Background How to harvest the purified corneal endothelial seed cells is very important for the corneal tissue engineering technology. Herein,to establish a good culture method and effective identification method of corneal endothelial cells ( CECs) is the key. Objective Present study wag to establish the cultivating and identifying approach of the rabbit CECs and detect the biological characteristics of passaged cells. Methods Rabbits CECs were isolated from Descemet's membrane peeled off completely in 30 New Zealand white rabbits and then digested with 0. 25% trypsin-0. 02% EDTA and primarily cultured in CECs medium containing 15% fetal bovine serum. The growth situate and cellular morphology of rabbit CECs were observed under the inverted phase-contrast microscope following the alizarin red staining. Rabbit CECs were identified by cellular morphology as well as in gene and protein level, including the detection of expressions of collagen type IV α2(COL4A2) ,vascular endothelial growth factor receptor 2 ( FLK1 ) , Na+-K+ ATPase alpha 1subunit ( ATP1 A1 ) , aquaporin 1( AQP1 ) , voltage-dependent anion channels ( VDACs) by reverse transcription-polymerase chain reaction ( RT-PCR ). The expression and distribution of neurone specific enolase ( NSE) , Na+-K+ ATPase, zonula occludens-1 ( ZO-1 ) were also detected by immunocytochemistry under the fluorescence microscope. The proliferation activity of the passage cells was dynamically observed by MTT assay,and Na+-K+ ATPase activity of different generations cells was detected by ATPase kit. Results Majority of the primarily cultured cells adhered in 24 hours, infused in 2-3 days with the hexagon shape in appearance. The morphology of the cells was very varied with passage. Alizarin red staining showed a well- defined and well-lined cellular morphology similar to the corneal cells in vivo. Target genes of C0L4A2, FLK1, ATPIAI ,AQPI and VDACs were positively expressed in the cells. However,the expression of CK12 was absent in the cells. NSE, Na+-K+ ATPase, ZO-1 positive cells were respectively observed under the laser confocal scanning microscopy. MTT results showed a gradually low growth curve with the passage. Quantitative results also revealed that the Na+-K+ ATPase activity was gradually declined in different generations of cells ( F = 77. 174, P = 0. 000 ). Conclusion The enzyme digestion is a better approach of isolating and culturing comeal endothelial cells. Cultured cells can be identified by cells staining, gene expression and protein level. Earlier generation of CECs are ideal seed cells for cornea tissue engineering.

Acupuncture Therapy ; Adult ; Cervical Vertebrae ; injuries ; Combined Modality Therapy ; Female ; Humans ; Male ; Massage ; Middle Aged ; Pharyngitis ; etiology ; therapy ; Spinal Diseases ; complications ; Young Adult

Objective To study the effects of Poly(I:C) on lung adenocarcinoma A549 cells viability and illuminate the mechanism of Poly (I:C)-induced apoptosis in A549 cells.Methods A549 cells were transfected with the complex of Poly(I:C) and lipofectamine 3000.The viability of A549 cells was tested by methyl thiazolyl tetrazolium (MTF) method.The apoptotic cells were tested by flow cytometry.The caspase proteins were tested by Western blotting and the expressions of interferon-β (IFN-β) and CXCL-10 were assayed by real-time PCR.After employing the pan-caspase inhibitor Z-VAD-FMK and caspase-8 inhibitor Z-IETD-FMK,the variation of Poly (I:C) proapoptosis in A549 cells was observed.RNA interfering experiments were employed to knock down melanoma differentiation related antigen 5 (MDA5) or retinoic acidinduced gene Ⅰ (RIG-Ⅰ),and the above indexes were tested.Results The viability of A549 cells was significantly reduced to 74.92％ ±--6.24％ after 200 ng/ml Poly (I:C) transfection compared with that before transfection (95.32％ ± 3.05％,t =2.883,P =0.041).The apoptotic rates induced by 100,200,400 ng/ml Poly(I:C) were 9.97％-± 0.88％,23.63％-± 1.41％,32.57％-± 2.39％,respectively.All of them were higher than that in the control group (0.74％-± 0.15％),with significant differences (t =4.489,P =0.002;t =11.616,P =0.000;t =16.932,P =0.000).Besides,the death receptor pathway proteins such as TNF-related apoptosis inducing ligand (TRAIL),cleaved-caspase-8 and cleaved-caspase-3 increased obviously.MDA5/RIG-Ⅰ pathway was also activated dramatically and the expressions of IFN-β,CXCL-10 were significantly up-regulated.The apoptotic rates reduced to 3.17％ ± 0.66％,5.35％ ± 0.64％ with pancaspase inhibitor Z-VAD-FMK and caspase-8 inhibitor Z-IETD-FMK pretreatment,compared with the control group (15.87％ ±0.93％),and the differences were statistically significant (t =8.643,P =0.001;t =6.824,P =0.002).Moreover,the expressions of TRAIL,IFN-β and CXCL-10 induced by Poly (I:C) were inhibited with MDA5 or RIG-Ⅰ depletion.Conclusion Poly(I:C) can reduce the survival rate of A549 cells and promote the apoptosis mainly by activating the death-receptor pathway mediated by MDA5/RIG-Ⅰ probably,which may involve in IFN-β,CXCL-10.

Objective The separation and purification of anthocyanin from blueberry was studied to determine the optimum purification conditions. Methods Static adsorption and desorption analytical test was conducted to screen the best macroporous resin from HPD-600, HPD-100A, DM130, D4020, and D3520 for purification of anthocyanins from blueberry. Effects of the sample liquid concentration, pH, adsorption flow rate, eluent ethanol concentration, eluent ethanol amount, and elution flow rate were investigated through dynamic adsorption-desorption test by single factor and orthogonal experiments. Results D3520 resin was the most effective resin for adsorption and desorption of anthocyanins from blueberry, when the amount of macroporous resin (g) and the amount of anthocyanin from blueberry (mL) was 1:5. The optimal conditions were as follows: The concentration of sample was 1.0 mg/mL, adsorption flow rate was 2.0 mL/min, and sample solution pH value was 3. Under the optimal conditions, eluent ethanol concentration 70%, elution flow rate 2.0 mL/min, eluent ethanol amount 60 mL, and the adsorption rate and desorption rate can reach 92.82% and 91.58% respectively. Conclusion The color value of purified products under the optimized conditions was increased by 11 times; The purity fraction of anthocyanin was 96.17% and the purity was increased by 5 times; And the yield of anthocyanin was 14.271%. This process effectively improves the efficiency of anthocyanin from blueberry purification, which is suitable for the future production of anthocyanin from blueberry.

Objective To explore the mobile changes of nitric oxide(NO) and the relationship between nitric oxide and glomerular hyperfiltration in experimental diabetic rats.Methods Experimental diabetes mellitus(DM) was induced in rats with streptozotocin(STZ).The levels of NO and NOS in renal tissue homogenate were assayed after establishment of diabetesat the 4 th,8 th,12 th week.At the same time, renal morphology in diabetic rats was examined by light microscope and image of computer.Results The contents of NO and NOS in renal homegenates were evidently increased at 4 th week,and decreased gradually from 8 th week(P

Background Aspheric intraocular lens (IOL) is designed to reduce the spherical aberration of the eye after cataract surgery and to obtain better visual quality.However,the selection of a personalized aspheric IOL is a problem to be solved.Objective This study was to compare the wavefront aberration and quality of vision of patients between the implantation of negative spherical aberration IOL and non-aberration IOL,and to investigate the relationship between corneal Q values and postoperative spherical aberration.Methods One hundred and four eyes of 90 patients with age-related cataract were randomized into two groups.Fifty-two eyes of 46 patients who received a Tecnis Z9001 IOL implantation were assigned as the negative spherical aberration IOL group and 52 eyes of 44 patients who received Akreos AO IOL without aberration were assigned as the non-aberration IOL group.The preoperative corneal Q values were measured and the mean Q value(Q)was computed.Then the patients in the two groups were further divided into 2 subgroups,respectively,based on their Q values were over or below (Q）.The corneal Q values,root mean square(RMS) of ocular spherical aberration,coma and total higher-order aberrations(HOAs) for 5 mm diameter pupil,scotopic contrast sensitivity with or without glare at 6 mm pupil diameter were measured 3 months after surgery.Results The pre-and post-operative corneal Q values were insignificantly changed (t =1.447,P =0.151).The spherical aberration in the negative spherical aberration IOL group was(0.059-±0.047)μm,and that in the non-aberration IOL group was(0.110±0.066)μm,with a statistically significant difference between them (t =-4.567,P=0.000).Scotopic contrast sensitivities at intermediate and high frequencies were significantly better in the negative spherical aberration IOL group than in the non-aberration IOL group (t =2.495,t =2.359,P ＜ 0.05).There was no significant difference in coma and HOAs between the two groups after operation (P ＞ 0.05).Weak positive correlations were seen between the pre-and post-operative corneal Q values and spherical aberration in the two groups(r=0.277,0.292,0.285,0.325,all at P＜0.05).However,no significant differences were found in spherical aberration,contrast sensitivity and scotopic contrast sensitivity between the different subgroups (P ＞ 0.05).Conclusions Negative spherical aberration IOL has lower spherical aberration and better scotopic contrast sensitivity than non-aberration IOL.The results suggest that it may be not enough to choose the corneal Q value only as the single reference criterion for selection of aspheric IOL.

This study aimed to evaluate the influence of quartz fiber post placement on the fracture resistance of endodontically treated premolars with different dental defects under dynamic loading. Fifty extracted single-rooted mandibular premolars were randomized into five groups. Each group was prepared according to numbers of residual walls ranged from 0 to 4. Then each group was divided into two subgroups with one restored with quartz fiber posts and the other without posts. In no-post groups, gutta percha point 2 mm below cemento-enamel junction was removed. Composite resin was adapted to the well and used to shape the core directly. Each tooth was restored with a complete metal crown. Dynamic loading was carried out in a masticatory simulator with a nominal load of 50 N at 2 Hz for 300 000 loading cycles. Then a quasi-statically load was applied in a universal testing machine 30° to the long axis with a crosshead speed of 1 mm⋅min(-1) until fracture. Data were analyzed with one-way analysis of variance and pairwise comparison (P<0.05). No specimens failed during dynamic loading. The fracture resistance enhanced with the increase of numbers of coronal walls and the differences were significant (P<0.05). Placement of fiber posts had a significant effect when fewer than two walls remained (P<0.05), but it had no significant influence in groups with two, three or four walls (P>0.05). Fiber post did not change failure mode, and the fracture pattern was mainly favorable. More dentin walls need to be retained in clinic. When no less than two walls remained, a fiber post is not always necessary.
Analysis of Variance ; Bicuspid ; Composite Resins ; Dental Restoration Failure ; Dental Stress Analysis ; Dentin ; physiology ; Humans ; Post and Core Technique ; Quartz ; Tooth Fractures ; prevention & control ; Tooth, Nonvital

Objective Dendritic cell-associatedC-type lectin-1 ( Dectin-1) is one of the most important receptors in antifungal innate immune response.This study was to construct a recombinant adenovirus vector expressing themurine Dectin-1gene and acquire a high-concentration adenovirus by amplification and purification. Methods The PCR amplification product CLEC7A-pIRES2-EGFP was cloned into the intermediate vector pDONR221, and then recom-bined with the backbone vector pAD/CMV/V5-DEST to produce a re-combinant plasmid pAD-CLEC7A-pIRE2S -EGFP.The recombinant plasmid was linearized with Pac I and transfected into human embryon-ic kidney ( HEK293) cells to produce recombinant adenovirus pAD-CLEC7Ap-IRES 2-EGFP. The adenovirus was propagated in the HEK293 cells and purified by filtering through the cellulose acetate membrane and concentrating column.Fluorescence microscopy and re-al-time PCR were used to determine the expression of the Dectin-1 gene. Results PCR identification, enzyme digestion, and sequen-cing results manifested theDectin-1 gene in the vector, with the final adenovirus titer of 5×1011 IU/mL.Fluorescence microscopy revealed green fluorescence and real-time PCR assay confirmed that the expression of Dectin-1 was improved by 8677.25 times. Conclusion A relatively high-titer adenovirus expressing Dectin-1 was acquired,which may help to further study the high expression of Dectin-1 in anti-fungal innate immunity in vitro and in vivo.

Objective The aim of this study is to evaluate clinical outcomes of patients with acute type A intranural hematoma of the aorta(IMH) received surgical treatment.Methods We analyzed 40 consecutive patients with acute type A aortic IMH in Fuwai hospital.The patients are from 2012.1.1 to 2015.12.31.The average age of patients is(56 ± 11) years.Clinical outcomes and morphological evolution by CT were analyzed for 2 years.Results Most of the patients were treated medically during their initial hospitalization.There were 2 patients died in in-hospital and no 2-year mortality.16 patients (40％) were received acute surgery,24 patients(60％)were received normal surgery.Conclusion Surgical treatment would be a favorable treatment option in type A acute IMH.

Objective To explore risk factors of unintentional injuries among internal-migrant children. Methods A 1 ∶〗1 matched case-control study was conducted between 332 internal-migrant children with unintentional injuries, based on the principle that same class, same gender and age was no more than two years old. The survey was based on the unified questionnaire. Epi Data 3.1 was used to establish database and SPSS 23.0 was used for univariate analysis and multi-conditional Logistic regression analysis. Results Multi-conditional Logistic regression analysis model indicated that caregiver put pressure on children before exam and the adjusted odd ratio (OR) was 2.086 and 95% confidence interval (CI) was 1.263-3.444, caregiver’s character was extravert type (OR=2.074, 95% CI:1.275-3.372) or middle type (OR=1.796, 95% CI: 1.158-2.784), caregiver’s educational background was illiteracy (OR=1.867, 95% CI: 1.280-2.722) or primary school (OR=2.458, 95% CI: 1.169-5.168), the neurotic behaviors of children (OR=1.466, 95% CI: 1.012-2.124) were risk factors of unintentional injuries among internal-migrant children. Conclusions The characteristics related to internal-migrant children’s caregiver and children’s neurotic behaviors are associated with the occurrence of unintentional injuries. Immediate and effective measures should be taken to ease the high injury prevalence and prevent the unintentional injuries among internal-migrant children.