Objective To establish a purified model of rat retinal ganglion cells (RGCs) cultured by serum-free medium,and provide a good cell model to investigate the damage of RGCs in glaucoma,retinal ischemia,and degenerative retinopathy. Methods Two monoclonal antibodies,anti-rat SIRP (OX-41) against rat macrophage and antibody against rat Thy-1 (OX-7),were used to purify and characterize RGCs from 1-3-day old Sprague-Dawley (SD) rats by means of two-step filtration.Purified RGCs were cultured in serum-free neurobasal medium containing B27 and ciliary neurotrophic factor (CNTF) meeting the neuronal cell's special requirements.Photomicrographs illustration,immunfluorescence staining of Thy-1,calcein-acetoxymethyl ester (calcein-AM) fluorescence images were used to observe and identify cultured retinal cells and purified RGCs. Results Among the primary cultured rat retinal cells,91% were retinal neurons.Protuberances of RGCs were seen after cultured for 24 hours.At the4th to 8th day,many cells had uniform configuration,large body,and long protuberances. At the 14th day,over 60% cells maintained viability.Immunoflurescence staining of Thy-1 showed the purity of RGCs was about 90%. The results of calcein-AM staining,which stained the living cells only,showed large cell body of RGCs and most of RGCs had a protuberance whose length was twice longer than the diameter of the cells. Conclusion RGCs cultured by serum-free medium has uniform size,good configuration,and high purity,which is adapt to the research of damage of RGCs caused by various factors and to evaluate the protective effects of neuroprotective agents.

Objective To investigate the intestinal absorption kinetics of breviscapine in rats.MethodsThe intestinal absorption in small intestine and colon of rats in situ was investigated using circular perfusion and HPLC methods.The in situ absorption of scutellarin in the small intestine was studied and the effects on the intestinal absorption were observed at the various concentrations and different pH values using the same methods.Results The results showed that there were significant differences in both absorptive percentage and absorption rate constant(ka)in the small intestine between the experimental groups of rats with and without bile duct being ligated.The absorption rate constants in small intestine and colon were(0.107 1?0.013 0)and(0.070 7?0.008 9)h-1,respectively.No saturation phenomenon occurred and ka was almost kept unchanged at different concentrations of breviscapine.The absorption of breviscapine was not influenced by pH values ranging from 6.0 to 7.4.Conclusion The results indicates that breviscapine is absorbed more in small intestine than in colon.The absorption of breviscapine complied with the passive diffusion mechanism and first order kinetics.In conclusion,these results suggest that breviscapine could be prepared in oral sustained-release dosage form.

0.05).But the P_ eff values were significantly increased in the presence of P-glycoportein(P-gp)inhibitor,verapamil or digoxin.CONCLUSION:Rhamnosylvitexin can be classified into high penetrating drug.Passive diffusion dominates the absorptive transport behavior of rhamnosylvitexin in HLF.There is not a preferential absorption zone in the intestine for rhamnosylvitexin.The absorption and secretion of rhamnosylvitexin in HLF are mediated by the efflux transport system,P-gp.

Objective To establish the normal data of the fetal thorax,and to evaluate its values in the diagnosis of fetal thorax malformation.Methods Totally 398 normal singleton fetuses at 16 to 36 gestational weeks(GW) were enrolled,2D-US and 3D-US VOCAL technique were used to measure the 2D data and 3D volumes on the transverse section at the level of the four-chamber view,and the correlation among all measurements with GW was analyzed.Thirty fetuses collected randomly were examined to analyze the reliability.Nine fetuses with congenital thoracic dysplasia (CTD) and 10 fetuses with congenital diaphragmatic hernia (CDH) were assessed and compared with the normal fetuses.Results ① In healthy controls,the fetal thoracic 2D measurements and 3D volumes increased along with the growth of the GW.The regression equations were listed as follows:thoracic transverse diameter (cm) =-0.002 GW2 + 0.301 GW-1.510;thoracic anteroposterior diameter (cm) =0.003GW2 + 0.046GW + 0.666;thoracic area (cm2) =0.071GW2-1.466 GW + 14.728;thoracic circumference (cm) =0.01GW2 + 0.313GW + 3.341;thoracic volume (cm3) =0.285 GW2-7.797GW + 66.592;lung volume (cm3) =0.178 GW2-5.317GW + 45.539;the ratio of lung volume to thoracic volume =0.005GW + 0.396.② The reliabilities of the data obtained by the same/two different operators were good.③ CTD group was obviously lower than the healthy controls in all thoracic measurements (all P ＜0.01).There was no statistical difference in the 2D data between the CDH group and healthy controls (P ＞0.05),while the 3D volumes and the ratio of lung volume to thoracic volume were obviously lower than those in the healthy controls (P ＜0.01).Conclusions 2D-US can evaluate the fetal thoracic development and malformation preliminarily,but 3D-US VOCAL technology plays an important role in distinguishing different types of thoracic malformations.

Objective To explore the change of function and expression of hematopoietic lineage cell specific protein-1 (HS1) and phosphorylated HS1 (p-HIS) and factors devoting to HS1 phosphorylation in platelet with sepsis.Methods Plasma with rich platelet was collected from 150 sepsis patients and 50 healthy subjects, and comparison of platelets adhesion and aggregation were detected by micro-pore method and platelet aggregation instrument.Meanwhile the ATP concentrations of washed platelet of two groups were detected by the kit to compare release reaction.And then total HS1 (t-HIS) and p-HS1 of platelet from two groups were compared by using western blot.Afterwards the specific inhibitors of Src and Syk were used to verify the HS1 activation regulated by Src and Syk in LPS-induced cell model.Results The significant differences were present between healthy subjects and sepsis patients in platelet counts, platelet distribution width (PDW) and mean platelet volume (MPV) (P ＜ 0.01).The data showed the sepsis patients had greater ability than healthy subjects in adhesion, aggregation and release reaction.Meanwhile the platelets of sepsis patients had higher concentration of t-HS1 and p-HS1 than healthy subjects, and the specific inhibitors of Src and Syk , PP2 and piceatannol, inhibited the increase in p-HS1 in LPS-induced cell model.Conclusions Function of platelet is closely related to HS1 in sepsis and it will be a target for sepsis therapy.

Objective To establish normative data for the fetal cisterna magna septa (CMS) at various gestational age,and to evaluate its clinical significance.Methods A total of consecutive fetal between 14 and 40 gestational week(GW) were included in this prospective study.The length and width of CMS were measured by two-dimensional ultrasonography.Regression analysis was used to study the relationship between the width and length of the fetal cisterna magna septa and gestational age.Twenty-five case of fetuses with the absence of CMS and 12 case of fetuses with the enlargement of CMS were retrospectively analyzed in the past six years in our hospital.Results ①The fetal CMS length and width increased gradually between 14 and 22 GW,then plateaued between 23 GW and 36 GW,and decreased after 37 GW.This ultrasonographic pattern was in agreement with normal development of rhombencephalon.②The absence of CMS in the fetuses were common in Dandy-Walker syndrome,holoprosencephaly,severe hydrocephalus,neural tube defects,rhombencephalon synapsis and Arnold-Chiari malformation.The enlargement of CMS in the fetuses may be shown in physiologic enlargement of posterior fossa.ConclusionsCMS is a potential new marker for normal development of rhombencephalon.The enlargement and absence of CMS are related to various malformations of central neural system,especially in the abnormalities of posterior fossa.

Objective To examine loss of heterozygosity (LOH) and microsatellite instability (MSI) of locus D8S532 on chromosome 8 and their influence on the expression of sFRP1 in the hepatocellular carcinoma (HCCs), which may provide an experimental evidence for clarifying the mechanism of sFRP1 gene and tumor development. Methods DNA was extracted from formalin-fixed paraffin-embedded tissues. Polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) and ordinary silver stain were used to study LOH and MSI of locus D8S532. Envision immunohistochemistry, Leica-Qwin computerized imaging system and Image-Pro PluS (IPP) version 4.5 professional imaging analysis software were used to assess the expression of sFRP1. Results The detection rates of LOH and MSI of locus D8S532 in the 36 specimens of HCC were 11.11% and 8.33% respectively. The down-regulation of sFRP1 was observed in 31 of 36 HCCs (86.11%) compared with non-carcinoma liver tissues, and the positive rate of sFRP1 protein of the HCCs was 52.78%( 19/36 ). The frequency of LOH was lower in the cases with positive expression of sFRP1 protein than those negative (0 vs 23.53%, P <0.05). Conclusion It was a common phenomenon that expression of sFRP1 protein is negative or low in Chinese with HCCs. The genetic instability of sFRP1 gene was one of causes, which lead to HCCs. LOH may play a major role in negative expression of sFRP1.

Objective To investigate the effect and mechanism of cell differentiation agent Ⅱ (CDA-Ⅱ) on the differentiation of human acute mycloid leukemia (APL) HL-60 cells.Methods The cell morphology and differentiation was detected by Wright-Giemsa staining,the expression of cell surface differentiation antigen CD11b of HL-60 was detected by flow cytometry,the differentially expressed genes were screened by gene expression profile chip (NimbleGen).Results The result of Wright-Giemsa staining showed that CDA-Ⅱ induced HL-60 differentiation towards mature stages in a time-dependent manner.After treated with CDA-Ⅱ,the percentage of CD11b-positive HL-60 cells significantly increased in a time-and dose-dependent manner.The result of gene expression profiling indicated differentially expressed genes including 113 up-regulation genes and 140 down-regulation genes.The up-regulation expression genes involved in six pathways including mineral absorption,complement and coagulation cascades,down-regulation expression genes involved in nine pathways including pyrimidine metabolism,RNA polymerase,purine metabolism and so on.Conclusion CDA-Ⅱ can induce HL-60 differentiation and make gene differentially expressed.The data provide the feasibility of CDA-Ⅱ in differentiation induction therapy for APL.

AIM:To study testicular pathological change and its pathogenesis. METHODS:Testicular structure of alloxan induced diabetic rats was observed under light microscopy (LM)and transmission electron microscopy(TEM). Activity of superoxide dismutase(SOD),glutathione peroxidase(GSH-PX)，nitric oxide synthase(NOS)and content of malondialdehyde (MDA),nitric oxide(NO) were detected biochemically in testicular homogenate. RESULTS:It is manifestated as atrophy of seminiferous tubule and germinal arrest under LM and expansion of mitochondria and smooth endoplasmic reticulum, cytoplasmic inclusion of sertoli cell under TEM.Activity of SOD, GSH-PX decreased while activity of NOS, content of MDA, NO increased in diabetic rats compared with control one. CONCLUSION:Disturbance of spermatogenesis and damage of sertoli cell are the main morphological change of diabetic testis, lipid peroxidation and NO may be involved in it.

Objective To construct and identify an eukaryotic expression plasmid containing rat hepatocyte growth factor(rHGF)gene and rat augmenter of liver regeneration(rALR)gene,so that to provide experimental basis for developing new treatments of hepatic fibrosis.Methods The gene fragments of rHGF and rALR were amplified from recombinant prokaryotic plasmid pUC18-rHGF and pUC18-rALR by polymerase chain reaction(PCR),respectively,then were spliced by overlap extension PCR with a linker,and the fusion gene rHGF-linker-rALR was constructed.The fusion gene was directionally inserted into the eukaryotic expression plasmid pcDNA3.1 between restriction sites of Kpn Ⅰ and Xba Ⅰ to construct the recombinant eukaryotic expression plasmid pcDNA3.1-rHGF-linker-rALR,and the new constructed recombinant plasmid was identified by double restriction digestion and DNA sequencing.Results DNA fragments of 2200 bp and 400 bp were observed after the electrophoresis of products amplified from recombinant prokaryotic plasmid pUC18-rHGF and pUC18-rALR,respectively,which was consistent with the theoretical value.The electrophoresis of fusion gene rHGF-linker-rALR obtained by overlap extension PCR technique showed only a 2 600 bp DNA fragment,which was in accordance with the expected value.Electrophoresis of products of pcDNA3.1-rHGF-linker-rALR digested with Kpn Ⅰ and Xba Ⅰ showed two DNA fragments with 2600 bp and 5400 bp,which were both consistent with the expected value.The sequences were confirmed correctly by DNA sequencing.Conclusion The recombinant eukaryotic expression plasmid pcDNA3.1-rHGF-linker-rALR is successfully constructed,which provides experimental basis for developing gene therapy of hepatic fibrosis.