Background How to harvest the purified corneal endothelial seed cells is very important for the corneal tissue engineering technology. Herein,to establish a good culture method and effective identification method of corneal endothelial cells ( CECs) is the key. Objective Present study wag to establish the cultivating and identifying approach of the rabbit CECs and detect the biological characteristics of passaged cells. Methods Rabbits CECs were isolated from Descemet's membrane peeled off completely in 30 New Zealand white rabbits and then digested with 0. 25% trypsin-0. 02% EDTA and primarily cultured in CECs medium containing 15% fetal bovine serum. The growth situate and cellular morphology of rabbit CECs were observed under the inverted phase-contrast microscope following the alizarin red staining. Rabbit CECs were identified by cellular morphology as well as in gene and protein level, including the detection of expressions of collagen type IV α2(COL4A2) ,vascular endothelial growth factor receptor 2 ( FLK1 ) , Na+-K+ ATPase alpha 1subunit ( ATP1 A1 ) , aquaporin 1( AQP1 ) , voltage-dependent anion channels ( VDACs) by reverse transcription-polymerase chain reaction ( RT-PCR ). The expression and distribution of neurone specific enolase ( NSE) , Na+-K+ ATPase, zonula occludens-1 ( ZO-1 ) were also detected by immunocytochemistry under the fluorescence microscope. The proliferation activity of the passage cells was dynamically observed by MTT assay,and Na+-K+ ATPase activity of different generations cells was detected by ATPase kit. Results Majority of the primarily cultured cells adhered in 24 hours, infused in 2-3 days with the hexagon shape in appearance. The morphology of the cells was very varied with passage. Alizarin red staining showed a well- defined and well-lined cellular morphology similar to the corneal cells in vivo. Target genes of C0L4A2, FLK1, ATPIAI ,AQPI and VDACs were positively expressed in the cells. However,the expression of CK12 was absent in the cells. NSE, Na+-K+ ATPase, ZO-1 positive cells were respectively observed under the laser confocal scanning microscopy. MTT results showed a gradually low growth curve with the passage. Quantitative results also revealed that the Na+-K+ ATPase activity was gradually declined in different generations of cells ( F = 77. 174, P = 0. 000 ). Conclusion The enzyme digestion is a better approach of isolating and culturing comeal endothelial cells. Cultured cells can be identified by cells staining, gene expression and protein level. Earlier generation of CECs are ideal seed cells for cornea tissue engineering.

To analyze the specific mechanism of Jinxueyuan granules, the relationship between the Jinxueyuan granules increased the saliva secretion of xerostomia model SD rats and excitement of receptors were studied in this experiment. In the study, three groups of xerostomia model rats were successfully established by using M-receptor blockers-4-diphenyl-acetoxy-N-methyl-piperidine (4-DAMP) and atropine, or adrenergic receptor blocker phentolamine; after the modeling, the medicine Jinxueyuan granules were gavaged. According to the clinical dose of Jinxueyuan granules and SD rats body surface area, the rats in atropine group were divided three dose groups respectively, namely low, medium and high dose of Jinxueyuan granules groups. The 4-DAMP group and phentolamine group were gavaged medium dose of Jinxueyuan granules. And the amount of salivary secretion for 150 minutes in all groups continuously were measured, and the effect of Jinxueyuan granules increased salivation and the relationship between characteristics and the receptors were observed; and submandibular gland tissue of the rats was isolated, then the effect of Jinxueyuan Granules for expression of the water channel protein aquaporin-5 (AQP5) in submandibular gland cells was analyzed by the Western blot technology. It was found that the saliva secretion of Jinxueyuan Granules groups was increased significantly, and compared with the saline control group, phentolamine group, 4-DAMP group and atropine group, difference was significant, P < 0.05. There was no significant difference between the low-dose of Jinxueyuan granules group and the saline group, but the medium dose of Jinxueyuan granules group had a significant difference, compared with the saline group (P < 0.05). In the time distribution of increasing saliva secretion, there was a significant difference between the saline and Jinxueyuan granules group in the saliva secretion (P < 0.05). After administration of Jinxueyuan granules, the expression of AQP5 protein in the submandibular gland cells expressing of treatment groups was increased, and compared with the blocker groups, there was a significant difference, P < 0.05. Except the atropine group, there was no significant difference in Jinxueyuan granules relieving the inhibition induced by blocks in phentolamine group and 4-DAMP group, compared with the saline group. Compared the AQP5 expression in three blockers groups, there was no significant difference in the efficacy of Jinxueyuan granules between phentolamine group and 4-DAMP group; but there was a significant difference between the atropine group and other groups (P < 0.05). Therefore, it was considered that the mechanism of Jinxueyuan granules increasing saliva secretion (effectiveness of nourishing Yin and generating body fluid ) possibly through the pathway mediated by muscarinic M receptor, especially M3 receptor, or adrenergic receptor, and increased expression of salivary gland AQP5 membrane, and then stimulate saliva production.
Animals ; Disease Models, Animal ; Drugs, Chinese Herbal ; administration & dosage ; Humans ; Male ; Rats ; Saliva ; secretion ; Salivary Glands ; drug effects ; secretion ; Xerostomia ; drug therapy ; metabolism

Acupuncture Points ; Acupuncture Therapy ; Adult ; Combined Modality Therapy ; Female ; Humans ; Intervertebral Disc Displacement ; therapy ; Male ; Massage ; Middle Aged ; Young Adult

Objective To investigate infection situation and knowledge of high‐risk human papilloma virus(HPV)among women working in entertainment places in Gaoqiao area ,and provide theory evidences for making health education measures related to high‐risk HPV .Methods According to the systematic random sampling method ,entertainment female in Gaoqiao area were extracted , and normal women who participated in community gynecology census were extracted as control group .Then questionnaire investiga‐tion and laboratory testing were carried out .Results The infection rate of high‐risk HPV in entertainment female was 35 .18% , while in normal woman was 15 .84% ,there was significant differences of infection rate between the two groups (P<0 .05) .There were were only 16 women(14 .81% )and 17 women(16 .83% )had heard of information about HPV in two groups respectively ,and there was no statistically significant difference(P>0 .05) .The cognitive situation of whether a person with family history of cervi‐cal cancer should be regularly attend screening among the two groups of women was different ,and had significant difference(P<0 .05) .Conclusion Women who worked in entertainment places had higher infection rate of high‐risk HPV ,both of the two groups had poor knowledge of HPV .So the propaganda and screening of HPV should be strengthen in order to raise consciousness in pre‐venting cervical cancer .

Objective In order to provide the basis for establishment of food allergy models , we compared the differences of sensitivity and alterations of intestinal flora of food allergy models in two strains of mice .Methods Forty 4-5-week old female BALB/c and Kunmimg mice were divided into control group ( n=10) and food allergy goup ( n=30), respectively.Ovalbumin (OVA) was injected to the mice to establish food allergy models .Serum OVA-specific IgE of the mice was assayed by ELISA .The jejunum tissue was examined by pathology with HE staining .The changes of fecal flora were detected by denaturing gradient gel electrophoresis (DGGE).Results (1)Among the sensitized 60 mice, OVA-sIgE levels were significantly increased in 27/30 BALB/c mice and 21/30 KM mice compared with those of control groups(P<0.001).Moreover, there were more evident inflammatory cell infiltration , epithelial cell shedding and cytolysis in the jejunal villi of BALB/c mice than those of KM mice.(2) After food allergy modeling, there were significant changes of intestinal flora in the BALB/c mice (P<0.001), while only significant change of evenness was found in the KM mice (P<0.05).(3)There were changes of abundance , Shannon index and evenness of intestinal flora in the model groups of BALB/c and KM mice.Conclusions BALB/c mice are more sensitive to OVA allergy than KM mice .The composition of intestinal flora is different among different strains of mice .The changes of intestinal flora after OVA challenge in BALB /c mice are more obvious than those in KM mice .

Acupuncture Therapy ; Adult ; Cervical Vertebrae ; injuries ; Combined Modality Therapy ; Female ; Humans ; Male ; Massage ; Middle Aged ; Pharyngitis ; etiology ; therapy ; Spinal Diseases ; complications ; Young Adult

Background Cyclosporine A (CsA) is an effective drug to prevent rejection response after penetrating keratoplasty (PKP).Appropriate dosage forms and right administrating route is very important for improving the bioavailability of CsA.Objective This study was to investigate the preventing effect of CsA microspheres via subconjunctive and anterior chamber injection and CsA eye drops on immune rejection after PKP.Methods Sixty eyes of 60 clean adult New Zealand white rabbits served as receipts,and 60 eyes of 30 clean adult pigmented rabbits were used as the donors.The receipts were randomized into the blank control group (only PKP group),subconjunctival CsA injection group,subconjunctival vector injection group,anterior chamber CsA injection group,anterior chamber vector injection group,and 1％ CsA eye drops group.PKP was performed on the all rabbits,and then the CsA microsphere (0.1 ml,12 g/L) or blank microsphere (0.1 ml) were respectively administered in the corresponding groups.The corneal grafts were examined by slit lamp microscope regularly,and rejection index (RI)was calculated based on the corneal opacity,edema and neovascularization.The intraocular pressure (IOP) of operative eyes was measured by Tono-pen tonometer before operation,3 days,1 week,2 weeks,3 weeks,1 month,2 months and 3 months after operation,respectively.Histopathological examination on corneal grafts was performed 1month and 3 months after operation.Results The IOP of all the rabbits lowed after operation,but there was no statistical difference in different time points and various groups (Ftime =29.210,P =0.000; Fgroup =0.254,P =0.938).The grafts of the blank control group,subconjunctival vector injection group and the anterior chamber vector injection group showed varied degrees of corneal opacity and neovascularization 2-3 weeks after operation,and the degree of graft opacity was aggravated obviously in the fourth week,with the RI 8.60±1.52,8.60±0.55 and 8.80±0.84 individually.Neovascularization of the grafts in the subconjunctival CsA injection group,anterior chamber CsA injection group,and 1％ CsA eye drops group was found 3 weeks after operation,and the RI were 4.40±0.89,3.20±0.84 and 3.00±0.71,showing a significantly lower than that of the control groups (P＜0.05).Mild inflammatory response of the grafts was seen in the anterior chamber CsA injection group,but it was lightened over time.The histopathological examination revealed obvious thickening of corneal grafts,neovascularization and infiltration of lots of inflammatory cells in the blank control group,subconjunctival vector injection group and the anterior chamber vector injection group.However,only slight new blood vessels and inflammatory response were seen on the subconjunctival CsA injection group,anterior chamber CsA injection group,and 1％ CsA eye drops group.Conclusions Administration of CsA in different methods can prevent the immune rejection after PKP in rabbit eyes,the effect of giving drugs via anterior chamber injection is better than that via subconjunctive pathyway.

BACKGROUNDCyclic adenosine monophosphate (cAMP) could activate chloride channels in bovine ciliary body and trigger an increase in the ionic current (short-circuit current, Isc) across the ciliary processes in pigs. The purpose of this study was to investigate how cAMP modulates Isc in isolated human ciliary processes and the possible involvement of chloride transport across the tissue in cAMP-induced Isc change.

METHODSIn an Ussing-type chamber system, the Isc changes induced by the cAMP analogue 8-bromo-cAMP and an adenylyl cyclase activator forskolin in isolated human ciliary processes were assessed. The involvement of Cl(-) component in the bath solution was investigated. The effect of Cl(-) channel (10 µmol/L niflumic acid and 1 mmol/L 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS)), K(+) channel (10 mmol/L tetraethylammonium chloride (TEA)), or Na(+) channel blockers (1 mmol/L amiloride) on 8-bromo-cAMP-induced Isc change was also studied.

RESULTSDose-dependently, 8-bromo-cAMP (10 nmol/L-30 µmol/L) or forskolin (10 nmol/L-3 µmol/L) increased Isc across the ciliary processes with an increase in negative potential difference on the non-pigmented epithelium (NPE) side of the tissue. Isc increase induced by 8-bromo-cAMP was more pronounced when the drug was applied on the NPE side than on the pigmented epithelium side. When the tissue was bathed in low Cl(-) solutions, the Isc increase was significantly inhibited. Finally, niflumic acid and DIDS, but not TEA or amiloride, significantly prevented the Isc increase induced by 8-bromo-cAMP.

CONCLUSIONScAMP stimulates stroma-to-aqueous anionic transport in isolated human ciliary processes. Chloride is likely to be among the ions, the transportation of which across the tissue is triggered by cAMP, suggesting the potential role of cAMP in the process of aqueous humor formation in human eyes.

8-Bromo Cyclic Adenosine Monophosphate ; pharmacology ; Chloride Channels ; antagonists & inhibitors ; Ciliary Body ; drug effects ; physiology ; Colforsin ; pharmacology ; Cyclic AMP ; physiology ; Humans ; In Vitro Techniques ; Potassium Channel Blockers ; pharmacology ; Sodium Channel Blockers ; pharmacology

Objective　 To investigated the possible roles of IL-18 in HBV infection, and the expression of IL-18 in the peripheral blood mononuclear cells (PBMC) of chronic hepatitis B. Methods 　In 15 cases of asymptomatic carriers, 30 cases of active and remissive phases of chronic hepatitis and 10 healthy individuals (as normal controls), IL-18 expression in PBMC were quantitatively analyzed with flow cytometric immunological method. The PBMC were separated routinely and stimulated with LPS (SIGMA) and Monensin (SIGMA) for 6 hours. Then the cells were harvested and fixed by PBS/4% paraformaldehyde. The treated cells were stored in liquid nitrogen for detection later. After immunologic stain, the expression of IL-18 in PBMC were examined by flow cytometry. Results　 ① IL-18 was the lowest in asymptomatic carriers. It was fewer in remissive phase than in normal controls (P＜0.01).There was no difference between active phase and normal controls(P=0.25).②Within the group of active phase, IL-18 was significantly different between each grade of inflammatory activity(P＜0.01),and correlated with serum ALT positively(r=0.63,P＜0.01). Conclusion　 IL-18 may relate to disease activity and the inflammation of liver.

Objective To explore the clinical value of renal dynamic imaging and urinary N-acetyl-?-D-glucosaminidase(NAG),apoptosis DNA fragment(ADF) in evaluating the damage degree of hydronephrotic kidneys(HnK) in children with hydronephrosis.Methods Level of glomerular filtration rate(GFR) was detected in 41 children with congenital hydronephrosis by renal dynamic imaging,and urine NAG,ADF in pelvis in HnK and healthy kidneys (as controls) were detected by enzyme-linked immuno-sorbent assay(ELISA).Patholo-gic changes of HnK in 41 children were graded intoⅠ~Ⅴ according to Elder standard. And GFR,urinary NAG and ADF of HnK were divi-ded into subgroup according to pathologic changes ,at the same time statistical analysis was performed within each groups. And the correlations of pathologic grades with GFR,urinary NAG and ADF of HnK were analyzed.Results 1.Kindneys GFR in healthy kidneys and Hnk were (174.33?20.43)?10-3 L/min,(143.86?17.51)?10-3 L/min respectinely,and there was significant difference between healthy kidneys and Hnk (P0.05).3.There was significant negative correlation between GFR levels of HnK and pathologic grades(r=-0.814 P0.05).Conclusions For hydronephrotic kidneys,urinary NAG can eva-luate impaired nephric tubule whereas renal dynamic imaging may evaluate the damage level of glomeruli;urine ADF may not indicate the damage level of diseased kidneys in children with congenital hydronephrosis.