Objective: To reduce the residual proteins and DNA of host cells in the preparation of H5N1 influenza A virus. Methods: Core 700 was firstly used to remove residual host cell proteins, and then Capto Q was used to remove host cell DNA. Several batches of H5N1 influenza A virus cultured in Madin-Darby canine kidney (MDCK) cells were purified using this method. The efficiency of purification was evaluated using many methods including quantitative real-time PCR, hemagglutination (HA) test and single radial immunodiffusion assay. Moreover, Benzonase nuclease was used for comparison. Results: Without the use of Benzonase nuclease, the overall removal rates of host cell DNA and residual proteins were 99.62% and 98.1%, and the HA antigen recovery rate was 66.96%. Conclusions This study established a purification strategy with good effect for cell-based influenza vaccines. It can efficiently remove host cell DNA and proteins and achieve a high HA recovery rate. The purification result is no worse than that of adding Benzonase nuclease, suggesting the potential of its application in actual vaccine production.

Objective To investigate whether vascular endothelial cells in choroidal neovascularization whether hypoxia condition can up-regulate SNAI1 and activate matrix metalloproteinase (MMP)2 and MMP9 therefore to participate in choroidal neovascularization(CNV).Methods Sixteen SPF male C57 mice aged 6-8 weeks were divided into control group and model group.CNV models were induced by retinal laser photocoagulation,and flatmount and frozen sections of retinal pigment epithelium (RPE)-choroid-sclera compound were prepared at 7 days after modeling.The CNV in flat-mount was examined by Isolectin B4 staining,and the location of SNAI1,MMP2 and MMP9 in frozen sections was determined by immunofluorescence technology.The expression of SNAI1,MMP2 and MMP9 at mRNA level in CNV was detected by real-time fluorescence quantitative PCR (real-time PCR).The use and care of experimental animals complied with Statement for the Use of Animals in Ophthalmic and Visual Research.The RF/6A cells were divided into normoxia group and hypoxia group and cultured for 24 hours in 5％ CO2condition and mix condition of 94％ N2,5％ CO2 and 1％ O2,respectively.The expression of SNAI1,MMP2 and MMP9 in the cells at mRNA and protein levels was detected by real-time PCR and Western blot assay,respectively.Small interfering RNA of SNAI1 (siSNAI1) was transfected into the cells,and then the expression of MMP2 in the cells at mRNA and protein levels was detected by real-time PCR and Western blot assay,respectively,and the migrating number of the cells was assayed by Transwell chamber assay.Results CD31 and SNAI1 positive-response cells were seen in RPE-choroid-sclera flat-mounts under the laser scanning confocal microscope.The relative expression levels of SNAI1mRNA and MMP2 mRNA in RPE-choroid-sclera tissues were higher in the model group than those in the control group (SNAI1 mRNA:1.291 ±0.060 vs.0.759±0.074,P =0.001;MMP2 mRNA:1.610±0.424 vs.0.772 ±0.080,P =0.044).The expression of MMP9 mRNA was not significantly elevated between model group and control group (P＞0.05).The relative expression level of MMP2 mRNA was higher in comparison with MMP9 mRNA in the model group (P＜0.01).The relative expressions of hypoxic induced factor 1α (HIF-1α),SNAI1 and MMP2 at mRNA level and protein level in RF/6A cells were significantly higher in the hypoxia group than those in the normoxia group (all at P＜0.05) and no considerable difference was seen in MMP9 mRNA expression between the two groups (P＞0.05).The relative expressions of MMP2 mRNA in the cells were 0.217±0.036 and 0.818±0.105,and those of MMP2 protein in the cells were 0.236±0.009 and 1.043±0.120 in the hypoxia+siSNAI1 group and only hypoxia group,respectively,with significant differences between them (P =0.002,0.003).The migrating number of the cells was (254.60 ±71.31)/field in the hypoxia+siSNAI1 group,which was significantly less than (534.10±96.21) /field in the control group (P =0.029).Conclusions The hypoxic environment at CNV can activate MMP2 by up-regulating the expression of SNAI1,which promotes the migration of vascular endothelial cells and therefore participates in CNV formation,and the intervention of SNAI1 activation under the hypoxic condition can inhibit this process.

Objective Detecting antibodies against RBCs,platelets,lymphocytes,and neutrophils by a solid-phase antibodies screening system.Methods mono-layer blood cell immobilized in the bottom of U-microplate respectively to prepare the solid-phase antibody screening system.Then detecting the antibodies exist or not in 2 150 random blood donor and 440transfusion patients' samples.Analyze the influence of antibodies against blood cells to the transfusion effect.Results 53RBC antibodies,22 platelet antibodies,13 lymphocyte antibodies,55 neutrophil antibodies were detected in random blood donor samples;31 RBC antibodies,38 platelet antibodies,24 lymphocyte antibodies,43 neutrophil antibodies and 115 mixed (two or more antibodies against RBC,platelets,lymphocytes or neutrophils exist at the same time) were detected in transfusion patients' samples after detection.233 suspected adverse reaction happened after transfusion,which antibodies were detected in 133 samples.Conclusion The antibodies against blood cells solid-phase screening system can applied in pre-and post-transfusion detection.The solid-phase screening method is more sensitivity than serologic tube test and flow cytometry.Existing of antibodies influence the transfusion effect.

Objective To investigate the feasibility of ADC values in the evaluation of normal fetal brain development by measuring ADC values changes in specific regions with advancing gestational age.Methods Forty fetuses(gestational age:24 to 41 weeks) with normal brain underwent DWI(b value were 0 and 600 s/mm2).ADC values of the frontal white matter(WM),occipital WM,thalamus,basal ganglia,and cerebellum were measured by post-processing software.The differences among different regions' ADC values were calculated by repeated measurements of ANOVA,and simple linear regression was used to evaluate the relationship between ADC values and gestational age.Results The mean ADC valuesof 40 fetuses were(1 800±214) ×10-6mm2/s in frontal WM,(1 400±100) ×10-6mm2/s in basal ganglia,(1 300±126) ×10-6mm2/s in thalamus,(1 700±133) ×10-6mm2/s in occipital WM and(1 400± 155) × 10-6mm2/s in cerebellum,respectively.There was significant difference in the ADC values among the five regions(F=80.813,P＜0.01).In pair-wise comparison,ADC values of basal ganglia,thalamus and cerebellum had no significant difference; however,others had significant difference between each other.With the increasing gestational age,ADC values of basal ganglia,thalamus,occipital WM and cerebellum decreased,and had significant negative correlations with gestational age(Pearson correlation coefficient were-0.568,-0.716,-0.830 and-0.700,respectively,all P＜0.01).In terms of ADC value,occipital WM declined fastest with gestational age,followed by cerebellum and thalamus,and the slowest was basal ganglia.Frontal ADC values showed no correlation with gestational age(P＞0.05).Conclusions Specific regions of fetal brain have specific ADC values,and ADC values of the region undergo regular change with advancing gestational age.ADC value is a specific quantitative parameter that could help to evaluate normal brain development and early diagnosis of fetal brain lesions.

BACKGROUND: There are different methods to isolate and culture human nucleus pulposus cells, and the differences in digestive enzymes components and digestion time quite are significant. So how to rapidly and efficiently harvest human nucleus pulposus cells has become a research hotspot. OBJECTIVE: To optimize the digestive enzymes components and digestion methods for the preparation of human nucleus pulposus cells. METHODS: Nucleus pulposus tissue specimens were selected from three adult discs in the Department of Orthopedics, China-Japan Union Hospital of Jilin University. The acute traumatic disc tissues that outstanding to the spinal canal were taken under aseptic conditions, and then the peripheral white annulus and jel y-like nucleus pulposus in the center could be seen. According to different mixed enzyme concentration ratio, the samples were divided into two groups. The enzyme Ⅰ group was treated with 0.2% Ⅱ col agenase; and the mixed enzymeⅡ group was digested with 0.25% trypsin for 30 minutes, and then treated with 0.2% Ⅱ col agenase. According to digestion time, each group was divided into three subgroups: 2 hours group, 4 hours group, and overnight group. Final y, suspended cel volume was decided as 2 mL to count cells. Dulbecco’s modified Eagle’s medium containing fetal bovine serum was used for cel culture in vitro. Trypan blue staining was performed to count total cel number and ratio of living cells. Methylthiazolyldiphenyl-tetrazolium bromide assay was used to detect the growth curve of nucleus pulposus cells. RESULTS AND CONCLUSION: Based on the two digestion enzyme concentration, the number of digested cells in the enzyme Ⅰ group was larger than that in the enzyme Ⅱ group after digested for 2 and 4 hours, but the difference was not significant (P > 0.05). Overnight, cellsurvival rate was decreased in the enzyme Ⅰ group after digested for 2 and 4 hours when compared with the enzyme Ⅱ group, and the difference was significant (P < 0.05). After digested for 4 hours, tissue blocks disappeared, and the number of cells reached maximum. The results indicate that enzyme Ⅰgroup composite with Ⅱ col agenase is benefit for the separation of nucleus pulposus cells, and the digestion time is appropriate to 4 hours. This condition has the advantages of simple operation, high efficiency and low cost, and it considered that digestion of nucleus pulposus tissues with 0.2% Ⅱ col agenase for 4 hours is the best condition to obtain nucleus pulposus cells.

BACKGROUND:Advancement in bioengineering based upon tissue engineering techniques may offer the possibility of repairing degenerative intervertebral disc.
OBJECTIVE:To summarize the research progress in the scaffolds of tissue engineered intervertebral disc.
METHODS:A computer-based retrieval was performed to search manuscripts describing tissue engineered intervertebral disc scaffolds published between January 1st, 1900 and December 31st, 2012 in PubMed database with the key words of“tissue engineering, intervertebral disc, scaffold”in English.
RESULTS AND CONCLUSION:Scaffold is an important part of tissue-engineered research. There are three kinds of materials for intervertebral disc scaffolds:natural biomaterials, synthetic materials, and composite materials. A variety of scaffold materials have their own advantages and disadvantages. Up to now, none of these scaffold materials is accepted as the most suitable one. The selection of scaffold materials is stil to be further studied. The study and development of nanoscale biomaterials is an inevitable trend. Otherwise, with the help of bionics, improving scaffolds is also an inexorable trend in progress of simulating human intervertebral disc. Furthermore, injectable scaffold is also an research hot spot, and the selection range of injectable scaffold materials mainly focuses on chitosan, typeⅡcolagen,hyaluronic acid,fibrin,elastin,and alginate.C urrently, studies on chitosan as a scaffold material are relatively more.

The present study examined the role of Wnt/β-catenin signaling pathway in the degeneration of nucleus pulposus cells and the protective effect of DKK1 on nucleus pulposus cells. The model of nucleus pulposus cell degeneration was induced by intra-disc injection of TNF-α, and the expression of β-catenin protein was detected by Western blotting. The cultured rabbit nucleus pulposus cells were divided into 4 groups. In group A, the cells were cultured with normal medium and served as control group. In group B, the cells were cultured with TNF-α and acted as degeneration group. In group C, the cells were cultured with TNF-α and transfected with Adv-eGFP and was used as fluorescence control group. In group D, the cells were cultured with TNF-α and transfected with Adv-hDKK1-eGFP, serving as intervention group. The expression of type II collagen, proteoglycan, β-catenin, and MMP-13 in each group was detected by immunocytochemistry and RT-PCR. The result showed that TNF-α increased the expression of β-catenin and MMP-13, and significantly inhibited the synthesis of type II collagen and proteoglycan, which resulted in the degeneration of nucleus pulposus cells. This effect could be obviously reversed by DKK1. We are led to concluded that TNF-α could activate the Wnt/β-catenin signaling pathway, and increase the expression of MMP-13, thereby resulting in disc degeneration. Specifically blocking Wnt/β-catenin signaling pathway by DKK-1 could protect the normal metabolism of intervertebral disc tissue. The Wnt pathway plays an important role in the progression of the intervertebral disc degeneration.

The present study examined the role of Wnt/β-catenin signaling pathway in the degeneration of nucleus pulposus cells and the protective effect of DKK1 on nucleus pulposus cells.The model of nucleus pulposus cell degeneration was induced by intra-disc injection of TNF-α,and the expression of β-catenin protein was detected by Western blotting.The cultured rabbit nucleus pulposus cells were divided into 4 groups.In group A,the cells were cultured with normal medium and served as control group.In group B,the cells were cultured with TNF-α and acted as degeneration group.In group C,the cells were cultured with TNF-α and transfected with Adv-eGFP and was used as fluorescence control group.In group D,the cells were cultured with TNF-α and transfected with Adv-hDKK1-eGFP,serving as intervention group.The expression of type Ⅱ collagen,proteoglycan,β-catenin,and MMP-13 in each group was detected by immunocytochemistry and RT-PCR.The result showed that TNF-α increased the expression of β-catenin and MMP-13,and significantly inhibited the synthesis of type Ⅱ collagen and proteoglycan,which resulted in the degeneration of nucleus pulposus cells.This effect could be obviously reversed by DKK1.We are led to concluded that TNF-α could activate the Wnt/β-catenin signaling pathway,and increase the expression of MMP-13,thereby resulting in disc degeneration.Specifically blocking Wnt/β-catenin signaling pathway by DKK-1 could protect the normal metabolism of intervertebral disc tissue.The Wnt pathway plays an important role in the progression of the intervertebral disc degeneration.

Objective To evaluate the contraception efficacy, mode of bleeding, side effects and other positive effects of drospirenone-ethinylestradiol (Yasmin) in healthy Chinese women. Methods This was a multicenter, randomized, control study of 768 healthy Chinese women who consulted about contraception. The subjects were randomized into Yasmin group (30 μg ethinylestradiol plus 3 mg drospirenone, 573 cases) or desogestrel group (30 μg ethinylestradiol plus 150 μg desogestrel, 195 cases) with the ratio of 3: 1. Each individual was treated for 13 cycles. Further visits were required at cycle 4, cycle 7, cycle 10 and cycle 13 of treatment. Weight, height, body mass index were evaluated at each visit. The menstrual distress questionnaire (MDQ) was given to the women at baseline, visit 3 (cycle 7) and visit 5 (after cycle 13). Results The values of basal features were similar between two groups (P> 0.05). The Pearl index (method failure) of Yasmin was 0. 208/hundred women year which was lower than that of desogestrel (0. 601/hundred women year). The mode of bleeding was similar between two groups after trial without showing any significant difference. According to MDQ subscale, the improvement of water retention and increasing appetite during inter-menstrual period and water retention and general well-being during menstrual period in the Yasmin group ( -0. 297, -0. 057, 0. 033, 0. 150 respectively) was more obvious than that in the desogestrel group ( - 0. 108, 0. 023, 0. 231, - 0. 023 respectively) with a significant difference (P < 0. 05 ). Some other values which improved in beth two groups, especially the improvement of breast tenderness and pain and skin abnormality in Yasmin group (18.0%, 89/494; 12. 6%, 62/494) was more distinct than that in desogestrel group (11.3%, 19/168; 5.4%, 9/168). The mean weight increased in desogestrel group (0. 57 kg) while it decreased in Yasmin group ( -0. 28 kg) with a significant difference (P < 0. 01 ). Conclusions Both Yasmin and desogestrel have good efficacy on contraception and similar modes of menstrual bleeding. Yasmin is better than desogestrel in terms of weight control and premenstrual syndrome of oral contraceptive.

Objective To explore expression and clinical value of VEGF-C and p63 in early esophageal carcinoma and intraepithelial neoplasia. Methods 146 cases were randomized into normal esophageal mucosa, low level intraepithelial tumor, high level intraepithelial tumor and early esophageal carcinoma. The expression of VEGF-C and p63 were detected by using the immunohistochemistry dyeing.Results The expression of VEGF-C immunohistochemistry dyeing had statistical differences among different levels(X~2= 47.455, P <0.001). Normal esophageal mucosa v.s. high level intraepithelial tumor (X~2=36.721, P <0.001), Normal esophageal mucosa v.s. early esophageal carcinoma (X~2=26.483, P <0.001), low level intraepithelial tumor v.s. high level intraepithelial tumor(X~2= 10.025, P<0.0083), low level intraepithelial tumor v.s. early esophageal carcinoma(X~2=16.734, P<0.001). There was a significant correlation between pathological classification and the expression amount of VEGF-C (r = 0.462, P <0.001). The expression of p63 had statistical differences among different levels(X~2=28.962, P <0.05). There was a significant difference on normal esophageal mucosa comparing with low level, high level intraepithelial tumor or early esophageal carcinoma (X~2=12.735, P =0.005, X~2=20.421, P<0.001, X~2=20.854, P<0.001). There was a significant correlation between pathological classification and the expression of p63 (r= 0.272, P<0.05). Conclusion There is a significant correlation in the express of either VEGF-C or p63 comparing with either intraepithelial tumor or early esophageal carcinoma. It may be an early warning indicator.