, which were similar at 6 h and 12 h after transplantation between the two groups, but which at 24 h and 72 h after transplantation in the experimental group were significantly higher than those in the control group (P < 0.05).③The results of TGF-β1 protein expression using immunohistochemical detection:The percentage of positive area of TGF-β1 of liver tissue at 12 h in the experimental group was significantly higher than that in the control group (P < 0.05).With the extension of time, it was gradually increased and significantly higher than that in the control group at 24 h or 72 h (P < 0.05).④The semi-quantitative results of TGF-β1 protein expression using Western blot detection:The TGF-β1 protein expressions began to increase at 6 h after liver transplantation in the experimental group and the control group, and the magnitude of increase was more obvious in the experimental group.The TGF-β1 protein expressions at different time (6 h, 12 h, 24 h, and 72 h) in the experimental group were significantly higher than those in the control group (P value was 0.003, 0.001, 0.001, and 0.001, respectively). ConclusionsThe elevated level of TGF-β1 of liver tissue after liver transplantation might suggest the enhanced cellular immune function, it might have certain significance for early diagnosis of acute rejection after liver transplantation.
ObjectiveTo construct eukaryotic expression vector of pEGFP-N3-TFPI-2, and to provide the base of studying the function of TFPI-2 gene. MethodsExtraction of total RNA from placental tissue was extracted at first, and then reverse transcriptase synthesis of cDNA was carried out. The cDNA fragment of TFPI-2 gene which was obtained by real time PCR (RT-PCR) was inserted into eukaryotic expression vector of pEGFP-N3. After double digestion with XhoⅠand KpnⅠ, the recombinant vector of pEGFP-N3-TFPI-2 was identified in 1% agarose gel electrophoresis and was tested by the sequence analysis. Then, the recombinant vector of pEGFP-N3-TFPI-2 (transfection group) and vector of pEGFP-N3 (blank control group) were transfected into Top10 competent cells with LipofectamineTM 2000, but no transfection-related treatment was performed in cells of untransfection group. Western blot method was used to test the expression of TFPI-2 protein in cells of 3 groups. ResultsThe purity of total RNA which were analysis by agarose gel electrophoresis and spectrophotometry were fit for PCR. After coding of TFPI-2 gene fragment and eukaryotic expression vector of pEGFP-N3, the recombinant plasmid of pEGFP-N3-TFPI-2 were got double digestion with XhoⅠand KpnⅠ, and was identified in 1% agarose gel electrophoresis, of which showing that there were 2 specific amplification of strips at 708 bp and 4 700 bp. Result of sequence analysis confirmed that the size of recombinant vector was consistent with the theoretical value. Results of Western blot showed that the expression of TFPI-2 protein in transfection group (0.657 3±0.032 5) was higher than those of blank control group (0.301 7±0.028 7) and untransfection group (0.314 3±0.026 6), P < 0.01. ConclusionsThe eukaryotic expression vector of pEGFP-N3-TFPI-2 has been constructed successfully, which laiding the foundation for the analysis about function of TFPI-2 gene.