Objective To explore the effect of connective tissue growth factor on the pathogenesis of hypertrophic scar and keloid tissue. Methods The content of hydroxyproline was determined and the expression of connective tissue growth factor gene was detected by the reverse transcription-polymerase chain reaction and image analysis technique in 5 normal skins, 15 hypertrophic scars and 7 keloid tissues. Results The contents of hydroxyproline in the hypertrophic scar(84.10±1.76) and keloid tissue (92.38±2.04) were significantly higher than that of normal skin tissue (26.52 ± 4.10) (P lt; 0.01). The index of connective tissue growth factor mRNA in the hypertrophic scar (0.78 ± 0.63) and keloid tissue (0.84 ± 0.04) were higher than that of normal skin tissue ( 0.09 ± 0.25) (P lt; 0.01). Conclusion Connective tissue growth factor may play an important role in promoting the fibrotic process of hypertrophic scar and keloid tissue.
【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.
【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.
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.
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.
With the method of PCR-SSCP combined sequencing,we analysed the mutations in the RB1 gene of 10 retinoblastoma samples. All 27 exons of RB1 gene have been examined. The PCR products were digested with some restriction endonuclease to the size of about 200 bp and then denatured into single strands. Loading the single strands of DNA into the 6% polyacrilamide gel,we followed the technology named SSCP to f/nd the abnormal bands in the SSCP gel. After that, the samples found with the abnormal bands in the SSCP gel were amplified again and sequenced to confirm the types and locations of the mutations in the RB1 gene. We have fonnd some kinds of mutations in the RB1 gene. （Chin J Ocul Fundus Dis,1994,10:17-20）
Objective To evaluate the expressive varieties of Nogo-A mRNA in injured optic nerves of rats. Methods Reverse transcription polymerase chain reaction (RT-PCR) method was used to hemi-quantitatively analyze the levels of Nogo-A mRNA in the optic nerves 3, 7, 9, 15, 21, and 25 days respectively after injury.Results The level of the expression of Nogo-A mRNA was low in the normal optic nerves, while it was significantly high in the optic nerves 3 days after in jury, and kept the high level still after 25 days.Conclusion The expression of Nogo-A mRNA in injured optic nerves is increased. (Chin J Ocul Fundus Dis，2003，19：201-268)
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 evaluate branched-chain DNA (b-DNA) signal amplification and semi-quantitative (Sq) RT-PCR in detection of free cancer cells in peritoneal flushing fluid of colorectal cancer patients during surgery. Methods The CEA mRNA in peritoneal flushing fluid in 48 cases of colorectal cancer were detected by b-DNA and SqRT-PCR. Peritoneal flushing fluid cytology (PLC) was conformed simultaneously to detect the free cancer cells. The peritoneal flushing fluid of 12 cases with colorectal benign disease were taken as negative control, GAPDH mRNA as internal control. Results In colorectal cancer patients, positive rate of free cancer cells by bDNA and SqRT-PCR （43.8%, 31.3%） was higher than that by PLC （4.2%）. The relative quantitative expressions of CEA mRNA were related to the Dukes staging， depth invasion and differentiation degree (Plt;0.05)， but irrelevant to tumor size，the patients’ age and gender (Pgt;0.05).Conclusion Both b-DNA and SqRT-PCR technologies have advantages and disadvantages to detect free cancer cells in peritoneal flushing fluid， which are related to clinicopathological factors.
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.