Effect of hesperidin on expression of inducible nitric oxide synthase in cultured rabbit retinal pigment epithelial cells.

OBJECTIVE: To study the effect of hesperidin on expression of inducible nitric oxide synthase (iNOS) in cultured rabbit retinal pigment epithelial (RPE) cells under the condition of high glucose in vitro. METHOD: Hesperidin was extracted from Pericarpium Citri Reticulatae by ultrasound and ethanol precipitation and was detected qualitatively by high performance liquid chromatogram. The third to fifth primary cultured rabbit RPE were selected. The cells were divided into 6 groups including the control group cultured in DMEM, the model group cultured in DMEM containing 33 mmol/L glucose without any drug and four experimental groups which were exposed to hesperidin at the concentration of 10, 20, 40 and 80 mg/L at 37 degrees C under 5% CO(2) for 2 h and then cultured in DMEM containing 33 mmol/L glucose. The proliferation of RPE was measured by the MTT assay. The levels of NO produced were measured by spectrophotometry. The changes of iNOS expressed in RPE cells were determined with immunohistochemistry. RESULTS: The growth rate of RPE cells was associated with the concentration of hesperidin. NO production induced by high glucose was significantly inhibited by hesperidin. iNOS expression in hesperidin-treated group was decreased compared with the control group (p <0.001). CONCLUSION: Hesperidin can increase the proliferation of rabbit RPE cells, and inhibit the level of NO and iNOS expression, so hesperidin can protect rabbit RPE cells.

Mutation frequency of IMPDH1 gene of Han population in Ganzhou City.

OBJECTIVE: Mutations in the inosine monophosphate dehydrogenase 1 gene (IMPDH1) have recently been discovered that IMPDH1 gene plays a critical role in pathogenesis of autosomal dominant retinitis pigmentosa (adRP). Aiming towards an understanding of the molecular background of retinitis pigmentosa (RP), this paper investigates the mutation frequency of IMPDH1 genes in the Han patients with adRP in Ganzhou City. METHODS: The whole blood samples were collected randomly from 56 adRP patients and 62 unrelated normal controls who were residents of Han population in Ganzhou City, and then their genomic DNA samples were extracted respectively. Genic polymorphism was examined by the polymerase chain reaction and restriction-fragment-length polymorphisms (PCR-RFLP). The statistical significance of the data was further analyzed by SPSS 14.0 software. RESULTS: Mutation rate of IMPDH1 gene had no significance between in adRP patients and in the normal control by exact probabilities in 2 x 2 table (p = 0.232). The mutation frequency of IMPDH1gene in the Han samples was 3.6%. CONCLUSION: The mutation frequency of IMPDH1 gene of the Han population in Ganzhou city was similar as approximately 2-5% of the adRP cases among Americans of European origin and Europeans.

In vitro passage and line establishment of human limbal stem cells / 中国组织工程研究

BACKGROUND: The investigation of culturing, passaging and establishing human limbal stem cells can strengthen the recognition of the stem cells and provide the enough cellular reserve for the basic and clinical research of limbal stem cell transplantation.OBJECTIVE: To explore a method of pessaging and establishing cell line of human limbal stem cells cultured in vitro.DESIGN: Randomized controlled observation.SETTING: Gannan Medical College.MATERIALS: The experiment was performed at Scientific Center of Gannan Medical College and the National Key Laboratory of Ophthalmology Hospital Affiliated to Sun Yat-Sen University from June 2003 to April 2004. Fresh human limbus corneae were isolated from two healthy donors. Procedures were performed according to the informed consent of the donors. Main reagents contained RPMI-1640 (Sigma R8755, containing L-glutamine) and 200 g/L fetal calf serum (FCS) (Gibco 16140-071). DMEM medium, chondroitin sulfatase and human epidermal growth factor (hEGF) were purchased from Sigma Co. USA; HEPES and DMSO were bought from Gibco, USA; 100% glycerinum was purchased from Yunjia Huangpu Pharmaceutical Product Limited Company, PRC; glutaraldehyde was bought from E.Merk, Germany; Alcohol, chlorhydric acid, acetone and methyl aldehyde were purchased from Beijing Chemical Agent Company, PRC; 0.25% parenzyme was bought from Shanghai Xinhua Pharmaceutical Factory, PRC.Above-mentioned reagents were analytical pure grade.METHODS: After digestion, human limbal tissues in limbal basilar part with an abundant pigment were cultured in the culture flask containing RPMI-1640 and 200 g/L FCS and in culture dish containing amniotic extracellular matrix (AECM) as the cultural supporter. Primary and passage cells were observed under light microscope and scanning electron microscope (SEM). The revival ratio of stem cell refrigeration of every generation was calculated by the trypanblau exclusion experiment.MAIN OUTCOME MEASURES: ① Observational results of limbal stem cells during the primary culture and serial subcultivation in vitro, and ② revival ratio of stem cell refrigeration.RESULTS: ①Findings of primary culture: Most limbal stem cells in the culture flask had the adherence and were arrayed uniformly sparsely to form monolayer and adhered to the bottom of culture flask under the inverted phase contrast microscope after 1-day culture. ② Findings of serial subcultivation: After human epidermal growth factor (hEGF) was added into the second passage, cells were scattered into the monolayer and adhered to grow quickly.Morphological variability of all the cells increased obviously when passage the 30th generation. The cellular volume was obviously increasing, and the round or irregular round cells gathered together. The 33rd generation human limbal stem cells still could vigorously differentiate, proliferate and grow in ACEM. ③ The revival ratio of stem cell refrigeration was 82.2%.CONCLUSION: The human limbal stem cell lines were preliminarily established by culturing and freezing the cells of 33 generations in vitro. The human limbal stem cell lines preferred to grow in the culture dish containing AECM as the cultural supporter.