Objective To detect expression of Ras association domain family 1A （RASSF1A） gene in the colonic carcinoma tissue and to analyze the relationship of this expression to its clinical features. Methods Immunohistochemistry and Western blot methods were employed for detecting the RASSF1A protein expressions in 34 colonic carcinoma tissues and corresponding normal colon tissues. RT-PCR was employed for detecting RASSF1A mRNA expression. Results ①The RASSF1A protein expression in the colonic carcinoma tissues was significantly lower than that in the normal colontissues by using immunohistochemistry〔35.3% （12/34） versus 97.1% （33/34）， P＜0.05〕.There were significant relati-onships of RASSF1A protein expressions to the tumor differentiation and TNM stage （P＜0.05）， in other words， the positive rates of RASSF1A protein in the moderately and well differentiated andⅠ＋Ⅱof TNM colonic carcinoma tissues were all higher （P＜0.05）. ② The RASSF1A protein expression in the colonic carcinoma tissues was significantly lower than that in the normal colon tissues by using Western blot 〔0.316 8±0.019 6 versus 0.914 4±0.177 6， P＜0.05〕， which was close to the result of RT-PCR〔0.158 9±0.223 7 versus 0.572 3±0.193 9， P＜0.05〕. Conclusions Absentexpre-ssion of RASSF1A gene in the colonic carcinoma tissue might play an important role in tumor genesis and tumor progre-ssion， and it might become useful early detection of the colonic carcinoma.
Objective To research the expressions of miR-196b and HoxB8 mRNA in colorectal cancer and theircorrelation with clinicopathologic features，and to explore the relationship between miR-196b and HoxB8 in vivo. Methods Expressions of RNA （including miR-196b and HoxB8 mRNA） and HoxB8 protein were detected respectively by using quantitative real-time reverse transcriptase PCR and Western blot in 30 cases of colorectal cancer and corresponding normalmucous membrane tissues. Results In colorectal cancer tissues，expressions of miR-196b and HoxB8 mRNA were higher than those of the corresponding normal mucous membrane tissues （P＜0.05）. Expression of miR196b mRNA was assoc-iated with lymph node metastasis，neoplasm stages （Ⅰ＋ⅡandⅢ＋Ⅳ），and distant metastasis （P＜0.05），on the otherhand，no significant differences were observed regarding tumor site，size，gross type，depth of invasion，tissue differentiation，age，and sex （P＞0.05）. Expression of HoxB8 mRNA was no significant differences concerning lymph node metastasis，tumor stages （Ⅰ＋Ⅱ，Ⅲ＋Ⅳ），distant metastasis，tumor site，size，gross type，depth of invasion，tissue differentiation，age，and sex （P＞0.05）. The expression of miR-196b mRNA was negatively correlated with HoxB8 mRNA expression （r=-0.458，P＜0.05），and HoxB8 protein expression with no obvious correlation （r=-0.236，P＞0.05） in colorectal cancer tissues. Conclusions The expressions of miR-196b and HoxB8 mRNA in colorectal cancer tissues are higher，the high expression of miR-196b mRNA is related to the tumorigenesis and progression of colorectal cancer as well as correlated with prognosis in colorectal cancer. The miR-196b inhibits the expression of HoxB8 mRNA by binding to the3′-UTR of target HoxB8 mRNA.
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.
Objective To investigate the expression of protein gene product 9.5 (PGP9.5) in human gastric cancer, and to find out the relations between its expression and carcinogenesis, invasion and metastasis of gastric cancer. Methods The expression of PGP9.5 was detected by immunohistochemistry (SP) and Western blot in 80 samples of gastric cancer and 8 samples of normal gastric tissues. Results ①Of 80 gastric cancer specimens examined, 56 cases (70.0％) showed staining with PGP9.5 in most tumor cells, whereas no PGP9.5 expression was detected in normal epithelium, which was consistent with the results of Western blot. ②The results of immunohistochemistry revealed that there were significantly correlations between the expression of PGP9.5 and the depth of invasion, the degree of differentiation, and the occurrence of lymph node metastasis (Plt;0.05), respectively; yet, there were no relation between the expression of PGP9.5 and age, gender, histopathologic type and TNM stage (Pgt;0.05). Conclusion PGP9.5 may play an important role in the invasion and metastasis of gastric cancer, and the invasion of gastric cancer could be detected by PGP9.5, which may be a useful molecular marker.
Objective To discuss the changes of c-kit/scf mRNA and protein in guinea pig gallbladder fed on high cholesterol diet. Methods Twenty guinea pigs were divided into two equal groups of 10 each：the control group and lithogenic group. Normal diet and high cholesterol diet was given to each group respectively. The period of stone permeation was six weeks. RT-PCR and Western blot were used to determin the expressions of c-kit and scf mRNA and protein. Results RT-PCR results showed that the expressions of c-kit mRNA（t=6.985，P＜0.01） and scf mRNA （t=6.028， P＜0.01）decreased significantly in lithogenic group compared with the control group. Western blot results showed that the expressions of c-kit protein （t=10.256， P＜0.01） and scf protein （t=9.586， P＜0.01）decreased significantly in lithogenic group compared with the control group. Conclusions The expressions of c-kit/scf mRNA and protein decrease during the formation of cholesterol gallstones in guinea pigs fed on high cholesterol diet. Inhibition of c-kit/scf pathway may play a role in the formation of cholesterol gallstones.
Objective To explore the expression and function of NDRG2 gene in human primary hepatocellular carcinoma and normal hepatic tissues. Methods The immunohistochemical ABC method， Western blot， and Real-time PCR were used to investigate the expression and content of NDRG2 in human hepatocellular carcinoma and hepatic normal biopsies. Results The NDRG2 protein located in cytoplasm. The positive rate was 16.67%（5/30） and 100%（30/30） in hepatocellular carcinoma and normal hepatic tissues, respectively. The relative content of NDRG2 protein in hepatocellular carcinoma and normal hepatic tissues were 0.029 0±0.005 9 and 0.109 2±0.002 8. There were significant differences between human hepatocellular carcinoma and hepatic normal biopsies both in staining positive rates and relative content（P＜0.05）. The Western blot also agreed with the result，the expression level of NDRG2 protein in hepatocellular carcinoma and normal hepatic tissues was 1.13±0.15 and 1.57±0.18， respectively, there was significant difference（P＜0.05）. Also, compared with normal hepatic tissues, the expression level of NDRG2 mRNA in carcinoma tissues was reduced significantly (0.89±0.15 vs. 1.48±0.17, P＜0.05). However, there were no significant differences in NDRG2mRNA expression between Edmondson-Steiner grades. Conclusions There possibly have difference in NDRG2 expression between human primary hepatocellular carcinoma and normal hepatic tissue. NDRG2 gene may take part in the pathogenesis of human primary hepatocellular carcinoma. Futher study will be needed to study its mechanism and function.
【Abstract】ObjectiveTo eliminate the interference of CEA-related substances in CEA measurement and increase the specificity of CEA in the detection of malignant digestive diseases. MethodsCEA level of peripheral blood and digestive juice (bile, gastric juice) from patients with benign or malignant digestive diseases was measured by ELISA, and semi-dry electrophoretic transfer method of Western blot technique to distinguish CEA and CEA-related substances. ResultsIn malignant diseases, the CEA level of digestive juice was significantly higher than that in the blood, and there was no difference of CEA level in digestive juice and blood in benign diseases. Meanwhile, the CEA level of digestive juice and blood in malignant diseases were significantly higher than that in benign diseases. A specific band (molecular weight about 210×103) was detected in all malignant diseases except four cases whose CEA level was too low (less than 5 μg/L), whereas no one of benign diseases had this specific band no matter how high or low the CEA level was. ConclusionThe specificity of CEA detection in malignant digestive diseases can be improved by using digestive juice as sample and combining with Western blot technique.
To obtain recombinant human β2-microglobulin (rhβ2M) with properties of good solubility and high purity from E.coli, prokaryotic expression conditions were optimized and protein purification was performed in this study. After testing the effect of different IPTG concentrations, temperatures and induction times on the production of rhβ2M, the optimum expression conditions were determined, i.e. joining IPTG to final concentration being 0.8 mmol/L and inducing time 6 h and at temperature of 25℃. Under the optimum induction conditions, the ratio of soluble rhβ2M to soluble bacterial protein was 63.7%. After purified by Ni Sepharose 6 Fast Flow, the purity of rhβ2M achieved a greater value of 95%. Western blot analysis revealed that rhβ2M possessed the antigen property that specifically interacted with anti-β2M antibody.