Objective To investigate the feasibility of using ultrasound‐mediated destruction of microbubbles ( US+ MB) to enhance the transplantation of endothelial progenitor cells ( EPCs) to confer chronic allograft vasculopathy (CAV) .Methods Bone marrow derived mononuclear cells were isolated and induced in vitro . The abdominal aorta transplantation was performed . Four groups were divided:control group without treatment (group A) ,injection with saline (group B) ,injection with EPCs (group C) ,group D ( US+MB+EPCs) was injected with EPCs and US was applied to MB prior to the infusion . All rats were killed during 8 weeks after transplantation to enable histological examination;SDF‐1α expression was detected by immunohistochemistry ,the expression of SDF‐1αand TNF‐αin the grafted aortas were detected with RT‐PCR . Results When 8 weeks after EPCs transplantation ,there was a significant improvement in aortic intima of Group D compared with Group B and C ,respectively ( P <0 .05) . In addition ,treatment of Group D significantly increased the expression of SDF‐1αand reduced the expression of TNF‐αin the grafted aortas . Conclusions US‐mediated MB destruction prior to EPCs transplantation into the grafted aortas can improves the effectiveness of endothelial repair and delay the progress of CAV .

Animals ; Conditioning (Psychology) ; drug effects ; Extinction, Psychological ; drug effects ; physiology ; Fear ; drug effects ; physiology ; Male ; Mifepristone ; pharmacology ; Rats ; Rats, Sprague-Dawley

Background and purpose: The literature on dose-volume parameters and pneumonitis is extensive. The results are inconsistent, both for the best predictive metrics and significant comorbid factors. This study aimed to investigate a prospective functional equivalent uniform dose (fEUD) with perfusion single photon emission computed tomography (SPECT) images as predictors of radiation pneumonitis (RP) in patients undergoing curative radiotherapy (RT). Methods: Functional lung imaging was performed using SPECT for perfusion imaging. Perfusion factors were defined as the mean percentile perfusion levels of the 4 areas, top to 75%, 75% to 50%, 50% to 25%, 25% to 0%, re-spectively. fEUD was calculated from perfusion factors and standard dose-volume parameters extracted from treatment planning computed tomography (CT) scans. Total lung (TL), ipsilateral (IL) and contralateral lung (CL) volumes minus gross tumor volume (GTV), whole-lung V5, V20, whole lung fEUD, IL and CL fEUD, and general equivalent uniform dose (gEUD) were analyzed to evaluate correlations between RP using Common Terminology Criteria for Adverse Events (CTCAE) version 4.03. Statistical significance was defined as P<0.05. Results: A total of 15 patients treated with intensity modulated RT or 3D conformal RT were analyzed, grades≥3 RP were observed in 6 patients. There was only a trend toward significance for unilateral (UL) fEUD of higher dose side (P=0.007). Whole-lung V5, V20 were almost identical between patients who developed pneumonitis and patients who did not, as the values were below the recommended thresholds from published papers. Unilateral fEUDs were linear with unilateral gEUDs (t=0.815, P=0.000). Conclusion: SPECT-based equivalent uniform dose appears to be a better predictor of RP compared to stan-dard dose-volume parameters. Planning constraints should aim to keep unilateral fEUD below 21 Gy.

OBJECTIVE:To study and evaluate the efficacy and safety of homemade sterilized powder for injection of rifampicin(weifunin) in the treatment of pulmonary tuberculosis(PT) and to compared with same import preparation . METHODS:Divide the 121 of bacteriological positive PT patients in the proportion of 1 to 1 with computer automatically into the trial group and the control group used with homemade rifampicin (weifuxin) and import rifampicin(nifu)respectively,the others regimens was same in 2 groups,to evaluate the sputum culture negative conversion rates,X-ray results and adverse drug event etc.after treatment 1 and 2 months . RESULTS: 116 cases were finished ,at the end of 2 month the sputum smear negative conversion rates were 86.21 % and 91.38% (X2=0.780,P=0.377), the sputum culture negative conversion rates were 91.38% and 93.10%(X2=0.120,P=0.729)in the trial group and the control group respectively .The X-ray remarkable effective rates of 2 groups were 82.76% amd 70.69%(X2=2.365,P=0.124)respectively, and the effective rates of the 2 groups were both be 96.55% .The adverse drug event rates were 8.20% and 11.67% respectively, there was on sighificant difference in each indexes .CONCLUSIONS:2 preparation were similar in efficacy ,safety and tolerance.

Human nerve growth factor (NGF) is a nerve cell growth regulation factor, which can provide nutrition for the neurons and promote the neurites outgrowth. In order to produce large-scale recombinant human nerve growth factor (rh-beta-NGF), we constructed a plasmid vector, which can stably express the rh-beta-NGF in the HEK293 cell lines. First, the plasmid of pCMV-beta-NGF-IRES-dhfr was constructed and transformed into HEK293 cells. Then MTX pressurized filter and limiting dilution methods were used to obtain monoclonal HEK293 cell lines. After stepwise reducing serum in culture media, the cells eventually adapted to serum-free medium and secreted rh-beta-NGF. SDS-PAGE analysis revealed that the expression product owned a molecular weight of about 13 kDa and a purity of more than 50%. The peptide mapping sequencing analysis demonstrated the sequences of rh-beta-NGF matched with the theoretical ones. Later we purified this protein by ion exchange and molecular sieve chromatograph. Finally, our experimental results exhibited that the recombinant cell lines can stably express rh-beta-NGF with a high efficiency of more than 20 pg/cell x day. In addition, this protein could successfully induce differentiation of PC12 cells. In summary, our recombinant HEK293 cells can express bio-active rh-beta-NGF with great efficiency and stability, which supply a valid basis to large-scale production of rh-beta-NGF.
Cell Differentiation ; Genetic Vectors ; HEK293 Cells ; Humans ; Nerve Growth Factor ; biosynthesis ; Plasmids ; Recombinant Proteins ; biosynthesis

There are some shortcomings of animal experiments applied in chemical toxicity testing, such as long period, large cost, species differences and dose differences, which limit the use of animal experiments' results in predicting human toxicity.Accordingly, we established a toxicity testing alternative screening system in line with the toxicity endpoints which required in chemical safety evaluation and risk assessment (genotoxicity, carcinogenicity, reproductive toxicity, acute toxicity and general toxicity) based on 3R principles (replacement, reduction, refinement) for animal experiments.This system covers most of the endpoints of toxicity assessment, and molecular biology technology was also applied to integrate the toxicity test, as well as some operation was optimized in order to shorten the experimental period, reduce experimental costs, improve animal welfare.Furthermore, the results from the screening system have higher clinical relevance because it is based on the toxicity mechanisms.

Objective To investigate the effects of 12-lipoxygenase (12-LO) and angiotensin Ⅱ (Ang Ⅱ) on the CIP/KIP family of cyclin-dependent kinase inhibitors (CKIs) p21,p27 and p57 related to cell hypertrophy.Methods Mesangial cells were treated with high glucose for 24 hours and 48 hours respectively.12(S)-hydroxyeicosatetraenoic acid [12(S)-HETE] and Ang Ⅱ were infused to rats by osmotic mini-pump for 1 week and 2 weeks respectively.Rats fed high fat diet were received low dose streptozotocin (STZ) to make type 2 diabetes (DN).The rats were divided into normal control group,DN group,DN+Ang Ⅱ type 1 receptor blocker (ARB) group or 12-LO inhibitor (CDC) group.DN+ARB rats were treated by losartan for 6 weeks,and DN+CDC rats were treated for 8 weeks.Urine albumin and protein expressions of p21,p27 and p57 were detected by ELISA and Western blotting respectively.Glomeruli injury and expressions of p21 and p27 were detected by PAS staining and immunohistochemistry respectively.Results High glucose increased p21 and p27 protein expression in mesangial cells significantly compared with the relative control (all P ＜ 0.05),but had no effect on p57.Ang Ⅱ increased p27 protein expression in gloneruli significantly (P ＜ 0.05),but had no effect on p21 and p57 protein expression.12(S)-HETE increased both p21 and p27 protein expression in glomeruli significantly (all P ＜ 0.05),but had no effect on p57 protein expression.Blood glucose,kidney/body weight,urinary protein,and glomerular p21 and p27 protein expressions were increased in DN group (all P ＜ 0.05) compared with those in control group,with little change of p57 protein expression (P ＜ 0.05).Moreover,glomerular hypertrophy and extra cellular matrix accumulation were observed in DN group.However,urine protein,kidney/body weight,renal injury,but not blood glucose,were decreased in DN+ARB group and DN+CDC group compared with DN group respectively (P＜ 0.05).Further DN+CDC rats had decreased both p21 and p27 protein expressions in glomeruli,but DN+ ARB rats only had decreased p27 protein expression (all P ＜ 0.05).Conclusions 12-LO may induce both p21 and p27 protein expression in DN glomeruli,but Ang Ⅱ may induce only p27 expression.

Objective To explore the effects of amniotic lacrimal duct stenting on the prevention of dry eye in castrated rabbits.Methods Thirtysix healthy male rabbits were selected,the third eyelid were cut off and antiinfection treatment were given,which were randomly divided into 3 groups (12 cases in each group),the castrated male rabbits models were made.Among them,group A was negative control group,group B was dry eye model group,group C was group of lacrimal amniotic membrane group.At 2 weeks before implantation of amniotic lacrimal duct stent,2 weeks,4 weeks and 6 weeks after implantation,the fluorescent (FL) examination,Western blot,Schirmer I examination,immunofluorescence staining and corneal confocal microscopy were performed.Results The levels of tear secretion and FL in the three groups among different time points were significantly different (F=7.126,P =0.009;F =9.658,P =0.016),and there were significant differences among three groups (F =12.582,P =0.005;F =13.187,P =0.013).The tendency of tear secretion and FL in the three groups were also significantly changed (F =8.531,P =0.007;F =10.652,P =0.019).The epithelial basal cells at 6 weeks after implantation in three groups were 3811 ±414,3820 ± 314,2789 ± 353,and the density of inflammatory cells was 266 ±28,266 ± 29,67 ± 13,there were significant differences among three groups (F =13.442,P =0.012;F =9.231,P =0.021).The K1 6 staining in the duct epithelium were negative,and the expression of α-SMA in the lacrimal duct tissue of group A,B and C was not changed at all time points after implantation of amniotic lacrimal stent,and there was no significant difference (F =14.681,P =0.002).Conclusion The amniotic lacrimal stent implantation has certain effect on the prevention of dry eye in rabbit.

Objective Recent studies have shown cardiac protection effects of erythropoietin (EPO).The present experiment was designed to investigate the effects of EPO on TGF-β1, nitric oxide synthase ( NOS), collagen contents induced by angiotensin Ⅱ ( Ang Ⅱ ) in rat cardiac fibroblasts (CFs) and explore the roles of PI3-K/Akt signaling pathway on related effects.Methods Neonatal rat CFs was isolated by collgenase and trypsinase digestion methods.PBS, EPO, Ang Ⅱ in the absence or presence of LY294002, an inhibitor of PI3-K, or L-NAME, an inhibitor of NOS, were added to CFs and cultured for 24 hours.The concentration of collagen Ⅰ and collagen Ⅲ in culture medium were quantitated by ELISA.The levels of nitric oxide (NO) and the activities of NOS as well as NOS isofonns were measured by chemical enzymic method.Western blot was applied to detecting the protein expressions of Akt, p-Akt, eNOS, iNOS, and TGF-β1.Results The concentrations of collagen Ⅰ and collagen Ⅲ in CFs culture medium were significantly increased while the level of NO was significantly decreased by Ang Ⅱ and these changes were significantly suppressed by EPO in a dose dependent manner.The effects of EPO on eNOS and NO could be blocked by LY294002.L-NAME could block EPO's effect on NO but not on the eNOS expression.The suppression effects on expressions of TGF-β1 and collagen by Ang Ⅱ in CFs were blocked by both LY294002 and L-NAME.conclusion EPO suppresses the upregulated expressions of TGF-β1 and increased production of collagen induced by Ang Ⅱ through activating the PI3-K/Akt signaling pathway in neonatal rat CFs.

OBJECTIVETo study the perioperative change in serum neutrophil gelatinase-associated lipocalin (NGAL) level among children with congenital heart disease (CHD) and pulmonary hypertension (PH) and its significance.

METHODSEighty children with CHD were divided into four groups according to pulmonary artery systolic pressure: non-PH, mild PH, moderate PH and severe PH groups. Serum NGAL levels were measured before operation, immediately after operation and 24 hours after operation. The relationship of serum NGAL level with PH and early prognosis was analyzed.

RESULTSThe mild, moderate and severe PH groups had significantly higher serum NGAL levels than the non-PH group, and the severer the PH, the higher the serum NGAL level (P<0.01). All groups showed significant decreases in serum NGAL levels after operation (P<0.01). Serum NGAL level was positively correlated with the degree of PH and length of stay in the intensive care unit (P<0.01).

CONCLUSIONSSerum NGAL level increases in children with CHD and PH, and it gradually decreases after operation for closing the abnormal shunt. Serum NGAL level may be used as a serological indicator for evaluating the degree of PH and surgical outcome.

Acute-Phase Proteins ; Adolescent ; Child ; Child, Preschool ; Female ; Heart Defects, Congenital ; blood ; surgery ; Humans ; Hypertension, Pulmonary ; blood ; Infant ; Lipocalin-2 ; Lipocalins ; blood ; Male ; Perioperative Period ; Proto-Oncogene Proteins ; blood