BACKGROUND:There are two types of epithelial stem cells in the ocular surface tissue:corneal epithelial stem cells and conjunctival epithelial stem cells. The corneal epithelial stem cells play an important role in renewal of corneal epithelial cells and maintenance of corneal transparency.
OBJECTIVE:To study the location of corneal epithelial stem cells using laser in vivo confocal microscopy and immunofluorescent staining.
METHODS:Patients with unilateral limbal stem celldeficiency who went to Henan Eye Institute from September 2009 to September 2012 were enrol ed in this study. Bilateral eyes were scanned by laser in vivo confocal microscopy, and the healthy eye was imaged as a control. The central cornea and limbus were scanned and images were recorded for statistical analysis. The eye bal s were obtained from Henan Eye Bank, China. Central cornea and limbus were dissected and embedded in the OCT compound for frozen section and the proper thickness of the section was 5-7μm. Immunofluorescent staining was used to detect the expression of p63, ABCG2, K3 and Connexin 43 in the epithelial layers of central cornea and limbus.
RESULTS AND CONCLUSION:Twenty-four patients diagnosed with unilateral limbal stem celldeficiency were recruited. Under confocal microscopy, in the affected eyes, the typical morphology of conjunctival epithelial cells and goblet cells was detected instead of corneal epithelial cells;in the limbus, a great amount of fiber scarring tissue was detected instead of Vogt palisade, rete pegs and pigment cells. Immunofluorescent staining showed the expression of p63, ABCG2 was mainly in the basal layer of limbal epithelium, especial y in the outer and middle parts, but the expression of p63 and ABCG2 was not detected in the epithelial celllayers of central cornea. K3 and Connexin43 were not expressed in suprabasal layers of limbal epithelium, but in central cornea, they were expressed highly in the whole epithelial celllayers. Laser in vivo confocal microscopy and immunofluorescent staining showed the corneal epithelial stem cells were located in the basal layer of outer and middle limbal epithelium, mainly in Vogt palisade and rete pegs.

AIM: To investigate the effect of 7-(4-methoxyphenyl)-5, 8a-diphenyl-1,2, 3, 7, 8, 8a-hexahydroimidazo[1,2-a] pyridine (TIP-6) on cell proliferation in human hepatoma cell line HepG2 and human normal hepatocyte cell line L02. METHODS: Typan blue assay was used to check the effect of TIP-6 on cell proliferation. The changes of cell morphology were observed by the phase contrast microscope. Flow cytometry (FCM) was used to check cell cycle. Autophagy and autophagic cell death were detected after acridine orange (AO) staining under fluorescent microscopy. Apoptosis was analyzed by Annexin V/7-AAD, DAPI staining and DNA ladder. NF-κB expression was detected with cellular immunochemistry. RESULTS: Cell proliferation inhibiting effect was appeared when treated with TIP6 from 60 μmol/L to 200 μmol/L, which was correlated with treated concentrations and time. The proliferation rates were just 12.10% and 18.75% (vs control) under 200 μmol/L 72 h in HepG2 and L02 respectively. Vacuolization were found more and more frequently with the increasing of TIP-6 concentrations and treated time prolonged. FCM results indicated that cells were blocked in G2/M phase, and more sensitive were found in HepG2 than L02. AO staining results indicated that the phenomenon of autophagy and autophagic cell death were occurred and appeared more potent with more TIP-6 and longer time treated. No apoptosis markers were found with Annexin V/7-AAD and DAPI staining, and no DNA ladders were found either, these indicated that TIP6 didnt induce apoptosis in these cells. NF-κB was found increased after treated with TIP-6, and the autophagic vacuole became more and more with the increasing of NF-κB protein, but the proliferation rates decreased at the same time. CONCLUSION: TIP-6 inhibited cell proliferation and induced autophagy and autophagic cell death in HepG2 and L02 cells. NF-κB activation may be involved in these effects.

Objective To study the correlation between non-alcoholic fatty liver disease (NAFLD)and glycosylated hemoglobin (HbA1 c) in middle-aged and aged population.Methods A total of 4 127 inservice workers and retirees aged 45 years old or above from one petrochemical enterprise in Ningbo were enrolled in our study.The waistline,body mass index,blood pressure,fasting blood-glucose,blood lipid profile,glutamyltranspeptidase,HbA1c and epigastrium B ultrasound were investigated.According to the quartile of HbA1c level,participants were divided into four groups,namely,Q1 group ≤5.2％,Q2 group ＞ 5.2％-5.4％,Q3 ＞ 5.4％-5.6％ and Q4 group ＞ 5.6％.The prevalence of NAFLD and clinical characteristics of each group were analyzed.Logistic regression was used to predict independent risk factors of NAFLD.Results The morbidity of NAFLD was 27.2％ with 31.9％ in male and 21％ in female,which was significantly higher in men.In Q1,Q2,Q3 and Q4 group,the prevalence of NAFLD were 18.5％ (178/961),22.8％ (185/812),25.6％ (280/1 095),38.1％ (480/1 259) respectively.With the increase of HbA1 c level,the morbidity of NAFLD increased synchronously.The age,systolic pressure,total cholesterol,low densitylipoprotein cholesterin and fasting blood-glucose were all elevated according to the increase of HbA1 c in 1 123 NAFLD patients.Multi-factor logistic regression analysis indicated that high HbAlc level was the risk factor of NAFLD (OR =1.67,95％ CI 1.15-2.43,P =0.007).Conclusion HbA1c is an independent risk factor of NAFLD and both of these are closely related to blood lipid metabolism disorder.

Objective To understand the epidemic characteristics of an outbreak of panresistance Klebsiella pneumoniae occurred between 2006 and 2009 in a university hospital of Shanghai, China. Methods A total of 57 panresistance K. pneumoniae isolates were collected from August 2006 to December 2009.Antibiotic susceptibility of the isolates were determined by Kirby-Bauer disc diffusion method and microbroth dilution (MBD). ESBLs-producing initial screen test and phenotypic confirmatory test and carbapenemase-producing modified Hodge test ( MHT) were performed to detect the resistance phenotype of the isolates. Be-ta-lactamases were studied by IEF, PCR and the product sequencing. While conjugation assay were conducted to understand the transferability of these genes. The genetic relationship between isolates was established by ERIC-PCR and multilocus sequence typing (MLST). Except for the antibiotics recommended by CLSI guideline in the routine test, the other antibiotics were added to find out the effective drugs to treat the infection. Results All 57 isolates were highly resistant to all examined antibiotics. All isolates produced ESBLs and carbapenemase. IEF revealed that each isolate produced four beta-lactamases. All isolates carried blaKPC-2,blaCTX-M-14,blaSHV12,blaTEM-1,qnrB and aac(6') - I b-cr. Forty-four of the 57 (77.2% ) isolates were successful to transfer their resistance genes to E. coli recipient J53 by conjugation assay. By RAPD, all 57 isolates were grouped into two genotypes that were further identified as members of MUST types 423 and 11.Sequence types 423(ST423) only occurred before May 2008 and ST11 occurred (52 isolates) after May 2008. Most of isolates of the outbreak were ST11 (91. 2% ). A part of isolates were susceptive to added antibiotics. Conclusion The outbreak of panresistance K.pneumoniae was caused by those isolates which carried multiple resistant genes. There is a different ability of dissemination between different ST types K. pneumoniae isolate. It was necessary to add the antibiotics to find out the effective drugs to treat the infection.

Objective To investigate clinical features and immunologic status in human immunodeficiency virus(HIV)/hepatitis C virus(HCV)co-infected and HIV mono-infected patients,and to assess the possible interactions between HCV and HIV.Methods Fifty-nine patients with HIV/HCV co infection were enrolled.The control group was consisted of 38 patients with HIV monoinfection.The liver function,peripheral blood T cell subgroups(CD4+and CD8+)cell count and HIV RNA level were compared between these two groups.Peripheral blood mononuclear cells(PBMCs) were analyzed by interferon-γ enzyme-linked immunospot(ELISPOT)using a panel of HIV antigens.Results The frequency of HIV/HCV co-infection was 60.8%.Both alanine aminotransferase(ALT)and aspartate aminotransferase(AST)in HIV/HCV co-infection group were significantly higher than those in HIV mono infection group(49.8 U/Lvs 23.6 U/L,49.1 U/L vs 32.3 U/L,P=0.000,0.013).Platelet count was lower in HlV/HCV co-infection group than in HIV group[(167.3±59.2)×109/Lvs(198.0±63.9)×109/L,P=0.040].CD4+cell and CD8+cell counts were not significantly different between co-infection group and HIV group.The HIV RNA level was lower in HIV/HCV co-infection group than in HIV group [(4. 046 ± 0. 541 ) log10 copy/mL vs (4. 394 ± 0. 507) log10copy/mL, P=0. 018]. The intensity of HIV-specific cytotoxic T lymphocyte (CTL) response to HIVGag overlapping peptides in HIV/HCV co-infection group (mean bank 30.85) was lower than HIV group (mean bank 44.34). The number of the HIV-specific CTL cell in HIV/HCV co-infection group (4.60±5.52) was slightly lower than HIV group (6.24±6.93) without significant difference.Albumin was negatively correlated with HCV RNA in HIV/HCV co-infection group (r=-0. 540). A positive correlation was found between platelet and peripheral blood CD4+ cell counts (P=-0. 040). No linear correlation was found between HCV viral load, HIV viral load and peripheral blood CD4+ cell counts. Conclusions The prevalence of HCV co-infection is relatively high. The cellular immunity status in these co-infected patients is relatively poor.

Objective To investigate the effect of short stature homeobox (SHOX) gene promoter-372G →A mutation on the promoter activity and its mechanism.Methods The luciferase report gene vectors containing human SHOX gene promoter-372G or -372A were contructed.Their transcription activities were detected in chicken chondrocytes.Double-stranded DNA probes containing-372G or-372A were produced by PCR,and used for detecting the affinity with nuclear transcription factors by electrophoretic mobility shift assay(EMSA).Results The transcription activity in a-372A promoter construct was significantly higher than that in the wild type-372G (P<0.01).The result of EMSA showed that-372A gene mutation resulted in loss of the binding affinity to nuclear transcription factors.Conclusion The-372A mutation increases SHOX promoter activity with decreased DNA binding affinity to transcription factors,which may contribute to impaired long bone growth in patients with idio pathic short stature.

Objective To establish a new method, applying high resolution melt, to discriminate the VEB-3 hypotype from the clinical gram negative isolates. Methods From January to December 2003,292 consecutive and non-repetitive gram-negative bacteria producing VEB extended spectrum β-lactamase (ESBL) were collected. Extract the DNA of clinical gram negative isolates with phenol-chloroform. PCR was performed to amplify the VEB gene with the DNA being template. After that, we amplify the fragment of VEB gene containing the position 168. Then we detect the high resolution melt curve and analyze them. At last, we analyze the results of sequence and high resolution melt( HRM ). Results VEB-1 and VEB-3 gene are markedly different through HRM analysis. Conclusion It is accurately and quickly for us to identify the VEB-3 from other hypotype through the technology of HRM.

OBJECTIVETo investigate difference between the appearance and the bony structure in the polysyndactyly of the fifth toe fused with the fourth toe.

METHODSFrom Jan. 2009 to Jan. 2014, 54 patients (65 feet) with polysyndactyly of the fifth toe fused with the fourth toe were treated. The appearance, X-ray and intraoperative finding were recorded and compared to classify the deformity. Then the extra toe was excised and syndactyly was separated. The malalignment and brachydactyly of the sixth toes were corrected simultaneously.

RESULTSAccording to the bone and joint type, the fifth toes were neoplastic toes without joints in 17 feet, or had poor bony and joint alignment with the sixth toes in 48 feet. So the fifth toes were excised in all the cases. The patients were followed up for 1 month to 4 years. The oblique deformity of sixth toes were corrected completely with improved length.

CONCLUSIONSThe polysyndactyly of the fifth toe fused with the fourth toe should be classified to design the excised toe (usually fifth toe) and correction procedure. The appearance and bony joint recovery are both important.

Humans ; Polydactyly ; pathology ; surgery ; Syndactyly ; pathology ; surgery ; Toe Phalanges ; abnormalities ; surgery ; Toes ; abnormalities ; surgery

Objective To observe the effects of large-dose ambroxol hydrochloride on lung ischemia-reperfusion injury (LIRI)and discuss the protection of ambroxol hydrochloride on Toll-like receptor 4 (TLR4)/nuclear transcription factor-kappa B (NF-κB ) after lung ischemia-reperfusion injury in rats.Methods We randomly assigned 60 healthy SD rats into four groups (n=15 for each):control group,ambroxol hydrochloride group (0.75 g/L),ischemia-reperfusion group (I/R),and I/R+ambroxol hydrochloride group.The ambroxol hydrochloride group and I/R+ambroxol hydrochloride group were injected large dose of ambroxol hydrochloride by intravenous injection.The control group and the I/R group received normal saline.The effects of ambroxol hydrochloride on lung ischemia-reperfusion (LIR)-induced pathological changes and inflammatory cytokines release level were examined.DNA ends situ labeling assay (TUNEL)was used to detect the apoptosis of cells.NF-κBp6 5 was detected by immunohistochemistry.In addition,the TLR4 signaling pathway activation in lung tissues was detected by Western blot analysis.Results Compared with those in the control group,some hemorrhage and inflammation changes of lung tissues were observed;the W/D ratio,inflammatory cytokines,apoptosis of cells,NF-κBp6 5 and TLR4 signaling pathway protein expression in I/R group was obviously increased.Compared with I/R group,some mild hemorrhage and inflammation changes of lung tissues were observed;W/D ratio,inflammatory cytokines,apoptosis of cells, NF-κBp6 5 activity, and TLR4 signaling pathway expression were all decreased significantly in I/R+ambroxol hydrochloride group.Conclusion Large dose of ambroxol hydrochloride can protect rats with lung ischemia-reperfusion injury by downregulating TLR4 signaling pathway.

Traditional Chinese medicine treatment is the key to restore the balance of Yin and Yang through gasification. So it is important to discuss the mode of gasificaiton of Qi and Chinese medicine and their relationship in the machanism of the Chinese medicine for diseases. Life is a mode of Qi and Qi movement and gasificaiton make people alive. The Chinese medicine works on the Qi gasification to balance the Yin and Yang to keep the peace inner and outsides.