BackgroundMüller cells has been recognized as being vital in both healthy and diseased retina.Recently,these cells even have been identified to be the source of retinal progenitor cells.In order to study the possible function of the retinal Müller cells,it is important to establish a practical procedure to obtain the purified cells. ObjectiveThis study was to simplify the procedure of primary culture and purification of retinal Müller cells in vitro. MethodsEyeballs of SPF newborn SD rats were enucleated and retinas were dissected free after soaking the eyeballs overnight in DMEM/F12 medium in room temperature.Then the retinas were mechanically dissociated into micro aggregates and cultured in DMEM/F12 medium containing 10％ FBS for 8-10 days.The floating retinal aggregates and debris were removed and the medium was changed in 2-3 days interval to get more purified flat cell population.Cultured cells were passaged after confluent.Immunofluorescence staining was used to detect the response to glutamine synthetase (GS) and Vimentin for the identification of the cultured Müller cells,and flow cytometry (FCM) was used to estimate the purity of the cells. Results Cultured Müller cells had large cellular body and richer cytoplasm.More than 95％ of the cells showed the positive response for GS with the brown staining in cytoplasm and cellular nuclei,and the positive stainiug also was seen for Vimentin in cytoplasm.FCM showed that 99.7％ of the cells were GS positive after 3 passages.Conclusions Modified procedure in this experiment is a simple and practical method for culturing retinal Müller cells.

Retina is subjected to many acquired and inherited neuronal degenerative diseases such as agerelated macular degeneration (AMD), diabetic retinopathy (DR) and retinitis pigmentosa (RP). All of these diseases are associated with the progressive damage and loss of photoreceptors, which is causing visual impairment and irreversible blindness. Stem-cell therapy is being widely considered as a promising treatment of these incurable retina diseases. However, in mammals including humans, there seems to be little or no recovery of lost cells. By contrast, nonmammalian vertebrates, such as amphibians and fish, have robust regenerative responses to injury, which can lead to the near complete restoration of the neurons lost through injury. Nevertheless ,over the past several years, studies have investigated that stem cells do exist in the adult mammalian eyes, and at least some types of neurons can be regenerated in the mammalian retina by stimulating with growth factors or transcription factors. These recent results suggest that some part of the regenerative program occurring in lower vertebrates remains in the mammalian retina.Here, the origin of various of adult retinal stem cells for the self-renewal and proliferation. and the relevant influencing factors were summarized.

Objective To establish a convenient and specific molecular method for cytomegalovirus(CMV) surveillance in recipients of allogeneic hematopoietic stem cell transplantation(allo-HSCT) and ana-lyze the correlation between cytomegalovirus genotypes and transplant-related complication to decrease CMV-related mortality through restricted endoenzyme digestion.Methods 135blood samples were regularly col-lected from79consecutive patients who received allo-HSCT between April2001and April2003.Of the79 patients observed,median age was32(range from11to60) and the ratio of male and female was1.63∶1.DNA extracted from whole blood was amplified with CMV sequence using Nested PCR and gB genotypes were identified through restriction enzyme analysis.Results Among the135clinical isolates from79pa-tients,42cases(66specimens) were Positive.The gB genotype from42cases(45samples) were identified as gB1,21(46.7%);gB2,14(31.1%);gB3,7(15.6%);gB4,3(6.7%),respectively,by restric-tion enzyme analysis.Among them,3patients were successively infected with CMV gB1and gB2.The inci-dence of GVHD in groups of CMV(+) and CMV(-) was81.0% and32.4%,respectively(P

Background Krüppel-like factor 6 (KLF6) is related to the physiological or pathological process,such as growth,cell differentiation,proliferation,apoptosis,angiogenesis,tissue repair,and so on.But in ophthalmology,it is less reported about the expressing level of KLF6 protein in lens epithelial cclls (LECs) or the effect of KLF6 on the proliferation of human LECs.Objective This study was to investigate whether KLF6 can inhibit proliferation of human LECs.Methods KLF6 eukaryotic expression plasmid (pEGFP-C2-KLF6) was constructed using reverse transcription PCR(RT-PCR) and identified by double enzyme digestion method and PCR.Human LECs strain (HLE-B3) was cultured and passaged using low glucose DMEM containing 10％ fetal bovine serum and then divided into 4 groups.KLE-B3 transfection reagents were added in the culture medium of all groups.In addition,no agent was used in the blank control group;only insulin-like growth factor-1 (IGF-1) was appended to the medium in only IGF-1 group ;null vector was transfected and IGF-1 was appended in the null plasmid transfection+ IGF-1 group;while pEGFP-C2-KLF6 eukaryotic expression plasmid was transfected into the cells,and simultaneously IGF-1 was added in the pEGFP-C2-KLF6 plasmid transfection+IGF-1 group.After 24 hours of intervene,water soluble tetrazolium salt-1 (WST-1) test was used to detect the growth status of the cells,and Western blot assay was used to assay the relative expressing level of KLF6 protein in the cells.In the other hand,the cells were cultured at the density of 1 ×104/piece,and 0,0.10 and 0.25 μg pEGFP-KLF6 were transfected into each piece of cells respectively,and then IGF-1 was added with a final concentration of 50 μg/L for 24 hours after cell culture.Expressions of Ki-67 protein and mRNA in the cell pieces were detected by immunocytochemistry and fluorescent semiquantitative PCR,respectively.Results The PCR product bands were consistent with KLF-6 gene in length,and the product fragments were corresponding with expectant ones via PCR and double enzyme digestion method,showing a successful construction of pEGFP-C2-KLF6 eukaryotic expression plasmid.After 24 hours of IGF-1 stimulation,the absorbance values of the cells were 0.86±0.00,2.10±0.01,2.24±0.12 and 1.06±0.02 in the blank control group,only IGF-1 group,null plasmid transfection + IGF-1 group and the pEGFP-C2-KLF6 plasmid transfection + IGF-1 group,with a significant difference among the 4 groups (F =38.322,P ＜ 0.05),and that in the pEGFP-C2-KLF6 plasmid transfection +IGF-1 group was significantly lowed in comparison with the only IGF-1 group and the null plasmid transfection+IGF-1 group (q=6.42,7.31,both at P＜0.05).Western blot assay showed that the relative expressing levels of KLF6 protein were statistically different among the four groups (F =591.858,P＜0.05),and those in the pEGFP-C2-KLF6 plasmid transfection+IGF-1 group were 1.47,2.04,3.27 folds higher than those in the blank control group,only IGF-1 group,respectively and null plssmid transfection+IGF-1 group,respectively.Immunocytochemistry revealed that the expressing intensity of Ki-67 protein was gradually weakened with the decrease of pEGFP-C2-KLF6 plasmid dose in the 0,0.10 and 0.25 μg pEGFP-C2-KLF6 plasmid transfection+IGF-1 groups.Fluorescence semiquantitative PCR results showed that the relative expression values of Ki-67 mRNA in the cells were O.15±0.08 and 0.11±0.03 in the 0.10 μg and 0.25 μg pEGFP-C2-KLF6 plasmid transfection+ IGF-1 groups,which were significantly lower than O.77± 0.12 of the 0 μg pEGFP-C2-KLF6 plasmid transfection group,with a statistically significant difference among the three groups (F =54.825,P＜0.05).Conclusions KLF-6 can effectively inhibit IGF-1-induced proliferation of human LECs,and it can be regarded as one of the regulatory factors of the proliferation of HLE-B3 cells.

Objective To study the chemical composition from leaves of Smallanthus sonchifolius.Methods Some chromatography methods were used in the isolation procedure,while the structures were determined on the aids of NMR and MS spectral analyses.Results A new compound,together with five known compounds,was isolated from the ethanolic extract of the leaves.The new compound is characterized as 5,8-dihydroxyl-(5H,8H)-?-ionol(Ⅰ).Other compounds are obtained for the first time from the title plant and identified as ent-kaurane-3?,16?,17-triol(Ⅱ),entkaurane-16?,17-diol-19-oic acid(Ⅲ),3,4-dihydroxybenzaldehyde(Ⅳ),1-pentacosanol(Ⅴ),and 1-octacosanol(Ⅵ),respectively.Conclusion Compounds Ⅰ-Ⅵ are isolated from the plants of genus Smallanthus for the first time.Compound Ⅰ is a ?-ionol derivate firstly isolated from genus Smallanthus.It is named as sonchifolol.

Background Cystoid macular edema(CME) is an important cause of visual impairment of central retinal vein occlusion(CRVO).Spectral-Domain optical coherence tomography(SD-OCT) has increased speed and higher resolution,offering a better chance of understanding the morphological changes and pathogenesis of CME. Objective This study was to survey the morphologic features of macular edema associated with CRVO by SD-OCT. Methods Clinical data of the patients with CRVO diagnosed in Department of Ophthalmology,Peking Union Medical College Hospital from March 2008 to August 2009 were retrospectively analyzed.SD-OCT features of macular edema induced by CRVO were analyzed and recorded.Results The average macular foveal thickness was(527.5±218.2) μm in macular edemas eyes.Main morphological changes included 55 cases(84.6%) of CME,15 cases of(23.1%) serous macular detachment(SMD),and 10 cases(15.4%) of simple macular edema,and these findings occurred at the same time in some eyes.Cystoid spaces in the parafoveal region were seen in the inner nuclear layer,outer plexiform layer and outer nuclear layer,and discontinuous or weak inner segment/outer segment(IS/OS) line was often seen in CME.The incidence of CME associated with incomplete posterior vitreous detachment(PVD) was 14.5%,and that of neural epithelial edema associated with incomplete PVD was 10.0%,showing an insignificant difference between them(χ2=0.000,P=1.000).The average area of SMD was 1838.4μm ×1428.1μm×190.1μm,and the incidence of partial PVD was higher(χ2=4.266,P=0.039).Conclusion SD-OCT can reveal the micro-morphological change of macular zone in macular edema eye.SD-OCT enabled visualization of its spatial extent in each retinal layer and the condition of IS/OS layer.Serous macular edema is related with partial PVD.

AIM: To assess the differentiation of rat mesenchymal stem cell (MSC) in the microenvironment of retinitis pigmentosa(RP) induced by the administration of sodium iodate. METHODS: In vitro cultured Lewis rat MSC were injected into the subretinal space of NaIO3 induced RP rat eyes (30g/L NaIO3 100mg/kg). To observe the trace and differentiation of MSC by immuno-fluorescent method successively in 5 weeks after the surgery.RESULTS: The majority of the transplanted cells stay in retinal pigment epithelium(RPE) layer and cones and rods layer. From the 2nd week after transplantation, the engrafted MSC expressed PCK and rhodopsin under fluorescent microscope.CONCLUSION: MSC can survive mainly in the outer layer of retina in the microenvironment of RP and differentiate forward the RPE cell and photoreceptor.

Adult ; Female ; Humans ; Male ; Middle Aged ; Otosclerosis ; surgery ; Stapes Surgery ; methods

Objective To investigate the clinical characteristics of Takayasu's arteritis (TA) with pulmonary hypertension (PAH) in order to improve the diagnosis and treatment earlier. Methods Twelve out of 191 patients with TA registered in Peking Union Medical College Hospital from 1987 to 2007 were diagnosed as PAH, the clinical data of 12 patients were analyzed. Results Ten patients were females. The range of age were from 14 to 47 years old, the average age was (27±10) years old. Eleven patients had the clinical manifestations or/and signs of pulmonary artery involvement. Seven patients presented with short breath after exercise or hemoptysis as the first manifestation, four patients with fatigue, four patients with intermittent claudication or pain or numbness of extremities, three patients with dizziness. Seven patients belonged to type Ⅰ+Ⅳ, one patient to type Ⅱ+Ⅳ, three patients to type Ⅲ+Ⅳ, one patient to type Ⅴ. Elevated ESR/CRP was found in ten patients. All patients took the glucocorticoid and DMARDs, stent implantation in pulmonary artery was done in one patient, Bentall was operated in another patient.The symptoms of all patients improved except one patient died for low cardiac output after operation. Conclusion PAH is one of the severe complications in late stage of TA, and other arteries are usually involved too. Because it is difficult to observe PAH in TA patients in early stage, CTA or pulmonary angiography and UCG should be taken in early stage. The stent implantation or dilating the artery should be considered aa a treatment, but at on the same time, glucocorticoid and DMARDs should be taken to avoid the relapse.

Objective To investigate the combined inhibition effect and the potential mechanism of rapamycin (mammalian target of rapamycin inhibitor) and PD98059 (mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MEK) inhibitor) on mouse colorectal cancer (CRC). Methods S-ICR mice were subcutaneously injected with 20 mg/kg of 1,2-dimethylhydrazine dihydrochloride in the nape for 20 weeks to induce CRC. From the 16th week, the mice were treated with alone or combined injection with 0.25 mg/kg rapamycin and 2.5 mg/kg PD98059. The drugs were administered for 8 weeks. Subsequently, the animals were sacrificed and dissected, the tumor sizes were measured, and the tumors were harvested for pathological assay. Furthermore, the phosphorylation of mTOR, p70S6K, and 4E-BPl proteins was detected by using immunohistoehemistry. Results The mice treated with rapamycin (44. 44 %) or PD 98059 (either alone (38.89%) or combination treatment (6.67%) were significantly less likely to develop cancer compared with mice receiving none of them (77.78%, P<0. 05). The average size of tumors was (6.15±2. 192), (8.85±3. 983), (2.917±0. 191), (16.36±6.855) mm3 respectively (P<0.05).The anti-cancer effect of the combination treatment was substantially significant. The proteins of phospho-mTOR, phospho-p70s6K and phospho-4E-BPl were significantly down-regulated after treatments (all P values < ,0.05). Conclusions Combined treatment was more effective than single-drug treatments of rapamycin or PD98059 alone for the prevention and treatment of mouse CRC. The mTOR signal pathway might be involved in the inhibitory mechanism.