ObjectiveTo screen compounds or drugs can affect the hypoxia induced-gene expression of retinal vascular endothelial cell based on gene expression microarrays and connectivity map (CMAP) technology.
MethodsTotally 326 up-regulated and down-regulated genes of hypoxic human embryonic retinal microvascular endothelial cells minduced by cobalt chloride in the previous study were converted into query signature format documents. Gene profile of the disease characteristics was then compared with that of control in CMAP website database, positive and negative compounds related to retinopathy of prematurity (ROP) were finally screened out.
Results44 and 18 compounds or drugs have positive and negative relationship with ROP respectively by searching CMAP database with differentially expressed genes. Ciclopirox, cobalt chloride, gossypol and withaferin A have positive relationship with ROP. Cyclic adenosine monophosphate, harmalol, naringin and probenecid have a negative effect on ROP.
ConclusionsCiclopirox, cobalt chloride, gossypol and withaferin A have a positive effect on ROP. However, cyclic adenosine monophosphate, harmalol, naringin and probenecid have a negative effect.
ObjectiveTo study the expression of lipid associated with neutrophil gelatinase associated lipocalin (NGAL) in nude mice orthotopic pancreatic cancer tissues and the relationship between the occurred and development of pancreatic cancer.
MethodsThe expressions of NGAL mRNA and protein of pancreatic cancer tissues and their adjacent tissues, and normal pancreatic tissues in nude mice were detected by using RT-PCR and immunohistochemical methods.
ResultsThe expressions of NGAL mRNA in pancreatic cancer tissues and adjacent tissues were significantly higher than that in normal pancreatic tissues (P < 0.05), and the expression of NGAL mRNA in pancreatic carcinoma tissues was significantly higher than that in para carcinoma tissues (P < 0.05). The strong positive expression rate of NGAL protein in pancreatic carcinoma tissues was significantly higher than thoes in para carcinoma tissues and normal pancreatic tissues (P < 0.05).
ConclusionsNGAL is highly expressed in pancreatic cancer tissues, and NGAL may be an important regulatory factor in the development of pancreatic cancer.
OBJECTIVE To probe the possibility of direct transfer of exogenous gene into peripheral nerve and its following expression in vivo. METHODS The PCMV beta plasmid containing cytomegalovirus (CMV) promoter and Escherichia Coli (E. Coli), beta-Galactosidease (beta-Gal) structural gene (lacZ gene) was constructed and injected into the rabbit sciatic nerve. The control group was injected PBS solution. The injected nerves were sampled and tested by beta-Gal enzyme activity assay of the 5-bromo-4-chloro-3-indolyl-beta-D-galactoside and beta-Gal histochemical stain. RESULTS In the control group, no beta-Gal enzyme activity was detected in the different stages after operation, and beta-Gal histochemical stains showed positive. In the experimental group, enzyme activity could be detected from 2 days to 30 days after operation, and the histochemical stains showed negative. CONCLUSION The exogenous gene can be transferred into peripheral nerve and expressed with bioactivity, thus the gene therapy to accelerate the recovery of nerve is practical.
OBJECTIVE To study the biocompatibility of skin reproductive membrane. METHODS According to ISO’s standards, the extractions of the skin reproductive membrane were prepared, and the acute systematic toxicity test, primary skin irritant test, cytotoxicity test, gene expression of type I collagen and fibronectin were detected to evaluate the biocompatibility of skin reproductive membrane. RESULTS All of those tests showed negative results. CONCLUSION The skin reproductive membrane has excellent biocompatibility in the level of the systematic, cellular and molecular biology.
【Abstract】 Objective To investigate the effect of retinoic acid (RA) on cell apoptosis and gene regulation of IGF-2in chondrocyte. Methods One 1-month-old Chinese rabbit weighted 500 g was used in this experiment. The chondrocyte from rabbit knee were cultured by enzyme digestion. Twenty-five μL all-trans-retinoic acid (ATRA) (1×10-6 mol/L) were added in the media of cultured chondrocyte for 24 hours as experimental group, while 25 μL DMEM were added as control group. The secretion of collagen Ⅱ was observed by immunohistochemistry method, cell apoptosis was detected by flow cytometry, IGF-2 mRNA and protein expression in chondrocyte were detected by RT-PCR and Western blot analysis. Results The expression of collagen Ⅱ was down-regulated by ATRA in the experimental group. The cell apoptosis in chondrocyte exposed to ATRA at 1 ×10-6 mol/L was 21% ± 2%, which increased 5 times compared with the control group（5% ± 1%）. The IGF-2 mRNA and protein level in the experimental group were decreased 75% and 57%, respectively, compared to the control group. There weresignificant difference between the experimental group and control group in each index (P lt; 0.05). Conclusion RA may down-regulate the secretion and cell prol iferation, but up-regulate the cell apoptosis in chondrocyte. The apoptotic effect may carry out through inhibiting the IGF-2 expression of chondrocyte.
ObjectiveTo observe the effect of preserving tibial residual fibers on the expressions of ligament remodeling related genes in rabbit anterior cruciate ligament (ACL) reconstruction model.
MethodsSixty healthy adult New Zealand white rabbits were randomly divided into 4 groups:normal control group (group A, n=6) , sham-operation group (group B, n=18) , non tibial remnant preserved group (group C, n=18) , and tibial remnant preserved group (group D, n=18) . At 2, 6, and 12 weeks after operation, the ligament tissue was harvested to detect the mRNA expressions of collagen type 1A1(COL1A1) , collagen type 3A1(COL3A1) , transforming growth factor β1(TGF-β1), vascular endothelial growth factor (VEGF), growth-associated protein 43(GAP-43) , and neurotrophin 3(NT-3) by real-time fluorescent quantitative PCR.
ResultsAt each time point, there was no significant difference in the mRNA expressions of COL1A1, COL3A1, VEGF, and NT-3 between group A and group B (P>0.05) . In group D, the mRNA expressions of COL1A1, COL3A1, TGF-β1, and GAP-43 significantly increased when compared with those of group C at 6 weeks after operation (P＜0.05) ; an increased level of VEGF mRNA was also detected in the group D at 12 weeks after operation (P＜0.05) ; and an increased level of NT-3 mRNA was also observed in group D at 2 and 12 weeks after operation (P＜0.05) .
ConclusionThere is a time-dependent manner of angiogenesis-promoting, repair-related, and nerve-related gene expressions after ACL reconstruction with preserving tibial residual fibers during the process of ligamentization. Furthermore, the remnant preservation in ACL reconstruction can promote the expressions of related genes in some time points.
Objective
To investigate the development and metastasis of malignant choroidal melanoma cell strain OCM-1-gfp modified with green fluorescent protein(GFP) and the factors which affected the tumor biological behaviors.
Methods
GFP was transfected into malignant melanoma cell strain OCM-1.Melanoma cells with high and stable expression of GFP were injected into subretinal space and the subcutaneous space of hind leg of Balb/c nude mouse respectively in order to establish orthotopic and heterotopic transplanted tumor models.The development and metastasis process of orthotopic tumor models was observed directly by fluorescence microscope，and the size of the hypodermal tumor was measured by vernier.The expressions of 13 genes in melanoma were detected by means of immunohistochemistry staining.
Results
Malignant choroidal melanoma cell strain OCM-1 stably expressed GFP and preserved the characteristics of parental generation，OCM-1-gfp may develop melanoma and continue to metastasize in nude mouse.Positive expression of most of the antibodies，including Rb，p53，p21，E2F，NFkappa;B，cyclin D1，proliferation cellular nuclear antigen（PCNA），bcl2、bclXL/S，bax，and epithelial growth factor（EGF）and its receptor（EGFR），was found.While the staining of inhibition gene p16 was negative.
Conclusions
GFP is the marker for observing the development and metastasis of malignant choroidal melanoma in vivo.The rate of tumor formation and development process in orthotopic models does not differs much from which in heterotopic models of malignant choroidal melanoma.The expressions of lots of genes in malignant choroidal melanoma developed from OCM-1-gfp including p16、p53、NFkappa;B，cyclin D，PCNA，EGF，and EGFR are abnormal.
(Chin J Ocul Fundus Dis, 2006, 22: 170-173)
Objective
To investigate proliferating cell nuclear antigen (PCNA) gene expression in retinal pigment epithelium (RPE) cells and inhibition of antisense oligonucleotides(AS-OND) encoding PCNA mRNA to gene expression and proliferation of RPE cells, so as to search for new genetic therapy way for pro1iferative vitreoretinopathy (PVR).
Methods
(1) Rabbit RPE cells cultured in vitro were detected for PCNA expression by streptoavidin-biotin-enzyme complex (SABC) immunohistochemistry at several times. (2) The liposome-mediated synthetic antisense oligodeoxynucleotides (AS-ODN) and sense oligodeoxynucleotides (S-ODN) encoding PCNA were delivered to the RPE cells at different concentrations, then PCNA expresstion were detected by immunohistochemistry. (3) Exposed to different concentrations of AS-ODN and S-ODN, growth activity and suppressive rate of RPE cells were measured by methyl thiazolyl tetrazolium (MTT) methods.
Results
(1) PCNA were expressed in RPE cells, culmination in 48 hours of culture. (2) PCNA expression were markedly suppressed in the RPE cells treated with 0.28 and 1.12 μmol/L PCNA AS-ODN. (3) 0.28 μmol/L and 1.12 μmol/L PCNA AS-ODN significantly inhibited proliferative activity of RPE cells in a dose-dependent manner, the arrest rates of cellular growth reached 53% and 81% respectively.
Conclusion
AS-ODN complementary to PCNA mRNA at some concentration can sequence-specifically suppress PCNA expression in RPE cells and cellular proliferative activity, and show potential application to further experimental study for PVR genetic medication.
(Chin J Ocul Fundus Dis, 2002, 18: 231-233)
【Abstract】ObjectiveTo study the effect of transfection with antisense DNMT3b gene eukaryotic expression vector on the expression of DNMT3b gene in human cholangiocarcinoma cell line QBC-939. MethodsThe constructed antisense DNMT3b gene eukaryotic expression vector was transfected into the human cholangiocarcinoma cell line QBC-939 by using lipofectamine transfection reagents, and positive cell clones were obtained by using G418 selection after transfection. Whether the constructed recombinant vector was transfected into QBC-939 cells successfully was confirmed by amplifying the exogenous neoR gene with PCR method. The expression of DNMT3b gene mRNA and protein were detected by semi-quantitative RT-PCR and FCM methods respectively. ResultsFollowing the transfection of antisense DNMT3b gene eukaryotic expression vector, the mRNA level of DNMT3b gene in QBC-939 cells of human cholangiocarcinoma decreased from 0.956±0.053 to 0.209±0.023, and the protein level of DNMT3b gene also decreased from (75.38±3.22)% to (29.87±3.46)%. There were very significant differences on the expression levels of DNMT3b gene between non-tranfections group and the antisense DNMT3b gene eukaryotic expression vector transfection group (P＜0.01). ConclusionTransfection with antisense DNMT3b gene eukaryotic expression vector significantly reduces the expression level of DNMT3b gene in human cholangiocarcinoma cell line QBC-939, and this study may provide a valid tool and method to investigate the function of DNMT3b gene and its role in cholangiocarcinoma.
ObjectiveTo explore the relationship between the expressions of fibronectin (FN) and phosphatase and tensin homology deleted on ehromosome ten (PTEN) in hepatocellular carcinoma (HCC) tissues and the clinical pathological features.
MethodsThe expressions of FN and PTEN were detected by using Western blot and immunohistochemistry respectively in 83 HCC tissues and para-carcinoma tissues, 47 hepatic cirrhosis tissues and 11 normal hepatic tissues. The correlations between the expressions of FN and PTEN and the clinicopathologic features of HCC patients were analyzed.
ResultsThe positive expression rate of FN protein in HCC tissues was significantly higher than those in para-carcinoma tissue, normal hepatic tissue, and liver cirrhosis tissues (P<0.05); meanwhile the expression of PTEN was opposite (P<0.05). The positive expression rate of FN protein in para-carcinoma tissues was also obviously higher than that liver cirrhosis tissues and normal hepatic tissues (P<0.05), meanwhile the expression of PTEN was opposite (P<0.05). The positive expression rate of FN protein was higher in HCC tissues with cancer embolus, lymphatic metastasis, positive AFP, and multiple tumor (P<0.05), but there were no statistically significant differences in FN protein expression, gender, age, HBsAg, degree of tumor differentiation, and size of tumor (P>0.05). The positive expression rate of PTEN was lower in HCC tissues with high-medium differentiation, cancer embolus, positive AFP, lymphatic metastasis, and tumor diameter ≥2 cm (P<0.05), there were no statistically significant differences in PTEN expression, gender, age, HBsAg, and the number of tumor (P>0.05).
ConclusionsThe abnormal expressions of FN and PTEN in HCC tissues which may play a role in promoting or inhibiting occurrence, development, invasion, and metastasis of HCC. The abnormal expressions of both can be used as molecular biological markers for the malignant degree, invasion, and metastasis of HCC.
