摘要:目的: 探讨激活转录因子(ATF1)在血管紧张素Ⅱ(AngⅡ)诱导血管平滑肌细胞(VSMCs)中NOX1基因表达增加的作用。 方法 :体外培养大鼠主动脉VSMCs,用荧光实时定量逆转录PCR(Realtime RTPCR)检测NOX1基因表达的量,Western Blot检测ATF1蛋白在AngⅡ的刺激是否引起NOX1基因的高表达并用RNA干扰(RNAi)技术转染VSMCs使ATF1基因沉默来观察NOX1的表达。 结果 :AngⅡ能够诱导 NOX1基因的表达增加以及增强ATF1的磷酸化及活性,ATF1基因沉默反过来可抑制AngⅡ诱导的NOX1基因表达的增加。 结论 :在大鼠的VSMCs中,ATF1是介导NOX1基因表达的一个必须的转录因子。Abstract: Objective: To detect the role of activating transcription factor (ATF1) involved in angiotensinⅡ(AngⅡ) stimulated NOX1 gene expression.Methods :Rat aortic vascellum smooth muscle cells(VSMCs) were cultured in vitro.Use Realtime RTPCR to measure the expression of NOX1 gene.Western Blot Analysis was carried out to test the activity of ATF1 protein. RNA interference was used and transfected into VSMCs to knockdown ATF1 gene expression, and then measured NOX1 gene expression.Results : AngⅡ stimulated NOX1 gene expression and phosphorylation of ATF1 Gene silencing of ATF1 attenuated the upregulation of NOX1 mRNA by AngⅡ. Conclusion :ATF1 is an essential transcription factor that mediates expression of NOX1 gene in VSMCs by AngⅡ.
Objective To investigate the effects of simvastatin on lung tissue in septic rats by observing the protein expression of nuclear factor kappa B ( NF-κB) and pathologic changes in lung tissue at different time points. Methods 90 healthy male Sprague-Dawley rats were randomly divided into three groups ( n =30 in each group) . All the rats received administration by caudal vein and capacity volume is 2 mL. The rats in the control group were treated with saline ( 2 mL) . The rats in the LPS group were treated with LPS ( 5 mg/kg ) . The rats in the simvastatin group were treated with LPS ( 5 mg/kg) and simvastatin ( 20 mg/kg) . Six rats in each group were killed randomly at 2, 4, 6, and 12 hours after the injection, and the right middle lobe of lung was taken out. Pathological changes of lung tissue wee investigated under light microscope. The expression of NF-κB in lung tissue was determined by immunohistochemistry ( IHC) method. Results Microscopic studies showed that there were not pathological changes in the lung tissue of rats in the control group. While in the LPS group, the alveolar spaces were narrowed and the alveolar wall were thickened. Furthermore, severe interstitial edema of lung and proliferation of epithelial cells were observed. In the simvastatin group, the degree of the infiltration of leukocytes and the lung interstitial edema were less severe than those in the simvastatin group. In the control group, the expression of NF-κB protein in most of lung tissue was negative. In the LPS group, the expression of NF-κB protein was detected at 2h, andreached the peak at 6h, then decreased at 12h. In the Simvastatin group, the NF-κB expression was significantly lower than that in the LPS group at all time points ( P lt; 0. 01) . Conclusion Simvastatin can ameliorate pathological lesions and decrease expression of NF-κB in lung tissue of septic rats.
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.
ObjectiveTo investigate the expression of transcription factor SOX11 in retinoblastoma (RB) and the relation with the genes and cellular pathways.MethodsA public data set gse59983 containing the full mRNA expression profile of cancer tissues in 76 RB patients was downloaded from the GEO Database at the National Center for Biotechnology Information. According to the expression of 15 marker genes, these genes were divided into cell cycle marker genome (group 1, 26 patients), vertebral photoreceptor marker genome (group 2, 4 patients) and rod photoreceptor marker gene (group 3, 46 patients). R2 bioinformatics platform (http://r2.amc.NL) was used to analyze the gse59983 public data set. The SOX11 expression in cancer tissues of patients in 3 groups were observed and the SOX11-related genes were identified. According to the gene correlation, the clustering heat map was drawn, and the related genes and pathways of SOX11 were preliminarily analyzed by Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis tool and Gene Annotation (GO) analysis method.ResultsSOX11 expression in cancer tissues of patients in group 1 was significantly higher than that of groups 2 and 3 (P＜0.001). SOX11 expression in cancer tissues of patients in group 3 was significantly higher than that in group 2 (P＜0.001). A total of 4524 genes significantly related to SOX11 expression were detected. Among them, there were 2139 positively correlated genes and 2385 negatively correlated genes. SOX11 and the 16 genes with the strongest correlation were screened and the clustering heat map was drawn. The clustering form of SOX11 and SOX11 significantly correlated genes was roughly the same as the original form. KEGG and GO analysis showed that SOX11 related genes were involved in regulating mitotic cell cycle, cell apoptosis, photoreceptor cell development, photoreceptor cell protection, etc.ConclusionThe expression of SOX11 in RB is significantly different in different types or periods. Its expression is significantly correlated with genes involved in the regulation of cell cycle.
Objective To explore the effect on apoptotic genes of pancreatic adenocarcinoma cell BxPC-3 from subcutaneous transplantation tumor in nude mice induced by 5-FU and sulfasalazine (SZ).Methods Changes of apoptosis-related genes 〔bcl-2, cyclinD1, Bax and NF-κB (p65)〕 in subcutaneous transplantation tumor treated by 5-FU, SZ alone or both at the levels of mRNA and protein were measured by RT-PCR and Western blot. Results NF-κB (p65) at mRNA relative content and protein expression in subcutaneously heterotopic transplantation tumor treated by 5-FU (7.5, 15 mg/kg), SZ (10, 20 mg/kg) alone or both showed significant difference, except for two subsets in SZ group, respectively, in comparison with each control group (P＜0.01). Meanwhile bcl-2 and cyclinD1 at the levels of mRNA and protein, and Bax protein level were significantly different from each control group (P＜0.01). The above-mentioned indexes were show obvious interaction of both by multiple factor analysis of variance. Conclusion Up-regulated level of Bax, down-regulated levels of bcl-2, cyclinD1 and NF-κB (p65) might be one of apoptotic mechanisms that SZ synergistically enhanced apoptotic effect on pancreatic adenocarcinoma cell BxPC-3 of subcutaneous transplantation tumor in nude mice induced by 5-FU.
Objective To detect the expression of forkhead box P3 （FOXP3 ）gene in esophageal squamous cell carcinoma(ESCC) and provide a new basis for immunotherapy of esophageal cancer. Methods Based on fluorescent TaqMan methodology, a realtime quantitative reverse transcription polymerase chain reaction (RT-PCR) for detecting the expression of FOXP3 was set up. In this method, a cloning vector pMD 18-T-FOXP3 was constructed as a standard plasmid. The specific expression of FOXP3 in 42 patients with ESCC and 30 healthy controls were measured by using GeneAmp 7500 Sequence Detection Systems. Results FOXP3 mRNA copy number in ESCC was significantly higher than that in healthy control tissue ［(72.20±23.10)×104copy/μg RNA vs.(0.68±0.34)×104 copy/μg RNA；Plt;0.05］. Conclusion A realtime quantitative RT-PCR method for detecting the expression of FOXP3 gene in ESCC has been successfully established. The expression level of FOXP3 is increased in ESCC compare with healthy controls.
Objective To explore the expressions of caudal-related homeodomain transcription factor-2 （CDX-2）and tumor suppressor gene KAI-1 in colon carcinoma tissues and to analyze their clinical significances. Methods Immu-nohistochemical SP method was used to detect the expressions of CDX-2 and KAI-1 in 50 cases of colon carcinoma tissues and corresponding adjacent tissues （from cancer tissue ≤2cm） and 25 cases of normal colon mucosa tissues. The relation-ships of the expressions of CDX-2 and KAI-1 to the clinicopathologic features were analyzed. Results ①The positive rates of CDX-2 expression and KAI-1 expression in the colon carcinoma tissues were significantly lower than those in the normal colon mucosa tissues 〔CDX-2：34% （17/50） versus 88% （22/25）， P＜0.05；KAI-1：30% （15/50） versus 92% （23/25）， P＜0.05〕 and adjacent tissues of colon carcinoma 〔CDX-2：34% （17/50） versus 54% （27/50）， P＜0.05；KAI-1：30% （15/50） versus 58% （29/50）， P＜0.05〕， which in the adjacent tissues of colon carcinoma were significantly lower than those in the normal colon mucosa tissues （P＜0.05）. ②The positive expressions of CDX-2 and KAI-1 were related to lymph node metastasis， depth of invasion， and degree of tumor differentiation （P＜0.05）. ③Spearman rank correl-ation analysis showed that the CDX-2 expression was positively correlated with KAI-1 expression （rs=0.544， P＜0.01）. Conclusions CDX-2 and KAI-1 may be closely related to the development， invasion， metastasis， and prognosis of colon carcinoma， the combination of CDX-2 and KAI-1 in evaluation of their function has a certain guiding significance in the treatment for colon carcinoma.
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)
ObjectiveTo investigate the predictive value of thyroid transcription factor-1 (TTF-1) in the treatment of advanced lung adenocarcinoma with different chemotherapy regimens.MethodsA total of 126 patients with advanced lung cancer were divided into three groups according to the chemotherapy regimen, namely a pemetrexed+nedaplatin group (PEM+NDP group), a pemetrexed+cisplatin/carboplatin group (PEM+DDP/CBP group) and a third-generation (3G) chemotherapy+cisplatin/carboplatin group (3G agent+DDP/CBP group). The predictive value of TTF-1 in the above three treatment regimens was analyzed. The patients were followed up by telephone or outpatient visit until April 2017.ResultsThere were no significant differences in disease control rate or objective response rate between the three different chemotherapy regimens (all P>0.05). The survival rate of PEM+NDP group was significantly higher than that of PEM+DDP/CBP group and 3G agent+DDP/CBP group (9.68%vs. 5.56% and 6.80%, both P<0.05). ECOG score and brain metastasis were independent risk factors for the prognosis of chemotherapy regimens. TTF-1 was an independent risk factor for PEM+NDP therapy.ConclusionTTF-1 is an independent risk factor for PEM+NDP chemotherapy, but not for 3G agent + DDP/CBP or PEM+DDP/CBP regimens.
Objective To observe the expression levels of nuclear factor kappa B （NF-κB）， vascular endothelial growth factor （VEGF）， and CD31 in portal vein and surrounding tissues of rats during the formation process of cavernoustransformation of portal vein （CTPV）， and try to search the relationship between NF-κB， VEGF， and the angiogenesisof portal areas， as well as the significance and the role of NF-κB and VEGF in the formation process of CTPV. Methods One hundred and ten Sprague-Dawley （SD） rats were randomly （random number method） divided into sham operation group and model group. The partial constriction operations on portal vein were performed in model rats with a blunt 21Gcaliber to establish CTPV animal models （model group）， while the exploratory operations on portal vein， not constriction，were performed in rats of sham operation group. All specimens （portal vein and surrounding tissues） were fixed in formalinand made into paraffin blocks. Each specimen was tested by immunohistochemistry for the expressions of NF-κB， VEGF， and CD31， then optical density （OD） of NF-κB expression and the mean integral optical density （IOD） of VEGF expressionwere measured by using Image Pro Plus 6.0 software， and microvessel density （MVD） was calculated under microscope. Results Nucleoplasm ratio of OD value of NF-κB， mean IOD value of VEGF， and MVD value in 1， 2， 3， 4， and 6 weeks after operation didn’t significantly differed from that of before operation in sham operation group （P＞0.05）， but higher at all time points after operation in model group （P＜0.01）. Compared with sham operation group， nucleoplasm ratio of OD value of NF-κB， mean IOD value of VEGF， and MVD value were significantly higher in 1， 2， 3， 4， and 6 weeks after operation in model group （P＜0.01）. NF-κB and VEGF， NF-κB and MVD， VEGF and MVD were positively correlated with each other （r=0.654 6，P＜0.01；r=0.620 7， P＜0.01；r=0.636 9， P＜0.01） in model group. Conclusion NF-κB and VEGF may relate to the formation of CTPV， and may involve in the angiogenesis.