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.
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.
Objective According to heparanase’s gene sequence of GenBank, to construct heparanase gene-targeted small interfering RNA (siRNA) and its expression vector and to observe its interference effect on the expression of heparanase gene in human malignant breast cancer MDA-MB-231 cell. Methods Heparanase gene-targeted hairpin siRNA was designed, two complementary oligonucleotide strands were synthesized and inserted into pGPU6/GFP/Neo vector, which was identified by sequence identify. Human malignant breast cancer MDA-MB-231 cell was transfected with the constructed vector with lipofectamine method. Fluorescence photograph was taken. Real-time PCR (RT-PCR) was performed to evaluate the level of heparanase mRNA expression. Results Four kinds of heparanase gene-targeted hairpin siRNA were designed, then were inserted into pGPU6/GFP/Neo vector after annealing. Sequencing indicated the construction was successful. Fluorescence photographs showed MDA-MB-231 cells were transfected successfully. RT-PCR showed that heparanase mRNA expression levels were inhibited significantly （Plt;0.05）. Conclusion The heparanase gene-targeted siRNA and its vector are successfully constructed and MDA-MB-231 cells are transfected successfully. Heparanase mRNA expression levels are significantly inhibited by siRNA vector, which provide a new method for the treatment of cancer.
ObjectiveTo evaluate the possible role of the expression of insulin-like growth factor-1 receptor (IGF-1R) in determining rectal cancer radiosensitivity. MethodsThe paired preradiation biopsy specimens and postoperative specimens were obtained from 87 patients with rectal cancer in the department of digestive tumor surgery, Jiangsu Province Hospital of Traditional Chinese Medicine, Affiliated Hospital of Nanjing University of Traditional Chinese Medicine from January 2009 to December 2010. The IGF-1R expression was examined by immunohistochemistry (IHC) and reverse transcription-polymerase chain reaction (RT-PCR). The tumor radiosensitivity was defined according to Rectal Cancer Regression Grade, then the relation between the IGF-1R expression and tumor radiosensitivity was evaluated. ResultsCompared with the preradiation biopsy specimens, IGF-1R expression significantly increased in the paired postoperative specimens of the residual cancer cells (Plt;0.001). The IHC result demonstrated IGF-1R overexpression was significantly associated with a poor response to radiotherapy (rs=0.401, Plt;0.001); RT-PCR detection of IGF-1R expression on preradiation biopsy specimens also showed that IGF-1R mRNA negative patients had a higher radiation sensitivity (rs=0.497, Plt;0.001). ConclusionDetection of IGF-1R expression may predict radiosensitivity of preoperative irradiation for rectal cancer.
【 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 observe the effect of gefitinib on expression of epidermal growth factor receptor (EGFR) in bile duct epithelial cells, and the feasibility of inhibiting hyperplasia of bile duct epithelial cells with gefitinib. Methods Sixty-one patients with hepatolithiasis having to be in hospital for surgery from the First People’s Hospital of Shuangliu county were selected, with 25-65 years old, average 46.92 years. The patients were randomly divided into therapy group and control group. There were 30 cases in therapy group, in which fine duct was placed on lesion bile duct during operation, and through whom gefitinib solution was perfused after operation. There were 31 cases in control group with only T tube drainage after operation. The bile duct sample was obtained respectively during the operation and 6 weeks and 12 weeks after operation. The histology and expression change of EGFR were observed by HE staining, immunohistochemistry and RT-PCR method respectively. Results There were no significant differences in pathohistology changes of bile duct and the EGFR protein and mRNA expression between therapy group and control group during operation. The hyperplasia of epithelium mucosae and submucosal gland in the therapy group were obviously decreased as compared with those in control group, the EGFR mRNA and protein expression in therapy group were weaker than those of control group （Plt;0.05） 6 weeks and 12 weeks after gefitinib treatment. Conclusion EGFR is overexpressed in the chronic proliferative cholangitis, and continuously local application of gefitinib after operation can specifically interrupt the activation and expression of EFGR and then effectively inhibit the hyperplasia of bile duct epithelial cells.
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.
OBJECTIVE To determine the characteristics and regularity of fibronectin mRNA expression in diabetic ulcers, and to investigate the relationship between the changes of fibronectin mRNA expression and pathogenesis of diabetic ulcer. METHODS Biopsies were removed from the margins of diabetic foot ulcers, included surrounding skin as experimental group, and the biopsies from normal skin of the same patients as control group. The mRNA expression of fibronectin was measured by quantitative RT-PCR technique. RESULTS The mRNA expression of fibronectin could be detected in both normal skin and diabetic foot ulcers, but the level of expression in diabetic ulcers was lower than that of normal skin. CONCLUSION The level of mRNA expression of fibronectin in diabetic ulcers is decreased, which suggest that the down-regulation of transcription may be one of the mechanisms of chronic impaired ulcers.
ObjectiveTo detect the 2019 novel coronavirus (2019-nCoV) in various biological specimens of novel coronavirus pneumonia (NCP), and preliminarily observe the status of 2019-nCoV in different systems of the body and its clinical significance.MethodsThe study design was a small-scale cross-sectional observational study. All the confirmed NCP cases being treated in the Second People’s Hospital of Yibin · West China Yibin Hospital, Sichuan University on February 2nd, 2020 were enrolled in this study. Two sets of primers were designed for 2019-nCoV-1ab and N regions using real-time reverse transcriptase-polymerase chain reaction (RT-PCR) assay. The 2019-nCoV in upper respiratory specimens, blood, feces and urine specimens of the NCP cases were detected on the single day.ResultsA total of 7 imported NCP cases (mild type) were included. The 7 patients were confirmed by the positive results of 2019-nCoV nucleic acid tests of upper or lower respiratory specimens between the 3rd day and the 7th day after fever onset, while 2 patients were found positive on the 3rd day after onset. The 2019-nCoV nucleic acid tests of the 7 patients were detected again on a single day between the 7th day and the 15th day after onset, and the results showed: the upper respiratory specimens of 5 patients were found negative (1 case was on the 7th day after onset); 2019-nCoV was not detected in the blood, feces or urine specimens of the 7 patients.ConclusionsFor mild type NCP patients, real-time RT-PCR test could detect 2019-nCoV between the 3rd day and the 7th day after onset, while 2019-nCoV might become negative since the 7th day after onset. 2019-nCoV was not detected in the blood, feces or urine of mild type NCP patients on the single day between the 7th day and the 15th day after onset. This study was only a preliminary observational study, which needed high-qualified studies to obtain more definitive conclusions.
ObjectiveTo improve the understanding of the diagnosis and therapy of chronic active Epstein-Barr virus (CAEBV) infection. MethodsData of 9 cases of CAEBV infection diagnosed between October 2008 and January 2013 were analyzed retrospectively,including clinical and auxiliary examination results,pathological data,especially EB virus (EBV) antibodies and DNA in peripheral blood mononuclear cells (PBMC) and infected tissue,and follow-up information. ResultsThe major manifestations of the 9 patients were fever,splenomegaly,hepatomegaly,lymphadenopathy,and others,including general fatigue,nausea,skin rash,jaundice,and so on.The abnormalities of auxiliary examination were as follows:anemia,leucopenia,neutropenia,thrombocytopenia,elevated LDH and HBDH levels,liver dysfunction and abnormal chest CT findings.EBV serologic tests revealed high IgA antibody levels against EB viral capsid antigen (VCA) in 6 patients,and 8 patients had positive IgG antibody levels against early D antigen (EAD).The mean load of EBV-DNA detected by real time polymerase chain reaction (PCR) in the PBMC was 3.07×105 copies/mL.Six of the nine patients presented a poor clinical course.One of them died of intracranial hemorrhage,one of them died of multiple organ failure,one of them died of EBV-associated hemophagocytic syndrome,and one of them died of severe pulmonary infection.Four patients developed lymphoma.One of them died of hepatic failure and one of them died of severe infection in the process of anti-tumor treatment. ConclusionThe clinical feature of CAEBV infection is varied.More attention should be paid to the disease because of its severe complications,poor prognosis and high mortality.