ObjectiveTo study the role of survivin gene in the research work of tumor. MethodsLiteratures about the molecular structure, function, mechanism,distribution of survivin gene and its role in the treatment of tumor were reviewed.ResultsSurvivin was a new member of inhibitor of apoptosis protein family, expressed in almost all the human tumors independent to the expression of bcl2. The expression of survivin was significantly correlated with the poor prognosis of tumor patients. Survivin inhibited the apoptosis of tumor cells via inhibiting the activity of caspase3, the effector molecule of the apoptosis signal transduction pathway. Inhibition of the expression of survivin gene could block its effect of inhibiting apoptosis and consequently get a therapeutic effect of tumors.ConclusionSurvivin is commonly expressed in human tumor tissues. It can be identified as an important prognostic parameter of poor outcome and a new therapeutic target in cancer.
ObjectiveTo construct the human small interfering RNA (siRNA) lentiviral vector who targeting inhibitor of differentiation-1 (Id1) gene, and to detect its efficiency of gene silence for the HepG2 cells.
MethodsThe most effective RNA interference sequences was screened from 4 kinds of siRNA vectors targeting Id1 gene (included pCGSIL-GFP-Id1-1, pCGSIL-GFP-Id1-2, pCGSIL-GFP-Id1-3, and pCGSIL-GFP-Id1-4), who was transfected to 293T cells. The selected siRNA vector was used to build lentiviral vector (Id1-RNAi-LV) and then infected human HepG2 cells. Then the expression levels of Id1 mRNA and its protein were detected by the real time PCR and Western blot method respectively.
ResultsExpression level of Id1 protein in pCGSIL-GFP-Id1-4 group was lower than those of pCGSIL-GFP-Id1-1 group, pCGSIL-GFP-Id1-2 group, and pCGSIL-GFP-Id1-3 group (P < 0.05), who had the best efficiency of gene silence. The Id1-siRNA lentiviral vector (Id1-RNAi-LV) was successfully constructed by using pCGSIL-GFP-Id1-4. The titer of lentiviral was 2.0×109 TU/mL.results of real time-PCR and Western blot showed that, the expression levels of Id1 mRNA and its protein in HepG2 cells of Id1-RNAi-LV group were lower than those of blank control group and negative control group (P < 0.05).
ConclusionsThe specific lentiviral can constantly down-regulate the expression of Id1 gene.
Objective To investigate the expression changes of nuclear factor kappa B (NF-κB) and matrix metalloproteinase-9 (MMP-9) in the cultured hepatocellular carcinoma cells 9204 (HCC9204) transfected with inhibitory kappa B alpha（IκB-α)vector. Methods After pcDNA3-IκB-α vector and pcDNA3 were transfected into HCC9204 by lipofectamine method, Western-blot and RT-PCR analysis were used to detect the expressions of NF-κB and MMP-9. Migration and invasion of tumor cells were assayed by fundus membrane invaded by them. Results When pcDNA3-IκB-α was transfected into HCC9204, the expression of NF-κB was decreased at the protein level, and the expression of MMP-9 mRNA and the invision and metastasis ability of transfected cells were obviously decreased. Conclusion When the activity of NF-κB is inhibited, the ability of invasion and metastasis in HCC9204 cells decrease, which could be related to the decreased the expression of MMP-9.
【Abstract】Objective To investigate the effects of selective cyclooxygenase-2 (COX-2) inhibitor nimesulide on the proliferation of colon adenocarcinoma cells in vitro and the expression of matrix metalloproteinase-2 (MMP-2). Methods The human colon cancer cell lines HT-29 and HCT-116 were employed in the study, grouped as nimesulide group, DMSO control group and blank control group. After treatment with nimesulide, the inhibitory effect of nimesulide on the proliferation of cancer cells was quantified by MTT assay, and the expression of MMP-2 in the cells was detected by quantitative zymography. Results Nimesulide inhibited the proliferation of HT-29 and HCT-116 cells in time and dosedependent manners. The inhibitory effect on HT-29 cells was ber than that on HCT-116 cells. Nimesulide downregulated the MMP-2 expression in HT-29 cells, whereas the expression in HCT-116 cells remained unchanged. Conclusion Nimesulide can obviously inhibit the growth of colon cancer HT-29 cells with positive COX-2 protein, suggesting that nimesulide may downregulate the expression of MMP-2 by inhibiting the activity of COX-2.
Diabetic macular edema (DME) is one of the main reasons causing blindness in patients with diabetic retinopathy. In recent years, with the recognition of the pathogenic role of vascular endothelial growth factor (VEGF) in DME, many clinical trials of intravitreal injection of anti-VEGF drugs have been carried out at home and abroad, proving that it has significant effects in improving visual acuity and reducing macular edema, and has become the first-line treatment of DME. However, there are still many challenges in routine clinical application of anti-VEGF drugs, such as frequent injections, insensitivity to treatment, and it is unclear whether repeated injections will cause damage to retina. The pathophysiological process of DME is very complicated, in addition to VEGF, there are many inflammatory factors and growth factors involved. Clinical trials of long-acting anti-VEGF agents, drugs of other targets and gene therapy are also being carried out. It is believed that with the in-depth research and progress of clinical trials, the gradual application of anti-VEGF drugs, other drugs and therapy in clinical practice are just around the corner, which is expected to provide more convenient and effective treatments for DME patients in the future.
Objective To review researches of the role of inhibitorof differentiation 2(Id2) in skeletal muscle regeneration. Methods The latest original literature concerning Id2 and its role in skeletal muscle regeneration was extensively reviewed. Results Id2 could form heterodimers by combining with E protein to prevent myogenic regulatory factors (MRFs) forming heterodimers by combining with E protein, to inhibit the transcription activity of MRFs anddifferentiation of skeletal muscle cell. Conclusion Id2 plays an important role in skeletal muscle regeneration.
Objective To study the effect of p38MAPK activity on tumor necrosis factor-α （TNF-α） mRNA and intercellular adhesion molecule 1 （ICAM1） mRNA expressions of isolated rabbit liver during early stage of cold preservation and reperfusion period. Methods Based on the cold preservation and reperfusion model of isolated rabbit liver, the animals were divided into inhibition group （n=12） with 3 μmol/L SB202190 (p38MAPK specificity inhibitor) in perfusate and control group （n=12） with no SB202190 in perfusate. Liver tissue samples were harvested at the time points of before resection, end of cold preservation, and different reperfusion period (10, 30, 60 and 120 min). Protein expression and activity of p38MAPK were detected by Western blot and immunoprecipitation respectively, expression of TNF-α mRNA was detected by RT-PCR, and expression of ICAM1 mRNA was detected by in situ hybridization. Results There was no obvious change of expression of p38MAPK protein in liver tissue both in two groups during the total period （P＞0.05）， and there was no statistically significant difference between two groups （P＞0.05）. At time points of end of cold preservation, 10, 30 and 60 min of reperfusion, the activity of p38MAPK in control group was significantly higher than that at the time points of before resection and 120 min of reperfusion (P＜0.01), and was also significantly higher than that in inhibition group at the same time points (P＜0.01). There was no significant difference in activity of p38MAPK among all time points in inhibition group (P＞0.05). The expressions of TNF-α mRNA and ICAM1 mRNA at the time points of before resection, end of cold preservation, and 10 and 30 min of reperfusion were significantly lower than those in 60 and 120 min of reperfusion in both two groups (P＜0.05, P＜0.01); The expressions of TNF-α mRNA and ICAM1 mRNA in inhibition group were significantly lower than those in control group at the time points of 60 and 120 min of reperfusion (P＜0.01). The activity of p38MAPK of liver tissue during cold preservation and reperfusion period was significantly correlated with the level of TNF-α mRNA and level of ICAM1 mRNA expression （r=0.996, P＜0.01； r=0.985, P＜0.01）. Conclusions These results suggest that p38MAPK pathway may regulate the expressions of TNF-α and ICAM1 at the level of transcription and the activation of p38MAPK can up-regulate TNF-α and ICAM1 expressions, which may be one of the important mechanisms to cause ischemia-reperfusion injury of isolated liver during cold preservation and reperfusion period.
ObjectiveTo detecte the expressions of phosphatase and tensin homolog deleted on chromosome ten (PTEN) gene and protein in peripheral blood mononuclear cells (PBMC) of patients with sepsis and to explore its role in the pathogenesis of acute obstructive suppurative cholangitis (AOSC).
MethodsThe PBMC and serum were separated from AOSC patients (n=25) before treatment and in 1 week after recure, and healthy volunteers (normal control group, n=15). The serum levels of lipopolysaccharide (LPS), tumor necrosis factor-α (TNF-α) and interleukin 10 (IL-10) were detected by enzyme linked immunosorbent assay (ELISA) methods. The mRNA and protein expression levels of PTEN, nuclear fator κB P65 (NF-κB), and inhibitor of NF-κB (IκB) were detected by real-time quantitative polymerase chain reaction (qRT-PCR) and Western blot, respectively.
ResultsThe levels of LPS, TNF-α, and IL-10 before treatment were significantly higher than those of normal control group (P < 0.05), the indicators were significantly decreased and close to normal levels in 1 week after recure. The mRNA and protein expression levels of PTEN and IκB before treatment were lower than those of normal control group (P < 0.05), NF-κB P65 was higher than those of normal control group (P < 0.05), while the phosphorylation levels of PTEN and IκB were higher than those normal control group (P < 0.05), and in 1 week after recure, the above indicators returned to normal levels.
ConclusionsSepsis shift may be associated with the occurrence of intestinal LPS, and caused the imbalance between proinflammatory and anti-inflammatory cytokines in the body. PTEN for phosphorylation activation of IκB or directly activation of NF-κB participate the originating process of sepsis, hinting a therapeutic potentialities in the early stage of sepsis.
ObjectiveTo study the effect of inhibitor of differentiation 1 (Id1) gene transfection on bone morphogenetic protein 2 (BMP-2) promoting the expressions of collagen type Ⅱ (COL Ⅱ) and aggrecan (ACAN) in intervertebral cartilage endplate cells (EPCs).
MethodsEPCs were harvested from the New Zealand white rabbits, the 2nd generation EPCs were used for experiment. The transfection efficiency of green fluorescent protein blank lentivirus, high expression of Id1 lentivirus, RNA interference (RNAi) Id1 lentivirus transfection in the EPCs were observed by the fluorescence microscopy, real-time fluorescence quantitative PCR, and Western blot. Blank vector, single BMP-2 gene, BMP-2 and Id1 genes were transfected into EPCs, respectively. The cell morphology and the expressions of COL Ⅱ and ACAN in each group were observed.
ResultsLentiviral transfection had no significant effect on the cell morphology. The EPCs were effectively transfected by the high expression Id1 lentivirus and RNAi Id1 lentivirus; the expression of Id1 mRNA was also significantly interfered. The expressions of COL Ⅱ and ACAN mRNA and synthesis of COL Ⅱ and ACAN protein were significantly higher in BMP-2 lentivirus and high expression Id1 lentivirus groups than control group (P<0.05). The expression of COL Ⅱ and ACAN protein were down regulated in the cartilage endplate cells when the expression of Id1 gene was decreased (P<0.05).
ConclusionUp-regulation of Id1 gene expression can enhance the effects of BMP-2 on the synthesis of COL Ⅱ and ACAN in EPCs.
ObjectiveTo observe RNA-Seq analysis of gene expression profiling in human retinal vascular endothelial cells after anti-vascular endothecial growth factor (VEGF) treatment.MethodsCultured the retinal vascular endothelial cells in vitro and logarithmic growth phase cells were used for experiments. The cells were divided into VEGF group and VEGF combined with anti-VEGF drugs group. The VEGF group cells were treated with 50 ng/ml VEGF for 72 h to simulate the high VEGF survival conditions of vascular endothelial cells in diabetic retinopathy. VEGF combined with anti-VEGF drug group cells was treated with 50 ng/ml VEGF and 2.5 μg/ml anti-VEGF drugs for 72 h to imitate the microenvironment of cells following the anti-VEGF drugs treatment, and whole transcriptome sequencing approach was applied to the above two groups of cells through RNA-Seq. Now with biological big data obtained as a basis, to analyze the differentially expressed genes (DEGs). And through enrichment analysis to explain the differential functions of DEGs and their signal pathways.ResultsThe gene expression profiles of the two groups of cells were obtained. Through analysis, 328 DEGs were found, including 194 upregulated and 133 downregulated ones. The functions of DEGs were influenced by regulations over molecular biological process, cellular energy metabolism and protein synthesis, etc. Among these genes, SI,PRX and HPGD were related to protein synthesis, BIRCT to cellular apoptosis, and ABLIM1 and CRB2 to retinal development, and ABCG1, ABCA9 and ABCA12 were associated with the cholesterol of macrophage and the transfer of phospholipid. GO enrichment analysis showed that DEGs mainly act in three ways: regulating biological behavior, organizing cellular component and performing molecular function. Pathway enrichment analysis showed that gene expressions of the two cell groups were differentiated in ECM receptor pathway, and Notch, mitogen-activated protein kinase, transforming growth factor (TGF)-β and Wnt signal pathways. Among them, the gene expression in TGF-β signal pathway attracts most attention, where the DEGs, such as CAMK2B, COL3A1, CYGB, PTGER2 and HS6ST2, among others, were closely related to fibrosis process.ConclusionThe anti-VEGF drugs may enhance the expression of CAMK2B, COL3A1, CYGB, PTGER2 and others genes related to TGF-β signal pathway and aggravate retinal fibrosis disease.
