BackgroundIt has been well-known that integrin linked kinase(ILK) plays an important role in the pathogenesis of neorascularization.But there are few researches to elucidate the relationship between ILK and retinal nevascularization. Objective This study was to explore the expression and significance of ILK on retinal neovascularization and new vessels regression in the oxygen-induced retinopathy(OIR) mouse model. Methods One hundred and twenty-eight 7-day-old C57BL/6J mice were randomly divided into the normal group and model group.The mice were rose in(75±2)％O2 environment with mother mice together for 5 days and then in normal environment for 5 days to establish the OIR models.The mice in normal group were rose in the normal environment for 21 days.The 17-day-old mice were sacrificed and retinal sections were prepared for histopathological examination and the numbers of the cellular nuclei of vascular endotheliumbreaking retinalinternal limiting membranewere calculated.Retinal sections and sheeting were prepared in 12,14,17 and 21 -day-old mice to examine the formation and regression of new blood vessel using ADP histochemistry staining,and then immunochemistry,real-time PCR and Western-blotting were used to detect the expressions of ILK and its mRNA in retina. ResultsThe numbers of the cellular nuclei of vascular endothelium breaking retinal internal limiting membrane were (45.64 ± 12.17 )in OIR group,and those of the normal group were( 0.35±0.14 )with a significant difference between two groups (t =22.85,P＜0.05).Retinal new blood vessel appeared in 14-day mice,and peaked in 17-day mice and then regression in 21-day mice.ILK protein was expressed mainly in retinal ganglion cell layer,inner nuclear layer,inner plexiform layer and photoreceptor layer.Real-time PCR showed that ILK mRNA expressing levels in retina in model group were( 1.00±0.22),(1.85±0.17),(1.58±0.43) and(1.53±0.36) respectively in 12-,14-,17- and 21-day mice.Westernblotting determined that the A value of the ILK/β-actin was increased in 12-,14-,17-day mice and decreased in 21-day mice,and the A values were significantly higher in model group than the control group in various aged mice ( t =2.97,P＜0.05 ;t =11.88,P＜0.01 ;t =16.84,P＜0.01 ;t =13.00,P＜0.01 ). ConclusionsThese results indicate a space-time corresponding relation between the expression of ILK and retinal neovascularization.The obvious positive expression of ILK may be highly correlated with retinal neovascularization.

Salvianolic acid A (Sal A) is one of the most effective compounds isolated from the root of Salvia miltiorrhiza. Up to now, several studies regarding the pharmacokinetic profiles of Sal A have been reported, however there is no such study reported in monkeys, the species which is more similar to human. The aim of this study is to develop a LC-MS method for the determination of Sal A in monkey plasma and apply it to the pharmacokinetic studies of monkeys. After single intravenous administration of Sal A, the plasma concentration-time curves were observed and the main pharmacokinetic parameters were calculated. The plasma concentration at 2 min (C2 (min)) values were (28.343 ± 6.426), (45.679 ± 12.301) and (113.293 ± 24.360) mg x L(-1) for Rhesus monkeys treated with Sal A at 2.5, 5 and 10 mg x kg(-1). The area under the concentration-time curve (AUC(0-∞)) values were (3.316 ± 0.871), (5.754 ± 2.150) and (13.761 ± 2.825) μg x L(-1) x h, respectively. Furthermore, this method was improved and applied to the simultaneous determination of Sal A, Sal B and Sal C, which provided useful information for preclinical studies and clinical trials of Sal A, Sal B and Sal C.
Administration, Intravenous ; Animals ; Caffeic Acids ; pharmacokinetics ; Chromatography, Liquid ; Drugs, Chinese Herbal ; pharmacokinetics ; Lactates ; pharmacokinetics ; Macaca mulatta ; Mass Spectrometry ; Plant Roots ; chemistry ; Salvia miltiorrhiza ; chemistry

Objective: To obtain the antibody against N terminal of 1A6/DRIM, and thereafter get the profile of 1A6/DRIM expression in different cell lines. Methods: The N terminal of 1A6/DRIM (aa 577 714) was cloned into pGEX 4T 3. Multiple antigenic peptides (MAPs)(aa638 661) was synthesized as the antigen with Fmoc/PyBOP method. Rabbits were immunized by injecting the MAPs and the immunized sera were analyzed with ELISA and Western Blot. The Western Blot and immunofluorescence were performed to analyze the expressing profiles of the 1A6/DRIM in different tumor cell lines. Results: The antibody specifically recognized the full length of 1A6/DRIM as a 310 kDa band, which was also recognized by C terminal monoclonal antibody shown by Western Blot. 1A6/DRIM is expressed in multiple tumor cell lines and mainly located in the nuclei. Conclusion: Preparation of the antibody with MAPs is a useful technique when the fusion proteins can not be induced in E coli . The antibody we got via MAPs has supplied a good tool for further studies on the functions of the novel gene 1A6/DRIM.

The AIDS epidemic in men who have sex wlth men (MSM) in recent years showed a sharp upward trend, looking for behavioral intervention strategies should be imperative. Fear appeals by fear prompted intervention received intervention information, provide a new breakthrough to achieve better effect of propaganda and intervention. After over 70 years development, the Fear Appeal generated from the driver model that proposed the fear decided the effectiveness of behavior intervention, to the extended parallel process model theory which integrated protection motivation theory and parallel process theory, both of which believed the fear is just one of the estimators, suggested fear is the key factor. The fear appeal theory is turning to be even more comprehensive and accurate. As an important theoretical basement, the fear appeal is still developing, and need more work to make it perfection.
Acquired Immunodeficiency Syndrome ; prevention & control ; psychology ; Fear ; Health Promotion ; methods ; Homosexuality, Male ; Humans ; Male

BACKGROUNDRheumatoid arthritis (RA) is a chronic, systemic autoimmune inflammatory disorder. Many methods have been used to observe the progress of RA. The purpose of this study was to observe the progress of RA in rats with 18F-fluorodeoxyglucose (FDG) positron emission tomography/computed tomography (PET/CT), magnetic resonance (MR) imaging and arthritis score, and analyze the relationships among different methods in evaluation of RA.

METHODSSixteen healthy Sprague Dawley (SD) rats about 8-week old were randomly assigned to a RA group and a control group. Bovine type II emulsified incomplete Freud's adjuvant was used to induce arthritis in the RA group. Arthritis score of the rats in two groups were recorded, and (18)F-FDG PET/CT, MR imaging were performed both on the corresponding rats every 3 days. All the rats were sacrificed at week 5, and histopathological examination was performed on rat knees stained with haematoxylin and eosin.

RESULTSThe arthritis score and the standard uptake value (SUV) of knee joints in RA rats increased with the progression of arthritis gradually. Both peaks of arthritis score and SUV appeared at 21 days after the first immune injection, then the arthritis score and SUV of knee joints decreased slowly. The arthritis scores of knee joints in RA rats were positively correlated with their SUV changes. The MR images were confirmed by the histopathological studies.

CONCLUSIONPET/CT can detect the earliest molecular metabolism changes of RA, and MR imaging can follow up the dynamical anatomical changes of RA, all of which indicated that PET/CT and MR imaging may be applied as useful tools to monitor the progress of RA.

Animals ; Arthritis, Rheumatoid ; diagnosis ; pathology ; Fluorodeoxyglucose F18 ; Magnetic Resonance Imaging ; Positron-Emission Tomography ; Radiopharmaceuticals ; Rats ; Rats, Sprague-Dawley ; Tomography, X-Ray Computed

Chymosin is an important industrial enzyme widely used in cheese manufacture. To improve expression efficiency of recombinant bovine chymosin in Kluyveromyces lactis strain GG799, we designed and synthesized a DNA sequence encoding bovine prochymosin gene (GenBank Accession No. AA30448) by using optimized codons. The synthesized prochymosin gene was amplified by two-step PCR method, and then cloned into the expression vector pKLAC1, resulting in pKLAC1-Prochy. pKLAC1-Prochy was linearized and transformed into K. lactis GG799 by electrotransformation. Positive clones were screened by YEPD plates containing 1% casein. A recombinant strain chyl with highest activities and multi-copy integration which was detected by using specifical integration primers was chosen and fermented in flask. Prochymosin was expressed in K. lactis successfully. SDS-PAGE analysis revealed that the purified recombinant bovine prochymosin had a molecular mass of 41 kDa. After acid treatment, molecular weight of chymosin is about 36 kDa, the same as native bovine chymosin. Activity tests showed that the chymosin activity of the culture supernatant was 99.67 SU/mL after 96 h cultivation. The activities of chymosin were not prominent increased when galactose was used as carbon source instead of glucose, which proved that the fermentation of recombinant strain does not need galactose inducing. The recombinant K. lactis strain obtained in this study could be further used to produce recombinant chymosin for cheese making.
Animals ; Cattle ; Chymosin ; biosynthesis ; genetics ; Enzyme Precursors ; biosynthesis ; genetics ; Gene Expression Regulation, Fungal ; genetics ; Genetic Vectors ; genetics ; Kluyveromyces ; genetics ; growth & development ; metabolism ; Protein Engineering ; Recombinant Proteins ; biosynthesis ; genetics

OBJECTIVETo investigate the difference of the antigenicity of xenogenic acellular dermal matrix (ADM) implants prepared by different methods.

METHODSThe split-thickness skin sheet from swine was processed by trypsin and Triton X-100 to make xeno-ADM. Twenty-five Japanese white rabbits were divided into 5 groups, i.e. xeno-ADM(1) (conjugated with glutaraldehyde), xeno-ADM(2) (conjugated with network) and xeno-ADM(3) (no conjugation, as control), in which the ADMs were and xeno-ADM(4) (conjugated) and allo-ADM (no conjugated as control), in which the ADMs were embedded into the subcutaneous place of rabbit ear and back after that the rabbits were pre-sensitized by xeno-ADM soluble protein antigen injections. The titers of anti ADMs antibody in rabbit serum were monitored during 2 - 32 post-operative weeks and the histological changes of the embedded ADMs were observed grossly and microscopically.

RESULTSThe serum titers of anti-xeno-ADM in xeno-ADM(4) group was the highest. Whereas regardless of the sensitizing effects, the titers in all groups ranged as follows: xeno-ADM(3) > xeno-ADM(2) > xeno-ADM(1) (P < 0.05 - 0.01). About 40% serum samples in allo-ADM group exhibited positive anti-allo-ADM protein antibodies. Histologically, Evident and lasting inflammatory reaction could be found in the xeno-ADM grafting sites, which was much stronger than that in allo-ADM group. The degradation and absorption gradient of ADM was ranked as follow: xeno-ADM(3) > xeno-ADM(2) > xeno-ADM(4) > xeno-ADM(1) > Allo-ADM. Foreign body megalocytic reaction might evoke in the surrounding of conjugated ADM.

CONCLUSIONThe immunogenicity in xeno-ADM was stronger than that in allo-ADM, which could induce the host to develop immune reaction restricted by IgG. Large sheets of degenerated ADM implants could lower down the antigen-antibody reaction and ameliorate the structural destroying and degeneration absorption of ADM induced by inflammatory immune reaction.

Animals ; Antigen-Antibody Reactions ; Dermis ; immunology ; transplantation ; Male ; Rabbits ; Skin Transplantation ; Swine ; Transplantation, Heterologous

miR-214 plays a major role in the self-renewal of skin tissue. However, whether miR-214 regulates the proliferation and differentiation of human hair follicle stem cells (HFSCs) is unknown. Primary HFSCs were isolated from human scalp skin tissue, cultured, and identified using flow cytometry. An miR-214 mimic and inhibitor were constructed for transfection into HFSCs. The MTS and colony formation assays examined cell proliferation. Immunofluorescence detected the localization and expression levels of TCF4, β-catenin, and differentiation markers. Luciferase reporter and TOP/FOP Flash assays investigated whether miR-214 targeted EZH2 and regulated the Wnt/β-catenin signaling pathway. Western blot determined the expression levels of enhancer of zeste homolog 2 (EZH2), Wnt/β-catenin signaling-related proteins, and HFSC differentiation markers in cells subjected to miR-214 transfection. miR-214 expression was remarkably decreased during the proliferation and differentiation of HFSCs into transit-amplifying (TA) cells. Downregulation of miR-214 promotes the proliferation and differentiation of HFSCs. Overexpression of miR-214 led to decreased expression of EZH2, β-catenin, and TCF-4, whereas downregulation of miR-214 resulted in increased expression of EZH2, β-catenin, and TCF-4 as well as TA differentiation markers. Immunofluorescence assay revealed that inhibiting miR-214 triggered the entry of β-catenin and TCF-4 into the nucleus. The luciferase reporter and TOP/FOP Flash assays demonstrated that miR-214 directly targets EZH2 and affects Wnt/β-catenin signaling. The miR-214/EZH2/β-catenin axis could be considered a candidate target in tissue engineering and regenerative medicine for HFSCs.
Antigens, Differentiation ; Blotting, Western ; Cell Proliferation ; Down-Regulation ; Flow Cytometry ; Fluorescent Antibody Technique ; Hair Follicle* ; Hair* ; Humans* ; In Vitro Techniques* ; Luciferases ; Regenerative Medicine ; Scalp ; Skin ; Stem Cells* ; Tissue Engineering ; Transfection

Objective To investigate the clinical efficacy and safety of Apatinib in treating the advanced esophageal cancer and their prognosis in elderly patients.Methods Totally 52 elderly patients with advanced esophageal cancer who met the inclusion and exclusion criteria at our hospital from March 2015 to August 2017 were retrospectively enrolled.They were treated with Apatinib.The clinical efficacy,adverse reactions and influencing factors for the prognosis were analyzed.Results The partial remission rate(PRR) was 25.0％ and the disease control rate(DCR)was 71.2％ in 52 patients.The main adverse reactions were hypertension,hand-foot syndrome and proteinuria,with the incidence of 50.0％,38.5％ and 36.5％,respectively.The degree of adverse reactions was mainly grade 1 ～ 2.The median progression-free survival(PFS)was 3.8 months,and the median overall survival(OS)was 7.0 months.Univariate analysis (log-rank test) indicated that the degree of adverse reactions(x2 =5.075,P =0.024) and the Eastern Cooperative Oncology Group (ECOG) score (x2 =7.550,P =0.006)were associated with OS in elderly patients with advanced esophageal cancer.Cox multivariate regression analysis showed that the degree of adverse reactions(HR =1.963,P =0.045)and ECOG score(HR =0.458,P =0.015)were the independent influencing factors for OS in elderly patients with advanced esophageal cancer.Conclusions Due to mild adverse reactions and a high safety,Apatinib still has a certain clinical efficacy in the treatment of elderly patients with advanced esophageal cancer.Moreover,patients with the low ECOG score and high degree of adverse reactions have better prognosis.

Objective To observe the clinical efficacy of Xingnao Kaiqiao Acupuncture(with the functions of awakening the brain and opening the orifices)combined with acupuncture at pericardium meridian points in the treatment of post-stroke sleep reversal(PSSR).Methods Sixty patients with PSSR were randomly divided into observation group and control group,30 patients in each group.Both groups were given conventional treatment,the control group was given oral use of Alprazolam,and the observation group was given the combination of acupuncture a at pericardial meridian points,and 10 days of treatment was one course of treatment.After 10 days of treatment,the clinical efficacy of the two groups was evaluated.The changes in the Pittsburgh Sleep Quality Index(PSQI)and Ascens Insomnia Scale(AIS)scores,as well as the Hamilton Depression Scale(HAMD)scores were observed before and after treatment in the two groups.The changes in cortisol levels at 0,8,and 16 o'clock were compared before and after treatment between the two groups.Results(1)After treatment,the PSQI scores of patients in the two groups were significantly improved(P＜0.05),and the observation group was significantly superior to the control group in improving PSQI scores,and the difference was statistically significant(P＜0.05).(2)After treatment,the AIS and HAMD scores of patients in the two groups were significantly improved(P＜0.05),and the observation group was significantly superior to the control group in improving the AIS and HAMD scores,and the difference was statistically significant(P＜0.05).(3)After treatment,the cortisol level of patients in the two groups at 0,8,and 16 o'clock was significantly improved(P＜0.05),and the observation group was significantly superior to the control group in improving the cortisol level at 0,8,and 16 o'clock was significantly superior to the control group,and the difference was statistically significant(P＜0.05).(4)The total effective rate was 86.67%(26/30)in the observation group and 80.00%(24/30)in the control group.The efficacy of the observation group was slightly superior to that of the control group,but the difference was not statistically significant(P＞0.05).Conclusion Xingnao Kaiqiao Acupuncture combined with acupuncture at pericardium meridian points for the treatment of PSSR can significantly improve the clinical symptoms of the patients,so as to improve the quality of life of the patients,and the therapeutic efficacy is remarkable.