【 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.
Objective
To investigate the effect of hypoxia on expressions of erythropoietin（EPO）mRNA and protein in retinal Muuml;ller cells cultured in vitro.
Methods
Retina tissues from the new-born Wistar rats were dissected into cell suspension after digested by pancreatin.Muuml;ller cells were separated and purified by mechanical concussion and blowing and striking method.The expression of EPO mRNA and protein under the condition of hypoxia was detected by semi-quantitative reverse transcriptase（RT）-polymerase chain reaction（PCR）and immunocytochemical method.
Results
Retinal Muuml;ller cells were cultured successfully，95% of which were positively stained by glial fibrillary acidic protein（GFAP）.Positively stained EPO protein was located in the cytoplasm and protuberance.The expression of EPO mRNA and protein was faint in the normal retinal Muuml;ller cells，but increased obviously and time-dependently after hypoxia.
Conclusion
Expression of EPO mRNA and protein increases in Muuml;ller cells after hypoxia，which may be one of the protective factors for the nerves in anoxic retinopathy.
(Chin J Ocul Fundus Dis, 2006, 22: 196-199)
【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.
OBJECTIVE To study the relationship between the changes of mRNA expression in wound tissues of diabetic ulcers and tissue repair. METHODS The mRNA expression of TGF-beta 1 and IL-6 in eight bioptic samples of diabetic ulcers were detected by RT-PCR and pathologic methods, and the surrounding normal skins from the same patients were measured as control group. RESULTS The mRNA expression levels of TGF-beta 1 were markedly decreased in the diabetic ulcers compared with control group, while the mRNA expression levels of IL-6 were increased at the same reaction conditions. CONCLUSION The different changes of mRNA expression level of TGF-beta 1 and IL-6 in wound tissue result in low production and decreased activity of TGF-beta 1 and IL-6, which lower the reparative ability of wound tissue.
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.
Objective To identify and isolate the variant gene associated with gastric adenocarcinoma and clone the fragment of variant gene.Methods By arbitrarily primer polymerase chain reaction (AP-PCR), DNA samples from 5 matched gastric adenocarcinoma and non-tumor gastric tissues were analysed. Results The produced AP-PCR profiles were different in each matched gastric adenocarcinoma and non-tumor gastric tissue. One differentiated amplified DNA fragments PW2.2 from a matched gastric adenocarcinoma were cloned. The result of Southern blot hybridization with PW2.2 as a probe showing that this fragment was also found in some other gastric adenocarcinoma samples. Conclusion AP-PCR fingerprinting assay can be used to identify and clone the variant genes associated with gastric adenocarcinoma.
【Abstract】 Objective To investigate the expression and significance of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptors in pancreatic cancer. Methods Thirty-two samples of pancreatic cancer tissue were collected from year 2002 to 2004. All of them were verified by histopathology and there were 9 cases of well-differentiated, 12 of moderately differentiated, and 11 of poorly differentiated, in which 12 cases were in the stage of Ⅰor Ⅱand 20 in the stage of Ⅲ or Ⅳ according to the TNM staging method. Eighteen normal pancreatic tissues were used as control group. The expressions of TRAIL receptors (death receptor 4, death receptor 5, decoy receptor 4 and decoy receptor 5) mRNA were assayed by semi-quantitive reverse transcription polymerase chain reaction (RT-PCR) in the pancreatic cancer tissues and the normal pancreatic tissues. Results The expressions of death receptor 4 (DR4) and death receptor 5 (DR5) were detected in all the pancreatic cancer tissues and the normal pancreatic tissues and the levels of DR4 and DR5 were significantly higher than those of the normal pancreatic tissues (P＜0.01). Decoy receptor 1 (DcR1) and decoy receptor 2 (DcR2) were also expressed in normal pancreatic tissues, whereas DcR1 and DcR2 were only expressed in 18 and 20 pancreatic cancer tissues, respectively. However, there were no significant difference of the expression of DcR1 and DcR2 between the pancreatic cancer tissues and the normal pancreatic tissues （Pgt;0.05）. The expression level of DR5 in pancreatic cancer tissue was correlated with tumor differentiation and clinical stage, and the levels in stage Ⅲ and stage Ⅳwere significantly lower than those of stageⅠand stageⅡ(P＜0.05). The expressions of DR4, DcR1 and DcR2 were not correlated with tumor differentiation and clinical stage (Pgt;0.05). Conclusion ①The expression of TRAIL receptors in pancreatic cancer tissues is prevalent, but the types of receptors expressed in different tissues were also different. High expression of death receptors may play an important role in TRAIL recptors regulated pancreatic cancer apoptosis. ②The expression of DR5 is correlated with the differentiation degree of pancreatic cancer cell and clinical stage of tumor. The expressions of DR4, DcR1 and DcR2 should not be considered as related indexes of differentiation degree or clinical stage of pancreatic cancer.
ObjectiveTo determine the level of CDH1 gene promoter hypermethylation in human gastric carcinoma by establishing MS-PCR method, and analyze retrospectively the possible statistical relationship between CDH1 gene promoter hypermethylation in human gastric carcinoma and HP infection, tumor differentiation, invasion, lymph nodal and distant metastasis, respectively.
MethodsThe bisulfite conversion MS-PCR method was adopted to examine the level of CDH1 gene promoter hypermethylation in 40 cases of human gastric carcinoma tissue collected between January 2008 and December 2009. The statistical relationship between CDH1 gene promoter hypermethylation in human gastric carcinoma and HP infection, tumor differentiation, invasion, lymph nodal and distant metastasis were examined respectively with SPSS statistical tools.
ResultsThe positive rate of CDH1 gene promoter hypermethylation in gastric carcinomas (67.5%) was higher than that in paired normal gastric mucosae (12.5%), and the difference was significant (P<0.05). In gastric carcinomas, the positive rate of CDH1 gene promoter hypermethylation in well differentiated or moderately differentiated groups (22.2%) was lower than that in poorly differentiated groups (80.6%), and the difference was significant (P<0.05). The positive rate of CDH1 gene promoter hypermethylation in HP positive groups (78.1%) was higher than that in HP negative groups (25.0%), and the difference was significant (P<0.05).
ConclusionCDH1 gene promoter hypermethylation may play an important role in the process of tumor carcinogenesis in gastric carcinomas. Meanwhile, the CDH1 gene promoter hypermethylation may lead to poor differentiation in gastric carcinomas. CDH1 gene promoter hypermethylation is related to HP infection in the original gastric carcinomas, which shows that HP may get involved in the process of tumor suppressor gene methylation/inactivation and tumor development process.
Objective To detect traces of microRNAs (miRNAs) in plasma and assess the expression stability of two common reference genes by stem-loop and poly A polymerase (PAP) real-time quantitative polymerase chain reaction (PCR) method, as miRNAs are the new bio-markers of tumor diagnosis and molecular targeted therapy, and its quantitative research is very important. Methods We extracted miRNAs from plasma of adult Sprague Dawley (SD) rats’ plasma, and detected the expressions of rno-miR-200b-3p and rno-miR-126-3p with stem-loop and PAP real-time PCR quantitative method, with rno-miR-103a-3p and U6 as internal controls. All the results were evaluated by 2△Ct method. Results Compared with PAP method, the stem-loop method reduced Ct value by 2-4 cycles and improved sensitivity by 10 times. In PAP method, the melting curve showed two peaks, a main peak and a small non-specific peak. Yet the melting curve of stem-loop method demonstrated a single specific peak. Furthermore, we validated the stability of internal references in the two real time PCR methods. U6 presented a more stable Ct value than rno-miR-103 in adult SD rats’ plasma samples. Conclusions Stem-loop real-time PCR is recommended as a major way to detect some samples with a low concentration of miRNAs, owing to its high accuracy and sensitivity. However, if a large number of tissue samples is going to be detected, PAP real-time PCR is more suitable and convenient than stem-loop method. U6 is more stable and repeatable than rno-miR-103a-3p as the reference gene to evaluate the semi-quantitative consequence of miRNAs.
