Background To optimize the culture method of human retinal microvascular endothelial cells is very important for the study of retinal angiogenesis disease.Human retinal microvascular endothelial cells have been successfully cultured in previous studies,but further improvement of the culture method to harvest higher yields and purity cells is still needed.Objective This study was to design a modified method to isolate and purify human retinal microvascular endothelial cells much easily and quickly,and to compare the expression of specific markers of vascular endothelial cells,factor Ⅷ and CD31/CD34 in the cells.Methods The use of human donor eyeballs was approved by the Ethic Commission of Zhongshan Ophthalmic Center of Sun Yat-sen University.The retina tissue from healthy donor was isolated and digested by the two-step digestion method with 2％ trypsin and 0.133％ collagenase Ⅳ.Human retinal microvascular endothelial cells were collected and plated in 60 mm dishes coated by 0.1％ fibronectin and cultured in endothelial cell-specialized medium supplemented with 10％ fetal bovine serum,0.3 mg/L β-endothelial cell growth factor (ECGF) and 100 ng/L sodium heparin.During the culturing,the growth situation of the cells was monitored by morphological observation,and immunohistochemical staining was performed to probe vascular endothelial cell-specific membrane protein CD31,CD34 and factor Ⅷ for identification of the cell purity.Results Human retinal microvascular endothelial cells were isolated successfully from the retina by the twostep digestion method.The primary cultured cells adhered to well 72 hours later and achieved confluence with the typical cobblestone appearance 9 to 10 days after cultured.The cells exhibited the blue nuclei and reddish cytoplasm by regular haematoxylin and eosin stain and showed a strong positive response for CD31,CD34 and factor Ⅷ by immunohistochemistry.The positive dye of CD31 and CD34 was lower than Ⅷ factor in both endothelial cells.Conclusions Modified culture method of human retinal microvascular endothelial cells can improve cell culture result and purify target cells.

BackgroundHuman retinal gliocytes play an important role in proliferative diseases,which are the basis of in vitro studies.Researchers have cultured human retinal gliocytes in the past.In our study,we found that the cells we cultured presented a unique shape different from those by other researchers.ObjectiveThis study was to design to produce a new culture and purification method for retinal gliocyte in vitro.Methods Retinal tissue was isolated from human eyeballs and digested using the two-step digestion method (2％ pancreatin and 0.133％collagenase Ⅵ) to harvest the retinal glio cytes.The cells were collected and cultured in endothelial cell-targeted nutrient culture containing 10％ fetal calf serum and supplemented with β-endothelial cell growth factor (ECGF) and sodium heparin,and the culture dishes were coated with fibronectin(FN) to promote the attachment of retinal gliocyte.During the culturing process,the gliocytes were identified by the observation of morphological characteristic and regular histological examination.The identification of the cells also was performed by immunochemistry targeting glial fibrillary acidic protein (GFAP),Vimentin,neuron specific enolase ( NSE ),S-100,CD34,and Ⅷ factor.Results Retinal gliocytes were isolated successfully from the human retina by the two-step digestion method.Primary cultured cells attached after 72 hours and achieved confluency between day 9 and 10 that were aligned petaliform in shape.Regular histological examination after H&E staining showed blue cell nuclei and light red cytoplasm.The target cells presented with strong responses for GFAP and Vimentin and no response for NSE,S-100,CD34 and Ⅷ factor.ConclusionsLarge amount of purified human retinal gliocytes can be obtained by two-step digestion and cultured in endothelial cells-targeted culture medium supplemented with β-ECGF and sodium heparin in plates coated with FN.The cultured cells expressed markers for retinal gliocytes.However,specific features of these cells remain to be further elucidated.

OBJECTIVETo investigate the curative effect of antiphlogistic agent series on treating chronic nonbacterial prostatitis (CNP).

METHODSOne hundred and sixty patients were randomized into 4 groups for an 8-week clinical observation: group A (oral antiphlogistic medicinal granules only), group B (oral antiphlogistic medicinal granules + retention enema), group C (oral antiphlogistic medicinal granules + rectal), and group D (antiphlogistic medicinal granules + rectally + hip bath). Single blind trials were employed.

RESULTSThe curative rates of the 4 groups were 37.5%, 57.5%, 52.5% and 82.5% respectively, while the total efficacy rates were 42.5%, 82.5%, 77.5% and 92.5% respectively. Compared with groups A, B and C, the curative rate of group D was significantly higher (P < 0.05). The difference in efficacy rates was slight between groups B and D (P < 0.05), but significant between groups A and C (P < 0.05).

CONCLUSIONSCombined treatment therapy can improve the effect of CNP treatment and clear away heat and toxic material. The antiphlogistic agent series, with the effect of motivating blood circulation and removing blood stasis, turned out to be an effective traditional Chinese medicine in treating CNP.

Adult ; Chronic Disease ; Humans ; Male ; Medicine, Chinese Traditional ; Middle Aged ; Prostatitis ; drug therapy

OBJECTIVETo observe the effect of total coptis alkaloids (TCA) on beta -amyloid peptide (A beta 25-35) induced learning and memory dysfunction in rats, and to explore its mechanism.

METHODSForty male Wistar rats were randomly divided into four groups: the control group, the model group, the TCA low dose (60 mg/kg) group and the TCA high dose (120 mg/kg) group, 10 in each. A beta 25-35 (5microl, 2 microg/microl) was injected into bilateral hippocampi of each rat to induce learning and memory dysfunction. TCA were administered through intragavage for consecutive 15 days. Morris Water Maze test was used to assess the impairment of learning and memory; concentration of malondialdehyde (MDA) in cerebral cortex was determined by thiobarbituric acid reactive substance to indicate the level of lipid peroxidation in brain tissues; activity of manganese-superoxide dismutase (Mn-SOD) in cerebral cortex was determined by xanthine-oxidase to indicate the activity of the enzyme; and NF- kappa B protein expression in cerebral cortex was measured by SP immunohistochemistry.

RESULTS(1) Morris Water Maze test showed that, during the 4 consecutive days of acquisition trials, the rats in the model group took longer latency and searching distance than those in the control group (P<0.01), which could be shortened by high dose TCA (P<0.05); during the spatial probe trial on the fifth day, the rats in the model group took shorter searching time and distance on the previous flat area than those in the control group (P<0.01), which could be prolonged after TCA treatment (for low dose group, P<0.05; for high dose group, P<0.01). (2) Analysis of cerebral cortical tissues showed that, compared with the control group, MDA level got significantly increased and Mn-SOD activity decreased in the model group (both P<0.01). After having been treated with TCA, the MDA level got significantly decreased (P<0.05 and P<0.01 respectively for low and high dose group), while relative increase of Mn-SOD activity only appeared in high dose group (P<0.05). (3) Immunohistochemistry analysis showed the protein expression of NF- kappa B got significantly increased after modeling, while high dose TCA can significantly inhibit it.

CONCLUSIONTCA could improve A beta 25-35 induced dysfunction of learning and memory in rats, and its protective mechanism is associated with its actions in decreasing MDA level, increasing Mn-SOD activity and inhibiting the expression of NF-kappa B in cerebral cortex.

Alkaloids ; administration & dosage ; pharmacology ; Amyloid beta-Peptides ; administration & dosage ; Animals ; Cerebral Cortex ; metabolism ; Coptis ; chemistry ; Dose-Response Relationship, Drug ; Hippocampus ; drug effects ; Injections ; Learning Disorders ; chemically induced ; psychology ; Male ; Malondialdehyde ; metabolism ; Maze Learning ; drug effects ; Memory ; drug effects ; Memory Disorders ; chemically induced ; psychology ; NF-kappa B ; metabolism ; Peptide Fragments ; administration & dosage ; Rats ; Rats, Wistar ; Reaction Time ; drug effects ; Superoxide Dismutase ; metabolism ; Swimming

Background The incidence of retinal vascular diseases increase annually,such as diabetic retinopathy,retinopathy of prematurity and age-related macular degeneration.The key of treatment for these diseases is how to evaluate retinal vascular change effectively and objectively.Retro-orbital injection of fluorescein isothiocyanatedextran (FITC-dextran) is a simple and effective method for observing C57BL/6J mouse retinal vessels.But,whether it is suitable for other mice and rats is seldom reported.Objective This experiment was to assess the feasibility of the observation of retinal vessels by retro-orbital injection of FITC-dextran in different genus of mouse and offer the reference for relevant study.Methods Twelve animals of C57BL/6J mice,Kunming mice,SD rats and Wistar rats were selected,respectively and divided into the experimental group and control group at average.The right eyes of the animals of the experimental group received the retro-orbital injection of 9 ml/kg FITC-dextran,and the right eyes of animals of the control group received PBS solution at the same volume and way.All the animals were sacrificed 10 seconds after injection and both eyes of each animal were obtained for retinal stretched preparation.The retrobulbar tissue and whole-mount retina were viewed under a fluorescence microscope.The use of the animals complied with Regulation for the Administration of Affair Concerning Experimental Animals by State Science and Technology Commission.Results Retinal blood vessels labeled by FITC-dextran could be observed in both eyes of C57BL/6J mice and Kunming mice to present with a green fluorescence in experimental group under a fluorescence microscope,but no any fluorescence-labeled retinal blood vessel was exhibited in the control mice.The retinal blood vessel could not be observed in all eyes of SD rats and Wistar rats after the injection of FITC-dextran both in the experimental group and the control group under a fluorescence microscope.The surrounding tissues of the right eyes of mice and rats dyed with green fluorescence of FITC-dextran in the experimental group,however,green fluorescence could not be seen in the surrounding tissues of the left eyes of mice and rats.Conclusions Retro-orbital injection of FITC-dextran is a suitable method of observing the retinal vessels of mouse but not rat.

Objective:To investigate the effect of fibroblasts in breast cancer microenvironment on the expression of TIGAR and Bcl-2 in breast cancer cells and their effect on breast cancer growth.Methods: In vitro experiments,the co-cultured model of human breast cancer cell line MDA-MB-231 with human fibroblast line CCC-ESF-1 was established.The effect of fibroblasts on the expression of TIGAR and Bcl-2 in breast cancer cells was tested with reverse transcription quantitative polymerase chain reaction(RT-qPCR) and Western blot.Annexin V flow cytometry and caspase-3 activity assay were employed to detect the apoptosis of breast cancer cells.In vivo experiments,human breast cancer transplanted tumor model in nude mice was established and the tumor volume of nude mice was meas-ured.Immunohistochemistry was used to detect TIGAR and Bcl-2 expression in the transplanted tumor tissues of nude mice.Results:The results showed that the co-cultured fibroblasts could up-regulate the expression of TIGAR and Bcl-2 in MDA-MB-231 cells and inhibited the apoptosis of MDA-MB-231 cells in vitro.The fibroblasts implanted with breast cancer cells could up-regulate TIGAR and Bcl-2 expression in breast cancer tissues of tumor-bearing nude mice in vivo,whereas highly expressed TIGAR and Bcl-2 accelerated the tumor growth of tumor-bearing nude mice.Conclusion:The fibroblasts in breast cancer microenvironment up-regulate the expression of TIGAR and Bcl-2 in breast cancer cells co-cultured with fibroblasts.Highly expressed TIGAR and Bcl-2 inhibit the apoptosis of breast cancer cells and promote the growth of breast cancer.

OBJECTIVETo discuss the proper surgical management of pancreatic benign and low-grade malignant potential neoplasm.

METHODSThe experience of 72 cases who accepted organ preserving pancreatectomy from January 1990 to May 2010 was analyzed retrospectively. There were 24 male and 48 female, aged from 15 to 68 years with mean age of 46 years. There were 9 cases underwent duodenum-preserving resection of the head of the pancreas, 29 cases underwent spleen-preserving distal pancreatectomy, 11 cases underwent middle segmental pancreatectomy, 23 cases underwent tumor extirpation of huge pancreatic cancer in pancreatic head and body.

RESULTSPancreatic fistula and biliary fistula in 1 case respectively were cured among who accepted duodenum-preserving resection of the head of the pancreas. Pancreatic fistula was found in 3 cases who accepted spleen-preserving distal pancreatectomy. Pancreaticobiliary anastomotic bleeding in 1 case was cured among who accepted middle segmental pancreatectomy. Pancreatic fistula was found in 5 cases among who accepted tumor extirpation of huge pancreatic cancer in pancreatic head and body, and liver metastasis was found in 3 cases at 6, 12, 16 months after surgery respectively.

CONCLUSIONSOrgan preserving pancreatectomy can obviously reduce operative injury to patients, its therapeutic effect is similar to that of classical operation, it is the first option of benign and low-grade malignant potential neoplasm.

Adolescent ; Adult ; Aged ; Female ; Follow-Up Studies ; Humans ; Male ; Middle Aged ; Pancreatectomy ; methods ; Pancreatic Neoplasms ; surgery ; Retrospective Studies ; Treatment Outcome ; Young Adult

BACKGROUNDBroad-spectrum antibiotic administration promotes intestinal colonization of exogenous fungal pathogens in healthy animals and has been recognized as one of the risk factors of invasive fungal infection in clinical settings. It is unclear whether broad-spectrum antibiotic treatment would change the intestinal mycobiota without exogenous fungal challenge in the context of sepsis.

METHODSWe established a rat model of double-hit sepsis using burn injury and endotoxin challenge. Rats with burn injury or double-hit sepsis received imipenem treatment for 3 days or 9 days, and their colon contents were sampled for selective fungal culture and isolation counts.

RESULTSImipenem treatment promoted the overgrowth of the commensal fungus Geotrichum capitatum in rats with burn injury. Imipenem treatment also promoted colon colonization by exogenous fungi in rats with burn injury and double-hit sepsis, including Trichosporon cutaneum, Candida albicans, Candida krusei, and Candida glabrata. A longer duration of imipenem treatment had a stronger impact on colon colonization by exogenous fungi.

CONCLUSIONImipenem treatment facilitates the overgrowth of commensal fungi and colonization by exogenous, potentially pathogenic fungi in the colons of rats with burn injury or double-hit sepsis.

Animals ; Anti-Bacterial Agents ; therapeutic use ; Burns ; complications ; microbiology ; Candida ; pathogenicity ; Colon ; microbiology ; Imipenem ; therapeutic use ; Male ; Rats ; Rats, Sprague-Dawley ; Sepsis ; drug therapy ; microbiology ; Trichosporon ; pathogenicity

OBJECTIVETo explore the mechanisms of electroacupuncture (EA) on postmenopausal osteoporosis (PMO).

METHODSSixty female SD rats aged 6 months were selected, resected double ovaries and fed for 90 days in order to make the model of experimental osteoporosis, and then, they were randomly divided into a model control group (without any therapy), a routine acupoints applying group, which were treated with EA at "Pishu" (BL 20), "Weishu" (BL 21), "Shenshu" (BL 23) and "Qihaishu" (BL 24), and an EA with tonification method (EAT) group at "Guanyuan" (CV 4) and "Zusanli" (ST 36), and an EA with dispersing blood stasis method (EAD) group at "Shenshu" (BL 23), "Geshu" (BL 17) and "Dazhu" (BL 11), and an EA with tonification and dispersing blood stasis method (EATD) group at "Guanyuan" (CV 4), "Zusanli" (ST 36), "Shenshu" (BL 23), "Geshu" (BL 17) and "Dazhu" (BL 11), 12 rats in each group. EA therapy was performed once a day, 30 min each time. Then these rats rested for 1 day after consecutive treatment for 6 days. The treatment lasted for 12 weeks. Enzyme linked immunosorbent assay (ELISA) was used to examine the level of TNF-alpha, and the level of E2, osteocalin (BGP) and carboxyterminal propeptide of type I procollagen (PICP), carboxyterminal telopeptide of type I lollagen (ICTP) by radioimmunoassay, and alkaline phosphatase by p-nitrophenylphosphate method, and Ca, P, Deoxypyridinoline (DPD) and creatinine (Cr) in urine were detected by automatic biochemistry analyzer.

RESULTSCompared with model control group, the level of DPD/Cr, Ca/Cr, and the serum contents of ALP, BGP, TNF-alpha and ICTP in all EA groups decreased significantly (all P < 0.01), the level of E2 and PICP in serum increased significantly (all P < 0.01). Compared with EATD group, the level of DPD/Cr and Ca/Cr in the other three EA groups had no statistical differences (all P > 0.05), but the level of ALP, BGP, TNF-alpha and ICTP in serum increased significantly (all P < 0.01), the level of E2 and PICP in serum decreased significantly (all P < 0.01).

CONCLUSIONAcupuncture can reduce significantly the level of DPD/Cr, Ca/Cr, and the serum contents of ALP, BGP, TNF-alpha and ICTP, increase significantly the level of E2 and PICP in PMO model rats and EA with tonification and dispersing blood stasis method is superior significantly to the other routine acupuncture methods.

Animals ; Bone and Bones ; metabolism ; Collagen ; metabolism ; Disease Models, Animal ; Electroacupuncture ; Female ; Humans ; Osteoporosis, Postmenopausal ; metabolism ; therapy ; Ovariectomy ; Rats ; Rats, Sprague-Dawley ; Tumor Necrosis Factor-alpha ; metabolism

OBJECTIVETo study the present situations of lung cancer in Wuhan and to explore the relationship between the potential years of life lost of lung cancer and air pollution, especially vehicle emissions.

METHODSData gathered between 1986 and 1995 in Wuhan city, including air pollution and tobacco production and data on lung cancer between 1991 and 2000 were collected extensively. Simple Correlation and Grey Relational Analysis were used to analyze the relationship of them.

RESULTSThere was a ascending tendency in variance of oxides of nitrogen (NOx). The degree of grey incidence (DGI) between the concentration of air pollutants and the male's or female's potential years of life lost of lung cancer (PYLL) were calculated respectively. In males, the values of DGI were 0.6702, 0.7071, 0.6199 on sulfur dioxide (SO2), NOx, total suspensions (TSP) respectively. In females,the values of DGI were 0.6188, 0.8555, 0.5842 according to the same order as listed above. Significant positive correlation was found between the concentration of NOx and with lung cancer in both males and females by spearman correlation test (rmale = 0.63523, P = 0.0484; rfemale = 0.76396, P = 0.0101).

CONCLUSIONWith the fast growing speed of the quantity of vehicles, pollution of vehicle emission-caused air pollution posed an important risk factor for lung cancer, despite the fact that tobacco smoking still played the leading role.

Air Pollution ; adverse effects ; China ; Female ; Humans ; Lung Neoplasms ; mortality ; Male ; Nitrogen Oxides ; analysis ; Sulfur Dioxide ; analysis ; Vehicle Emissions ; analysis