Objective To review the research progress of heterotopic ossification (HO) pathogenesis.Methods Recent articles about HO including the risk factors and pathogenesis were reviewed and comprehensively analyzed. Results The pathogenesis of HO is not completely understood, but the extracellular factors, signaling pathways, and transcription factors in the pathogenesis of HO are understood deeply, such as bone morphogenic protein, Smad signaling, and core binding factor α1/runt-related transcription factor 2, which are probably involved in HO. Furthermore, some related microRNAs are also probably involved in HO. Conclusion The pathogenesis of HO should be further investigated so as to lay a foundation for preventing and treating HO.
ObjectiveTo determine the optimizing parameters in transfecting the SV-40-PED cells mediated by oligofectamine. Methods With a change of Decoy oligodeoxynucleotides(ODNs)/oligofectamine in ratio and the transfection time, the uptake rate and the mean fluorescence intensity of SP1 ODNs in the SV-40-PED cells were measured by flow cytometry to evaluate the transfection efficiencies. 4 μl oligofectamine with different concentrations of ODNs(2.5，5.0，7.5，10.0 and 12.5 μl) were put into 100 μl of DMEM without serum and antibiotics. the (SV-40-PED) cells were transfected after 20 min at room temperature. the final concentration of SP1 decay ODNs were 50，100，150，200 and 250 nmol/L. Transfection effieiency was detected at 26 h after transfection. The intracellular distribution ofSP1 ODNs was determined with a fluorescence microscope. The lactate dehydrogenase (LDH) activity in the supernatant was measured to assess the cytotoxicity.Results The uptake of SP1 ODNs into the SV-40-PED cells was significantly improved by oligofectamine. The cell appearance did not change much in the groups of 50, 100 and 150 nmol/L. In the groups of 200 and 250 nmol/L, the cell reverted after being shrinked and altered to round. At 26 h after the transfection, there was no marked change in the cell form at the concentration of 250 nmol/L. There was floatation at 48 and 72 h after the transfection. Under the fluorescence microscope, we observed fluorescent materials distributed in the cell nucleus in the successfully-transferred groups. We could see the nucleoli clearly in the groups of 200 nmol/L and 250 nmol/L. There was a ber fluorescence intensitywith a higher concentration and the fluorescent materials gathered at the cell nucleus. At the final concentration of 250 nmol/L, the LDH level was 137.12±3.92 U/L in the 72hgroup, which was significantly higher those that in the 26h group（49.61±17.13 U/L）and the 48h group（120.26±8.42 U/L）(Plt;0.01). At 26 h after the transfection, there were no statistical differences at the above LDHlevels in the different-concentration groups（Pgt;0.05）. Conclusion Transfection efficiency is the highest when the final concentration of the SP1 decoy ODNs is 250 nmol/L during the incubation of for 24 h in transfecting the SV-40-PED cells.
Objective To investigate the possible effects of phosphorylated signal transduction and activator of transcription3 (STAT3) in the formation of choroidal neovascuarization (CNV) induced by photocoagulation in rats. Methods The CNV model in rats induced by photocoagulation was established, and the expression of phosphorylated STAT3 at the early stage in CNV were observed by immunofluorescence. To set up the hypoxia model, the specific inhibitor of Janus kinase 2 (JAK2), AG490 was mixed into cell culture fluid and then cultured for 0,1 hour,3,6,12,and 24 hours.Retinal pigment epithelial (RPE) cells proliferation activity were detected by flow cytometry (FCM).the expression of hypoxiainducible factor (HIF)1α and vascular endothelial grow factor (VEGF) mRNA were detected by reverse transcriptase polymerase chain reaction (RT-PCR); the expression of HIF1α protein was detected by Western blot; the content of VEGF in the supernatant of cell culture fluid was measured by enzyme linked immunosorbent assay (ELISA). Results Phosphorylated STAT3 highly expressed in CNV areas in rats 3 days after the photocoagulation. The proliferation activity of human RPE cells under hypoxia condition significantly decreased after inhibition of JAK2/STAT3 signal transduction pathway (t=1.472, 3.566,2.391,6.420; P=0.054,0.038,0.042,0.016). The expression of HIF-1α and VEGF mRNA increased gradually with increasing time of hypoxia;while the expression of HIF1α and VEGF mRNA and the activation of HIF1α protein in cultured human RPE cells with the JAK/STAT3 signal transduction pathway blocked by AG490 were suppressed obviously under hypoxia condition (t=0.07,0.02,0.01, P<0.05); the content of VEGF in RPE cells supernatant decreased significantly (t=1.330,1.106,2.828,7.742,5.610,6.894; P=0.082,0.063,0.014,0.002,0.016,0.011). Conclusion STAT3 may be involved in CNV formation, which may partly dependent on JAK2/STAT3 signal transduction pathway regulating the expression of HIF-1α and VEGF in RPE cells.
Objective To investigate the expression of E26 transformation-specific-1（E26ts-1），matrix metalloproteinase-1（MMP-1）and tissue inhibitor of matrix metalloproteinase-1 （TIMP-1） in choroidal melanoma and the correlation with the tumorprime;s infiltration and metastasis. Methods Immunohistochemistry was used to detect the expression of E26ts-1，MMP-1 and TIMP-1 in 78 cases of choroidal melanoma who were divided into shuttle-cells，paraepithelial-cells and mixed-cells type according to the configuration of tumor cells.The patients were followed up and their average existing time was calculated.The results were statistically computed with statistic SPSS 10.0 package. Results In the 78 cases，shuttle-cells type was found in 21，paraepithelial-cells type in 34，and mixed-cells type in 23. Expression of TIMP-1was low in uveal melanoma，while expression of E26ts-1 and MMP-1 was obviously found in the three types of choroidal melanoma;the sequence of expression intensity was shuttle-cells，mixed-cells and paraepithelial-cells type.Among 37 cases who had been followed up，the shuttle-cells type was in 18 with the average existing time of （78.33plusmn;24.69）months，the mixed-cells type was in 10 with the average existing time of（61.44plusmn;20.46）months，and the paraepithelial-cells type was in 9 with the average existing time of（36.76plusmn;12.19）months.The existing time was negative correlated with the intensity of expresion of E26ts-1 and MMP-1. Conclusion The high expression of E26ts-1 and MMP-1and low expression of TIMP-1may relate to the choroidal melanomaprime;s infiltration and metastasis. (Chin J Ocul Fundus Dis, 2006, 22: 174-176)
Objective To investigate the inhibitive effect of E2F decoy oligodeoxynucleotides (E2F decoy ODNs) on cultured human retinal pigment epithelial (HRPE) cells.Methods E2F decoy ODNs or scramble decoy ODNs at varied concentrations were put into the HRPE cells mediated by lipofectamineTM2000. The proliferative activity of HRPE was detected by methythiazolyl-terazollium assay, and the competitive combinative activity of E2F decoy ODNs and transcription factor E2F was detected by electrophoresis mobility-shift assay. Results The proliferation of HRPE was inhibited markedly by E2F decoy ODNs at the concentration of 0.2 μmol/L (P=0.002) in a dose-dependent manner but not by scrambled decoy. The results of electrophoresis mobility-shift assay showed that the combinative activity of transcription factor E2F was abolished completely by E2F decoy ODNs. Conclusions E2F decoy ODNs may sequence-specifically inhibit the combinative activity of transcripti on factor E2F,and inhibit the proliferation of HRPE cells.(Chin J Ocul Fundus Dis，2004，20：182-185)
Objective To explore the expression and activation of transcription factor E2F1 in cultured human retinal pigment epithelium (RPE) cells. Methods Cultured human RPE cells were divided into two groups after synchronization: one was cultured in Dulbecco′s modified Eagle′s medium (DMEM) without serum; the other was cultured in DMEM supplemented with 20% serum of newborn calf. The expressions of E2F1 protein in two groups were detected by Western blot analysis. The E2F1-DNA binding activities were measured by gel mobility-shift assay(EMSA). Results E2F1 protein of 60 000 molecular weight was detected in the nuclear extract of human RPE cells, and serum stimulation could increase its expression（P＜0.001）. EMSA exhibited the increased binding activity of E2F1 in the serum-stimulated RPE cells with DNA. Conclusions E2F1 is expressed in the nuclei of human RPE cells. Serum stimulation can increase its protein expression as well as binding activity, so as to play a regulation role of gene transcription. (Chin J Ocul Fundus Dis, 2002, 18: 224-226)
Objective To investigate the expression of transcription factor e2f-1 in the different development stages of gastric cancer, the relationships between clinicopathologic characteristics and e2f-1 expression status, as well as its influences on the prognosis. Methods The operative samples from primary lesion of 121 patients who underwent radical resection for gastric cancer were detected by SABC immunohistochemical staining. The relationships of e2f-1 expression with clinicopathologic characteristics and with the prognosis were observed by univariate, multivariate and relative analyses. Results The total positive expression rate of e2f-1 in all patients was 38.8% （47/121）. With the advancement of gastric cancer, the level of e2f-1 expression in TNMⅠ-Ⅳ stage was gradually decreased (r=－0.320， Plt;0.05)： Ⅰa stage with 62.5% （10/16）， Ⅰb with 47.1% （8/17）， Ⅱwith 55.0% （11/20）， Ⅲa with 40.0% （8/20）， Ⅲb with 27.3% （6/22）， Ⅳ with 15.4% （4/26）. The expression of e2f-1 was significantly negative correlated with tumor diameter, depth of infiltration, lymph node metastasis ratio, and N stage （Plt;0.05）. The multivariate analysis revealed that either histology type, or survival time was respectively an independent factor for e2f-1 expression （Plt;0.05）. Log-Rank test showed the relative factors to survival included N stage, tumor diameter, tumor position, lymph node metastasis ratio, depth of infiltration, and TNM stage （Plt;0.05）. Cox survival analysis found that both of later N stage and e2f-1 higher expression were independent prognostic factors （Plt;0.05）. The higher e2f-1 expression was related to a poor survival in TNM stageⅠand Ⅱ patients （r=－0.304, Plt;0.05）, the prognosis of patients with e2f-1 positive expression was worse than that of patients with negative expression （χ2=13.437， Plt;0.05）, and there was no statistic relationship between the expression of e2f-1 and prognosis in stage Ⅲ and Ⅳ patients （Pgt;0.05）. Conclusions e2f-1, as a useful marker, seems to be an indication for the malignant behavior in relatively earlier gastric cancer, in which the e2f-1 positive expression shares a significantly poor survival. And the lower expression of e2f-1 has been identified in later advanced gastric cancer, the more malignances in advanced gastric cancer might associate with a lower expression of e2f-1.
Objective To elucidate the role of the transcription factor liver activator protein (LAP, a member of the C/EBP family) in the expression of α1(I) collagen gene in activated hepatic stellate cells (HSCs). Methods Rat HSCs were prepared from SD rats by in situ perfusion and singlestep density Nycodenz gradient. Two chimeric luciferase reporter gene plasmids containing the human collagen α1(I) gene promoter fragments (-804～+1 452 or -804～+222) were constructed. Culture-activated HSCs were co-transfected with the reporter gene contructs and mammalian vector expressing LAP using the cationic-liposome mediated method, and the promoter activity was determined by measuring luciferase activity. Results The luciferase reporter gene construct containing the first intron of α1(I) collagen gene (-804～+1 452, was called as PGL3-col) had a higher level of gene expression, as compared with the construct lacking the first intron 〔was called as PGL3-col (△intron)-in activated HSCs (315±45 U/mg protein vs 220±70 U/mg protein, Ｐ＜0.05). Transient transfection of the vector expressing LAP significantly increased basal transcription from PGL3-col and PGL3-col (△intron) reporter gene vectors (587±62 U/mg protein vs 315±45 U/mg protein and 326±52 U/mg protein vs 220±70 U/mg protein respectively, both Ｐ＜0.05). Conclusion The transcription factor LAP transactivates collagen α1(I) gene in activated HSCs, and the first intron is important for α1(I) collagen gene transcription activity in activated HSCs.
ObjectiveTo observe the effects of Th9 cell relative factors, including PU.1, interferon regulatory factor 4 (IRF4) and interleukin 4 (IL-4), in rats with pulmonary fibrosis. MethodsNinety SD rats were randomly divided into 3 groups, ie. a normal group, a pulmonary fibrosis group, and a dexamethasone treatment group, with 30 rats in each group. Ten rats in each group were sacrificed respectively on 7th, 14th, 28th days. Model rats were induced by injecting bleomycin into trachea. Real-time PCR was applied to detect mRNA expression of PU.1 and IRF4 in bronchoalveolar lavage fluid. IL-4 in the peripheral blood was measured by ELISA. ResultsIn the normal group, the lung tissue was normal without inflammatory reaction and fibrosis at any time points. In the pulmonary fibrosis group, at the early stage the lung tissue showed alveolar inflammation with a large number of macrophages and other inflammatory cells infiltratation in the pulmonary interstitial and alveolar cavity; on 14th day, part of the alveolar structure disappeared, inflammatory cells infiltrated slightly, while the alveolar septum was mildly widened and fibroblasts proliferated; on 28th day, alveolar structure was destructed, partial alveolar walls were collapsed, alveolar septuml was significantly widened, extracellular matrix was hyperplastic, a wide range of fibrosis occured. In the dexamethasone treatment group, the alveolar structure exsisted completely, and the inflammatory cell infiltration, widened alveolar septum and fibrosis were significantly lighter than those in the pulmonary fibrosis group. PU.1 mRNA was significantly lower in the pulmonary fibrosis group compared with the normal group. Compared with the pulmonary fibrosis group, PU.1 mRNA were lower on 14th day and 28th day in the dexamethasone treatment group (P < 0.05). PU.1 mRNA increased from 7th day, reached peak on 14th day, and declined on 28th day. IRF4 mRNA was significantly lower in the pulmonary fibrosis group compared with the normal group. Compared with the pulmonary fibrosis group, IRF4 mRNA was lower on 28th day in the dexamethasone treatment group (P < 0.05). There was a positive correlation between the content of IRF4 mRNA and IL-4 on 14th day in the pulmonary fibrosis group (r=0.044, P < 0.05). ConclusionPU.1 and IRF4 play a role in inflammation leading to pulmonary interstitial fibrosis, and IL-4 may regulate Th9 cells through activating IRF4.
ObjectiveTo lay a theoretical foundation for the research of regulation of Hyperpolarization activated cyclic nucleotide gated channel 1 (HCN1) gene expression and its involvement in the pathogenesis of Mesio-temporal lobe epilepsy (MTLE) and other related diseases, the bioinformatics methods were used to analyze sequence characteristic, transcription factors and their binding sites in the promoter region of human HCN1 gene, and the physicochemical properties, signal peptides, hydrophobicity, transmembrane regions, protein structure, interacting proteins and functions of HCN1 proteins.MethodBiological software and website, such as Protparam, Protscale, MHMM, SignalP 5.0, NetPhos 3.1, Swiss-Model, Promoter 2.0, AliBaba2.1 and EMBOSS were used to analyze and predict physicochemical properties, structural functions, localized expression, phylogenetic relationships and protein interactions with human HCN1 protein, and promoter, CpG island and transcription factor characteristics of HCN1 gene.ResultsThe evolutionary analysis of HCN1 protein showed that the genetic distance between human and Pongo abelii was the smallest, indicating the closest genetic relationship between human and Pongo abelii. Human HCN1 protein was an unstable hydrophilic protein located on the plasma membrane, which contained two transmembrane structure. However, the predicted results showed that there was no signal peptide and nuclear localization sequence in this protein. The secondary structure of HCN1 protein was mostly random coil and alpha helix, and it contained multiple potential phosphorylation sites. The ontology analysis results of HCN1 protein were showed as follows. The cellular component of HCN1 protein was located in the plasma membrane (GO:0005886); the molecular functionof HCN1 protein were cyclic adenosine monophosphate binding (GO:0030552) and voltage-gated ion channel activity (GO:0005244); the biological process of this protein were reacting to cAMP (GO:0071320) and transmembrane transport of potassium (GO:0071805). The analysis results of String database showed that the proteins that had close interaction with human HCN1 protein mainly included the ten proteins (HCN2, HCN4, PEX5L, MARCH7, KCTD3, GNAT3, SHKBP1, KCNQ2, FLNA and NEDD4L). These proteins were mainly involved in regulation of ion transport and transmembrane transport of potassium (GO:0071805). The HCN1 gene was located at 5p12 and contained 8 exons and 7 introns.There were at least three promoter regions in the nucleotide sequence of 2 000 bp from the upstream of the HCN1 gene to the 5 'flanks, and contained a 158 bp CpG island in the promoter region and one TATA boxes and one CAAT boxes in the 5' regulation region ofHCN1 gene; niceteen transcription factors, including NF-κB, NF-1, AP-1, TBP, IRF-1, c-Ets-1, Elf-1, HNF-3, HNF-1, YY1, GATA-1, RXR-α, GR, AP-2αA, ENKTF-1, C/EBPβ, C/EBPα, c-Fos and c-Jun, binding in the promoter region of the HCN1 gene were predicted by both softwares (AliBaba2.1 and PROMO2).ConclusionThe analysis results provide important information for further studies on the role of HCN1. Bioinformatics analysis of the promoter region can improve the research efficiency of gene promoters, and provide theoretical basis for subsequent experiments to construct expression vectors of HCN1 gene promoters and identify their functions.