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 compare gene express difference ofkeloid and normal skin tissues by using the suppression subtractive hybridization (SSH) so asto find the differential express gene in keloid. Methods mRNA extracted fromkeloid and normal skin tissues was used as the template to synthesis cDNA of keoid and normal skin. The cDNA of keloid served as a tester, the cDNA of normal skin as a driver. cDNA was digested with RsaⅠ. Adaptor-ligated tester cDNA was prepared. Then first hybridization, second hybridization and PCR amplificationwere done. Differentially expressed cDNA was selectively amplified during thesereactions. After SSH, the PCR mixture was ligated with T-vector. The positive clones were selected and the insert gene fragments were analyzed. Southern hybridization identified the keloid differential express genes. The positive clones ofSouthern hybridization were selected, and these sequences were analyzed. The results were compared with that of GeneBank. Results Thirteen differential genes were found in keloid, of which 11 gene clones have been known their function, and 2 clones have not known their function. 〖WTHZ〗Conclusion Keloid differentially expressed gene was screened successfully by SSH.
For observation of the change of transforming growth factor-beta 1 (TGF-beta 1) gene expression in the process of skin wound healing, the following experiments were performed. Sixteen Wistar rats were chosen. At each side of the rat’s back, a 1 cm x 1.5 cm middle-thick skin wound was made. After 3, 6, 9 and 12 days, the specimens were taken from the wounds. For each specimen, half of it was used for RNA extraction, and underwent dot blotting; and the other half was frozen immediately and underwent in situ hybridization. The probes were dig-labeled PDGF-BB cDNA probe and TGF-beta 1 probe. The results showed that TGF-beta 1 gene was expressed mainly in fibroblast, epithelial cell and capillary endothelial cell. The peak of TGF-beta 1 mRNA content was in the 6th day postoperatively. After that, the content of TGF-beta 1 decreased to normal. It was suggested that TGF-beta 1 gene expression was in close relation with healing process. TGF-beta 1 may play an important regulatory role in the skin wound healing.
Objective To investigate the influnce of L-arginine (L-Arg) and L-nitro-arginine-methyl-ester(L-NAME) to purified retinal ganglion cells(RGCs) apoptosis of rats cultured in different consistencies of L-Arg and L-NAME. Method RGCs from Sprague Dawley (SD) neonatal rats(postnatal 1~5 day) were cultured in assimilative culture solution in vitro and RGCs were purified by Thy1.1 with sheep anti rat FITC monoclonal antibody. RGCs were cultured in different consistencies of L-Arg and L-NAME: 1×10-6, 1×10-5,1×10-4, 1×10-3, 1×10-2 and 1×10-1 mol/L for 24 hours and 48 hours, respectively. The changes of bcl-2, bax and p53 mRNA in RGCs in different consistencies of L-Arg and L-NAME were demonstrated qualitatively and quantitatively by in situ hybridization, and their apoptosis were detected by terminal deoxynucleotidyl transferase mediated dUTP nick end labeling(TUNEL) method, respectively. Results After 24 hours in vitro, the purification rate of RGCs in the experiment arrived at 97 %. After 48 hours, there were a few apoptotic cells expression in the control group. Apoptotic cells expression in L-Arg≥1×10-3 mol/L and L-NAME≥1×10-1 mol/L groups increased that had a significant difference with the control group (Plt;0.05). In the group of L-Arg≥1×10-3 mol/L and L-NAME≥1×10-1 mol/L, the expression of bcl-2 mRNA in RGCs became weaker and weaker as the consistencies were increased, but the expression of bax and p53 mRNA in RGCs became higher and higher and had a significant difference with control group (Plt;0.05). Conclusion Lower concentration of L-Arg can promote the growth of purified RGCs in vitro and higher concentration of L-Arg can promote the apoptosis of RGCs. (Chin J Ocul Fundus Dis, 2002, 18: 137-139)
Objective Using molecular biology method to detect and genotype human papilloma virus (HPV) in women taking physical examination in West China Hospital, Sichuan University, to explore the infection status and genotype distribution of HPV in normal women in Chengdu area, and to provide basis for early effective prevention and control of cervical cancer and domestic research and development of HPV vaccine. Methods Flow fluorescent hybridization technique was used to detect and genotype HPV-DNA in 25 148 healthy women taking physical examination in West China Hospital, Sichuan University between May 1st, 2018 and May 31st, 2019. The overall positive HPV infection rate, HPV genotype distribution, and characteristics of HPV infections were analyzed and calculated, and the HPV infection rates of different age groups were calculated and compared by chi-square test using SPSS 17.0 software. Results The overall positive rate of HPV infection was 12.19% (3 066/25 148). The high-risk HPV genotypes infection rate was 8.69% (2 186/25 148), and the top five subtypes with the highest infection rates were HPV52, HPV53, HPV58, HPV16, and HPV39. The low-risk HPV genotypes infection rate was 4.66% (1 171/25 148), and the top five subtypes with the highest infection rates were HPV61, HPV81, HPV43, HPV44, and HPV6. Single subtype infections were the main infections with a proportion of 81.74% (2 506/3 066), and the most common multiple infections were double infections which accounted for 13.96% (428/3 066). In different age groups, the HPV infection rate of group 60-69 was the highest (12.87%), while that of group 70-89 was the lowest (10.88%), but the difference among different age groups was not statistically significant (χ2=4.035, P=0.544). Conclusion According to the results of this study in women taking physical examination in West China Hospital, Sichuan University, we suggest adding HPV52, HPV53, and HPV58 which have the highest infection rate in high-risk HPV subtypes to the evaluation of domestic HPV vaccine screening and the cervical cancer prevention and control system.
ObjectiveTo observe the expression of connective tissue growth factor (CTGF) in injured model of retinal pigment epithelial (RPE) cells and the promoting effect of CTGF on migration of RPE cells.MethodsCultured monolayer-confluent human RPE cells were scraped with a trephine and a cotton stick, and set up the injured model of RPE cells with round scraped area. Immunohistochemistry and in situ hybridization(ISH) were used to detect the expression of CTGF protein and mRNA in injured RPE cells at distinct time points after injury. The number of RPE cells migrated to injured area was measured and the effect of CTGF on migration of RPE cells and the effect of dexamethasone (DEX) on the promoting process of CTGF were observed.ResultsThe results of immunohstochemistry and ISH indicated the weak positive expression of CTGF in RPE cells at the edge of scrape 6 hours after injury, and the positive expression increased gradually as time goes by after the injury. Strong positive expression of CTGF in RPE cells at the edge of scrape was found 24 and 48 hours after injury. Rebuilt human CTGF stimulated migration of RPE cells in a dose-depended manner, and DEX significantly inhabited the migration.ConclusionCTGF involves in the procedure of repair of injury of RPE cells, which may play an important role in the pathogenesis of intraocular proliferative diseases such as proliferative vitreoretinaopathy.(Chin J Ocul Fundus Dis, 2005,21:306-309)
Objective To investigate the association of the expression of CD15 mRNA with the invasion and prognosis of hepatocellular carcinoma (HCC) and the expression of nm23H1 mRNA. Methods In situ hybridization and immunohistochemistry methods were used to detect the expression of CD15 mRNA and protein nm23H1 mRNA in HCC.Results In 99 cases of HCC, the positive rate of CD15 mRNA,its protein and nm23H1 mRNA were 38.4%, 36.4% and 76.8%, respectively. The expression of CD15 mRNA was consistent with its protein and negatively correlated with the expression of nm23H1 mRNA. The expression of CD15 mRNA and its protein, nm23H1 mRNA were associated with the invasiveness and metastasis of HCC and the prognosis of HCC patients. Conclusion The detection of CD15 expression could be a new pathological biology index to judge the metastasis and prognosis of HCC.
Objective To identify the expression of pleiotrophin （PTN） mRNA and protein in the colorectal cancer tissues， and to explore the clinical value of it. Methods The expressions of PTN mRNA and protein in colorectal cancer tissues （colorectal cancer group） as well as normal colorectal tissues （normal control group） were tested by using in-situ hybridization and immunohistochemistry. Results The positive rates of PTN mRNA 〔63.9% （53/83） vs. 40.7%（22/54）〕 and protein〔60.2%（50/83） vs. 33.3%（18/54）〕 in colorectal cancer group were all significantly higher than those of normal control group （P=0.008， P=0.002）. There were no significant relationship between expressions of PTN mRNA and protein with gender， age， and type of tumor （P＞0.05）， but in tissues of Ⅲ-Ⅳ stage and poor differentiation，the positive rates of PTN mRNA and protein were all higher （P＜0.05）. Conclusions The over expressions of PTN mRNA and protein in colorectal cancer tissues may directly related to the invasion and metastasis. Meanwhile， it can be used as an index to predict metastasis and prognosis of colorectal cancer.
Objective It is reported that transforming growth factor β1 (TGF-β1) has the protective effects on the articular cartilage in osteoarthritis (OA). To investigate the significance of the expressions of matrix metalloproteinase 9 (MMP-9), TGF-β1 mRNA and corresponding proteins in OA. Methods The specimens of articular cartilage and synovium were collected from voluntary donators, including 60 cases of OA (experimental group) and 20 cases of traumatic amputation,cruciate l igament rupture, discoid cartilage injury, and menisci injury (normal control group). The pathological changes were observed by HE staining. MMP-9 and TGF-β1 protein expressions were detected by immunohistochemical technique, and the mRNA expressions of MMP-9 and TGF-β1 were detected through in situ hybridization technique; and their correlation was analysed. Results HE staining showed: shrinkage, necrosis, and irregular arrange of the articular chondrocytes, extracellular matrix fracture, hypertrophy and hyperplasia synovium, infiltration of lymphoid and mononuclear cells and prol iferation of many small blood vessels in the experimental group; regular arrangement of the articular chondrocytes, the homogeneously staining matrix, and synovial tissue without chronic inflammation and significant prol iferation in the normal control group. The mRNA and protein expressions of MMP-9 and TGF-β1 were positive in 2 groups. The positive-stained cells included chondrocytes, synovial l ining cells, and vascular endothel ial cells, fibroblasts, and inflammatory infiltrated cells in subsynovial layer. The expressions of mRNA and corresponding protein of MMP-9 and TGF-β1 in the experimental group were significantly higher than those in the normal control group (P lt; 0.01). There was a positive correlation between MMP-9 mRNA and protein expression (r=0.924, P=0.000), and between TGF-β1 mRNA and protein expression (r=0.941, P=0.000) in the experimental group. There was a negative correlation between the expression of MMP-9 protein and TGF-β1 protein (r= — 0.762, P=0.000), and between the expression of MMP-9 mRNA and TGF-β1 mRNA (r= — 0.681, P=0.000) in the experimental group. Conclusion The higher expression of TGF-β1 can protect articular cartilage by down-regulating the expression of MMP-9 of chondrocytes and synoviocytes in OA, which may delay the biological behavior of OA such as occurrence and progress, etc.
Objective To research the gene expression of transforming growth factor β1 (TGF-β1) in zone Ⅱ flexor tendon wound healing of rabbit. Methods Sixty New Zealand white rabbits forepaws(left side) underwent complete transection and the middle digit flexor digitorum profundus tendon in zone Ⅱ were repairedby Kessler methods as the experimental group. The normal right forepaws served as the control group. The tendons and tendon sheaths were harvested at 1, 7, 14, 21, 28and 56 days after repair(n=10). The expression patterns ofTGF-β1 wereanalyzed by in situ hybridization and immunohistochemistry staining methods. Results The in situ hybridization examination revealed thatTGF-β1 mRNA expression upregulated at 1 day, reached the peak levels at 1421 days and remained high levels up to 56 days in the experimental group. The expression ofTGF-β1 mRNA in control group was lowerthan that in the experimental group, showing statistically significant difference (Plt;0.05). The results of immunohistochemical staining was similar to that of in situ hybridization. Conclusion The normal tendon and tendon sheath cells are capable ofTGF-β1 production. The cytokine is activated in tendon wound condition. The upregulation of this cytokine in both tendon and tendon sheath cells are coincidence with both extrinsic and intrinsic mechanisms for tendonrepair.