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OBJECTIVE(S): To investigate and test the hypotheses that FGF-2 enhanced myocardial differentiation with rat bone marrow mesenchymal stem cells (BMSCs). MATERIALS AND METHODS: Lentiviral vectors carrying the FGF-2 gene were transfected into rat BMSCs firstly. According to the different inducing agents, they were divided into the following four groups: group A (BMSCs blank control group), group B (FGF-2 induction group), group C (Lenti-FGF-2-GFP lentivirus transfection group), and the group D (Lenti-control-GFP lentiviral transfer). Then several kinds of experimental methods such as real-time PCR, immunocytochemical staining, immunofluorescence staining, Western blot, and transmission electron microscopy were used to elucidate the effects by which FGF-2 adjusts myocardial differentiation in rat BMSCs. RESULTS: The results of real-time PCR showed that GATA-4 and Nkx2.5 were expressed in all groups of cells. Compared with the experimental control group, the expression of GATA-4 and Nkx2.5 genes was the strongest after induction of 2 weeks in each induction group, and gradually decreased after induction of 4 weeks. Among them, the relative expression levels of GATA-4 and Nkx2.5 genes in Lenti-FGF-2-GFP were highest at all time points. The expressions of cTnI, cTnT, Cx43, and Desmin were detected by immunocytochemical staining and immunofluorescence staining. After 4 weeks of induction, cTnI, cTnT, Cx43, and Desmin were positively expressed in the cytoplasm of cells. Statistical analysis showed that the integrated optical density (IOD) values of the markers in the Lenti-FGF-2-GFP were the strongest. Cx43 and cTnI were weakly positive or negative in the experimental control group. There was a significant difference in the positive expression of each marker in each induction group and the experimental control group. Western blot analysis showed that Tromyosin (Tm) and Desmin were expressed in the blank group, FGF-2 drug-induced group, Lenti-FGF-2-GFP, and empty virus control transfection group after 4 weeks of induction, among which FGF-2 lentivirus transfected. The expression levels of Tm and Desmin were the highest in the staining induction group. Statistical analysis showed that the positive expressions of Tm and Desmin in each experimental group were statistically significant. Transmission electron microscopy showed that the nucleus of the cells transfected and induced by FGF-2 was located at the center of the cells. Myofilaments, rough endoplasmic reticulum, and mitochondria, and ribosomes were seen in the cytoplasm. CONCLUSION: These results indicate that FGF-2 can transfect and induce differentiation of BMSCs into cardiomyocyte-like cells. Lentivirus-mediated FGF-2 transfection induces the differentiation of bone marrow mesenchymal stem cells into cardiomyocyte-like cells better than FGF-2 direct induction.

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We investigated the influence of signal transducer and activator of transcription-3 (STAT3) on the spinal cord tissue grafts of rat fetuses with spina bifida aperta. In particular, we hoped to identify whether transfection of the STAT3 overexpression plasmid increases the survival of spinal cord transplantation in order to improve therapeutic efficacy. The fetal rat model of spina bifida aperta was established using retinoic acid and treated with a microsurgical injection of bone marrow mesenchymal stem cells (BMSCs). The animals were divided into either the blank control group, negative control group or the experimental group. The optical density (OD) value of BMSCs viability was determined using the Cell Counting Kit-8 (CCK-8). The expression of STAT3, phosphorylated STAT3 (pSTAT3), neural markers and apoptosis-related factors were evaluated using real-time PCR and Western blot. The OD value in the experimental group was highest at eight hours after transplantation using CCK-8. The expression of pSTAT3, glial fibrillary acidic protein, neuron-specific enolase, neurofilament and nestin in the experimental group was significantly higher compared to the blank control group and negative control group (P<0.05). However, STAT3 expression in the experimental group was statistically significantly decreased (P<0.05). The relative expression of caspase-8 and bcl-2 in the experimental group were significantly lower compared to the blank control group and negative control group (P<0.05). Transfection of the recombinant lentivirus-mediated STAT3 overexpression plasmid with BMSCs can help improve the efficiency of transforming into neural cells and provide new seed cells for the treatment of congenital spina bifida aperta.

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OBJECTIVE: This study aimed to observe the metastatic behavior of head and neck squamous cell carcinoma cells after knocking down heat shock protein (Hsp) 27. METHODS: The experiment was divided into three groups: the lentivirus vector plasmid of pLenti-shRNA-Hsp27 was transfected into UM-SCC-22B cells as experimental group (shHsp27 group), routine culture of UM-SCC-22B cells as blank control (ctrl group), UM-SCC-22B cells transfection of pLenti-shRNA-ctrl lentivirus vector as negative control (shctrl group). Through real-time quantitative polymerase chain reaction (RT-qPCR) and Western blot assay to detect the mRNA expression of Hsp27 in three groups. MTS assay was performed to detect cell-proliferation changes, wounding healing assay was performed to detect cell-migration changes, and Matrigel Transwell invasion assay was performed to detect cell-invasion changes. RESULTS: The expression of Hsp27 in shHsp27 group decreased signifi-cantly; MTS assay showed that UM-SCC-22B before and after Hsp27 knockdown had similar proliferation rates after being cultured for 24 or 48 h. Compared with the ctrl group, the shHsp27 group decreased the metastatic behavior by 4.38-fold in migration and 2.03-fold in cell invasion. CONCLUSIONS: Stably transfected lentivirus vector plasmid of pLenti-shRNA-Hsp27 can efficiently decrease Hsp27 expression and reduce the metastasis ability of UM-SCC-22B.

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Lentiviral expression vectors for calcitonin gene-related peptide (CGRP) were used to transfect rat bone marrow mesenchymal stem cells (MSCs). After assessing the biological characteristics of proliferation and aging in MSCs transfected with CGRP, we observed the effects of the CGRP-modified rat MSCs on the migration and proliferation of rat vascular smooth muscle cells (VSMCs) in vitro. Rat MSCs were isolated, cultured in vitro, and identified by flow cytometry. A CGRP recombinant lentivirus was transfected into MSCs. The transfection efficiency was determined by fluorescence microscopy and flow cytometry, and CGRP in MSCs was detected by real-time quantitative PCR, ELISA, and immunofluorescence. The proliferation and senescence of CGRP-modified MSCs were evaluated by MTT assay and beta-galactosidase staining. VSMCs were isolated, cultured in vitro, and identified by immunofluorescence. CGRP-modified MSCs and VSMCs were cocultured in a Transwell system. The proliferation and migration of VSMCs were evaluated by scratch testing and the MTT method. Rat bone marrow MSCs showed a spindle-shaped morphology, adherent growth in vitro, positive CD29 and CD90 expression, and negative CD45 expression. CGRP was stably expressed in MSCs after 48 h of recombinant lentivirus transfection. CGRP mRNA and protein secretion in CGRP recombinant lentivirus-transfected MSCs were higher than that in control MSCs. Immunofluorescence showed that CGRP protein could be expressed in CGRP-modified MSCs. The proliferation ability and senescence rates did not differ between lentivirus-transfected MSCs and untransfected MSCs. Rat VSMCs expressed α-SMA protein and exhibited a spindle-shaped morphology and adherent growth in vitro. In Transwell coculture experiments, scratch testing of VSMCs showed that CGRP-modified MSCs could reduce VSMC proliferation and migration. The CGRP gene can be stably expressed in MSCs after CGRP recombinant lentivirus transfection. CGRP recombinant lentivirus transfection has little effect on the proliferation or senescence of MSCs, and CGRP-modified MSCs can inhibit the proliferation and migration of VSMCs. These results lay a foundation for research on the use of CGRP gene-engineered MSCs in restenosis therapy.

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AIM: Hypercoagulation and fibrinolysis inhibition in the alveolar cavity are important characteristics in acute respiratory distress syndrome (ARDS). Alveolar epithelial cells type II (AEC II) have been confirmed to have significant role in regulating alveolar hypercoagulation and fibrinolysis inhibition, but the mechanism is unknown. Nuclear factor-κB (NF-κB) signaling pathway has been demonstrated to participate in the pathogenesis of these two abnormalities in ARDS. The purpose of the present study is to explore whether controlling the upstream crucial factor IκB kinase (IKK)ß could regulate coagulation and fibrinolysis factors in LPS-stimulated AEC II. MATERIALS AND METHODS: An IKKß gene regulation model (IKKß+/+ and IKKß-/-) was prepared using lentiviral vector transfection. The models with wild type cells were all stimulated by lipopolysaccharide (LPS) or saline for 24 h. Expression of the related proteins were determined by western-blotting, ELISA and revere transcription-PCR respectively. Tissue factor (TF) procoagulant activity and nuclear p65 protein level were also detected. RESULTS: IKKß increased in IKKß+/+ cells but decreased in IKKß-/- cells. LPS stimulation promoted the expression of p-IκBα, p65, p-p65 and p-IKKß as well as TF and plasminogen activator inhibitor (PAI)-1, at the mRNA or protein level, and this was significantly enhanced by IKKß upregulation but weakened by IKKß downregulation. TF procoagulant activity presented the same changes as the molecules above. ELISAs showed additional increases in the concentrations of as thrombin antithrombin, procollagen III propeptide, thrombomodulin and PAI-1 in IKKß+/+ cell supernatant under LPS stimulation, however they decreased in IKKß-/-. The level of as antithrombin III however, appeared to show the opposite change to those other factors. Immunofluorescence demonstrated a greatly enhanced expression of p65 in the nucleus by IKKß upregulation, which was reduced by IKKß downregulation. CONCLUSIONS: IKKß could regulate the expression and secretion of coagulation and fibrinolysis factors in LPS-stimulated AEC II via the NF-κB p65 signaling pathway. The IKKß molecule is expected to be a new target for prevention of coagulation and fibrinolysis abnormalities in ARDS.

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This study aims to explore the regulatory mechanism of hypoxia-inducible factor HIF-1α on Kv3.4. Oral squamous cell carcinoma (OSCC) cell lines SCC3 and CAL27 were used in this study. Western blotting and qRT-PCR methods were used to detect Kv3.4 expression levels in OSCC and their adjacent tissues. The expression changes of Kv3.4 and HIF-1α in a hypoxic environment were detected in cell lines. The stable OSCC cell lines with knockouts of HIF-1α and Kv3.4 were constructed. Transwell and CCK-8 assays were used to detect changes in the invasion, migration and proliferation ability after transfection. Chromatin immunoprecipitation and luciferase reporter gene assays were used to determine the regulatory and binding sites of HIF-1α on Kv3.4. The expression level of Kv3.4 in oral cancer tissue was higher than normal oral epithelium's regular value. The expression level of HIF-1α and Kv3.4 increased under hypoxia. Knocking out HIF-1α and Kv3.4 could reduce the invasion, migration and proliferation of cells. A down regulation of HIF-1α will reduce the Kv3.4 expression level. Overexpressing Kv3.4 after knocking down HIF-1α partially restored the proliferation and invasion of cell lines. Therefore, HIF-1α regulates the invasion, migration and proliferation of oral cancer cells by regulating Kv3.4 expression.

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OBJECTIVE: To establish a PC12 cell line with stable expression of human apolipoprotein E( ApoE4) gene by transfection with a lentiviral vector carrying human ApoE4 gene and to investigate the effect of maltol aluminum on the viability of transfected PC12 cells. METHODS: The lentiviral vector carrying human ApoE4 gene was transfected into PC12 cells. PC12 cells with overexpression of ApoE4 gene and negative control vector were obtained after puromycin screening.The mRNA relative expression of R-Apo E and( or) H-Apo E-FLAG of cells in PC12,PC12-NC and PC12-ApoE4 groups were detected by real-time fluorescent quantitative polymerase chain reaction,and the effect of cell construction was identified. PC12-ApoE4 cells and PC12 cells were exposed to maltol aluminum solution at concentrations of 0. 00,100. 00,200. 00 and 400. 00 μmol/L respectively for 24 hours,and cell viability was detected by Cell Counting Kit-8( CCK-8)assay. RESULTS: PC12-ApoE4 and PC12-NC cells under the fluorescence microscope showed fluorescence expression,suggesting that transfection was successful. The expression of PC12 cells showed no fluorescence. The relative expression of H-Apo E-FLAG gene mRNA( the median amount) of PC12-ApoE4 cells was 148. 74,which was higher than the R-Apo E gene in PC12 cells( 1. 00) and PC12-NC cells( 1. 01)( P < 0. 01). After exposure to maltol aluminum,the cell survival rates in terms of the main effect and interaction effect of dose and cell type were statistically significant( P < 0. 01),among them,the cell viabilities were decreased in the concentration range of 0. 00-400. 00 μmol/L with the dose of maltol aluminum exposure increased,showing dose-effect relationship( P < 0. 01). CONCLUSION: The cell line stably expressed human ApoE4 gene was constructed successfully. There was interaction between the effects of maltol aluminum and ApoE4 gene on the survival rate of PC12 cells,and ApoE4 gene could enhance the cytotoxicity of maltol aluminum on PC12 cells.

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Heat shock protein 70 (HSP70) maintains Ca(2+) homeostasis in PC12 cells, which may protect against apoptosis; however, the mechanisms of neuroprotection are unclear. Therefore, in this study, we examined Ca(2+) levels in PC12 cells transfected with an exogenous lentiviral HSP70 gene expression construct, and we subsequently subjected the cells to ischemia-hypoxia/reoxygenation injury. HSP70 overexpression increased neuronal viability and ATPase activity, and it decreased cellular reactive oxygen species levels and intracellular Ca(2+) concentration after hypoxia/reoxygenation. HSP70 overexpression enhanced the protein and mRNA expression levels of sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPase (SERCA), but it decreased the protein and mRNA levels of inositol 1,4,5-trisphosphate receptor (IP3R), thereby leading to decreased intracellular Ca(2+) concentration after ischemia-hypoxia/reoxygenation. These results suggest that exogenous HSP70 protects against ischemia-hypoxia/reoxygenation injury, at least in part, by maintaining cellular Ca(2+) homeostasis, by upregulating SERCA expression and by downregulating IP3R expression.

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Objective:To explore the expression pattern and function of miR-126 in human colon cancer and the underlying mechanisms. Methods:hTe expression pattern of miR-126 in high-density human colon cancer tissue microarray was analyzed by in situ hybridization. Further more, the biological function of miR-126 in colon cancer in vitro was investigated by establishing a stable miR-126 over-expression cell lines. Result:hTe expression of miR-126 was lower in the tumor tissue, especially in metastasis tissue. hTe down-regulation of miR-126 was more obvious in the patients who displayed bad prognosis (P=0.025). Over-expression of miR-126 in colon cancer cell was able to inhibit cell proliferation, promote cell apoptosis and reduce the invasive ability. MiR-126 significantly enhanced the sensitivity of the colon cancer cell to chemotherapeutic drug. It has been shown that IRS1, SLC75A and TOM1 were the potential target genes of miR-126 in colon cancer. Conclusion:MiR-126 was able to inhibit the development of colon cancer and its level was closely related with the prognosis of patients with colon cancer. The potential target genes for miR-126 might include IRS1, SLC7A5 and TOM1. Therefore, miR-126 might be a therapeutic target for colon cancer diagnosis and treatment.