Background Human limbal allograft transplantation or limbal autograft transplantation are the primary approaches to the severe corneal-blindness,but their application in clinic were limited because of the defects of donor material.With the development of tissue engineering technology,transplantation of in vitro cultured limbal epithelial stem cells is being an advanced management.Objective The aim of this work was to expand human limbal epithelial stem cells ex vivo under the guidance of confocal microscope and to lay the foundation for fabricating ex vivo cultured cell sheets.Methods Ten eyes of ten patients were examined with the Heidelberg Retina Tomography Ⅲ Rostock Cornea Module(HRT3-RCM)to elucidate the structure of the human corneoscleral limbus and to correlate limbal epithelial dimensions.According to the analysis of the images of limbal epithelia,the limbal tissues provided by Eye Bank of Henan Eye Institute were cut into suitable explants.Then,this study was conducted to expand limbal epithelial stem cells ex vivo on denuded amniotic membrane.The phenotypes of primary cultured cells were evaluated by morphology and immunofluorescent staining with antibodies for limbal epithelial stem cell markers (p63,cytokeratinl9)and differentiation markers(keratin 3,involucrin).This experimental procedure was approved by the Ethic Committee of Henan Provincial People's Hospital.The written informed consent was obtained from subjects before initiation of any examination.Results The palisade morphology of human limbus was imaged clearly on the laser scanning in vivo confocal microscopy and many hyperreflective cells were observed in palisade basal cells.The cell-island phenomenon was seen in the basement membrane under the laser scanning in vivo confocal microscopy.The oblique sections of limbus showed many papilla-like epithelial columns below the superficial limbal epithelia.Throughout the experiment duration,the epithelial cells grew well with the migration rates from limbal tissue (68.62± 16.94)％ and the migration time(5.83 ±2.04)days,which depended on the tissue freshness.Compared with the second and forth batch of tissue,the migration rates of the third and sixth batch of tissues were significantly higher(P＜0.05),and the migration time was evidently longer in the forth and sixth batch of tissue compared with the first,second,third and fifth batch(P＜0.05).The positively expressing rates in the cultured corneal stem cells were 4.05％ and 36.52％ for p63,26.07％ and 40.55％ for CK19,57.88％ and 40.81％ for K3,64.66％ and 59.19％ for involucrin.Conclusion Human limbal epithelial stem cells can be successfully and purposefully obtained from the limbal tissue based on the guidance confocal miscroscope.The cultured corneal stem cells can grow well on the denuded amniotic membrane

Objective To investigate the curative effect of brucellar spondylitis,so as to provide scientific proof for improving the curative level of the disease.Methods Epidemiological information was collected from 113 patients diagnosed as brucellar spondylitis,who were divided into 5 groups according to different drugs and drug combinations of doxycycline,gentamicin,sulfamethoxazole and rifampicin.Then the curative effect was investigated.Twenty-one patients who had greater paoas muscle abscess or Para vertebral abscess,intraspinal abscess of spinal canal,necrotic intervertebral disk and major osteolasia received the minimal invasive surgery and the focus removal surgery.Results The occurrence of the disease in female was much higher than that in male.Grazing and breeding beasts was the principal route of infection.Lumbars was mostly involved.they usually was infected in the adjacent 2 piece.L4 was the most common and seriuous one.The curative effect of doxycycline group was better than that without doxycycline(72.60% vs 35.00%,X2=15.14,P＜0.05).Doxycyeline+gentamicin+sulfamethoxazole was reeommended as the first choice.However,the curative effect did not increase despi~course of the treatment prolonged.The heMing rate and effective rate after 1 course was 52.21%(59/113)and 92.04%(104/113).that after 2 courses 58.41%(66/113)and 95.58%(108/113),that after 3 courses 59.29%(67/113)and 95.58%(108/113).The healing rate in different course did not present differences(P＞0.05).21 patients undergoing surgery were followed-up,12 patients were after 2 years and 9 patients were between 1-2 years.The healing rate was 95.24%(20/21),1 case was healed basically,the effective rate was 100%.None reoccurred.Conclusions There are characterized features in clinical epidemiology of the brucell spondylitis.Long term,adequate in dosage,combination and multi-approach use of antibiotics is the most reliable way to treat and prevent it from recurring.But fof the patients soitable for surgery.the minimal invasive surgery or the focus removal could shorten the course of therapy,decrease the complications and increase the cure rate.

Animals ; Aorta ; pathology ; Apolipoproteins E ; deficiency ; Atherosclerosis ; metabolism ; pathology ; Female ; Glycosides ; isolation & purification ; pharmacology ; Intercellular Adhesion Molecule-1 ; metabolism ; Mice ; Plants, Medicinal ; chemistry ; Polygonum ; chemistry ; Vascular Cell Adhesion Molecule-1 ; metabolism

BackgroundIt is important to measure the corneal curvature, anterior chamber depth (ACD) and axial length accurately for calculating IOL power. The interchange outcomes from different measuring methods and apparatus will cause unreliable IOL power. ObjectiveThe present study was to compare the differences of corneal curvature, anterior chamber depth (ACD) measured by IOLMaster and Orbscan Ⅱbefore and after laser in situ keratomileusis(LASIK) and further compare the axial length measured by IOLMaster and A-ultrasound. Methods One hundred and thirty eyes from 65 consecutive myopic patients before LASIK and 56 eyes of 28 cases with 1-month follow-up duration after LASIK in Henan Eye Institute were enrolled in this study. The K value, ACD between IOLMaster and Orbscan Ⅱ as well as results of axial length between IOLMaster and A-ultrasound were compared by using paired t test. The agreements of the measured values among IOLMaster, Orbscan Ⅱ and A-ultrasound were evaluated using Bland-Altman plot. ResultsBefore LASIK,the K value measured by IOLMaster,Orbscan Ⅱ were ( 43.32 ± 1.52 ) D and ( 42.99 ± 1.45 ) D respectively with the difference value of( 0. 33 ±0. 03 ) D, showing a significant difference(t=10. 380,P=0.000) and a positive relation between them(r=0.971,P=0.000). After LASIK,the K value measured by IOLMaster, Orbscan Ⅱwere(39. 02±2. 14) D and ( 38.91 ±2. 04) D with the difference value (0. 12±0. 33 ) D, presenting a significant differences between them (t =2.715, P =0.009). Bland-Altman plots indicated the disagreement in K value and uninterchangeable. Before LASIK, the ACD measured by IOLMaster,Orbscan Ⅱ and A-ultrasound were ( 3.72 ± 0. 22 ) mm, ( 3.69 ±0. 22 ) mm and ( 3.75± 0.27 )mm respectively and no significant differences were found between them (P ＞ 0. 05 ). Axial length measured by IOLMaster significantly prolonged in comparison with A-ultrasound(25.59± 1. 01 mm vs 25.22±0.99 mm ) , and the difference was( -0. 37 ±0. 30 ) mm, showing significant difference ( t =- 14. 098, P =0. 000 ) and positive correlation ( r =0. 954, P =0. 000 ). Axial length values measured by IOLMaster were ( 25.54 ± 1.05 ) mm in preoperation and ( 25.48 ± 1.01 ) mm in postoperation with the difference (0.052±0. 412)mm, showing statistically insignificant difference between them (t=0. 946,P=0. 348). ConclusionsKeratometries measured by IOLMaster,Orbscan Ⅱ are much more different. Therefore,these two methods are not recommended to use interchangely. ACD measured by IOLMaster,Orbscan Ⅱ and A ultrasound are proved to obtain the similar results and is clinically interchange. Axial length measured by IOLMaster is longer than that measured by A-ultrasound.

OBJECTIVETo observe different effects of moxibustion on extracellular potassium ion in acupoint under physiological and pathological status and provide experimental evidence for exploring action mechanism of moxibustion on acupoint local.

METHODSForty female SD rats were randomly divided into a blank group, a blank-moxibustion group, a model group and a model-moxibustion group, 10 cases in each one. The complete Freund's adjuvant(CFA) was adopted to establish model of adjuvant arthritis (AA) in the model group and model-moxibustion group. No treatment was given in the blank group and model group while moxibustion was applied at "Zusan-li" (ST 36) for 30 min in the blank-moxibustion group and model-moxibustion group. The tissue fluid in "Zusanli" (ST 36) was collected with microdialysis and real-time analyzed by electrolytic analyzer. The change of concentration of potassium ion in "Zusanli" (ST 36) was observed.

RESULTS(1) Under physiological status, the concentration of extracellular potassium ion in the blank group was not changed within 150 min (P > 0.05); before the moxibustion, the concentration of extracellular potassium ion in the blank-moxibustion group was (1.21 +/- 0.31) mmol/L, and after treatment it was gradually increased and reached its peak at (2.38 +/- 0.42) mmol/L after 60 min (P < 0.05), then it was reduced. 150 min after the treatment, concentration of potassium ion was slightly higher than that before moxibustion as well as that in the blank group. The concentration in the blank-moxibustion group at 60 min was statistically significant compared with that in the blank group (P < 0.05). (2) Under pathological status, the concentration of extracellular potassium ion in the model group was not changed within 150 min, differences of which at each time point was not statistically significant (all P > 0.05). Before the moxibustion, the concentration of extracellular potassium ion was (1.09 +/- 0.12) mmol/L in the model-moxibustion group, and it was immediately increased to (1.96 +/- 0.18) mmol/L after moxibustion. 60 min and 90 min after the moxibustion, it still maintained a higher level, which was (1.87 +/- 0.29) mmol/L and (1.59 +/- 0.16) mmol/L respectively (both P < 0.05). The differences of each time point after moxibustion in the model-moxibustion group were statistically significant compared with those in the model group (all P < 0.05).

CONCLUSIONThe moxibustion could increase the concentration of potassium ion in rat's acupoint local under physiological status but time of effect is short; with moxibustion at "Zusanli" (ST 36) under pathological status, the concentration of local potassium ion is obviously increased and maintains for a long time.

Acupuncture Points ; Animals ; Arthritis, Experimental ; metabolism ; therapy ; Disease Models, Animal ; Female ; Humans ; Moxibustion ; Potassium ; metabolism ; Rats ; Rats, Sprague-Dawley

Benzodiazepines(BDZs)are used in clinics for anxiolysis,anticonvulsants,sedative hypnosis,and muscle relaxation.They have high consumptions worldwide because of their easy availability and potential addiction.They are often used for suicide or criminal practices such as abduction and drug-facilitated sexual assault.The pharmacological effects of using small doses of BDZs and their detections from complex biological matrices are challenging.Efficient pretreatment methods followed by accurate and sensitive detections are necessary.Herein,pretreatment methods for the extraction,enrichment,and preconcentration of BDZs as well as the strategies for their screening,identification,and quantitation developed in the past five years have been reviewed.Moreover,recent advances in various methods are summarized.Characteristics and advantages of each method are encompassed.Future directions of the pretreatment and detection methods for BDZs are also reviewed.

To investigate the relationship and significance of Wnt/b-catenin signaling pathway with caspase-3, XIAP, HSP27and Grp-78. The HCC cell line HepG2 was transfected with small interfering RNA (siRNA) directed against b-catenin. After 72 and 96 h, protein was extracted and the protein expressions of b-catenin, caspase-3, XIAP, Grp-78 and HSP27 were detected by Western blot. b-catenin protein expression was inhibited at both time points and the expression at 96 h was higher than that at 72 h (F = 160.72, P is less than to 0.01). Interestingly, Caspase-3 protein expression was decreased at 72 h and increased to normal at 96 h (F = 136.10, P is less than to 0.01), while p-caspase-3 protein expression increased at 72 h and decreased to normal at 96 h (F = 98.65, P is less than to 0.01). XIAP protein expression decreased at 72 h (F = 37.29, P is less than to 0.01) and increased at 96 h. Grp-78 protein expression increased at 72 h and decreased to normal at 96 h ( F = 58.72, P is less than to 0.01). HSP27 protein expression showed no change following transfection ( F = 1.91, P is more than to 0.05). Wnt/b-catenin signaling pathway is related to the protein expressions of caspase-3, XIAP and Grp-78, but not related to HSP27 protein expression in HCC. Wnt/b-catenin signaling pathway may participate in the regulation of HCC apoptosis, proliferation and differentiation through affecting these factors.
Carcinoma, Hepatocellular ; Caspase 3 ; Catenins ; Humans ; Liver Neoplasms ; Wnt Signaling Pathway ; beta Catenin ; metabolism

OBJECTIVETo study the changes of root canal curvatures and apical transportation index when the curved root canals were prepared by hand-use ProTaper, stainless steel K-files or nickel-titanium K-files.

METHODSForty-five extracted molars, with the degree of root canal curvature from twenty to forty, were divided into fifteen sections in curvature sequence. After that, three canals of each section were randomly distributed to three different instrumentation groups. One group of them was prepared by hand-use ProTaper with crown-down technique. The other two groups were instrumented by stainless steel or nickel titanium K-files with modified step-back technique to working length. The standard digital radiographs were taken to record the profiles of root canals and the positions of the tips of the instruments. The change of curvature degree between pre- and post-instrumentation and the apical transportation indexes (ATI) among three groups were analyzed respectively using SAS statistical software.

RESULTSThe change of curvature degree between pre- and post-instumentation for stainless steel K-files group was largest, and the ATI of stainless steel K-files group was significantly greater than that for nickel titanium K-files group (P < 0.05). The canals prepared by hand-use ProTaper was smallest in the change of curvature and ATI among the three groups (P < 0.01).

CONCLUSIONWhen preparing curved molar canals, hand-use ProTaper with crown down technique can obtain better shaping effect and higher efficiency.

Dental Instruments ; Dental Pulp Cavity ; Molar ; Nickel ; Root Canal Preparation ; Root Canal Therapy ; Stainless Steel ; Titanium

Objective To understand the distribution and drug resistance of pathogenic bacteria from diabetic foot patients of different grades of Wagner classification,and provide basis for the rational use of antimicrobial agents in clinical practice.Methods Clinical data,pathogens isolated from wound secretion,and antimicrobial susceptibility testing result of diabetic foot patients who were admitted in the department of endocrinology in a hospital between January 2014 and December 2016 were analyzed retrospectively,patients were divided into gram-positive (G+) bacteria infection group,gram-negative (G) infection group,and mixed infection group according to the isolation of bacterial species,relationship between patients' clinical characteristics and Wagner grades was analyzed.Results There were 103 patients with diabetic foot,39 with G+ bacterial infection,50 with G-bacterial infection,and 14 with mixed infection.The age and disease course of patients with mixed infection was significantly elder and longer than the other two groups of patients(both P＜0.05);glycosylated hemoglobin (HbA1c) and procalciton in mixed infection group were both lower than the other two groups(both P＜0.05);the percentage of neutrophils in G+ infection group was higher than the other two groups(P＜0.01).A total of 132 specimens were detected,103 strains were isolated,the positive rate was 78.03％,isolation rates of G+ and G-bacteria were 33.33％ (44/132) and 44.70％ (59/132) respectively.The major G+ strains were Staphylococcus aureus (n =19) and Staphylococcus epidermidis(n=11),the major G bacteria were Citrobacterbraakii(n=12),Escherichia coli(n=11),and Pseudo monas aeruginosa (n =11).As the Wagner grade increased,the proportion of mixed infection continued to rise.The main multidrug-resistant organisms were methicillin-resistant Staphylococcus aureus and extended-spectrum betalactamase-producing G-strains.Conclusion For diabetic foot patients,especially those with higher Wagner grades,etiological examination and antimicrobal susceptibility testing of ulcer secretion should be timely performed,so as to provide reference for rational use of antimicrobial agents in clinic.

Objective To screen the mutation of certain gene of a 10-years-old boy with multiple xanthomas and very high level of cholesterol who could be diagnosed as homozygous familial hypercholesterolemia (FH),to explore the relationship between the genotype and phenotype,and to discuss the molecular pathologic mechanism.Methods The basic information of life styles were asked from the boy and his familial members.The blood was drown to examine the lipid and genes.The boy was examined with electrocardiogram examination,ultrasonography and coronary CT angiography (CTA) to evaluate the degree of atherosclerosis.Peripheral blood DNA of the boy and his parents were extracted by phenol-chloroform method and investigated for mutations of promoter and all 18 exons of low density lipoprotein receptor(LDLR) gene.Screening was carried out by using Touch-down polymerase chain reaction (PCR) and single strand conformation polymorphism(PCR-SSCP),combined with DNA sequence analysis.In addition,the apolipoprotein B100 gene(apoB100) for known mutations (R3500Q) which caused familial defective apoB100 was screened by PCR-DNA sequence analysis.Results 1.The level of cholesterol of his parents were higher than the normal.2.Several clinical manifestations of atherosclerosis were detected from that boy.Increased intima-media thickness and plaques were detected in the common carotid artery.Mitral valve regurgitation was found by echocardiography.Coronary stenosis was confirmed by CTA.3.No mutations R3500Q of apoB100 was observed.4.A homozygous mutation in exon13 of the LDLR gene (D601Y) were identified in the boy and his parents harbour D601Y heterozygous mutation due to a single base pair substitution of G for T in the codon for residue 1864.Conclusions The final diagnosis of the boy with multiple xanthomas was homozygous FH.His disease was caused by D601Y homozygous mutation in exon13 of the LDLR gene inherited from his heterozygous parents.