Objective To investigate the effect and mechanism of epigallocatechin-3-gallate (EGCG) on restenosis of the vein graft. Methods Totally 90 Sprague-Dawley rats were randomly divided a the control group, a vein graft group and an EGCG+vein graft group. At week 1, 2 and 4, the intimal and tunica thickness of the venous graft wall was evaluated by hematoxylin-eosin staining, and the expression of Ki-67 was assessed by immunohistochemistry analysis, and then the expression of hairy and enhancer of split-1 (HES1) was measured by Western blot assay. Results At week 2, the intimal thickness (46.76±4.89 μmvs. 8.93±0.82 μm, 46.76±4.89 μmvs. 34.24±3.57 μm), tunica thickness (47.28±4.37vs. 16.33±1.52 μm, 47.28±4.37vs. 36.27±3.29 μm), positive cell rate of Ki-67 (21.59%±2.29%vs. 1.12%±0.22%, 21.59%±2.29%vs. 15.38%±1.30%), expression of HES1 respectively increased in the experimental group than those in the control group and the EGCG+vein graft group (P<0.05, respectively). At week 4, the intimal thickness (66.38±6.23 μmvs. 8.29±0.79 μm, 66.38±6.23 μmvs. 48.39±4.23 μm), tunica thickness (63.27±6.18 μmvs. 15.29±1.49 μm, 63.27±6.18 μmvs. 44.63±4.49 μm), positive cell rate of Ki-67 (33.19%±3.03%vs. 1.09%±0.19%, 33.19%±3.03%vs. 24.37%±2.73%), expression of HES1 increased in the experimental group than those in the control group and EGCG+vein graft group (P<0.05, respectively). Conclusion EGCG may inhibite restenosis of vein graft by inhibiting Notch signal pathway.
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.
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.
ObjectiveTo explore the relationship between nuclear factor κB (NFκB) and the occurrence, metastasis, and treatment of colon cancer. MethodsThe literature on the structure and the property of molecular biology of NFκB, the relationship between NFκB and apopotosis, malignant tumor and colon cancer were reviewed.ResultsNFκB had action of antiapopotosis. The occurrence of malignant tumor had close relation with the oncogene by NFκB, the metastasis of malignant tumor was that cell of cancer escaped the killing and supervising of immunity by NFκB. NFκB affected the occurrence and metastasis of colon cancer by regulating cmyc, Cox2, ICAM1.Conclusion NFκB has important action in the occurrence and metastasis of colon cancer. It will become a new target of treatment.
ObjectiveTo explore the mechanism of lung injury in Sprague-Dawley (SD) rats induced by acute organic phosphorus pesticides (AOPP) by observing the changes of the blood serum nuclear factor (NF)-κB consistence, NF-κB level of lung tissue and lung coefficient. MethodNinety-six healthy male SD rats (six weeks old) were randomly divided into group A (control, n=48) and group B (poison, n=48). The rats of group B were given omethoate by gavage (45 mg/kg), and the rats of group A accepted normal saline. Then the rats were killed at designated observing points (30 minutes; 3, 6, 12, 24, and 48 hours), and the lung coefficient, blood serum NF-κB consistence and NF-κB level of lung tissue were measured. At the same time, we observed the pathological changes of the rats' lung tissue. ResultsCompared with group A, blood serum NF-κB consistence, NF-κB level of lung tissue and the level of lung coefficient in group B were significantly higher (P<0.01). The lung tissues of group A were normal at each time point, but in group B, the lung pathological changes gradually appeared 30 minutes later with pulmonary interstitial engorging, alveolar septum widening and some alveolus being full of red blood cells, and this situation reached its peak at hour 12. Then it gradually mitigated from 24 to 48 hours. ConclusionThere are significant increases in blood serum NF-κB consistence and NF-κB level in lung tissues in rats with lung injury induced by omethoate poisoning. The NF-κB may play a role in the process of lung injury induced by organophosphorus pesticide.
ObjectiveTo investigate the expression of mitochondrial transcription factor A (TFAM) in colon cancer and the effect of its expression on proliferation of colon cancer cell. MethodsThirty cases of colon cancer in the First Affiliated Hospital of Sun Yat-sen University from March 2013 to April 2013 were studied. TFAM mRNA was detected both in colon cancer tissue and para-cancer tissue by real-time PCR. TFAM mRNA and protein were detected in normal colon cell strain and colon cancer strains SW480, HT-29, and HCT116 by real-time PCR and Western blot, respectively. The proliferation of SW480 cells was evaluated after up-regulating TFAM. ResultsThe expression of TFAM mRNA in the colon cancer tissue was significantly higher than that in the para-cancer tissue (P < 0.000 1). The expressions of TFAM mRNA were obviously increased in the SW480, HT-29, and HCT116 cells as compared with the normal colon cell strain (P value was 0.000 8, 0.002 3, and 0.000 6, respectively), among which the most notable increase was detected in the SW480 cells. The expressions of TFAM protein were obviously increased in the SW480, HT-29, and HCT116 cells as compared with the normal colon cell strain (P value was 0.000 2, 0.003 8, and 0.001 6, respectively), among which the most notable increase was detected in the SW480 cells. After up-regulating TFAM by plasmid transfection, the proliferation of the pcDNA3.1-TFAM-SW480 cell was increased significantly as compared with the pcDNA3.1-SW480 cell at 96 h and 120 h after transfection by the MTT test (P < 0.000 1). The proliferation of the pcDNA3.1-TFAM-SW480 cell was increased significantly as compared with the pcDNA3.1-SW480 cell at 48 h after transfection by the BrdU test (P < 0.001 0). ConclusionTFAM expression is high in colon cancer. Up-regulated TFAM could promote the proliferation of colon cancer cells.
目的：研究中药当归芍药散对外周血T淋巴细胞转录因子GATA-3和T-bet mRNA表达的影响，初步探讨其用于治疗不明原因反复自然流产的可能性。方法：体外分离提取11例志愿者外周血单核细胞，在含有或不含当归芍药散的培养液中培养24h，用实时定量PCR技术检测GATA-3和T-bet mRNA的表达。结果：用10 μg/mL浓度当归芍药散处理后,单核细胞中GATA-3 mRNA的含量与对照组比较显著增高（P＜0.05)。当归芍药散处理细胞后，T-bet mRNA的表达水平呈降低趋势，当浓度为100 μg/mL时，与对照组比较有显著性差异（P＜0.05)。结论：当归芍药散可上调转录因子GATA-3 mRNA的表达或下调T-bet mRNA的表达,从而可能通过调节Th2/Th1平衡向Th2偏移、对于Th1反应异常增强的不明原因反复自然流产有一定的治疗潜能。
ObjectiveTo investigate the regulatory effect of long chain non-coding RNA (lncRNA) metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) adsorbing microRNA-124 (miR-124) on osteogenic differentiation of mesenchymal stem cells (MSCs).MethodsC3H10T1/2 cells derived from mouse embryos were cultured in vitro, then randomly divided into control group (group A), lncRNA MALAT1 no-load plasmid group (group B), lncRNA MALAT1 overexpression plasmid group (group C), lncRNA MALAT1 small interfering RNA (siRNA) group (group D), and lncRNA MALAT1 siRNA negative control group (group E). The cells were transfected into plasmids and siRNA, then induced to differentiate into osteoblasts. Alkaline phosphatase (ALP) and alizarin red staining were used to detect the osteogenic differentiation of cells in each group, real-time fluorescence quantitative (qRT-PCR) analysis was used to detect the expressions of lncRNA MALAT, miR-124, and osteogenesis-related genes such as Runt-related transcription factor 2 (Runx2), osteopontin (OPN), and osteocalcin (OCN) in each group. Double luciferase reporter gene was used to detect the targeting regulation of lncRNA MALAT1 to miR-124.ResultsThe relative contents of ALP positive cells, mineralized nodule, and the relative mRNA expressions of lncRNA MALAT1, Runx2, OPN, and OCN in group C were significantly higher than those in other groups (P<0.05), while in group D significantly lower than in other groups (P<0.05); the relative expression of miR-124 in group C was significantly lower than that in other groups(P<0.05), while in group D significantly higher than in other groups (P<0.05). There was no significant difference in these indexes between groups A, B, and E (P>0.05). The results of double luciferase reporter gene assay showed that lncRNA MALAT1 targeting down-regulated the expression of miR-124.ConclusionLncRNA MALAT1 can targeting down-regulate the expression of miR-124 and promote the osteogenic differentiation of MSCs.
ObjectiveTo investigate the role of the forkhead/Fox transcription factor 2 (Foxc2) over-expression in regulating osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) by Wnt-β-catenin signaling pathways in vitro so as to provide the experimental basis for repairing osteonecrosis of the femoral head. MethodsThe recombinant lentivirus carrying green fluorescent protein (group A) or Foxc2 (group B) were used to transfect the fifth generation rabbit BMSCs, and untransfected BMSCs served as a control (group C). The cell viability was measured with water soluble tetrazolium-1 (WST-1) regent at 72 hours after transfection. After 2 weeks of transfection, the expression of β-catenin in BMSCs was detected by real time fluorescence quantitative PCR, Western blot, and immunofluorescence staining. Meanwhile, the β-catenin inhibitors XAV-939 (0, 0.1, and 1.0 μmol/L) was added in group B; at 2 weeks after osteogenic and adipogenic induction, the gene and protein expressions of collagen type I (COL I), osteocalcin (OCN), and peroxisome proliferator activated receptor gamma 2 (PPARγ-2) were detected by real time PCR and Western blot. ResultsWST-1 results showed that the cell viability of group B (130.85%±0.15%) was significantly higher than that of group A (100.45%±0.35%) (t=7.500, P=0.004) at 72 hours after transfection. At 2 weeks after transfection, the gene and protein expressions of β-catenin in group B were significantly higher than those in group A (P<0.01). After XAV-939 was added in group B, the mRNA and protein expressions of OCN and COL I gradually decreased; the mRNA and protein expressions of PPARγ-2 significantly increased (P<0.05), showing a dose-dependent manner. ConclusionThe over-expression of Foxc2 gene in BMSCs may promote osteogenic differentiation by Wnt-β-catenin signaling pathway.
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.