Objective To investigate the transfection and expression of recombinant plasmid human vascular endothelial growth factor 165/pcDNA3. 1 （hVEGF165/pcDNA3. 1） in myocardial cells, and to build foundation for gene therapy and cell therapy of coronary artery disease （CAD）. Methods Myocardial cells were cultured in vitro and transfected by hVEGF165/pcDNA3.1 with liposome; then transient expressed protein was detected by reverse transcriptase-polymerase chain reaction （RT-PCR）, immunochemistry and Western blotting. Results A strap as hVEGF165 was obtained by RT-PCR, the protein of hVEGF165 was found in myocardial cells by immunochemistry and in supernatant by Western blotting. Conclusion The recombinant plasmid hVEGFI65/pcDNA3. 1 can be expressed in myocardial cells, and may be used in studying CAD by gene therapy and cell transplantation.
Objective To investigate the detection of peritoneal free cancer cells and its clinical significance. Methods The peritoneal free cancer cells, the positive rates of CK20 protein and CK20 mRNA expressions of peritoneal lavage fluid were detected by peritoneal lavage cytology (PLC), flow cytometry (FCM) and real-time fluorescent quantitative RT-PCR in 50 cases of gastric cancer patients, respectively. The sensitivity of three kinds of detection method to peritoneal free cancer cells was compared. Results The positive rates of peritoneal free cancer cells, CK20 protein and mRNA expression of peritoneal lavage fluid were 20.0% (10/50), 36.0% (18/50) and 58.0% (29/50), respectively. The positive rate of CK20 mRNA expression detected by real-time fluorescencequantitative RT-PCR in peritoneal lavage fluid was significantly higher than those of the CK20 protein expression detected by FCM and peritoneal free cancer cells detected by PLC (Plt;0.05 or Plt;0.001). The difference of positive rate of CK20 protein expression and peritoneal free cancer cells was not significant (Pgt;0.05). The positive rate of CK20 mRNA expression of peritoneal lavage fluid was related to the tumor invasion depth, differentiation degree, TNM stage, and lymph node metastasis (Plt;0.05). Conclusion Real-time fluorescence quantitative RT-PCR is an effective method for the detection of peritoneal free cancer cells.
ObjectiveTo establish a method that can eliminate the pollution of endogenous nucleic acid in the real-time quantitative polymerase chain reaction (PCR) reaction system, which can be used to reduce or eliminate the false positive rate of real-time PCR assay in detection of postoperative intracranial bacteria infection.MethodsAt first, eliminated the pollution of endogenous nucleic acid in the real-time PCR reaction system. Then, with mixed bacteria DNA as a template, multiple PCR was used to specifically identify the gram-negative bacteria. Meanwhile, evaluated the text line and sensitivity of the multiple PCR after eliminating pollution in detecting the DNA of the mixed bacteria.ResultsThe method established could quickly eliminate the pollution of endogenous nucleic acid in the real-time PCR reaction system, and it didn’t affect the Taq enzyme activity and the amplification efficiency in PCR system, with the minimum detection limit of 102 CFU/mL (Staphylococcus aureus and Pseudomonas aeruginosa), which was the same to the culture method. The enzyme cutting method had no significant effect on the activity and amplification efficiency of the enzyme in PCR system, It had no effect on PCR reaction system and primer specificity (Ct=32, ΔRn=200). However, the filtration method significantly reduced the PCR amplification efficiency (Ct=32, ΔRn=150).ConclusionsThis method can easily and rapidly eliminate the pollution of endogenous nucleic acid in the real-time PCR reaction system, and greatly reduce the false positive of PCR detection. It is able to timely and accurately diagnose the intracranial bacteria infection, which is significant for clinical testing.
【 Abstract 】 Objective To study the mRNA expression of BC047440 gene in multiplicate malignant tumor tissues and the corresponding adjacent tissues, and to investigate its roles in the carcinogenesis and development of malignant tumors. Methods Forty-eight cases of malignant tumor tissues and their adjacent non-cancerous tissues were examined. The mRNA expression of BC047440 gene in those tissues of liver cancer, cholangiocarcinoma, gastric cancer, carcinoma of large intestine, glioma, and breast cancer were measured by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR). Results ① The mRNA expressions of BC047440 gene in liver cancer, gastric cancer, cholangiocarcinoma and carcinoma of large intestine were significantly higher than those in their adjacent non-cancerous tissues (Plt;0.05 or 0.01). BC047440 gene were highly expressed in both glioma and its adjacent tissues (Pgt;0.05), and poorly expressed in both breast cancer and its adjacent tissues (Pgt;0.05). ② There were close relationships between BC047440 gene expression and clinicopathologic findings of liver cancer, including tumor size and portal vein invasion (Plt;0.05). ③ There were also close relationships between BC047440 gene expression and different clinical stages in alimentary canal cancers (Plt;0.05). Conclusion The over expression of BC047440 gene may be related with the growth, infiltration and metastasis of some malignant tumors, including liver cancer, cholangiocarcinoma, gastric cancer, carcinoma of large intestine and glioma.
Objective To investigate the expression of ADAM9 in breast cancer and its clinical significance. Methods The expressions of ADAM9 in normal breast tissues and breast cancer tissues were detected by reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemistry, and whose relationship with clinicopathologic features was analyzed. Results The expression of ADAM9 mRNA increased in the breast cancer tissues, but which was not detected in the normal breast tissues. The expression of ADAM9 protein in the breast cancer tissues was significantly higher than that in the normal breast tissues (Plt;0.05), and which in the metastatic lymph nodes was significantly higher than that in the negative lymph nodes or corresponding primary lesions (Plt;0.05). The expression of ADAM9 in the breast cancer tissues was correlated with the lymph node metastasis and histological grade (Plt;0.05). Conclusion ADAM9 is overexpressed in the breast cancer tissues, which might involve in the pathological progression of breast cancer.
【Abstract】Objective To investigate whether liver resection for hepatocellular carcinoma (HCC) causes dissemination of liver tumor cells into blood circulation. Methods Fourteen patients with HCC, but without evidences of metastasis, were enrolled for the study. Blood samples of peripheral blood before skin incision and after abdominal wall suture, and of hepatic venous blood and portal venous blood after liver parenchyma dissection, were obtained. AFPmRNA was detected by reverse transcription polymerase chain reaction assays, the change of the level of its expression during operation was assessed by semi-quantitative analysis. Results The rate of its expression before and after operation in peripheral blood, and during operation in portal venous blood and in hepatic venous was 42.9%, 35.7%, 42.9% and 57.1% respectively. There were no differences between them. However, the level of its expression in hepatic venous blood was significantly higher than others (P＜0.05). Conclusion Liver resection for HCC induces releases of cells from the liver, probably including tumor cells, into blood circulation.
【Abstract】Objective To explore the changes of expression of AFP mRNA in human hepatocellular carcinoma （HCC） tissues after oral Xeloda therapy.Methods Total RNA was extracted from HCC tissue samples collect after operation and nested reverse transcription polymerase chain reaction （RT-nested PCR） assay was performed to determine the expression of AFP mRNA in this study.Results The final product of AFP mRNA amplified by RT-PCR was 174 bp and by RT-nested PCR was 101 bp. The AFP mRNA is positive in 12 of 21 patients (positive rate 57.14%) amplified by RT-nested PCR assay in Xeloda treatment group which is much lower than control group: 18 of 20 patients (positive rate 90.00%),P＜0.05.The serum AFP value of Xeloda treatment group 〔(23.2±12.8) μg/L〕 is much lower than that of control group 〔(39.6±24.3) μg/L〕 four weeks after operation (P＜0.05). However, There was no difference between two groups in serum AFP value before operation.Conclusion Xeloda can effectively suppress the expression of AFP mRNA in human HCC tissues and lower it’s product serum AFP value.The clinical application of Xeloda in HCC patients deserve further study.
We have devised a highly sensitive, specific, and quantitative assay for multidrug resistance (mdr1) mRNA expression based on the reverse transcription-polymerase chain reaction (RT-PCR). mdr1 mRNA levels were detected in 30 human primary hepatocellular carcinoma (PHC) tissue and adjacent liver tissue. Five of the patients had received chemotherapy before hepatectomy. The results show that the level of expression of mdr1 gene is higher in tumor tissue than in adjacent liver tissue. mdr1 gene is overexpressed in PHC after chemotherapy. Furthermore, mdr1 gene expression in the treated tumor adjacent liver tissue is higher than that in untreated tumor adjacent liver tissue. Our results indicated that overexpression of mdr1 gene may be responsible for the intrinsic and acquired drug resistance of PHC.
【Abstract】Objective To investigate the expression of the mRNA of cancer-testis antigen 9 (CT9) gene in hepatocellular carcinoma. Methods The expression of CT9 mRNA was detected through RT-PCR in HCC tissues and their adjacent non-HCC tissues from 45 HCC patients. From CT9 RT-PCR positive products, 3 samples were selected randomly and were sequenced. ResultsCT9 mRNA was detectable in 51.1%(23/45) of HCC samples, and no expression of CT9 mRNA was detected in the adjacent non-HCC tissues. In addition, the RTPCR products were proved to be CT9 cDNA by DNA sequencing. No relationship was found between the expression of CT9 mRNA and clinical factors such as age, sex, tumor size, degree of tumor differentiation, serum αfetoprotein level and infection of hepatitis B virus or hepatitis C virus （Pgt;0.05）. ConclusionCT9 mRNA is expressed with high percentage and specificity in hepatocellular carcinomas. The CT9 gene product is a potential target for antigenspecific immunotherapy of HCC.
ObjectiveTo investigate the clinical significance of CK20 mRNA expression in blood of patients with colorectal cancer. MethodsThe expressions of CK20 mRNA in blood of twenty healthy volunteers, ten patients with colorectal polyp and sixtyone patients with colorectal cancer were detected by RT-PCR. ResultsThe positive rate of CK20 mRNA in peripheral venous blood and portal venous blood of patients with colorectal cancer were 41.0%(25/61) and 45.9%(28/61), which was not significantly different (Pgt;0.05). The expression of CK20 mRNA in patients with colorectal cancer was associated with clinical TNM stage of tumor, local lymph node metastasis, distance metastasis, and the depth of invasion (Plt;0.05). No expression of CK20 mRNA was detected in blood of twenty healthy volunteer’s and ten patients with colorectal polyp. ConclusionCK20 is a specific marker for detecting blood micrometastasis of colorectal cancer. The expression of CK20 mRNA in blood of patients with colorectal cancer is related with TNM stage, invasion, and metastasis of colorectal cancer.