Objective To investigate the effects of intrathecal transplantation of bone marrow stromal cells (BMSCs) on inter cellular cell adhesion molecule-1 (ICAM-1) expression and blood spinal cord barrier following spinal cord ischemia reperfusion injury.Methods Ninety Sprague Dawley rats were randomly divided into three groups:sham (Sham group),ischemia-reperfusion injury (I/ R group),and BMSCs transplantation (BMSCs group).Spinal I/R injury was induced by clamping the aortic arch between left common carotid artery and left subclavian artery for 14 min in I/R group and BMSCs group.Sham group was subjected to exposure of aortic arch but without occlusion.I/R group and BMSCs group were intrathecally injected with phosphate buffered saline (PBS) or BMSCs (2 × 106) two days before injury.At 1 d,3 d,and 7 d after injury,neurological function was evaluated and damaged lumbosacral seg ment was removed for measurement of blood spinal cord barrier permeability and ICAM-1 protein expression.Results Compared with Sham group,neurological function score was significantly lower:1 d (F =38:59,P =0.001),3 d (F =31.34,P =0.001),and 7 d (F =27.71,P =0.001) ; ICAM-1 expression was increased 1 d (F =34.33,P =0.001),3 d (F =29.76,P =0.001),and 7 d (F =23.65,P =0.001),and blood spinal cord barrier permeability was higher:1 d (F =42.57,P =0.001),3 d (F =32.75,P =0.001),and 7 d (F =26.89,P =0.001) in I/R group.Compared with I/R group,neurological function score was increased:1 d (F =16.62,P =0.001),3 d (F =21.54,P =0.001),and 7 d (F =12.84,P =0.002) ; ICAM-1 expression was decreased:1 d (F =19.84,P =0.018),3 d (F =17.38,P =0.008),and 7 d (F =22.46,P =0.007),and blood spinal cord barrier permeability was lower:1 d (F =22.38,P =0.016),3 d (F =27.59,P =0.009),and 7 d (F =23.25,P =0.001) in BMSCs group.Conclusions Intrathecal transplantation of BMSCs inhibited ICAM-1 expression and decreased blood spinal cord barrier permeability,and then attenuated spinal cord ischemia-reperfusion injury.

Objective To investigate the protective effect of hypoxic-preconditioned bone marrow mesenchymal stem cells (BMSCs) on spinal cord tissue after ischemia reperfusion injury.Methods Healthy adult Sprague Dawley (SD) rats weighing 200 ～ 250 grams (g) were randomly divided into 3 groups with 6 animals in each group:The sham group received simple surgical manipulation without ischemia/reperfusion treatment;The spinal cord ischemia/reperfusion group (Control group) only received spinal cord ischemia/reperfusion surgery.The hypoxic preconditioned BMSC transplantation group (HP-MSCs group) was injected with hypoxic preconditioned BMSCs 2 days before ischemia/reperfusion.The control group,HP-MSCs group received spinal cord ischemia/reperfusion for 10 min and observed for 48 h.The permeability of the blood-spinal cord barrier was examined with Evans blue (EB),and the histomorphology changes were observed with hematoxylin and eosin (HE) staining.Results EB red fluorescence was significantly weakened in the HP-MSCs group than that in the Control group (P ＜ 0.05),and more intact motor neurons were found in the lumbar spinal cords in the HP-MSCs group than that in the Control group (P ＜0.05).Conclusions The hypoxic-preconditioned BMSCs could effectively attenuate spinal cord ischemia reperfusion injury,it may be associated with protective effect of the blood-spinal cord barrier integrity.

ObjectiveTo evaluate the effect of low glycemic index(LGI)diet and exercise on plasma glucose and lipid profiles in newly diagnosed type 2 diabetic patients. MethodsSeventeen newly diagnosed type 2 diabetic patients with FPG ≤ 10mml/L treated by LGI diet and exercise only for two months.Fasting plasma glucose (FPG),2 hours postprandial glucose(2hPG),glycosylated hemoglobin A1 C(GHbA1C),and lipid profiles were measured.The results of FPG,2hPG,GHbA1C,and lipid profiles were compared. ResultsTwo months after treatment,the level of fasting glucose(6.19 ± 0.60)mmol/L,postprandial 2h plasma glucose(8.59 ± 0.90)mmol/L,TG(1.15 ± 0.45)mmol/L,TC(4.98 ± 0.77)mmol/L,LDL(3.20 ± 0.71)mmol/L were significantly lower than (7.84 ± 1.19)mmol/L,(13.97 ± 3.35)mmol/L,TG(1.79 ± 0.75)mmol/L,TC(5.46 ± 0.27)mmol/L,LDL (3.57 ± 0.28)mmol/L,HDL(1.59 ± 0.30)mmol/L was significantly higher than(1.42 ± 0.26)mmol/L,the differences were statistically significant(all P＜0.05);HbA1c(6.49 ± 0.57)% was slightly lower than(7.29 ±0.77)%,but the difference was not significant(P＞0.05);No hypoglycemia was observed during the treatment. ConclusionThe exellent glycemic control and improvement of lipid profile could be achieved by low glycemic index diet and exercise only.Furthermore,no hypoglycemia occurred during the treatment.

Objective To investigate the expression of Notch1 and its ligand DLL4 in human gastric carcinoma tissues and its correlation with tumor angiogenic metastasis.Methods Immunohistochemical SP method was used to detect the expression of Notch1,DLL4 and VEGF in 45 gastric carcinoma tissues and paired adjacent normal gastric mucosa,and the relationship between them and clinico-pathological parameters were analyzed.Results The positive expression rate of Notch1,DLL4 and VEGF in gastric carcinoma were higher than that in normal gastric mucosa(P

Objective To investigate the effect of glucose-based peritoneal dialysis fluids and icodextrin-based peritoneal dial-ysis fluids on the expression of TLR2 and TLR4 on huamn peritoneal mesothelial cells .Methods Human peritoneal mesothelial cell line 5 - 10 generations(HMrSV5) was cultured in DMEM /F12 medium supplemented with 10% (v/v) fetal calf serum (FCS) .Cell viability and cell proliferation were assessed using M TT method .The experiment were divided into 5 different groups :group A (control group) ,1 .5% dextrose group ,2 .5% dextrose group ,4 .25% dextrose group and 7 .5% Lcodextrin group .Icodextrin group (aikau dextrin) ,TLR2 and TLR4 expression were detected by Western blot .Results Treatment with different concentrations of glucose-based peritoneal dialysis fluids for 24 h did not affect the expression of TLR2 and TLR4 protein .In addition ,after stimula-tion for 48 h ,1 .5% dextrose ,2 .5% dextrose ,4 .25% dextrose decreased TLR2 expression by (5 .5 ± 2 .8)% ,(31 .4 ± 7 .5)% , (54 .9 ± 1 .9)% respectively ,TLR4 expression by (32 .9 ± 17 .6)% ,(47 .7 ± 13 .5)% ,(66 .4 ± 13 .5)% respectively .Stimulation for 72 h ,decreased TLR2 expression by (29 .4 ± 14 .7)% ,(38 .9 ± 9 .9)% ,(63 .5 ± 16 .5)% respectively ,TLR4 expression by(59 .5 ± 16 .8)% ,(63 .1 ± 9 .5)% ,(79 .2 ± 14 .0)% respectively .There was no significant change in TLR2 and TLR4 protein expression on 7 .5% icodextrin group .Conclusion Glucose-based peritoneal dialysis fluids ,but not icodextrin-based peritoneal dialysis fluids downregulates expression of TLR2 and TLR4 by HM rSV5 .

Objective To investigate the effects of allele-31 C>T on the binding activity to IL-1βpromoter of the nuclear transcription factor C/EBPβand PU.1 induced by Mycobacterium tuberculosis infection.Methods The electrophoretic mobility shift assay ( EMSA) was performed to explore whether the nuclear transcription factor C/EBPβand PU.1 could bind to -31 region in IL-1βpromoter.The C/EBPβ-and PU.1-expressing vectors were constructed and co-transfected into HeLa cells with IL-1βpromoter luciferase vector.The expression of C/EBPβand PU.1 was confirmed using Western blotting assay, and the promoter activity was determined using Dual-Glo Luciferase system under various transfection conditions. Lentivirus-mediated RNA interference was used to explore the effects of C/EBPβand PU.1 on IL-1βexpression.GraphPad Prism 5.0 was used for data analysis.Results EMSA results showed that both C/EBPβand PU.1 could bind to -31 region in IL-1βpromoter.Both C/EBPβand PU.1 induced by Mycobacterium tuberculosis infection could increase IL-1βpromoter activity, especially for the -31 T allele (t=22.33 and 7.98,P<0.01), and there was a synergy on the promoter activity between C/EBPβand PU.1.The promoter activity decreased significantly when C/EBPβand/or PU.1 were silenced by lentivirus-mediated RNA interference (q=5.79, 6.23 and 11.66,P<0.01).Conclusion The allele-31 C>T can induce IL-1βpromoter activity and gene transcription through regulation of binding activity to C/EBPβand PU.1 induced by Mycobacterium tuberculosis infection.

OBJECTIVE:To establish the method for determining 10 residual organic solvents in norvancomycin hydrochloride raw material. METHODS:Headspace gas chromatography was performed on the column of nitro modified polyethylene terephthal-ate glycol as stationary phase capillary column;the oven temperature program started at 40 ℃ for 3 min and increased at a rate of 8 ℃/min up to 150 ℃ for 10 min;the temperature was 200 ℃ with carrier gas of high-purity nitrogen gas,the constant flow rate was 5 ml/min with split ratio of 15∶1;the headspace vial equilibrium temperature was 85 ℃ with equilibrium time of 40 min,and the volume was 1 ml. RESULTS:The concentration of n-pentane,acetone,ethanol,benzene,acrylonitrile,toluene,xylene,chlo-robenzene,styrene,divinylbenzene had good linear relationship with its peak area values(r=0.995 7-0.999 9);the RSDs of preci-sion,repeatability tests was ≤6.6%;average recovery was in the range of 94.3%-106.6%(RSD=0.5%-4.5%,n=9). CONCLU-SIONS:The method is fast,sensitive and accurate,and can be used for the determination of residual organic solvents in norvanco-mycin hydrochloride raw material.

Abstract: Objective To construct a shuttle vector pHT315-AaCPR100A with two spore-producing-dependent promoters and the target gene AaCPR100A in Escherichia coli-Bacillus thuringiensis. Methods The forward promoter of Cry3A, named Pro-1 (+), was amplified by PCR using pSVP27A plasmid as the template, and the target gene AaCPR100A was amplified using Aedes aegypti RNA reverse conversion cDNA as the template. The plasmid pHT315 was linearized by digestion with Hind Ⅲ and Sal Ⅰ. The forward promoter and the target gene were inserted into the linearized vector pHT315 successively by in-fusion cloning according to the transcription direction. The synthesized plasmid containing the Cry3A reverse promoter sequence was used as the template, and the Pro-1 (-) reverse promoter was amplified by PCR. The intermediate vector containing the forward promoter and the target gene was linearized by EcoR I restriction enzyme, and the reverse promoter was inserted downstream of the target gene by in-fusion cloning in the direction of transcription. Results By agarose gel electrophoresis, the forward promoter, target gene AaCPR100A and reverse promoter bands were clear and of good quality, which could be used for in-fusion cloning experiments. The two spore-producing-dependent promoters and target gene fragments were connected by In-fusion cloning. The recombinant vector pHT315-AaCPR100A was verified by PCR. The forward promoter, target gene fragment and reverse promoter were successfully amplified in the recombinant vector. Nucleotide sequencing verified that the sequencing results of the bidirectional promoter sequence and the target gene sequence were basically consistent with the sequence alignment results, which met the requirements of the construction of vector elements and proved that the recombinant vector was successfully constructed. Conclusions Based on the above results, this study proves that the recombinant shuttle vector with two spore-producing-dependent promoters can be successfully constructed by in-fusion cloning technology, laying the foundation for the construction of engineered Bacillus thuringiensis expressing dsRNA of AaCPR100A.

The aim of the study is to establish a platform to deliver therapeutic proteins into target cells through a polyarginine-based cell penetrating peptide. To facilitate the expression of therapeutic proteins, a pSUMO (Small Ubiquitin-like Modifier)-R9-EGFP (enhanced green fluorescence protein) prokaryotic expression vector was constructed. After induction, the fusion protein SUMO-R9-EGFP was efficiently expressed. To validate the cell penetrating ability of the fusion protein, HepG2 cells were incubated with the purified R9-EGFP or EGFP protein as control, internalization of the fluorescent proteins was examined by either flow cytometry or confocal microscopy. The result obtained by flow cytometry showed that the R9-EGFP fusion protein could efficiently penetrate into the HepG2 cells in a dose and time-dependent manner. In contrast, the fluorescence was barely detected in the HepG2 cells incubated with EGFP control. The fluorescence intensity of the R9-EGFP treated cells reached plateau phase after 1.5 h. The result obtained by confocal microscopy shows that R9-EGFP efficiently entered into the HepG2 cells and was exclusively located in the cytoplasm, whereas, no fluorescence was detected in the cells incubated with the EGFP control. The heparin inhibition experiment showed that heparin could inhibit penetrating effect of the R9-EGFP protein by about 50%, suggesting that the penetrating ability of the fusion protein is heparin-dependent. In summary, the study has established a platform to deliver therapeutic proteins into target cells through a polyarginine-based penetrating peptide.
Cell-Penetrating Peptides ; biosynthesis ; genetics ; pharmacology ; Genetic Vectors ; genetics ; Green Fluorescent Proteins ; biosynthesis ; genetics ; Hep G2 Cells ; Humans ; Peptides ; genetics ; metabolism ; Protein Transport ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; pharmacology

The aim of this project is to establish a fibroblast growth factor-21 (FGF-21) signaling pathway targeted cell model, for screening a class of FGF-21 receptor agonists as anti-diabetic candidates. FGF-21 requires beta klotho transmembrane proteins as co-receptor for the activation of tyrosine kinase FGF receptor (FGFR) signaling, thereby activating a series of intracellular signaling pathways and regulating gene transcription for glucose metabolism. Firstly a recombinant plasmid expressing co-receptor beta klotho and EGFP reporter genes was constructed. After introducing the recombinant plasmid into package cells, the cell culture supernatant was used to infect 3T3-L1 cells, which were then screened for stably expressing beta klotho gene. Administration of FGF-21 increased the expression of GLUT1 and stimulated GLUT1-mediated glucose uptake. This novel cell model can be conveniently used in high-throughput drug screening of FGF-21 or FGF-21 analogues.