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 construct the lentiviral vector containing homo sapiens forkhead box C2 (Foxc2) gene and to detect its expression in bone marrow mesenchymal stem cells (BMSCs) of rabbits. Methods Human Foxc2 gene coding region fragment was obtained by RT-PCR and then cloned into the plasmid of LV-green fluorescent protein (GFP) to prepare Foxc2 lentiviral plasmid. Foxc2 lentiviral plasmid, pGC-LV, pHelper1.0, and pHelper2.0 were co-transfected into 293T cells to obtain recombinant virus containing Foxc2 gene. The lentiviral titer was detected. BMSCs were isolated from bone marrow of rabbit and infected with Foxc2 recombined lentiviral, then the optimum multiplicity of infection (MOI) was determined by detecting the intensity of fluorescence expression. The expression of Foxc2 in the infected BMSCs was determined at 1, 3, and 7 days after transfection by inverted fluorescence microscope and Western blot. After osteogenic induction, Alizarin red staining was done to observe the formation of mineralized nodule. Results The Foxc2 recombinant lentiviral vector was constructed and was confirmed by restriction enzyme digestion and sequencing analysis. It could efficiently transfect 293T cells and express in 293T cells. The lentiviral titer was 2 × 108 TU/mL. The optimum MOI was 200. The inverted fluorescence microscope observation showed that the Foxc2 gene expressed in 84.5% ± 4.8% of infected BMSCs at 3 days after transfection. The expression of Foxc2 in infected BMSCs was stable and high, and increased gradually within 7 days after transfection by Western blot. At 2 weeks after osteogenic induction, Alizarin red staining showed that there were a large number of red calcified matrix deposition in the cytoplasm. Conclusion Foxc2 recombined lentivirus with high viral titer is successfully constructed and packaged, and the Foxc2 gene can be transfected into BMSCs with stable and high expression of Foxc2 in infected cells, and these cells may be applied for gene therapy of avascular necrosis of the femoral head.
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 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 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.
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.
Diabetic retinopathy (DR), a neurovascular complication of diabetes, presents a multifaceted pathogenesis that encompasses numerous biological processes and molecular mechanisms. Endoplasmic reticulum stress (ERS) plays a critical role in the maintenance of cellular homeostasis, and diabetic neuro-microangiopathy is driven by high glucose, which activated ERS through the promotion of protein misfolding, oxidative stress, and disturbances in calcium homeostasis. ERS activates the unfolded protein response, thereby influencing the onset and progression of DR through modulating mitochondria-associated endoplasmic reticulum membranes, autophagy, apoptosis, microvascular function, oxidative stress, and inflammation pathways. Currently, the principal interventions against ERS comprise the modulation of the ERS signalling axis and its interactions with associated pathological processes such as autophagy, oxidative stress, and inflammation, through pharmacological and molecular mechanisms. These interventions are directly or indirectly shown to inhibit persistent ERS and are demonstrated to ameliorate DR. With the in-depth study of ERS and the research and development of various drugs for ERS, it is expected to bring novel insights and strategies for DR management in the future.
目的 探讨主要组织相容性复合体(MHC)Ⅱ类基因的反式转录因子（CⅡTA）在多器官功能障碍综合征（MODS）中对MHCⅡ类基因的调控机理。方法 18只雄性家猪随机分为实验组（n=9）和对照组(n=9)。实验组给予失血性休克、再灌注损伤、内毒素血症等复合干扰因素，建立二次打击猪MODS模型； 对照组仅进行麻醉和动静脉插管。7 d后处死存活动物。切取实验组造模成功动物和对照组动物的脾脏组织，用Trizol法提取总RNA。设计CⅡTA和猪MHCⅡ类基因(SLA-DQA)引物序列，逆转录构建cDNA，行实时荧光定量PCR检测。UVP计算机图像分析系统绘出标准曲线并得出2组CⅡTA mRNA和SLA-DQA mRNA的拷贝数。以Pearson法分析MODS动物CⅡTA mRNA和SLA-DQA mRNA表达的相关性。结果 实验组动物死亡7例，有8例发生MODS。对照组动物CⅡTA mRNA的拷贝数为（3.516±1.237）×103，实验组MODS动物为（0.367±0.088）×103，差异有统计学意义（P=0.000）； 对照组SLA-DQA mRNA拷贝数为（5.330±3.053）×103，实验组为（1.376±1.006）×103，差异亦有统计学意义(P=0.002)。MODS动物中CⅡTA mRNA和SLA-DQA mRNA的表达呈正相关(Pearson值为0.499，P=0.017)。结论 MODS模型复制满意。MHCⅡ类基因在MODS中表达下降与CⅡTA的调控有关。
ObjectiveTo observe the effect of lncRNA-metastasis-associated lung adenocarcinoma transcript 1(MALAT1)on colorectal cancer cells-induced angiogenesis, and explore the potential underlying mechanism. MethodsMALAT1 was overexpressed in colorectal cancer cells SW48 by plasmids transfection, then SW48 cells were cultured at normoxia or hypoxia conditions. The culture media was collected, and the concentration of vascular endothelial growth factor (VEGF) in the media was measured by the enzyme-linked immuno sorbent assay (ELISA), and the human umbilical vein endothelial cells (HUVEC) were incubated with the media collected above. Meanwhile, the expression of hypoxia-inducible factor-1α(HIF-1α) in SW48 cells was detected by western blot. ResultsOverexpression of MALAT1 increased the VEGF level in the culture media, normoxia:the MALAT1 group (514±32) mg/L vs. the control group (110±14) mg/L, P < 0.05; hypoxia:the MALAT1 group (928±18) mg/L vs. the control group (230±21) mg/L, P < 0.05. Meanwhile, the tube formation activity of HUVEC was enhanced, and the expression of HIF-1αwas elevated in the MALAT1 group by western blot. ConclusionOverexpression of MALAT1 could promote colorectal cancer cells-mediated angiogenesis, it may be developed as a new drug target for colorectal cancer treatment.