The antisense block of CROC-1 gene expression makes cells grow slower

AIM:To establish FL-CROC- 1 ^- cell line in which CROC- 1 gene expression was blocked and study the role of CROC- 1 gene in cell growth. METHODS:The appropriate length cDNA fragment of the recently identified human gene CROC- 1 which encodes ubiquitin-conjugating enzyme like protein(Ubc-like protein) was cloned into the reconstructed eukaryotic expression vector pMAMneo-amp^- by antisense strategy. The recombinant plasmid which can express CROC- 1 antisense RNA was selected by restriction enzyme map analysis. The antisense expression recombinant plasmid pMAM-antiCROC- 1 was then transfected into human amnion FL cells by a modified calcium phosphate-mediated transfection procedure and selected with MEM medium containing 400 μg/mL geneticin. Finally ,the growth rate of the G418 resistant FL-CROC- 1 ^- cell line was determined.RESULTS:When the antisense inhibition of CROC-1 gene expression was induced by dexamethasone,the growth rate of the FL-CROC-1^- cel line was obviously slower as compared with that of the control FL cell and FL-MAMneo cell which was established by transfection of plasmid pMAMneo-amp^-(P<0.05).CONCLUSIONS:FL-CROC- 1 ^- cell line was successfully established in this study and the result of FL-CROC- 1 ^- cell growth suppression suggests that CROC- 1 gene encoded Ubc-like protein plays a positive regulation role in cell growth.     

茄子抗黄萎病遗传的初步研究

选取６份对黄萎病具有抗感差异的茄子材料，利用完全双列杂交设计轮配法配制１５个组合，调查人工灌根后的抗病指数。结果表明：抗性遗传不符合“加性－显性”模型，存在上位性作用，且至少受２对显性基因组控制。     

Effect of pioglitazone on pre-adipocytes differentiation and GILZ expression

AIM: To investigate the roles of pioglitazone on differentiation and expression of GILZ in 3T3-L1 pre-adipocytes. METHODS: The morphological changes during 3T3-L1 cell differentiation were observed. The cells were treated with pioglitazone at concentrations of 1×10^-4～1×10^-2 mmol/L for 48 h, then the relative content of triglyceride were analyzed by oil red O staining at 2nd, 4th and 6th day during adipogenesis. The mRNA expression of peroxisome proliferator-activated receptor γ2 (PPARγ2) and lipoprotein lipase (LPL) was measured by real-time PCR. GILZ protein expression was determined by Western blot after the cells were treated with pioglitazone at concentrations of 1×10^-4 ～1×10^-2 mmol/L for 48 h.RESULTS: Oil-red O staining showed that the relative contents of triglyceride in adipocytes were increased with the increase in the pioglitazone concentration. Compared with the control, the relative contents of triglyceride in group 1×10^-3 mmol/L and group 1×10^-2 mmol/L were significantly increased (P < 0.05). The mRNA expression of PPARγ2 and LPL was also increased with the increase in the pioglitazone concentration. When pioglitazone concentration was more than 1×10^-3 mmol/L, compared with the control, the mRNA expression of PPARγ2 and LPL significantly increased (P < 0.01). The protein expression of GILZ was decreased with the increase in the pioglitazone concentration.CONCLUSION: Pioglitazone down-regulates GILZ expression, and up-regulate PPARγ2 expression and the downstream functional factor such as LPL.     

Effect of inhaled nitric oxide on adhesion molecule CD11b expression in lung neutrophils of meconium aspiration rabbits

AIM:To evaluate effects of inhaled nitric oxide(iNO) on adhesion molecule CD11b expression on lung neutrophils in experimental meconium aspiration syndrome(MAS) rabbits treated with conventional mechanical ventilation under room air or 100%O₂. METHODS:Animals were randomly allocated to 8 groups(n=48) of 6 each: two MAS model groups(under room air or 100%O₂ without iNO treatment), 6 treatment groups were treated with continuous NO inhalation at a dose of 0.2×10^-6mol/L, 0.33×10^-6mol/L or 0.67×10^-6mol/L respectively for 12 hours under room air or 100%O₂. Mean systemic arterial pressure(SAP) and methemoglobin (MeHb) were performed at basement time, 0, 2, 4, 12 hours. Expression of CD11b on neutrophils in the bronchoalveolar lavage fluid(BALF) was detected with flow cytometry. RESULTS:SAP, MeHb at different time among different groups were within the normal scale. CD11b expression on the neutrophils in the BALF significantly decreased in groups of inhalation 0.33×10^-6 mol/L or 0.67×10^-6 mol/L NO, compared with the two MAS model groups. (x±s: under 21%O₂, 0.33×10^-6 mol/L NO, 121±20 υs 392±204; 0.67×10^-6 mol/L NO, 112±30 υs 392±204;under 100%O₂, 0.33×10^-6 mol/L NO, 113±24υs293±65; 0.67×10^-6 mol/L 102±114 υs293±65, P<0.05). 0.2×10^-6mol/L NO inhalation did no effect on CD11b expression. (x±s:21%O₂, 190±101 υs 392±204; 100%O₂, 222±85 υs 293±65; P>0.05). No statistic difference was observed between groups inhaled 0.33×10^-6 mol/L NO and 0.67×10^-6mol/L NO. CONCLUSION:0.33×10^-6 mol/L or 0.67×10^-6 mol/L NO inhalation down-regulated the CD11b expression on the neutrophils in BALF to reduce the sequestration of neutrophils in rabbit lung.     

Construction of atherosclerosis full-length fully human antibody libraries by mammalian cell surface display technique

AIM: To construct atherosclerosis (As) full-length fully human antibody libraries by mammalian cell surface display technique for looking for the atherosclerotic therapeutic antibody. METHODS: The vector pcDNA-DHL was constructed by remounding the vector pcDNA5/FRT. The human total RNA from human peripheral blood mononuclear cells was isolated, and then reverse transcripation of RNA to cDNA was performed. The genes encoding the heavy chain variable regions and kappa light chains of the antibodies were amplified by PCR, and the product of PCR was connected to the vector pcDNA-DHL and the vector pDHL-As. The library DNAs were transfected into Flp-In^TM-CHO (FCHO) cells, the expression of the full-length human antibodies on the surface of cells was analyzed by flow cytometry. The cells that were successfully transfected with pDHL-As were screened with hygromycin, and then the full-length antibody library of atherosclerosis was obtained. RESULTS: The vector pcDNA-DHL were constructed successfully. The heavy chain gene library constructed showed a diversity of 1.79×10⁵, and the kappa light chain gene library had a diversity of 1.80×10⁵. Theoretically, the diversity of antibody library is 2.32×10¹⁰. The antibody library of atherosclerosis pDHL-As expressed on the surface of Flp-In^TM-CHO cells. CONCLUSION: The full-length human antibody cell library of atherosclerosis was constructed successfully, and 45 FCHO cells that expressing anti-oxidized low density lipoprotein antibodies were selected.     

High-efficient genetic transfection of CD41+,UT7, U937 and MDA-MB-435 cells with a recombined murine stem cell retroviral vector

AIM: Gene transduction with a recombined murine stem cell retroviral vector has been investigated to find an effective way of gene transduction and to offer theory and experimental basis for the recombined murine stem cell retroviral vector used for gene transduction. METHODS: 1. Construction of retrovirus vector: EC1-4 gene (repeats 1-4 of cadher in-5 extracellular domain) and mutant (Ser 222A) MEK1 gene were cloned into retrovirus vector pMSCV after cut by Bgl Ⅱ and EcoR 1 restriction endonuclease. 2. Obtaining CD41⁺ cells and cell culture: Cells expressing CD34⁺ from cord blood were isolated. The inducement of cells expressing CD41 from CD34⁺ cells was performed by using TPO and cells CD41⁺ were selected by FACS. NIH 3T3 cells were cultured in high sugar DMEM medium and U937 in RPMI 1640 medium. UT7 cells which is cytokine-dependent cell line were grown in Iscove's modified Dulbeco's medium supplemented by GM-CSF. 3. Determination of viral titers: Retroviral vectors were transferred to packaging cell line 293. Retroviral containing supenatant was collected after transfection. The viral titers was tested on infection of NIH 3T3 cells by FACS analysis. 4. Western blot: Transduced CD41⁺, UT7, U937 and MDA-MB-435 cells were analyzed by western blot to examine expression of transduced genes. RESULTS: A packaging cell line 293 produces high-titer MEK1 pMSCV retroviruses (3.1×10⁷) and EC1-4 pMSCV retroviruses (1.0×10⁸). With 8-folds dilution retroviruses, 60.73% GFP positive cells have been obtained in MEK1 pMSCV transduced UT7 cells, 72.56% in U937 cells and 30.57% in CD41⁺ cells, respectively. GFP positive cells have reached up 97.54 % in EC1-4 pMSCV transducted MDA-MB-435 cells. Phosphorylated MEK1 has been decreased in experiment group when TPO has stimulated CD41⁺ and UT7 cells or serum has stimulated U973 cells. This indicates that is a dominant negative effect of mutation MEK gene. EC1-4 gene transduced MDA-MB-435 cells have expressed EC1-4. CONCLUSION:The recombined murine stem cell retrovirus can effectively mediate gene transduction of CD41⁺,UT7,U937 and MDA-MB-435 cells,and transduced genes can be stably expressed.     

The effect of antisense bcr-abl oligonucleotides on the growth of K562 cells

AIM:To study the effect of bcr- abl gene antisense phosphorothioate oligonucleotides(Aspo) on K562 cell line and explore its significance in chrenic myelogeneous leukemia (CML) gene therapy.METHODS:Cells were exposed to oligomeis, observed by inverted microscope.Cells inhibitory rate were determined by 0.4 trypan blue exclusion . CFU-K562 were cultured in 0.8 % methylcellulose . P210 was measured by flow cytomety RESULTS: K562 cells were treated with Aspo, they still grew in clone state and show antisense sequence specific and dose dependent. When the concentration of Aspo was more than Spznol/L, the growth of cells was inhibited and P210 was down regulated or completely suppressed, and the greatest growth inhibition was at 120h . There was signifi-cant inhibition of cell proliferation in a rang‘cells number from 1×10⁴/mL to 5×10⁴/mL after treatment with 10unol/L Aspo. b2a2 Aspo was also effect on K562 cells which expressing b3a2 mRNA.CONCLUSION: bcr-abl Aspo has a specific growth inhibition effect on K562 cells, and worths further study in CML gene therapy.     

Antagonistic effects of cyproheptadine and anisodamine on [Ca2+]i elevation induced by TNFα in endothelial cell strains

AIM: To study the effects of cyproheptadine (Cyp) and anisodamine (Ani) on the changes of intracellular free Ca²⁺ concentration ([Ca²⁺]_i) induced by tumor necrosis factor (TNFα) in single endothelial cells, and to explore the mechanisms of TNFα mediated shock and antishock actions of Cyp and Ani. METHODS: Human umbilical vein endothelial cell strains (ECV304) were seed in 35 mm tissue culture dish with 2 mL DMEM culture medium. The cultured cells were loaded by Fluo-3/AM. The spatial distribution and the dynamic changes of [Ca²⁺]_i in single endothelial cell was determined by laser scanning confocal microscopy (LSCM). RESULTS: [Ca²⁺]_i in single endothelial cell after stimulation of TNFα rapidly increased in a dose-dependent manner and approached the peak value within 60 seconds, afterwards, decreased and kept above the basal level. The confocal scanning image showed that [Ca²⁺]_i elevation was more obvious in nuclear than in cytoplasma, and decreased slowly. Cyp (3×10^-5, 6×10^-5 mol/L) and Ani (2×10^-5, 4×10^-5 mol·L^-1) markedly inhibited TNFα (1.2×10^-9 mol·L^-1)-induced [Ca²⁺]_i elevation. CONCLUSIONS: TNFα markedly induces elevation of [Ca²⁺]_i in single endothelial cell, it may be an important mechanism of TNFα-induced shock and tissue injury. Cyp and Ani obviously suppress TNFα-induced [Ca²⁺]_i elevation, which probably is one of the mechanisms of their antishock effects.     

AngⅡ/AT1R pathway leads to down-regulation of endothelial nitric oxide synthase phosphorylation

AIM:To investigate the mechanism of angiotensinⅡ (AngⅡ)/angiotensinⅡ type 1 receptor (AT₁R) pathway activating protein phosphatase 2A (PP2A) which leads to down-regulation endothelial nitric oxide synthase (eNOS) phosphorylation level in mesenteric arteries of rats. METHODS:The mesenteric arteries of adult male SD rats (weighing 160~180 g; n=90) were isolated under aseptic conditions. Firstly, to determine the effect of angiotensinⅡ down-regulated eNOS (Ser1177) phosphorylation level, the mesenteric arteries were randomly divided into normal control (control) group and AngⅡ group. The mesenteric arteries in AngⅡ group were incubated with AngⅡ at 1×10^-7 mol/L, 1×10^-6 mol/L and 1×10^-5 mol/L for 6 h, 12 h and 24 h, respectively. Secondly, to investigate the molecular mechanism by which angiotensinⅡ activated PP2A leading to down-regulation eNOS (Ser1177) phosphorylation level, the mesenteric arteries were randomly divided into control group, AngⅡ group and candesartan (CAN; a specific AT₁R blocker)+AngⅡ group. The mesenteric arteries were pretreated with 1×10^-5 mol/L CAN for 1 h, then incubated with 1×10^-7 mol/L AngⅡ for 12 h in CAN+AngⅡ group. The protein levels of eNOS, p-eNOS (Ser1177), PP2Ac, p-PP2Ac (Tyr307) and protein phosphatase 2A inhibitor 2 (I₂^PP2A) in the arteries were determined by Western blot. The activity of PP2A in the arteries was detected by PP2A activity kit. RESULTS:Compared with the control group, the protein level of p-eNOS (Ser1177) in the mesenteric arteries was decreased after incubated with AngⅡ for 6 h, 12 h and 24 h (P<0.05). The decreasing tendency of p-eNOS (Ser1177) showed concentration-dependently, especially in 12 h and 24 h groups. The expression of eNOS protein showed no significant difference in each group. Compared with the control group, the mesenteric arteries of the rats were incubated with AngⅡ at 1×10^-7 mol/L for 12 h in vitro, the protein levels of p-eNOS (Ser1177) were down-regulated (P<0.05); pretreatment with CAN significantly increased the protein level of p-eNOS (Ser1177) (P<0.05); the protein levels of eNOS showed no significant difference in each group. Compared with the control group, the protein levels of p-PP2Ac (Tyr307) and I₂^PP2A were decreased after the mesenteric arteries were treated with AngⅡ at 1×10^-7 mol/L for 12 h (P<0.05). Candesartan pretreatment restored the protein levels of p-PP2Ac (Tyr307) and I₂^PP2A (P<0.05), however the expression of PP2Ac protein showed no significant difference in each group. Compared with the control group, the activity of PP2A was increased in the mesenteric arteries incubated with AngⅡ at 1×10^-7 mol/L for 12 h (P<0.05). Candesarten pretreatment inhibited the activity of PP2A significantly (P<0.05). CONCLUSION:AngⅡ increases PP2A activity via AT₁R pathway, thus leading to down-regulation eNOS (Ser1177) phosphorylation level in mesenteric arteries. The molecular mechanism of PP2A activation may be associated with decreasing the protein levels of p-PP2Ac (Tyr307) and I₂^PP2A.     

Inhibitory effect of ampelopsin combined with mitomycin on proliferation of gastric carcinoma cells

AIM: To investigate the synergistic effects of ampelopsin (AMP) and a chemotherapeutic drug mitomycin (MMC) on the proliferation of gastric cancer cell line SGC-7901.METHODS: SGC-7901 cells were cultured in vitro and divided into 4 groups: control group, AMP group, MMC group and AMP+MMC group. Cell proliferation was measured by MTT method. The apoptotic index was examined by flow cytometry. The expression of apoptotic proteins, Bcl-2 and survivin, was detected by Western blotting.RESULTS: AMP at the concentrations ranging from 2.2 mg/L to 14.84 mg/L exerted inhibitory effect on the growth of SGC-7901 cells. Cell proliferation in AMP (14.84 mg/L) group was inhibitory by (60.85±1.13) %, significantly higher than that in control group (P<0.05). The inhibitory rates of cell proliferation varied from (17.40±0.30) % to (72.23±1.36) % when the concentrations of MMC increased from 1×10^-3 g/L to 1×10^-2 g/L. AMP combined with MMC showed a synergistic effect on the growth of SGC-7901 cells. The inhibitory rates of cell proliferation varied from (21.83±2.50) % to (46.70±1.45) % when the concentrations of MMC increased from 1×10^-3g/L to 5×10^-3g/L. The inhibitory effect of AMP plus MMC was higher than that of MMC or AMP alone. The protein levels of Bcl-2 and survivin were inhibited in AMP group, MMC group and AMP+MMC group, and were significantly lower in AMP+MMC group than those in AMP group or MMC group.CONCLUSION: AMP enhances the inhibitory effect of MMC on the growth of SGC-7901 cells. The mechanism is related to the inhibition of apoptotic protein expression.     

Effects of PKC and ROCK on vasodilation induced by nifedipine

AIM: To investigate the effects of Rho-associated kinase (ROCK) and protein kinase C (PKC) on the relaxation of isolated rat aortic rings induced by nifedipine and the mechanisms. METHODS: The changes of tension in vascular rings induced by nifedipine under the basic condition and pre-contracted by norepinephrine (NE, 10^-6 mol/L) or KCl (60 mmol/L) were observed. The effects of ROCK and PKC on the vasodilation induced by nifedipine were studied using the vascular ring perfusion device. RESULTS: Nifedipine (10^-10 mol/L, 10^-9 mol/L, 10^-8 mol/L, 10^-7 mol/L, 10^-6 mol/L and 10^-5 mol/L) had no significant relaxation effect on isolated aortic rings under basic condition. Nifedipine induced dose-dependent relaxation in both endothelium-intact and endothelium-denuded aortic rings pre-contracted by 10^-6 mol/L NE and 60 mmol/L KCl (P<0.05). No obvious difference between endothelium-intact group and endothelium-denuded group was observed. After incubation of the PKC inhibitor staurosporine (STA, 10^-8 mol/L) and PKC agonist phorbol 12-myristate 13-acetate (PMA, 10^-7 mol/L), STA increased the relaxation induced by nifedipine, while PMA reduced the effect of nifedipine on blood vessels (P<0.05). After the incubation of the ROCK inhibitor fasudil (10^-6 mol/L) and ROCK agonist angiotensin Ⅱ (Ang-Ⅱ, 10^-9 mol/L), fasudil increased the relaxation induced by nifedipine, while Ang-Ⅱ reduced the effect of nifedipine on blood vessels (P<0.05). The relaxation induced by nifedipine was not statistically inhibited by BaCl₂ (10^-4 mol/L), tetraethylammonium (10^-3 mol/L), glibenclamide (10^-5 mol/L) and 4-aminopyridine (10^-3 mol/L). In calcium-free and high-potassium solution, pre-treatment with nifedipine (10^-9 mol/L, 5×10^-8 mol/L and 10^-6 mol/L) inhibited calcium-induced contraction of the aortic rings (P<0.05). However, nifedipine pre-treatment did not affect the contraction induced by NE in Ca²⁺-free medium. CONCLUSION: Nifedipine exhibits vasodilatation effect in a dose-dependent manner and the vasodilatation activity is endothelium-independent. The vasodilatation effect of nifedipine may be related to the inhibition of extracellular calcium influx, and inhibition of PKC and ROCK enhances the vasodilatation effect of nifedipine.     

Screening of tissue factor targeting peptides alternately from phage display peptide library

AIM: To screen tissue factor (TF) targeting peptides by establishment of a new and effective phage display method to acquire the peptides specifically bound to the transmembrane receptor. METHODS: Five rounds of panning were alternately conducted by targeting TF and HT-29 cell line which showed the detectable TF expression (screen for targeting receptor and cell alternately with phage display, STRCA). The 30 phage clones were assessed by enzyme-linked immunosorbent assay (ELISA). DNA sequencing was performed for the phage clones. The affinity of synthetic peptides was verified with competitive inhibition ELISA. The repeated experiment was conducted to verify the reliability of the results. RESULTS: The phages were effectively enriched after 5 rounds of panning with the improvement of the recovery rate from (2.25×10^-4)% to (1.32×10^-2)%. In 30 individual phages, ELISA positive rate was 76.7%, and the repetition of A, B, C and D peptides showed 23.3% (7/30), 23.3% (7/30), 26.7% (8/30) and 10.0% (3/30),respectively. E peptide constructively consisted of A and B. The five synthetic peptides were verified by ELISA, and the IC₅₀ of each peptide showed 3.25 nmol/L, 6.72 mol/L, 3.24×10³ mol/L, 2.08×10² mol/L and 45.77 mol/L,respectively. The positive phages were selected again in the second experiment to compare the results of the first experiment and the repeatability was 33.3%. CONCLUSION: STRCA can select TF targeting peptides with high affinity, which has the potential to become a therapeutic screening of transmembrane receptor-binding peptides.     

Effects of thymosin α1 on the development and maturation of thymocytes

AIM: To investigate the effect of Thymosin α₁ on the development and matutation of thymocytes. METHODS: The proportion of CD4⁺CD8⁺ thymocytes and the expression of smoothened (Smo) of the hedgehog (Hh)-signaling in CD4^-CD8^-thymocytes were examined to observe the effect of thymosin α₁ on thymocyte development and matutation. RESULTS: Flowcytometric analysis showed that thymosin α₁ showed activity at a low dose of 30 μg/kg, and 30 μg/kg thymosin α₁ accelerated the replenishment and maturation of thymocytes according to the expression of Smo of the Hh-signaling in CD4^-CD8^-thymocytes, the potent negative regulator of proliferative responses. CONCLUSION: Thymosin α₁ can accelerates thymocyte development from CD4^-CD8^- to CD4⁺CD8⁺.     

Effects of lentivirus-mediated transfection of shRNA targeting Adra1d gene on calcium and calmodulin in rat aortic vascular smooth muscle cells

AIM:To investigate the effects of lentivirus-mediated transfection of shRNA targeting α_1D-adrenergic receptor (Adra1d) gene on calcium ion (Ca²⁺) and calmodulin (CaM) in vascular smooth muscle cells (VSMCs) of rat aorta. METHODS:Single oligonucleotide sequences of shRNA targeting rat Adra1d gene were design and synthesized, and then the shRNA was constructed and cloned into GV248 vector. The U6-shRNA carrier and expression vector were transfected into 293T cells together and packed with lentivirus, and the supernatant was collected and concentrated by overspeed centrifugation. The VSMCs of rat aorta were transfected with recombinant lentivirus vector. The interference effects were identified by RT-qPCR and Western blot. The concentration of Ca²⁺ in VSMCs was detected by laser confocal inspection, and the expression of CaM at mRNA and protein levels in the VSMCs was determined by RT-qPCR and Western blot. RESULTS:The lentiviral shRNA expression vector was successfully constructed. The titer of the concentrated virus was 3×10¹¹ TU/L. The mRNA and protein expression levels of Adra1d in the rat aortic VSMCs were significantly reduced after transfection. The interference efficiency of Lv-shRNA4-Adr to Adrald gene was greater than 85%. After target silencing of Adra1d gene, compared with scrambled group, the Ca²⁺ fluorescence intensity of rat aortic VSMCs was significantly increased. Moreover, the mRNA and protein expression levels of CaM were also increased significantly. CONCLUSION:A lentiviral shRNA expression vector targeting rat Adra1d gene was successfully constructed, which significantly increased Ca²⁺ concentration and CaM expression in rat aortic VSMCs.     

Effects of bilberry anthocyanins on MMP2 and collagen I in human fetal scleral fibroblast in vitro

AIM:To investigate the effects of bilberry anthocyanins on matrix metalloproteinase 2 (MMP2) and collagen type I (collagen I) expression in human fetal scleral fibroblasts (HFSF) in vitro and to provide experimental data for the prevention and treatment of myopia by bilberry anthocyanins. METHODS:HFSF were treated with bilberry anthocyanins at different concentrations (0, 10^-6, 10^-5, 10^-4, 10^-3, 10^-2, 10^-1 and 1 g/L). The effects of bilberry anthocyanins on the viability of HFSF for different time (6 h, 8 h, 12 h, 24 h and 48 h) were measured by MTT assay. The cell cycle distribution and apoptosis of HFSF with highest viability (10^-1 g/L bilberry anthocyanins for 12 h) were analyzed by flow cytometry. The mRNA expression of MMP2, COL1A1 and COL1A2 in the HFSF was detected by RT-qPCR. The protein expression of MMP2 and collagen I was determined by Western blot. RESULTS:The cell viability was the highest after treatment with bilberry anthocyanins at a concentration of 10^-1 g/L for 12 h. Compared with blank control group, 10^-1 g/L bilberry anthocyanin group showed increased cell numbers in S and G₂ phases (P<0.05), but no significant difference of the apoptotic rate was observed. The mRNA expression of MMP2 was decreased (P<0.05), and that of COL1A1 was increased (P<0.05). No significant difference of COL1A2 expression was seen. The protein expression of MMP2 was decreased (P<0.05), and collagen I protein was increased (P<0.05). CONCLUSION:Bilberry anthocyanins inhibit the expression of MMP2 and increase the expression of collagen I in the HFSF.     

Construction and in vitro analysis of recombinant adenovirus vector carrying red fluorescent protein reporter gene

AIM: To provide important tools for gene therapy and gene vaccine research by constructing an adenovirus vector containing red fluorescent protein ( RFP ) reporter gene with the approach of in vitro recombinant ligation. METHODS: The RFP gene fragment of pTurboRFP-N was digested and ligated into pShuttle transfer vector to construct recombinant vector pShuttle-TurboRFP-N. I- Ceu I/PI- Sce I were used to double digest recombinant vector pShuttle-TurboRFP-N and backbone of vector pH5'040.pkGFP-II. The target fragment was collected and ligated, and recombinant adenovirus vector AdH5'.040.CMV.RFP-N was obtained. After linearization, the vector was transfected into AD293 cells by liposome for virus packaging. The efficiency of virus packaging and RFP expression level in AD293 cells were examined using fluorescent microscope. In addition, the biological activity and titer of the virus were tested. Human lung cancer cell line A549 and breast cancer cell line MDA-MB-231 were infected with recombinant adenovirus vector AdH5'.040.CMV.RFP-N and control adenovirus vector AdH5.CMV.EGFP respectively. The infection efficiencies of the 2 vectors to different cell lines were compared by evaluating the expression levels of RFP and enhanced green fluorescent protein (EGFP). RESULTS: The recombinant adenovirus vector AdH5'.040.CMV.RFP-N was correctly constructed and confirmed by enzyme digestion. The virus was packaged by the vector in AD293 cells and had the ability to infect the target cells. The target gene in eukaryotic cells was also expressed. The number of recombinant adenoviruses and the titer of the virus after amplification and purification were 3.6×10¹⁵ vp/L and 1×10¹³ pfu/L,respectively. The infection efficiencies of recombinant adenovirus vector Ad5'.040.CMV.RFP-N to human lung cancer cell line A549 and breast cancer cell line MDA-MB-231 were higher than those in control adenovirus vector AdH5.CMV.EGFP (P<0.05). CONCLUSION: We have constructed recombinant virus vector carrying RFP reporter gene and provide an important tool for gene therapy and gene vaccine research. The reporter gene can be highly expressed in AD293 cells and has high infection efficiency to cancer cells. RFP is a good substitution and supplement to green fluorescent protein.     

Effect of the combination of positive end expiratory pressure and nitric oxide inhalation on oleic acid-induced acute lung injury in canine

AIM:To observe the effects of the combination of positive end expiratory pressure (PEEP) and 80×10^-6 nitric oxide (NO) inhalation on oleic acid induced acute lung injury (ALI) in canine.METHEDS:30 dogs were divided into 6 groups. Oleic acid was injected through Swan-Ganz catheter to induced ALI. Pulmonary and systemic hemodynamics,blood gas were measured in dogs before and after injection of oleic acid and the period of inhaled NO for 1-6 h. The methemoglobin(MHb) concentrations were measured. Histology and ultrastructure of the lung tissue were observed. RESULTS:(1) The combination of PEEP and 80×10^-6 NO inhalation rapidly reduced mean pulmonary arterial pressure (MPAP) and pulmonary vascular resistance (PVR), increased PaO₂/FiO₂, reduced A-aDO₂ without inducing significant change on systemic hemodynamics. Arterial blood levels of MHb did not change significantly. (2)The combination group was showed the lightest ALI change by HE stain and electron microscopy. CONCLUSION:The combination of PEEP and inhalation of 80×10^-6 NO significantly and rapidly increased PaO₂/FiO₂ without producing lung injury induced by high FiO₂ and high PEEP.     

Detection and clinical significance of myeloid-derived suppressor cells in peripheral blood of patients with Parkinson disease

AIM: To detect the myeloid-derived suppressor cells (MDSCs) in peripheral blood from the patients with Parkinson disease (PD) and its clinical significance. METHODS: The patients (n=80) diagnosed PD from January 2016 to March 2017 in our hospital and 20 healthy volunteers were selected as the subjects. According to the Hoehn-Yahr staging, 80 PD patients were staged, of whom 22 were I, 24 were Ⅱ, 20 were Ⅲ, 14 were IV, and 0 was V. Peripheral blood (5 mL) samples from the patients with PD and the healthy volunteers were collected and the mononuclear cells were isolated. The levels of CD14⁺CD11b⁺ cells and CD14^-CD11b⁺ cells in the peripheral blood were detected by flow cytometry. The two populations of the cells were sorted by magnetic beads. The mRNA levels of arginase 1 (ARG1), interleukin-10 (IL-10) and cyclooxygenase 2 (COX-2) were detected by qPCR. The expression of surface membrane proteins CD14 and CD11b, and immunosuppressive factors ARG1, IL-10 and COX-2 was determined by Western blot and ELISA. RESULTS: No significant change of CD14⁺CD11b⁺ cells between the patients with PD and normal controls was observed, but the cells with CD14^-CD11b⁺ increased significantly in the patients with PD compared with the control people (P<0.05). The CD14^-CD11b⁺ cells in peripheral blood of the patients were related to the stage of Hoehn-Yahr. The CD14^-CD11b⁺ and CD14⁺CD11b⁺ cells showed high levels of IL-10 and COX-2, and the high level of ARG1 was only expressed in the CD14^-CD11b⁺ cells. The expression of ARG1 in the CD14^-CD11b⁺ population from PD patients was significantly different from that of CD14⁺CD11b⁺ population and normal subjects (P<0.05). CONCLUSION: The CD14^-CD11b⁺ cells and ARG1 expression level in peripheral blood of the PD patients can be used to evaluate the pathogenesis and staging. Immunosuppression may play an important role in the pathogenesis and development of PD.