AIM: To optimize the conditions for in vitro culture of retinal pigment epithelium (RPE) cells, we characterized expressions of various growth factors in RPE cells, including tumor necrosis factor (TNF-α), vascular endothelial growth factor (VEGF), β fibroblast growth factor (βFGF), transforming growth factor β2 (TGFβ2), and interferon-γ (IFN-γ). We also studied expressions of caspase-3 under different concentrations of fetal bovine serum (FBS) with insulin-transferrin-sodium selenite (ITS) supplement. METHODS: First, we investigated if expressions of TNF-α, VEGF, βFGF, TGFβ2, IFN-γ, and caspase-3 in FBS and ITS with of concentration. Second, we cultured primary RPE cells from eyes of forty C57 BL/6 mice in standard dulbecco's modified eagle's medium (DMEM) containing 20,40,100mL/L FBS and 20,40,100mL/L FBS together with 10g/L ITS. Immunohisto-chemical staining and cell counting were performed to verify the existence and growth condition of RPE cells. Expressions of TNF-α, VEGF, βFGF, TGFβ2 and IFN-γ were determined using cells and supernatant from passage-3 to -4 primary RPE cell after 48 hours of culture with RT-PCR and enzyme-linked immunosorbent assays (ELISA). The expression of casepase-3 was determined via Western blotting. The major outcome measurement is the expression level of growth factors in cultured RPE cells and the experiment design is to expose the RPE cells to different culture medium. RESULTS: TNF-α, VEGF, βFGF, TGFβ2, but not IFN-γ, were expressed and the expressions increased with concentration. No expression of the aforementioned genes was detected in presence of ITS. The primary cultures of RPE cells were successfully established. TNF-α, VEGF, βFGF, TGFβ2 (but no IFN-γ) and the active caspase-3 were detected in 20,40,100mL/L FBS or 20,40,100mL/L FBS combined with 10g/L ITS; the expressions were upregulated with increasing concentration of FBS. There is no significant difference in the expression of growth factors between these groups. However, significant differences were shown among different concentration of FBS (P＜0.01）. The lowest expression was observed in 20mL/L FBS or 20mL/L FBS combined with 10g/L ITS medium with RPE cells. But RPE cells were shown in better growth condition in 20mL/L FBS combined with 10g/L ITS.CONCLUSION: TNF-α, VEGF, βFGF, TGFβ2 and caspase-3 were expressed in RPE cells and supernatants. The production of above 20mL/L FBS combined with 10g/L ITS in DMEM may be the ideal cell culture medium that supports the normal growth of RPE cells.

Objective To investigate the prevalence of clonorchiasis among residents of Huachuan Country,Heilongjiang Province and to provide a basis for development of control strategies.Methods From 2011 to 2012,cluster random sampling was performed to survey the incidence of clonorchiasis in Huachuan Country.Fecal specimens were collected and examined the clonorchis sinensis eggs by Kato-Katz method.A questionnaire survey was conducted to collect related information such as age,gender,occupation and eating habits.The infection characteristic was analyzed.Results Totally 884 patients with clonorchiasis were found among 2248 residents,and the infection rate was 39.32％.The infection rate in male[47.15％(611/1296)] was significantly higher than females [28.68％(273/952),x2 =34.55,P ＜ 0.01].The infection rate increased with age,which was higher in the 20-69 years old people,with the highest infection rate in the 50-59 years old groups[45.34％ (219/483)].Of the occupational distribution,farmers had the highest infection rate [47.24％ (420/889)],followed by cadres and staffs[38.38％(190/495)].Of residents with fresh fish eating history,the prevalence of clonorchiasis was 53.38％(150/281).Conclusions The prevalence of clonorchiasis is still high in Huachuan County.To reduce the prevalence of clonorchiasis,comprehensive prevention measures,health education and group chemotherapy should be carried out.

To explore the effect of advanced glycation end-products(AGEs)on cell viability and level of reactive oxygen species(ROS)in MIN6 cells. After intervention of various concentrations(100,200, and 400 mg/L)of AGEs for some time, cell viability was detected by MTT assay. 2', 7'-dichlorofluorescein diacetate(DCFH-DA)was used as a reactive oxygen species capture agent. The fluorescent intensity of 2', 7'-dichlorofluorescein(DCF), which was the product of cellular oxidation of DCFH-DA, was detected by flow cytometry. The level of ROS and insulin secretion was thus measured. Viability of MIN6 cells was inhibited by AGEs in a dose and time dependent manner(P＜0.05).Intracellular fluorescent intensity of DCF was markedly elevated in the AGEs groups as compared with that in the control group(P＜0.05).Insulin secretion was decreased in the AGEs groups than that in the control group(P＞0.05). The results suggest that AGEs inhibit the viability and induce oxidative stress in MIN6 cells by overproduction of ROS.

OBJECTIVETo assess how trace element selenium and B27 supplements affect the neural stem cell (NSc) differentiation in vitro.

METHODSThe development and differentiation of NSc from the newborn rat were observed with primary culture and subculture during treating by sodium-selenite, and selenium-methyl-cysteine (SMC). The immunocytochemistry techniques were used to identify the NSc and mature protein expression with neuron marker beta-tubulin, astrocyte marker GFAP, and oligodendrocyte marker CNPase. The neurosphere morphology and neurite outgrowth were observed.

RESULTSAdding the complete B-27 serum-free supplement, Selenium could promote the neurosphere viability, development and differentiation. Without selenium and B-27, neurosphere could not survive and differentiate. Without B-27 in the medium but there containing selenium, the neurosphere could promote the viability and development into neuron, astrocyte and oligodendrocyte, as compared with the no-containing B-27 and selenium groups, these differentiated cells might have more quantity, more branches and better morphological nerve net. The count of the neuron, astrocyte and oligodendrocyte was 11.2/Hp, 16.1/Hp and 9.3/Hp.

CONCLUSIONSThe selenium should be very important for neural stem cells' survival. Selenium could promote the neurosphere cells differentiation and development.

Animals ; Animals, Newborn ; Cell Differentiation ; drug effects ; Cell Survival ; drug effects ; Cells, Cultured ; Culture Media, Serum-Free ; pharmacology ; Cysteine ; analogs & derivatives ; pharmacology ; Female ; Glial Fibrillary Acidic Protein ; metabolism ; Immunohistochemistry ; Male ; Neurons ; cytology ; drug effects ; metabolism ; Organoselenium Compounds ; pharmacology ; Rats ; Rats, Wistar ; Selenium ; pharmacology ; Selenocysteine ; analogs & derivatives ; Sodium Selenite ; pharmacology ; Stem Cells ; cytology ; drug effects ; metabolism ; Tubulin ; metabolism

OBJECTIVETo explore cytological parameters that may identify the primary sites of metastatic adenocarcinomas in serous fluid.

METHODSSerous fluid specimens from 89 cases of metastatic adenocarcinomas (40 metastatic adenocarcinomas of lung, 6 metastatic adenocarcinomas of breast, 21 metastatic ovary adenocarcinomas, 22 metastatic gastrointestinal and pancreatic adenocarcinomas) were studied by using multiple morphologic parameters. Immunocytochemical S-P method was used to detect the expression of CA125, CA199, SPB and TTF-1 in 75 cases.

RESULTSMetastatic adenocarcinomas of different primary sites displayed certain different morphologic features, including the total amount of tumor cells, size of clusters, ratio of clusters over single cells, configuration of tumor clusters and the background of the smear. Cell clusters of small to medium sizes represented 95% and 100% in the metastatic adenocarcinomas of lung and breast, respectively. Most of the ovarian metastatic adenocarcinomas (85.7%) presented some large cell clusters and larger amount of cells, whereas certain metastatic gastrointestinal and pancreatic adenocarcinomas (45.5%) presented smaller number of cells and predominantly to be single cell in distribution (40.9%). Psammoma bodies were found in metastatic adenocarcinomas of lung and ovary. SPB and TTF-1 expression supported the diagnosis of adenocarcinoma of pulmonary origin. CA125 expression supported an ovarian origin. Although CA199 was seen in all groups of metastatic adenocarcinomas, nevertheless, its appearance in tumor cells in ascitic fluid specimens supported gastrointestinal and pancreatic origins.

CONCLUSIONMorpho-logic features of the cytological smear, immunohistochemical staining and clinical history are equally important in determining the primary sites of metastatic adenocarcinomas in serous fluid.

Adenocarcinoma ; metabolism ; secondary ; Ascitic Fluid ; metabolism ; pathology ; Breast Neoplasms ; metabolism ; pathology ; Colonic Neoplasms ; metabolism ; pathology ; Female ; Humans ; Lung Neoplasms ; metabolism ; pathology ; Male ; Nuclear Proteins ; metabolism ; Ovarian Neoplasms ; metabolism ; pathology ; Pancreatic Neoplasms ; metabolism ; pathology ; Pleural Effusion, Malignant ; metabolism ; pathology ; Proteins ; metabolism ; Stomach Neoplasms ; metabolism ; pathology ; Thyroid Nuclear Factor 1 ; Transcription Factors ; metabolism

OBJECTIVETo evaluate the value of cytomorphologic and immunocytochemical approaches in the diagnosis of hematologic neoplasms in serous effusion.

METHODSThe cytospin and Thinprep smears of effusion specimens were prepared from 23 cases of lymphoid malignancies with histological confirmation and 30 cases of benign effusions used as control. Morphological assessment of the cellular components was conducted, including the ratio of mesothelium to lymphocyte, karyomorphism of lymphoid cell and the presence of apoptosis and mitosis. Immunocytochemical study was performed in all the cases, with flow cytometry in one case.

RESULTSAmong the 23 tumor cases, 14 represented disease relapse, and in the remaining nine cases, the serous effusion was the primary manifestation. The proportion of mesothelium was low in the tumor group, being less than 10% in 20 cases (87.0%, 20/23). It was more than 10% in most of benign cases (20/30, 66.7%). Lymphoid cells were prominent (> 80% cells) in 69.6% of the tumor cases, and the cellular component in some control cases (63.3%, 19/30) showed fewer lymphocytes. Nipple-like projection of lymphocytic nuclei could be detected in almost all the tumor cases (91.3%, 21/23), but was occasionally found in the control group (26.7%, 8/30). Apoptosis and mitosis were obvious in lymphomatous effusion, but observed in only 6.7% of the control cases. Significant difference of the previously mentioned cytomorphologic features existed between the tumor and control groups (P < 0.01). The results of immunocytochemical staining in cell block were identical to the corresponding immunohistochemistry, and one case of mantle cell lymphoma was confirmed by flow cytometry. The cytologic findings seen in all the 23 studied cases were in agreement with the corresponding histologic diagnosis.

CONCLUSIONSSome cytomorphologic features, including decreased number of mesothelium, increased number of lymphoid cells, nuclear nipple-like projection, and the presence of apoptosis and mitosis, are very useful for diagnosing lymphoid malignancy in serous effusion. Immunocytochemistry is an important approach to the cytodiagnosis and classification of lymphoma.

Adult ; Aged ; Aged, 80 and over ; Apoptosis ; Ascitic Fluid ; pathology ; Cyclin D1 ; metabolism ; Cytodiagnosis ; methods ; Female ; Humans ; Immunohistochemistry ; Interferon Regulatory Factors ; metabolism ; Lymphocytes ; pathology ; Lymphoma ; complications ; metabolism ; pathology ; Lymphoma, Large B-Cell, Diffuse ; complications ; metabolism ; pathology ; Male ; Middle Aged ; Mitosis ; Pleural Effusion, Malignant ; etiology ; metabolism ; pathology ; Young Adult

OBJECTIVETo explore the diaphragmatic toxicity in doxorubicin (DOX)-treated rats and the related mechanisms, as well as the effects of Shengmai Injection (SMI, ) on the diaphragmatic dysfunction.

METHODSThirty Sprague-Dawley male rats were randomly divided into three groups: control, DOX-treated and DOX+SMI treated groups. DOX was given to rats in DOX and DOX+SMI groups in 6 equal doses [2.5 mg/kg, intraperitoneal injection (i.p.)], on alternate days, over a period of 2 weeks for a cumulative dose of 15 mg/kg. SMI was given to DOX+SMI rats in 12 doses (3 mL/kg, i.p.) for a period of 2 weeks before the administration of DOX and 2 weeks during the administration of DOX. The rats in the control group received equal volume of normal saline. Subsequently, the twitch and tetanic characteristics and force-frequency relationships, and the malondialdehyde (MDA) levels and the superoxide dismutase (SOD) activities, as well as the mRNA content and proteins of inducible nitric oxide synthase (iNOS) were determined.

RESULTSThe DOX-treated rats had decreased the peak twitch tension (Pt), maximal tetanic tension (P0) and force-frequency relationship as compared with the control rats (P<0.01), while the diaphragm contractility in rats treated with SMI were significantly higher than that in DOX-treated rats (P<0.01). The DOX-treated rats had increased MAD levels and decreased SOD activities (P<0.05), and SMI decreased the MDA levels and increased the SOD activities in DOX-treated rats (P<0.05). Ultrastructure of diaphragm in the DOX-treated rats revealed typical alterations including fracture of diaphragm fibers, and edema and degeneration of mitochondria; these changes were relieved by SMI treatment. The mRNA content and protein of iNOS in DOX-treated rats were remarkably higher than those in control rats (P<0.01), while SMI decreased the mRNA expression level of iNOS in DOX-treated rats (P<0.05).

CONCLUSIONSLipid peroxidation is responsible for DOX-induced diaphragm toxicity. SMI protects diaphragm muscles and their function from DOX impairment, and these beneficial effects may be somehow correlated with the decrease in expression of iNOS and lipid peroxidation.

Animals ; Biomechanical Phenomena ; drug effects ; Blotting, Western ; Diaphragm ; drug effects ; pathology ; physiology ; ultrastructure ; Doxorubicin ; adverse effects ; Drugs, Chinese Herbal ; pharmacology ; Gene Expression Regulation ; drug effects ; In Vitro Techniques ; Injections ; Male ; Malondialdehyde ; metabolism ; Muscle Contraction ; drug effects ; Nitric Oxide Synthase Type II ; genetics ; metabolism ; Oxidative Stress ; drug effects ; RNA, Messenger ; genetics ; metabolism ; Rats ; Rats, Sprague-Dawley

AIMTo investigate the effects of calcitonin gene-related peptide (CGRP), gastrin 17 (G17), bombesin (Bom), met-enkephalin (Met-enk), neuropeptide Y (NPY) and somatostatin (SS) on GMBF and the role of endogenous NO in increased GMBF induced by neuropeptides in rats.

METHODSBy hydrogen gas clearance technique to measure gastric mucosal blood flow (GMBF) and arterial infusion close to stomach or intracerebroventricular (icv) to microinject neuropeptides.

RESULTS(1) Arterial infusions of CGRP and G17 (5, 50 and 100 pmol x min(-1)) increased GMBF significantly in dose-dependent manners. CGRP had more effective effect on increasing GMBF than that of G17. Intravenous pretreatment of L-nitro-L-arginine methyl ester (L-NAME) to inhibit the synthesis of endogenous NO could abolish completely or partially the increases in GMBF response to CGRP or G17 respectively. (2) Arterial infusions of Bom and Met-enk (50 and 100 pmol x min(-1)) increased GMBF significantly. The increases in GMBF induced by Bom or Met-enk were abolished completely or partially by pretreatment of L-NAME respectively. (3) Arterial infusion of NPY (5, 50 and 100 pmol x min(-1)) led to reduction of GMBF significantly in a dose-dependent manner. SS (50 and 100 pmol x min(-1)) also reduced GMBF significantly. (4) icv microinjection of CGRP (10 microg) and G17 (10 Microg) increased GMBF significantly. The increases in GMBF induced by icv microinjection of CGRP or G17 were blocked completely or partially respectively by pretreatments with L-NAME. (5) icv microinjection of NPY (10 microg) decreased GMBF significantly.

CONCLUSIONNeuropeptides play important roles in the regulation of GMBF in rats and NO is involved in the increase of GMBF induced by some neuropeptides.

Animals ; Gastric Mucosa ; blood supply ; drug effects ; Male ; Neuropeptides ; pharmacology ; physiology ; Nitric Oxide ; physiology ; Rats ; Rats, Sprague-Dawley

BACKGROUNDHypertension is a complex biological trait that influenced by multiple factors. The encouraging results for hypertension research showed that the linkage analysis can be used to replicate other studies and discover new genetic risk factors. Previous studies linked human chromosome 14 to essential hypertension or blood pressure traits. With a Chinese population, we tried to replicate these findings.

METHODSA linkage scan was performed on chromosome 14 with 14-microsatellite markers with a density of about 10 centi Morgen (cM) in 147 Chinese hypertensive nuclear families. Multipoint non-parametric linkage analysis and exclusion mapping were performed with the GENEHUNTER software, whereas quantitative analysis was performed with the variance component method integrated in the SOLAR package.

RESULTSIn the qualitative analysis, the highest non-parametric linkage score is 1.0 (P = 0.14) at D14S261 in the single point analysis, and no loci achieved non-parametric linkage score more than 1.0 in the multipoint analysis. Maximum-likelihood mapping showed no significant results, either. Subsequently the traditional exclusion criteria of the log-of-the-odds score-2 were adopted, and the chromosome 14 with lambda s > or = 2.4 was excluded. In the quantitative analysis of blood pressure with the SOLAR software, two-point analysis and multipoint analysis suggested no evidence for linkage occurred on chromosome 14 for systolic and diastolic blood pressure.

CONCLUSIONThere was no substantial evidence to support the linkage of chromosome 14 and essential hypertension or blood pressure trait in Chinese hypertensive subjects in this study.

Adult ; Aged ; Asian Continental Ancestry Group ; genetics ; Chromosomes, Human, Pair 14 ; Genetic Linkage ; Humans ; Hypertension ; genetics ; Lod Score ; Middle Aged

Objective To explore the clinical data of the pediatric malignant solid tumor and to summarize the experiences of the pediatric malignant solid tumor treatment in Guangxi.Methods The clinical data of 323 cases of pediatric malinant solid tumor,treated surgically in the First Affiliated Hospital of Guangxi Medical University from Dec.1988 to Dec.2008.Pathology,sex,age,clinical characteristics,diagnosis and treatment were analysed respectively.Results The most frequent histological types were malignant lymphoma,Wilms tumor,neuroblastoma and malignant germ cell tumor.There was a male prevalence with a sex-ratio of 1.99 ∶ 1.The peak incidence in embryo tumor was under 3 years,and epithelium cancer was between the ages of 10-13 years.The main symptoms were palpable mass (56.3) and a few were found in health examination ;64.2％ of the pediatric malignant solid tumor was of stage Ⅲ or Ⅳ ;the complete tumor resection rate was 78.4％.Conclusions The characteristics of pediatric malignant solid tumor is different from those of adults.To detect as soon as possible and treat the pediatric malignant tumor as soon as possible depends on screening.Combined modality therapy can improve the patients complete resection rate and survival quality of the patients.