Development of cell model of polymer/liquid crystal and effect of their elasticity on adhesion of rBM-MSCs

AIM: To develop the cell model of polymer/liquid crystal and to study the effect of their elasticity on the adhesion of rat bone marrow mesenchymal stem cells (rBM-MSCs). METHODS: Using the method of solvent evaporation induced phase separation, the cell model of polymer/liquid crystal was constructed. The surface morphology and phase separation structure were determined by polarized optical microscopy (POM), scanning electron microscopy (SEM) and small angle X-ray scattering (SAXS). rBM-MSCs were separated and expanded by adherent culture. The surface markers of rBM-MSCs were detected by flow cytometry. The cells were induced to osteogenic differentiation and adipogenic differentiation for 2 weeks. After 3 passages, the cells were divided into 4 groups, including total PU control group, 10% membrane group, 30% membrane group and 50% membrane group. The cells were then incubated with rhodamine phalloidin for cytoskeleton staining and were observed under the confocal laser scanning microscope after cultured for 24 h. RESULTS: The cell model of polymer/liquid crystal was constructed successfully using the method of solvent evaporation induced phase separation. Flow cytometry results showed that the rBM-MSCs positively expressed CD29, CD44 and CD90, and negatively expressed CD34 and CD45. After stained with alizarin red S and oil red O, the calcium nodule and lipid droplets in rBM-MSCs were observed obviously. The cytoskeleton staining result indicated that the area in total PU control group, 10% membrane group and 30% membrane group were greater, and the actin microfilaments were also clearer than that in 50% membrane group. CONCLUSION: The cell model with suitable content of liquid crystal made a contribution to the rBM-MSCs' adhesion, but too much liquid crystal inhibits cell adhesion.     

Relationship between Th17 cells infiltrated in lungs and pulmonary vascular reconstruction under hypobarric hypoxia

AIM: To investigate the changes of retinoid-related orphan receptor γt(RORγt) mRNA and interleukin-17(IL-17) protein in the lung tissue under hypobaric hypoxia, and the relationship between Th17 cells and hypoxic pulmonary vascular reconstruction. METHODS: Male BALB/c mice(n=50) were randomly divided into control group and 3 d, 7 d, 14 d and 28 d hypobaric hypoxia groups. The mice in hypobaric hypoxia groups were housed in a hypobaric hypoxia chamber(simulated altitude of 6 000 m) for 3 d, 7 d, 14 d or 28 d. The mice in control group were housed in normal pressure and oxygen environment. The hemodynamic data were recorded by cardiac catheterization. The hypertrophy of right ventricle was evaluated by the ratio of weight of the right ventricle to the weight of the left ventricle plus interventri-cular septum, and the right ventricular weight over body weight. The spleen was collected and the proportions of the Th17(CD4⁺IL-17⁺RORγt⁺) cells were detected by flow cytometry. The serum levels of IL-4, IL-6 and IL-17 and the change of IL-17 in the lung tissue were measured by ELISA. The mRNA expression of RORγt in the spleen and lung tissues was measured by RT-qPCR. RESULTS: Compared with control group, the mouse right ventricular systolic pressure, the hypertrophy index of right ventricle and the serum IL-17 level were significantly elevated in hypoxia groups, which was consistent with the results of flow cytometry. The mRNA expression of RORγt in the lung tissue was also significantly increased in 7 d, 14 d and 28 d hypoxia groups. The expression of IL-17 in the lung tissue was significantly increased in 14 d and 28 d hypoxia groups. CONCLUSION: Hypoxia promotes differentiation of Th0 cells to Th17 cells in the spleen. The Th17 cells infiltrated in the lung tissue under hypobarric hypoxia are involved in pulmonary vascular reconstruction.     

Effects of inhibiting myosin light chain kinase on endothelin-1 induced proliferation and apoptosis of pulmonary arterial smooth muscle cells in rats

AIM: To investigate the effect of inhibiting myosin light chain kinase(MLCK) on endothelin-1(ET-1) induced proliferation and apoptosis of rat pulmonary artery smooth muscle cells(PASMCs). METHODS: Rat PASMCs were cultured and stimulated with ET-1. The cells were randomly divided into control group, ET-1 group and ET-1+MLCK inhibitor group(ET-1+M). Western blotting, MTT assay, [³H]-TdR incorporation and flow cytometry were employed to test the expression of myosin light chain(MLC) and MLCK, cell proliferation, cell cycle and apoptotic rate of PASMCs,respectively. The phosphorylation of MLC was determined by glycerol-PAGE coupled with Western blotting. RESULTS: Compared with control group, the protein expression of MLCK and MLC phosphorylation significantly enhanced after ET-1 stimulation. ET-1 markedly induced the proliferation and decreased the percentage of apoptotic rate in the PASMCs. However, pretreatment with ML-7, a MLCK inhibitor, significantly reversed the above effects induced by ET-1. CONCLUSION: MLCK inhibitor effectively inhibits the ET-1-induced proliferation and the cell cycle progression.     

Transplantation of bcl-2 gene-modified bone marrow mesenchymal stem cells improves cardiac function and angiogenesis in rabbit ischemic car-diac insufficiency model

AIM: To investigate the effects of transplantation of bone marrow mesenchymal stem cells (BMSCs) modified by bcl-2 gene on myocardial cell apoptosis, angiogenesis and cardiac function in the rabbit after acute myocardial infarction (MI).METHODS: The rabbit BMSCs were isolated, cultured and purified in vitro. The BMSCs were transfected with adenovirus or adenovirus-Bcl-2. The rabbit model of MI was established by ligation of left anterior descending branch. The rabbits were injected with Ad-Bcl-2-BMSCs (MI+Bcl-2-BMSCs group), Ad-BMSCs (MI+BMSCs group) and DMEM (MI group) in infarction marginal zone 2 weeks after ligation. The cardiac function was evaluated by echocardiography.The apoptosis of myocardial cells was measured by TUNEL. The mRNA expression of VEGF was detected by real-time PCR. The expression of CD31 was examined by immunohistochemical staining, and new blood capillaries were counted at 4 weeks after BMSCs transplantation. The correlation of the above values with cardiac function was analyzed.RESULTS: The cardiac function was better, the apoptotic rate was lower, the mRNA expression of VEGF and the capillary density were higher in both MI+Bcl-2-BMSCs group and the MI+BMSCs group than those in MI group, and those in MI+Bcl-2-BMSCs group increased more obviously.The left ventricular ejection fraction (LVEF) had a negative correlation with the myocardial cell apoptosis rate. A positive correlation with the mRNA expression level of VEGF and the capillary density was also observed.CONCLUSION: The transplantation of BMSCs modified by bcl-2 gene significantly reduces the myocardial cell apoptosis, promotes angiogenesis, improves heart function of the rabbits with MI.     

Protective effect of BNDF on vascular endothelial cells with H2O2-induced oxidative injury

AIM: To study the protective effect of brain-derived neurotrophic factor (BDNF) on vascular endothelial cells with H₂O₂-induced oxidative injury. METHODS: Human umbilical vein endothelial cells (HUVECs) were cultured in vitro, and the oxidation injury model of HUVECs was established by treatment with H₂O₂. The oxidatively injured HUVECs were cultured with different concentrations (1, 10 and 100 μg/L) of BDNF. At the same time, the control group (no injury), PBS treatment after H₂O₂ injury group and TrkB inhibitor group (with 100 μg/L BDNF and 1: 1 000 TrkB inhibitor) were also set up. The viability of the HUVECs was detected by MTT assay. The levels of LDH, MDA, SOD and GSH were measured. The releases of NO, ET-1 and ICAM-1 were analyzed by ELISA. The changes of ROS production and cell apoptosis were evaluated by flow cytometry. The protein levels of TrkB, p-TrkB, cleaved caspase-3, Bcl-2 and Bax were determined by Western blot. RESULTS: Compared with uninjured control group, in H₂O₂ oxidative injury plus PBS treatment group, the viability of the cells was decreased significantly, the LDH and MDA levels were increased significantly and the activities of SOD and GSH were decreased significantly. The NO secretion was decreased, and the ET-1 and ICAM-1 concentrations were increased significantly. The ROS content and apoptotic rate were increased significantly. The protein levels of cleaved caspase-3 and Bax were increased but Bcl-2 protein expression was decreased significantly. Compared with PBS treatment group, in H₂O₂-injured HUVECs treated with different concentrations of BDNF, the cell viability was gradually increased, the LDH and MDA levels were decreased and the activities of SOD and GSH were increased gradually. The secretion of NO was increased but ET-1 and ICAM-1 were decreased gradually. The ROS content and apoptotic rate were decreased significantly. The TrkB and p-TrkB levels were significantly increased significantly, the protein expression of cleaved-caspase 3 and Bax was decreased gradually and the Bcl-2 protein expression increased gradually. The role of BDNF was inhibited by TrkB inhibitor. CONCLUSION: BDNF protects HUVECs from oxidative injury by binding with TrkB to activate the BDNF-TrkB signaling pathways.     

Sodium dichloroacetate improves radiosensitivity of U251 cells

AIM: To study the change of radiosensitivity of U251 cells after treated with sodium dichloroacetate (DCA) and further to explore the possible mechanism.METHODS: The U251 cells were divided into 4 groups: control group, DCA group, X-ray irradiation without DCA pretreatment (IR) group and X-ray irradiation with DCA pretreatment (DIR) group. MTT assay was applied to determine the cell viability. The intracellular reactive oxygen species (ROS) were detected by DHE fluorescence. The expression level of Bcl-2 was assessed by Western blot. The percentage of apoptosis of cells was determined by flow cytometry. RESULTS: No difference between control group and DCA group in cell viability (P>0.05) was observed. However, the cell viability of both IR group and DIR group was markedly reduced compared with control group (P<0.05). Furthermore, the viability of DIR group was significantly decreased compared with IR group (P<0.05). The percentage of ROS positive cells was obviously increased in DIR group compared with IR group (P<0.05). The expression level of Bcl-2 was sharply decreased in DIR group (P<0.05) and the percentage of apoptosis of cells was significantly elevated (P<0.05) in DIR group compared with IR group.CONCLUSION: The better antitumor effect was obtained by improving the radiosensitivity through pretreating the cells with DCA, and the possible mechanism was down-regulation of the Bcl-2 expression by developing the intracellular ROS.     

Hsa-miR-218 inhibits growth of cervical cancer HeLa cells by targeting to LASP1

AIM: To study the effect of hsa-miR-218 on cervical cancer HeLa cell growth and the underlying molecular mechanism.METHODS: The lentivirus expression vector pmiR-218 targeting to hsa-miR-218 was constructed. pmiR-218 was transfected into HeLa cells. The number of viable HeLa cells was counted by the method of Trypan blue exclusion. The inhibitory rate of cell activity was detected by WST-8 assay. The expression of LIM and SH3 protein 1(LASP1) at mRNA and protein levels was determined by real-time PCR and Western blot. The interaction between miR-218 and LASP1 was examined using a luciferase reporter assay.RESULTS: The lentivirus expression vector pmiR-218 targeting to hsa-miR-218 was constructed successfully and confirmed by DNA sequencing. Over-expression of miR-218 inhibited the activity of HeLa cells with the inhibitory rates of 15%, 26% and 65% at 24 h, 48 h and 72 h, respectively. The difference between transfection group and blank control/negative control group was statistically significant. The luciferase activity was reduced when co-transfection with miR-218 mimics and LASP1-3,UTR plasmid. The relative expression of miR-218 was increased after transfection with pmiR-218. Over-expression of miR-218 down-regulated the LASP1 expression at mRNA and protein levels by 25% and 75% respectively. Compared with blank control group and negative control group, the difference was statistically significant(P<0.05).CONCLUSION: pmiR-218 effectively inhibits the growth of HeLa cells in a time-dependent manner. miR-218 targets to the 3,UTR of LASP1, thus down-regulating the expression of LASP1 in HeLa cells.     

miR-21 inhibits apoptosis of Schwann cells following peripheral nerve injury

AIM: To explore the relationship and molecular mechanism between microRNA-21(miR-21) and Schwann cells (SC) following peripheral nerve injury. METHODS: The mRNA expression of miR-21 and phosphatase and tensin homologue deleted on chromosome ten (PTEN) in animal model were detected by real-time PCR. The over-expression of miR-21 and inhibition of miR-21 expression in the Schwann cells according to transfection of lentiviral vectors were performed, the nonspecific miRNA was used as a negative control (NC). The cell apoptosis was measured by flow cytometry. The mRNA expression of miR-21 and PTEN in the cells was detected by real-time PCR. The protein expression of PTEN and cleaved caspase-3 was determined by Western blot. RESULTS: The level of miR-21 was significantly higher and the mRNA level of PTEN was significantly lower in the model of nerve injury than those in control group. miR-21 over-expression decreased the number of apoptotic Schwann cells compared with NC-SC. The mRNA expression of PTEN was down-regulated by over-expression of miR-21. The protein expression of PTEN and cleaved caspase-3 was down-regulated by over-expression of miR-21(P<0.05). CONCLUSION: miR-21 may play an important role in the peripheral nerve injury through inhibiting apoptosis of Schwann cells by down-regulating the expression of PTEN.     

洋葱苯丙氨酸解氨酶基因AcPAL2的克隆与表达分析

利用简并PCR技术和RACE技术克隆得到了一条洋葱的苯丙氨酸解氨酶(PAL)基因全长cDNA序列。该cDNA序列全长2349 bp,编码长685个氨基酸残基的多肽序列,命名为AcPAL2。Blast分析表明该序列与虎眼万年青Galtonia saundersiae、野蕉Musa balbisiana的相似性均较高。Real-time PCR表达及花青素含量分析表明,红皮洋葱该基因表达量最大,而黄皮和白皮洋葱表达量极低;在红皮洋葱中该基因在膨大初期大量表达,并迅速降低至一定程度后趋于相对平稳表达,且与花青素的积累过程相一致。     

Selection of Adequate Species for Degraded Areas by Oil‐Exploitation Industry as a Key Factor for Recovery Forest in the Ecuadorian Amazon

One of the requirements for the forest restoration of soils disturbed by the oil‐exploitation industry is that saplings be able to endure soil‐adverse conditions. In this study, saplings of 20 species susceptible to be used in reforestation programs were evaluated for their ability to grow on substrates derived from soils disturbed by petroleum extractions in the Ecuadorian Amazon. Seeds of each species were planted in germination trays. Once seedlings reached 5 cm in height they were transplanted to plastic bags with three treatment substrates: two derived from petroleum‐exploitation activity (soils from mud and drill cutting cells and from areas surrounding oil wells) and a control soil. Plant survival rate, stem height, and diameter were measured on a weekly basis until 14 weeks after transplantation, when we harvested the plants and also measured plant biomass and calculated the Dickson quality index for each species. Oil‐exploitation by‐product substrates impaired the performance of many saplings, with the substrate from mud and drill cutting cells being the one that most affected plant performance. Only saplings of five native species in the Amazon basin—Apeiba membranaceae, Cedrelinga cateniformis, Inga densiflora, Myroxylon balsamum, and Pouroma cecropiifolia—exhibited high or similar Dickson quality index values in all soil treatments and performed better than the rest. The use of these five species in remediation of soils disturbed by petroleum extraction in the Amazon basin could prove important because of their high potential to adapt to these disturbed sites. Copyright © 2016 John Wiley & Sons, Ltd.