The innervation and distribution of 5-HT cells in gastric wall of rabbit and rat were studied by means of histochemical techniques of monoamine fluorescence and cholinesterase. And the morphological relation between them was investigated using the consecutive method for demonstrating the fluorescence and ChE on the same section. At the fundic gland, 5-HT cells were in contact with both the adrenergic and cholinergic axonal terminals. There are adrenergic and cholinergic terminals to innervate the gland cells of fundic gland, partial fundic gland the dual axonal terminal are identical in their localization and morphological outline. The loyer of muscle and smooth muscles surrounding the arteriole in various stomach layers were innervated by both adrenergic and cholinergic terminals, some of them are superimposed at the samelocalization. There are a lot of nsChE nerve ending network in gastric lamina propria beneath epithelium of gastric mucous membrane, it was discussed about their sensory nature in this paper.

Fifty two adult male rats were selected for the investigation the adrenergic and cholinergic innervation of rat heart by means of histochemical demonstration of catecholamine fluorescence and acetylcholinesterase (ACHE). Consecutive method was employed on the same section to demonstration the relation between the distribution of the sympathetic and parasympathetic nerves in various parts of rat heart, e. g. atrium, ventricular myocardium, valves, epicardium, endocardium, atrioventricular node and coronary arteries. Adrenergic and cholinergic terminals innervated all parts dually. By comparing the photographs demonstrating the fluorescence CA and AChE on the same section treated by the consecutive method, we found that the location, the density and morphology of both types of nerve terminals were more like. In other words, under light microscopy the localization of both terminals can hardly be distinguished from each other. Such kind of morphological relation may strongly support the results of interaction between sympathetic and parasympathetic nervous systems in physiological and pharmacological experiments of heart.In the cardiac ganglia there are some small intense fluorescence ceils (SIF-cells) lying besides the postganglionic cholinergic cells of the parasympathetic nervous system. Both kinds of cells were shown in close contact with each other in the same section with consecutive method. This morphological relation provided an evidence that catecholamine containing SIF-cells may control and regulate the neurotransmission of parasympathetic cholinergic neurons.

Objective:To construct a fusion protein of extracellular domain peptide fragment of muscle specific kinase ( MuSK) and fluorescent protein mCherry ,and used as antigen in the detection of antibodies against MuSK ( MuSKAb ) in the sera of patients with myasthenia gravis ( MG).Methods:The mCherry gene was amplified by PCR from vector pRSET-B and cloned into pGEM-T Easy Vector,and furthermore, cloned into Eukaryotic expression vector pMT /BiP/V5-His ( MuSK), which contains MuSK extracellular domain 22-452 amino acid peptide fragment gene to construct the fluorescent fusion protein gene MuSK -mCherry.The recombinant vector was subsequently transfected into drosophila S 2 cells for expression.The expressed fusion proteins were verified in confocal mi-croscope ,and used as antigen in the detection of MuSKAb in sera of MG patents in fluorescence immunoprecipitation test .Results:The fluorescent fusion protein MuSK-mCherry was successfully constructed and expressed.The MuSKAb in sera of patents with MG could be detected in fluorescence immunoprecipitation test using the constructed MuSK-mCherry fusion protein as antigen.Conclusion: It is available to use the constructed fluorescent fusion protein MuSK-mCherry as antigen in fluorescence immunoprecipitation test for the detection of MuSKAb in sera of patents with MG.

Objective:To detect the expression of microRNA-218 (miR-218) in human acute lymphocyte leukemia (ALL) T lymphocytes ( CCRF-CEM) ,explore its effects on the biological features of CCRF-CEM cells and the expression of its target gene c-kit, so as to provide new insights for leukemia treatment.Methods: Using the quantitative real-time polymerase chain reaction ( qRT-PCR) ,we detected the expression of miR-218 in the normal peripheral blood T lymphocytes and CCRF-CEM cells.Forty-eight hours after the miR-218 mimic was transfected into the CCRF-CEM cells,the expression of miR-218 in the CCRF-CEM cells was detected by qRT-PCR.The effect of miR-218 on the CCRF-CEM cell viability was detected using MTT.The effect of miR-218 on the proliferation and apoptosis of CCRF-CEM cell was analyzed using flow cytometry.c-kit gene was identified to be a target gene of miR-218 by luciferase reporter enzyme system,and the effect of miR-218 on the expression of KIT protein in cells were determined using Western blot.Results:As shown by qRT-PCR,compared with that in the normal peripheral blood T lymphocytes,the expressions of miR-218 in ALL T lymphocytes cell lines were significantly decreased ( P<0.01 ) .Compared with the control group, the expression of miR-218 increase significantly in CCRF-CEM cells transfected with miR-218 mimic for 48 hours ( P<0.01).MTT showed that the cell viability decreased significantly after the over-expression of miR-218 in the CCRF-CEM cells ( P<0.05 ) .Flow cytometry showed that the S-phase fraction significantly declined after the over-expression of miR-218 ( P<0.01 ) , and meanwhile the apoptosis of cells also significantly increased (P<0.01).Luciferase reporter gene assay showed that,compared with the control group,the relative luciferase activity significantly declined in the miR-218 mimic transfection group (P<0.01).Compared with the control group,the expression of KIT protein in the CCRF-CEM cells transfected with miR-218 mimic for 48 hours significantly decreased ( P<0.01).Conclusion:The expression of miR-218 decreases in ALL T lymphocytes cell lines.MiR-218 can negatively regulate the expression of KIT protein,inhibit the proliferation and increase the apoptosis of CCRF-CEM cells.Treatment based on the enhanced expression of miR-218 may be a promising strategy for leukemia.

Objective To study the estradiolNGF regulatory cascade in retinal endothelial cells. Methods Retinachoroids vascular endothelial cell line RF/6A from rhesus was cultured in M199 medium contain 20% FBS.mRNA of ER,NGF and its receptors,TrkA and P75 NTR were detected with RTPCR.The cells were incubated with NGF,VEGF,antibodies against NGF or VEGF,and K252a(100nmol/L),the specific inhibitor of TrkA,separately or in different combination.MTT based cell counting assay was used to study the viability of the cells.The apoptosis was evaluated by FACS,mass migration by wound healing assay,and tubogenesis by AngioMatrix assay. Results We amplified the specific fragments of cDNA of ER,NGF and NGF receptor TrkA using RTPCR.10?nmol/L1??mol/L estradiol augments the proliferation and increases the viability of RF/6A in a dosage dependent manner.In the wound healing based migration assay,we found the similar alteration.This effects of estradiol was partially blocked by NGF neutralized antibody and K252a.Apoptosis rates were at the similar level among the groups.For the tubogenesis of RF/6A,we found no augmentation by NGF,and no blocked augmentation by estradiol.Conclusion NGF,first identified as the survival factor of nerve system,now also seemed to be an activator of retinal endothelial cell,is under the regulation of estrogen.The proliferation and migration,but not the tubogenesis of retinal endothelial cells are regulated by this regulatory cascade.

Objective:To investigate the association of single nucleotide polymorphisms (SNPs) of receptor-associated protein at the synapse ( rapsyn ) with myasthenia gravis ( MG ).Methods: The genomic DNA was extracted from peripheral blood cells , sampled from 132 patients with MG and 153 control individuals.The 8 exons of rapsyn gene were amplified by PCR ,then the products of PCR sequenced directly.Each sequence was compared with wild-type rapsyn gene , and the association between mutation and clinical symptoms of MG analysed.Results:No mutation was found in the exons 1,2,4,5,6,7,and 8 of rapsyn gene both in MG patients and control group compared with the wild-type rapsyn gene.However,a new SNP,L222R[CTG>CGG(2)] or T665G,was found in exon-3.The allele and genotype frequencies of SNP L 222R met Hardy-Weinberg genetic equilibrium (P>0.05),indicating the group repre-sentativeness.The allele frequencies of G were not statistically different between patient and control groups ( P>0.05 ).There were differences in the 3 genotypes TT , TG and GG between patient ( 42.4% vs 48.5% vs 9.1%) and control ( 49.0% vs 33.3% vs 17.6%) groups ( P<0.05 ).The genotype frequencies of GG were statistically higher in control group than that in patient group , showing a recessive model of inheritance.Conclusion: The SNPs in the rapsyn gene are associated with MG in this study.L222R ( T665 G) is a new SNP found and allele G might be a protective factor for MG.

Objective To assess the feasibility of quantitative measurement of subcutaneous and supraclavicular adipose tissue with iterative decomposition of water and fat with echo asymmetry and least square estimation-iron quantification sequence (IDEAL-IQ).Methods Totally 87 normal young female volunteers (20-35 years old) were recruited and divided into body mass index (BMI)＜24 kg/m2 group (n=72) and BMI≥24 kg/m2 group (n=15).Fat fraction (FF) and T2* relaxation rate (R2*) of supraclavicular adipose tissue,chest wall adipose tissue,abdominal wall adipose tissue and liver were measured,respectively.The differences of FF and R2* value of chest wall,subcutaneous and supraclavicular adipose tissue were compared between two groups,and the correlation between FF,R2* value of supraclavicular adipose tissue and BMI was respectively analyzed.Results FF of supraclavicular adipose tissue ([80.99 ± 7.73]％) was lower than that of chest wall subcutaneous adipose tissue ([93.04 ± 1.55] ％,P＜0.001).R2* of supraclavicular adipose tissue ([65.52±23.59]Hz) was higher than that of chest wall subcutaneous adipose tissue ([38.82±7.11]Hz,P＜0.001).The differences of FF and R2* values of supraclavicular adipose tissue,chest wall,abdominal wall subcutaneous adipose tissue and liver were significant between BMI＜24 kg/m2 group and BMI≥24 kg/m2 group (all P＜0.05).There was positive correlation (r=0.601,P＜0.001) between FF of supraclavicular adipose tissue and BMI and negative correlation (r=-0.409,P =0.001) between R2* of supraclavicular adipose tissue and BMI.Conclusion IDEAL-IQ technique can quantitatively assess the difference between subcutaneous and supraclavicular adipose tissue in healthy young women.FF and R2 * value has significant difference between the above mentioned positions.

Objective To observe the influence of the staining phenomenon after fluorescein sodium staining on eye irritation in normal rabbits.MethodsIn the experimental rabbit eye irritation test conducted with sodium chloride eye drops, Siwei Zhenceng Bingpeng eye drops, sodium hyaluronate eye drops, sodium cromoglycate eye drops, and compound aspartate eye drops (4 in each group, half male and half female), the left eyes of rabbits were administered normal saline (self-negative control) and the right eyes were administered the experimental medicine; the eyes were stained with 1% sodium fluorescein, and eye irritation was observed and scored using slit lamp microscope for 31 days. Morphological changes of corneal epithelial staining were recorded and the incidence of staining was calculated. After the observation, the eyeballs and Hasselblad glands were examined histopathologically, and the staining rate of the left eye was compared with that of the right eye which was administered the corresponding medicine.ResultsNeither eye had any irritation symptoms; the scores were 0, and the total incidences of corneal staining were 3% (left) and 1% (right), respectively. There was no significant difference between the two groups (P > 0.05). Corneal epithelial staining showed single-spot staining, scattered dot, localized, or large areas of fusion staining. No histopathological changes were found in the eyeballs or Hasselblad glands, and the results were evaluated as non-irritative.Conclusion The irregularity of corneal epithelial staining in rabbits did not influence the results of the ocular irritation test.