Objective To investigate the mRNA expression of ciliary neurotrophic factor on the retina during injury and repair of optic nerves in rats. Methods Thirty-five healthy SD rats were randomly divided into 3 groups: 5 in the control group, 15 in the simply transected optic nerve group and 15 in the optic nerve-sciatic nerve anastomosis group. The simply transected and optic nerve-sciatic nerve anastomosed models were set up, and the retinal tissues of all of the rats were taken out after 3, 7 and 14 days, respectively; and the mRNA expression of CNTF in the 3 groups were observed by semiquantitative reversal transcription-polymerase chain reaction method. Results A minimum expression of CNTF mRNA was found in the retinae of the control group, and the increased rates of expression were found in the retinae of the simple transection of optic nerve group with the increase rate of 100%, 594%, and 485% on the 3rd, 7th, and 14th day respectively after the operation, while in optic nerve-sciatic nerve anastomosis group, the increase rates were found to be 258%, 752% and 515% on the 3rd, 7th, and 14th day respectively after the operation. Conclusion Retinal neurons can respond to axonal reaction of retinal ganglion cells by up-regulate endogenous CNTF after the injury of the optic nerves, which may provide a theoretic base for the application of the exogenous CNTF. (Chin J Ocul Fundus Dis，2004，20：355-357)
Objective To investigate the expression of T cell receptor (TCR) Vβ8.3 gene on CD4+ T lymphocytes in the rats with experimental autoimmune uveoretinitis (EAU). Methods Eighteen Lewis rats were divided into EAU, complete Freund′s adjuvant, and the control group. Inter photoreceptor retinoid-binding protein (IRBP) R16 peptide was synthesized using Fmoc procedure for induction of EAU. Magnetic absorption cell sorting (MACS) me thod was used to isolate the CD4+T lymphocytes from the spleen of the rats. Flow cytometry was used to monitor the efficiency of isolation. The expression of TCR Vβ8.3 gene segment on CD4+T lymphocytes was determined by fluorescent quantitative polymerase chain reaction. Results EAU was successfully induced in the Lewis rats immunized with IRBP R16 peptide. The proportion of CD4+T lymphocytes isolated by means of MACS was statistically higher than that before isolation (P＜0.001). The expression of TCR Vβ8.3 gene segment on CD4+ T lymphocytes in EAU rats was significantly higher than that in the control (P＜0.05). Conclusions There is a predominant usage of antigen-specific TCR Vβ 8.3 gene in EAU rats induced by IR BP R16 peptide, which may serve as a target for immunotherapy of EAU. (Chin J Ocul Fundus Dis，2004，20：165-167)
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.
Objective To investigate the effect of the 8-bromum-cyclic adenosine monophosphate (8-Br-cAMP) on the telomerase activity and changes of cell cycle in retinoblastoma （RB） cells. Methods The cultured RB cells were divided into the experimental group (8-Br-cAMP) and control group. After cultured for 24, 48 and 72 hours in vitro, the telomerase activity of RB cells was detected by polymerase chain reaction enzyme-linked immunosorbent assay (PCR-ELISA) and the changes of cell cycle were detected by flow-cytometry. Results The difference of telomerase activity was significant between the experimental groups and control group (Plt;0.01). There was a negative correlation between the A value of absorbance and the time in the experimental groups (r=-0.778 9, F=33.936, Plt;0.01). The changes of the cell cycle were that the percentages increased in G1 phase and decreased in S phases. Conclusion 8-Br-cAMP may weaken telomerase activity, affect the cell cycle, and inhibit the proliferation of RB cells. (Chin J Ocul Fundus Dis，2004，20：358-360)
OBJECTIVE: To construct eukaryotic expression plasmid of mouse myogenic regulatory factor MyoD gene for further study on MyoD gene function in molecular regulatory mechanism in skeletal muscle repair. METHODS: The plasmids PEMMBC2 beta 5 containing full cDNA length of MyoD inserted in EcoRI restriction site, were first propagated in Escherichia coli DH5a, then extracted and purified with the Wizard Plus Minipreps DNA Purification System (Promega, USA). The coding sequence of MyoD in PEMMBC2 beta 5 was confirmed by agarose gel electrophoresis and DNA sequence analysis. After plasmids PEMMBC2 beta 5 and plasmids pcDNA3-neo were prepared by digestion with EcoRI, the MyoD cDNA fragment was inserted into EcoRI site in pcDNA3-neo eukaryotic expression vector, and pcDNA3/MyoD was formed. The pcDNA3/MyoD, digested with restriction enzymes, was found to contain the MyoD cDNA sequence by agarose gel electrophoresis analysis. RESULTS: The extracted and purified PEMMBC2 beta 5 contained the correct nucleotide sequence for the full length of MyoD cDNA fragment. The MyoD cDNA fragment had been inserted into the eukaryotic expression plasmid pcDNA3-neo, which formed the pcDNA3/MyoD. CONCLUSION: The pcDNA3/MyoD, a eukaryotic expression plasmid, for MyoD is constructed successfully.
ObjectiveTo establish 16HBE cell lines stably expressing glutathione S-transferase mu 5 (GSTM5) gene, and explore the mechanism of GSTM5 nuclear translocation. MethodsRecombinant lentiviral expression vector containing GSTM5 gene was constructed and lentivirus was produced. After lentivirus infection of 16HBE cells, 16HBE-GSTM5 cell lines were obtained by screening with puromycin. Expression of GSTM5 in different cells was examined by RT-qPCR and Western blot. The nuclear translocation of GSTM5 was observed by confocal laser scanning microscope, after the 16HBE-GSTM5 cell lines were treated with tumor necrosis factor-α (TNF-α; 10 ng/ml) for 0.5 hour. ResultsLentiviral expression plasmids, PLVX-puro-3*flag-SBP-GSTM5-C and PLVX-puro-GSTM5-SBP-3*flag-N, were constructed and lentiviral particles were successfully packed. After infected with lentivirus and screened by puromycin, two cell lines, 16HBE-GSTM5-SBP-3*flag-N and 16HBE-3*flag-SBP-GSTM5-C, were obtained. GSTM5 expression in these two cell lines was significantly higher compared with the control group and parental cells. After treated with TNF-α for 0.5 hour, the nuclear translocation of GSTM5 in 16HBE-GSTM5-SBP-3*flag-N was much more obviously than that in 16HBE-3*flag-SBP-GSTM5-C. ConclusionThe N-terminal region of GSTM5 is critical for nuclear translocation induced by TNF-α, which is mediated by a novel and non-classical nuclear localization signal.
Abstract In order to understand the expression change of epidermal growth factor (EGF) gene and its distribution in tissue duringwound healing, 12 Winstar rats were divided into 4 groups. In each side of the back of every rat, 4 wounds of 1.5cm×1.5cm in size were made. After 4,8,12,16 days, one group of rats was sacrificed and tissues from wound were collected. By DIG-labelled probe hybridization in situ technique, EGF gene mRNA was detected. It was shown that EGF gene expression was evident during the whole stage of woundhealing, and the peak was in 8th day. It suggested that the promotion of EGF gene expression may lead to wound healing earlier.
Objective To investigate the molecular mechanism of apoptosis in cultured human retinal pigment epithelial (RPE) cells. Methods The growth media of confluency human RPE cells were replaced with a daunoblastinacontaining one at a dose of 180mu;g/L,and the cells were incubated for 12 hr at 37℃.After incubation with the drug,the medium was withdrawn,fresh medium was added and incubation was carried out for an additional 24 hr.Apoptosis was monitored by light microscopy,enzyme linked immunosorbent assay(ELISA)and terminal deoxynucleotidyl transferase mediated biotin-dUTP nick-end labelling(TUNEL)staining.The expression of bax and bcl-2 were evaluated by immuncoytochemical staining with anti-human bcl-2 and bax antibodies. Results After the RPE cells treated with daunoblastina,shrinkage of cytoplasm and nucleus was identified.The ratio of nucleus to cytoplasm was increased.TUNEL staining showed that many cells were positive staining.The amount of apoptotic cells was directly proportional to the drug dose.The integral optical desity values for expression of bax inereased by 22.0%(Plt;0.05), and that of bcl-2 did not change significantly(Pgt;0.05). Conclusions During human RPE cell apoptosis induced by daunoblastina,overexpression of bax or low bcl-2/bax ratio were demonstrated.The results suggest that bax and bcl-2 gene expression could play a role in regulation of RPE cell apoptosis. (Chin J Ocul Fundus Dis, 1999, 15: 153-156)
Objective To search for the significant early diagnostic and prognostic indicators in cholangiocarcinoma. Methods Literatures about the variation of various kinds of genes in cholangiocarcinoma and their clinical significances were reviewed.Results There are several genes mutation, deletion and overexpression in cholangiocarcinoma, K-ras gene was most deeply investigated. Conclusion K-ras mutation detection may be of help in the early finding and prognosis of cholangiocarcinoma.
OBJECTIVE: To construct eukaryotic expression vector of rat myogenin gene for further study on its functions in skeletal muscle denervated atrophy and repair. METHODS: The cloning vectors (containing full length of myogenin cDNA and two restriction sites: Hind III and Xho I) were first cut by two restriction endonuclease: Hind III and Xho I, and the same as the eukaryotic expression vector; then, the myogenin cDNA and the digested vector were ligated by T4 DNA ligase, and recombinant eukaryotic expression vector was formed. Its length was certificated by agarose gel electrophoresis analysis, digestion with Hind III and Xho I, PCR; and the rightness of the myogenin cDNA sequence was confirmed by sequencing. RESULTS: The results of agarose gel electrophoresis analysis, digestion, and PCR confirmed the right length of inserted DNA, which was the same as the myogenin cDNA, and the sequencing result of pcDNA3-myogenin was identical with the reported. CONCLUSION: pcDNA3-myogenin a eukaryotic expression vector, is successfully constructed.