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 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.
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.
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 evaluate the feasibility of reconstructionof urothelium tissue in vivo using tissue-engineering technique. Methods The urothelium cells were obtained from young rabbit, bladder by mechanical and enzyme digested methods. After expanded in vitro, the 4th to 5th generation urothelium cells were seeded onto the surface of 8 Polylatical／glycolic acid copolymer polymer，the polymer matrix without seeding cells served as control group. A total of 8 cell-polymer scaffolds and 4 simply scaffolds were separately implanted into subcutaneous pockets of athymic mice. Theexperiment groups included cell-polymer scaffolds 4 weeks and cell-polymer scaffolds 8 weeks. The control group included simply scaffold 4 weeks and simply scaffold 8 weeks.After 4 and 8 weeks, the specimens were obtained and examined by gross inspection, histologically and immunohistochemically. Results The results of HE and Masson staining showed that the polymer were covered by urothelium cells layers and cells layers increased markly in experimental group. Immuocytochemical studies revealed that the cells were stained positively for anti-cytokeratins (AE1/AE3) in experimental group. Fiber tissue deposition were found on the surface of polymers in control group by HE and Masson staining. Immunocytochemical staining of implants showed the negative result for cytokeratins in control group. Conclusion It is feasibility that reconstruction of urothelium tissue using tissue-engineering -technique,whichprovides basic understandings for further development of the bladder and ureteral tissue engineered research.
Objective：To observe the expression of gene and protein l evel of unfolded protein, glucoseregulated protein 78 (GRP78), after retinal d etachment (RD); to find out the relationship between UPR and the cell damage after RD. Methods：Eightyeight Wistar rats were divided into 2 groups: con trol group (11 rats) and RD group (77 rats). In RD group, subretinal injection with 10 mg/ml hyaluronic acid sodium was performed on the left eyes of the rats t o set up RD model, and the left eyes and retinal tissue were collected 1/2 day, 1 day, 2, 4, 8, 1 6 and 32 days after RD; there were 11 rats in each subgroup. The expression of G RP78 mRNA in retina tissue was detected by semiquantitative reverse transcript i on polymerase chain reaction (RT－PCR), the expression of GRP78 protein level wa s detected by Western blotting, and the distribution of GRP78 in each retinal lay er was observed by immunofluorescence labeling method and confocal microscopy. Results：The expression of retinal GRP78 mRNA significantly in creased in 1/2 day , 1 day, 2, and 4 days subgroups after RD (Plt;0.05). The expression of GRP7 8 protein significantly increased in each subgroup after RD compared with which in the control group, and reached the peak in 8, 16, and 32 days subgroups. The expres sion of GRP78 protein was detected in all of the retinal layers after RD. Conclusion：The protection mechanism of UPR starts up after RD, and l eads the correc t pucker of the protein and reduces cellular injury by upregulating the expres s ion of GRP78, which provide the theoretic basis for reducing the cellular injury and improving the visual function in patients with RD.
【 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 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.
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.