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141.
AIM To investigate the mechanism of long noncoding RNA (lncRNA) FEZF1-AS1 regulating microRNA-363-3p (miR-363-3p) on the viability and apoptosis of lipopolysaocharide (LPS)-induced vascular endothelial cells. METHODS Human umbilical vein endothelial cells (HUVECs) were cultured in vitro . pcDNA-NC, pcDNA-FEZF1-AS1, anti-miR-NC, anti-miR-363-3p, miR-NC and miR-363-3p mimics were transfected into the HUVECs and LPS stimulation was applied for 24 h. RT-qPCR was used to detect the expression of FEZF1-AS1 and miR-363-3p. The cell viability was measured by MTT assay. The apoptotic rate was analyzed by flow cytometry. The dual-luciferase reporter experiment was used to verify the targeted regulation of FEZF1-AS1 and miR-363-3p. Western blot was used to determined the expression of cyclin D1, Ki67 and cleaved caspase-3. RESULTS Compared with control group, the expression level of FEZF1-AS1 in LPS group was significantly reduced (P <0.05), and the expression level of miR-363-3p was significantly increased (P <0.05). Compared with pcDNA-NC+LPS group, the cell viability in pcDNA-FEZF1-AS1+LPS group was significantly increased (P <0.05), the apoptotic rate was significantly reduced (P <0.05), the protein levels of cyclin D1 and Ki67 were significantly increased (P <0.05), and the protein level of cleaved caspase-3 was significantly reduced (P <0.05). Compared with anti-miR-NC+LPS group, the cell viability in anti-miR-363-3p+LPS group was significantly increased (P <0.05), the apoptotic rate was significantly reduced (P <0.05), the protein levels of cyclin D1 and Ki67 were significantly increased (P <0.05), and the protein level of cleaved caspase-3 was significantly reduced (P <0.05). Dual-luciferase reporter experiment confirmed that FEZF1-AS1 targeted miR-363-3p. Compared with miR-NC+pcDNA-FEZF1-AS1+LPS group, the cell viability in miR-363-3p+pcDNA-FEZF1-AS1+LPS group was significantly reduced (P <0.05), the apoptotic rate was significantly increased (P <0.05), the protein levels of cyclin D1 and Ki67 were significantly reduced (P <0.05), and the protein level of cleaved caspase-3 was significantly increased (P <0.05). CONCLUSION Over-expression of FEZF1-AS1 promotes the viability and inhibits apoptosis of LPS induced vascular endothelial cells by inhibiting the expression of miR-363-3p. 相似文献
142.
SONG Guo-bin XI Guo-ping LI Yan-hua LI Jia-shan LIU Jian-chun CHAI Zhi XIAO Bao-guo ZHANG Guang-xian MA Cun-gen 《园艺学报》2017,33(12):2113-2120
AIM: To explore the neuroprotective effect of fasudil combined with bone marrow-derived neural stem cells (BM-NSCs) on the mice with experimental autoimmune encephalomyelitis (EAE). METHODS: Female C57BL/6 mice (8~10 weeks old, n=32) were immunized with myelin oligodendrocyte glycoprotein 35-55 (MOG35-55) to establish chronic EAE model. The mice were randomly divided into control (ddH2O) group, fasudil group, BM-NSCs group, and fasudil+BM-NSCs group. The clinical score and body weight were recorded every other day. The expression of neurotrophic factors was determined by immunofluorescence staining. RESULTS: In comparison with ddH2O group, fasudil combined with BM-NSCs delayed onset and ameliorated severity of EAE. The numbers of brain-derived neurotrophic factor, glial cell-derived neurotrophic factor, nerve growth factor, neurotrophin-3 and ciliary neurotrophic factor positive cells in fasudil group, BM-NSCs group and fasudil+BM-NSCs group were all increased in various extents. In particularly, the expression of these neurotrophic factors in fasudil+BM-NSCs group was significantly higher than that in the mice treated with fasudil or BM-NSCs alone (P<0.01). CONCLUSION: Fasudil combined with BM-NSCs promotes the expression of neurotrophic factors and improves microenvironment of central nervous system, thus playing a positive role in neural restoration and regeneration through a synergistic and superimposed effect. 相似文献
143.
XIA Zi-rong LI Qing XIA Zhen LI Ju-xiang HONG Kui WU Yan-qing WU Qin-hua CHENG Xiao-shu 《园艺学报》2017,33(12):2238-2244
AIM: To investigate the effects of xeroderma pigmentosum group D (XPD) gene on the proliferation of human umbilical arterial smooth muscle cells (HUASMCs) induced by oxidized low-density lipoprotein (Ox-LDL). METHODS: The recombinant plasmid pEGFP-N2/XPD was transfected into HUASMCs by liposome. The cells were divided into blank control group, pEGFP-N2 group, pEGFP-N2/XPD group, Ox-LDL group, Ox-LDL+pEGFP-N2 group and Ox-LDL+pEGFP-N2/XPD group. The proliferation rate of the cells was detected by MTT and EdU assays. The apoptotic rate and cell cycle distribution were analyzed by flow cytometry. The protein levels of XPD, caspase-3, Bcl-2 and Bax were determined by Western blot. RESULTS: Compared with blank control group, the expression of XPD was increased in pEGFP-N2/XPD group (P<0.05). According to the results of MTT and EdU assays, the cell proliferation in pEGFP-N2/XPD group was reduced compared with blank control group (P<0.05). Compared with Ox-LDL group, the cell proliferation in Ox-LDL+pEGFP-N2/XPD group was significantly inhibited (P<0.05). According to the results of flow cytometry, the cell proportion of S phase decreased and the G0/G1-phase cell proportion increased significantly in pEGFP-N2/XPD group and Ox-LDL+pEGFP-N2/XPD group compared with blank control group and Ox-LDL group, repectively (P<0.05). Compared with blank control group and Ox-LDL group, the protein level of Bcl-2 decreased and the protein levels of Bax and cleaved caspase-3 increased in pEGFP-N2/XPD group and Ox-LDL+pEGFP-N2/XPD group, respectively (P<0.05). CONCLUSION: XPD inhibits the proliferation of HUASMCs and promotes their apoptosis, and reduces the promoting effect of Ox-LDL on the proliferation of HUVSMCs. XPD may be the target for treatment of atherosclerosis. 相似文献
144.
MicroRNAs (miRNAs)are a class of non-coding, endogenous, single-stranded small RNA molecules composed of 19~25 nucleotides. miRNAs are widely involved in the process of human life activities. Recent studies have shown that part of miRNAs regulate the vascular endothelial function and angiogenesis. High expression of miRNA-21 is found to play important roles in the cell proliferation, cell apoptosis, cell growth and death of vascular endothelial cells. This review will focus on the recent progress related to miRNAs in vascular endothelial function and angiogenesis, providing a new insight in cardiovascular disease prevention, clinical diagnosis, prognosis and target therapeutics. 相似文献
145.
AIM: To establish a method for obtaining specific cells in solid tumor tissue by sorting of CD11b+ myeloid cells in hepatic metastases from colorectal cancer.METHODS: Tumor tissues were prepared into single cell suspension by mechanical method combined with enzyme digestion, and then the CD11b+ myeloid cells were isolated by flow cytometry. The sorted cells were identified by immunocytochemistry. The viability and morphologiy of the sorted cells were evaluated by Giemsa and Typan blue staining. The cell purity was evaluated by flow cytometry.RESULTS: Sufficient numbers of CD11b+ cells with high purity were isolated by sorting with flow cytometry from the single cell suspension prepared by mechanical and enzyme digestion. The purity of the cells was confirmed by statistical analysis (P<0.05). The positive rates of the cells before and after sorting were significantly different (P<0.01). The positive cells were verified by immunocytochemical method. Meanwhile, the sorted cells had complete morphology and good activity.CONCLUSION: The CD11b+ myeloid cells in solid tumor tissue can be isolated by flow cytometry from the machine-enzyme digestion suspension with high purity, good activity and complete morphology. 相似文献
146.
AIM: To explore the effect of poncirin on the growth of AGS gastric cancer cells and the underlying mechanism.METHODS: The effect of poncirin on AGS cell viability was measure by MTT assay. The cell cycle distribution and cell apoptosis were analyzed by flow cytometry. Nuclear staining with DAPI was used to reflect the morphological change of the AGS cells treated with poncirin. The protein levels of extrinsic apoptosis pathway-related proteins such as FasL, caspase-8, caspase-3 and PARP, and mitochondria-mediated intrinsic apoptosis pathway-associated proteins such as Bak, Bcl-xL, Bax and caspase-9 were determined by Western blot.RESULTS: Poncirin inhibited the viability of AGS gastric cancer cells in a time-and concentration-dependent manner (P<0.05). Poncirin induced accumulation of G1 DNA content and significantly increased total apoptosis in the AGS cells. Nuclear staining showed a dose-dependent increase in the number of apoptotic cells after treated with poncirin.The protein level of FasL was upregulated in a dose-dependent manner by treatment with poncirin. Poncirin significantly activated caspase-8 and caspase-3. Moreover, poncirin significantly induced the cleavage of PARP in a dose-dependent manner (P<0.05). In addition, the protein levels of Bcl-xL, Bax and Bak were unchanged after treated with different doses of poncirin. Furthermore, caspase-9 was not activated by poncirin treatment in the AGS cells.CONCLUSION: Poncirin has the anti-cancer effect via extrinsic apoptosis pathway to inhibit the growth of AGS gastric cancer cells, possibly making it a therapeutic agent for human gastric cancer treatment. 相似文献
147.
CHEN Xiao-yan DENG Chun-yu KUANG Su-juan YANG Hui RAO Fang SHAN Zhi-xin LIN Qiu-xiong JIANG Li 《园艺学报》2017,33(1):128-132
AIM: To investigate the primary culture method for coronary artery smooth muscle cells (CASMCs), and to establish the endoplasmic reticulum stress (ERS) model in CASMCs of SD rats. METHODS: CASMCs were cultured by tissue explant method. The morphological characteristics were observed under optical microscope. The marker proteins of CASMCs, including α-SMA and SM-MHC, were identified by immunofluorescence technique. The protein expression levels of BiP and CHOP, the marker molecules of ERS, were determined by Western blot. RESULTS: The spindle-shaped CASMCs climbed out from the edge of coronary artery tissues after 6 d, and formed the typical "hill and valley" growth pattern of CASMCs at 9~10 d. The result of immunofluorescence technique showed that α-SMA and SM-MHC were positively expressed. The results of Western blot showed that the protein expression of BiP and CHOP in TG (1 and 2 μmol/L) treatment groups was increased compared with control group. Compared with control group, the protein expression of BiP and CHOP was significantly increased after 1 μmol/L TG treatment for 24 and 48 h. CONCLUSION: CASMCs can be successfully cultured by tissue explant method. ERS model of CASMCs was established by 1 μmol/L TG treatment for 24 h. 相似文献
148.
AIM:To study the role of microRNA-219 (miR-219) in regulation of transforming growth factor-β receptor type 2 (TGFBR2) in renal fibrosis. METHODS:The renal fibrosis patients (n=70) were selected in this stu-dy, and 20 cases of healthy people were selected as control group. RT-qPCR was used to detect the expression of miR-219 in the serum of the patients with renal fibrosis and control group, and the expression of miR-219 in NRK49F cells after stimulation with angiotensin Ⅱ(AngⅡ) was detected. The protein expression of α-smooth muscle actin (α-SMA) in the NRK49F cells transfected with miR-219 mimics after stimulation with AngⅡ was determined by Western blot. The potential target gene TGFBR2 of miR-219 was screened and verified by the method of luciferase reporter gene. RT-qPCR and Western blot were used to detected the effect of miR-219 mimics on the expression of TGFBR2 at mRNA and protein levels, and the mRNA expression of α-SMA, connective tissue growth factor (CTGF), type I collagen α1 (COL1A1) and COL3A1 in the NRK49F cells was also detected, respectively. The unilateral ureteral occlusion (UUO) mouse model was established and the expression of miR-219 in the renal tissue was monitored. The morphological change of renal fibrosis was observed in the UUO mice after injection of miR-219, and the mRNA expression levels of COL1A1 and COL3A1 were detected. RESULTS:The expression level of miR-219 in the patients with renal fibrosis was significantly lower than that in control group, and the expression of miR-219 in the UUO mice was decreased significantly (P<0.01). The expression level of miR-219 was significantly decreased in the NRK49F cells after AngⅡ stimulation, and miR-219 mimics inhibited the protein expression of α-SMA(P<0.01). miR-219 mimics had a targeted regulatory effect on TGFBR2 gene, which inhibited the mRNA and protein expression of TGFBR2. miR-219 mimics inhibited the mRNA expression of α-SMA, CTGF, COL1A1 and COL3A1. miR-219 also down-regulated the mRNA expression of COL1A1 and COL3A1 in the UUO mice and inhibited the process of renal fibrosis. CONCLUSION:miR-219 inhibits the development of renal fibrosis by inhibiting the expression of TGFBR2, which may become a new target for the diagnosis and treatment of renal fibrosis. 相似文献
149.
AIM:To study the effect of C/EBP homologous protein (CHOP) on the apoptosis of renal tubular epithelial HK2 cells. METHODS:The serum mRNA levels of CHOP in the patients with acute kidney injury and healthy controls were detected by qPCR. In vitro, renal tubular epithelial HK2 cells were divided into control group, negative group (transfected with negative control siRNA), si-CHOP group (transfected with CHOP siRNA), and induced by transforming growth factor-β1 (TGF-β1). The viability of the cells was measured by MTT assay, and the apoptotic rate was analyzed by flow cytometry. The protein levels of nuclear antigen Ki-67, proliferating cell nuclear antigen (PCNA), caspase-3 and cleaved caspase-3 were determined by Western blot. RESULTS:Compared with the healthy controls, the serum mRNA levels of CHOP in the patients with acute kidney injury were increased significantly (P<0.05). Transfection with CHOP siRNA significantly decreased the expression of CHOP in the renal tubular epithelial HK2 cells (P<0.05). Knock-down of CHOP expression by siRNA significantly increased the viability of renal tubular epithelial HK2 cells (P<0.05), decreased the apoptotic rate (P<0.05), increased the expression of Ki-67 and PCNA (P<0.05), and down-regulated the protein level of cleaved caspase-3 (P<0.05). CONCLUSION:The serum mRNA levels of CHOP were increased in the patients with acute kidney injury. Knock-down of CHOP expression inhibits the apoptosis of renal tubular epithelial cells by regulating the expression of proliferation-and apoptosis-related proteins. 相似文献
150.
AIM: To investigate the role of microRNA-29b (miR-29b)-mediated TGF-β/Smad signaling pathway in the activation of hepatic stellate cells (HSC) and its effect on the progression of hepatic fibrosis in rats.METHODS: Hepatic liver fibrosis rat model was established, and its HSC were isolated. Normal rat HSC were also obtained and identified in vitro. RT-qPCR and Western blot were used to detect the alterations of miR-29b, TGF-β/Smad signaling pathway-related proteins and liver fibrosis marker proteins in the acquired cells. Finally, the direct targeting binding of miR-29b to TGF-β1 was identified by dual-luciferase reporter assay system.RESULTS: With the activation of HSC, the expression of miR-29b gradually decreased (P<0.01), while the expression of collagen type I and α-smooth muscle actin gradually increased (P<0.01). At the same time, the expression of Smad2/3/4 was significantly increased, and the expression of Smad7 was significantly decreased (P<0.01). Dual-luciferase reporter assay showed that miR-29b bound directly to "UCUCUCCGU" in the 3'UTR of TGF-β1, indicating that TGF-β1 was a downstream target gene of miR-29b.CONCLUSION: miR-29b may be involved in the inhibition of HSC activation and migration, thereby inhibiting the process of liver fibrosis. The biological function of miR-29b may be through the direct targeting of TGF-β1, thus regulating and inhibiting the TGF-β/Smad signaling pathway. 相似文献