Objective To study the role of glucagon-like peptide-1 (GLP-1) analogue liraglutide played in the proliferation of CD4+CD25 T cells in normal people and newly-onset type 1 diabetic patients,and to evaluate the possible immune regulatory role of liraglutide in the therapy of type 1 diabetes.Methods CD4+ CD25-T cells of 10 normal people and 10 newly-onset type 1 diabetic patients were separated from peripheral blood by MACS immunomagnetic beads and stimulated by Human T-Activator CD3/CD28 Dynabeads to proliferate.CFSE labeling technique was used to evaluate the proliferation of CD4+ CD25-T cells by flow cytometry.Liraglutide of different concentrations(0,25,50,and 100 nmol/ml) was added to the proliferation system,then the proliferation of CD4+CD25-T cell was measured.Results (1) Liraglutide suppressed the proliferation of CD4+ CD25-T cells from either normal people or type 1 diahetic patients with dose-dependent manner (P ＜ 0.05).(2) Under the different concentrationsofliraglutide,the proliferation ofCD4+CD25 T cells from diabetic patients was mueh more robust than that of normal people (P＜0.01).(3) The inhibitory effects of liraglutide on CD4+ CD25-T cells proliferation in normal people and diabetic patients were similar (P＞0.05).Conclusion The proliferation of CD4+ CD25 T cells in type 1 diabetic patients was more robust than normal people,which indicated cellular immune dysfunction in type 1diabetes.Liraglutide inhibits the proliferation of CD4+ CD25-T cells of type 1 diabetic patients in vitro.The immunosuppression effect of liraglutide may have potential value in the treatment of type 1 diabetes.

Background The construction of tissue-engineered corneal endothelium needs the functional seeding cells,so how to culture a large amount of functional corneal endothelial cells (CECs) is an urgent problem to be solved.Objective The aim of this study was to evaluate the role of aqueous humor on bovine CECs in vitro.Methods Aqueous humor of 1.2 ml was collected from the anterior chamber of bovine and sterilized,and the liquid supernatant was obtained.The bovine CECs were isolated from bovine cornea and then cultured in low glucose Dulbecco Modified Eagle Medium with 10％ fetal bovine serum (FBS) in vitro.Aqueous humor was added into the medium with the final concentration of 2.5％,5.0％,l0.0％,15.0％ and 20.0％,respectively,and no aqueous humor was added in the control group.Cell counting kit-8 (CCK-8) assay was used to detect the absorbency value of CECs for the evaluation of cell proliferation.Progression of the cell cycle was analyzed by flow cytometry (FCM).After confluence of the cells was reached,1 ml plastic spear tip was used to scratch the cell single layer,and the cells were incubated consequently in medium with 10％ FBS and with or without aqueous humor for 24 hours.Healing area of the cell single layer was measured.The cells were incubated at a density of 6 × 105 cells/ml and cultured using medium with or without 10.0％ aqueous human for 5 days,and the number of the cells was analyzed by DAPI fluorescence technique.Results Under the phase-contrast microscopy,the confluent CECs showed a slabstone-like and hexagonal appearance.CCK-8 assay revealed that the absorbance values of CECs was significantly different among the various culture groups (F=4.051,P =0.007),and the absorbance value in different concentrations of aqueous human culture groups was significantly higher than that in the control group (P ＜ 0.01).FCM showed that the percentage of the cells in S-G2 phases was (34.80-±3.13)％ in the 10.0％ aqueous humors group and (23.06±1.13)％ in the control group,showing a significant difference (t =-5.729,P=0.005).The scratch test showed that the healing area of the cell signal layer was (0.116±0.019) mm2 in the 10.0％ aqueous humors group and (0.358 ±0.049) mm2 in the control group,showing a significant difference (t =13.842,P =0.000).The density of cells in the 10.0％ aqueous humor group was (1439± 1 10)/field,which was more than (1162±45)/field in the control group (t =-11.020,P=0.000).Conclusions Aqueous humor at the concentration of 10.0％ promote the growth and proliferation of bovine CECs.The result suggests that 10.0％ aqueous humor can be used as a promoting agent during the culture of CECs.

Objective To study the expression and pathologic significance of CD147,cyclophilin A (CyPA)and cyclophilin B(CyPB)in psoriatic lesions.Methods Immunohistochemical method was ap- plied to detect the expression of CD147,CyPA and CyPB in skin specimens of 15 patients with psoriasis pustulosa(PP),20 patients with progressive psoriasis vulgaris(PPV),and 20 patients with inactive psori- asis vulgaris(IPV).Immunoreactivity intensity distribution index(IRIDI)was calculated to assess the expres- sion intensity of CD147,CyPA and CyPB.Results CD147,CyPA and CyPB were detected in all speci- mens.The IRIDI scores of CD147 and CyPA were significantly higher in keratinocytes of psoriatic lesions than in those of the control specimens(all P0.05).The IRIDI score of CyPB in T lymphocytes of PP lesions was significantly elevated than that in PPV lesions,which was in turn higher than that in IPV lesions(all P0.05).Conclusion CD147,CyPA and CyPB may play a role in the occurrence and development of psoriasis.

0.05);and there were significant diffe-rences between the final diagnosis and pre-hospitalized diagnosis in all patients with VE(?2=47.08 P

Background Clinical studies indicated that the pathogenesis of most corneal dystrophy is associated with the mutation of the transforming growth factor beta-induced (TGFBI) gene.However,the molecular mechanism of mutated TGFBI gene in corneal dystrophy is unclear. Objective The present study was to investigate the expression of the TGFBI gene in human corneal tissue and cells in vitro.MethodsHuman corneal epithelial cells and keratocytes were cultured and passaged,and donor corneal tissue was obtained for the section preparation.RT-PCR was used to detect the expression of TGFBI mRNA in human corneal tissue and cells.Immunofluorescence was used to test the expression of the TGFBI protein in the human corneal tissue,and immunohistochemistry was used to test the expression of the TGFBI protein in human corneal epithelial cells and corneal stromal cells.ResultsRT-PCR analysis showed that TGFBI mRNA could be detected as a 1274 bp band in human corneal tissue and corneal stromal cells,but no TGFBI mRNA was observed in corneal epithelial cells.Immunofluorescence assay revealed that corneal stromal cells were positive ly expressed for the TGFBI protein,but the corneal epithelial cells did not express the TGFBI protein.Immunohistochemistry indicated that the expression of TGFBI was detected the red fluoressence in the cytoplasm of corneal stromal cells;however,no positive response was found in corneal epithelial cells.ConclusionsThe expression of the TGFBI gene occurs in human corneal stromal cells but not in the corneal epithelial cells.This result might be of helpful for studying the function and role of TGFBI gene in pathogenesis of corneal dystrophy.

Background Induced pluripotent stem cells (iPSCs)can differentiate into various types of somatic cells without causing ethical controversy and immune rejection in clinical activity,which is similar to differentiation ability of embryonic stem cells.So,iPSCs may be used as seed cells for tissue engineering corneal endothelial reconstruction.Objective The present study was to survey the morphologic change of iPSCs after coculture with corneal endothelium cells(CECs) under the atomic force microscopy(AFM).Methods Rabbit CECs and human MMC-iPSCs were isolated and cultured respectively.The iPSCs were identified with the marker by immunochemistry.iPSCs passaged for 7 days were then cultured with 60％ confluent CECs to establish the co-culture model.The surface morphology and cellular membrane ultrastructure of differentiated iPSCs after induced by CECs were examined by AFM combination with inverted microscope,and compared with CECs and undifferentiated iPSCs.Results Thelengthand width were(66.93±10.48)μm and (44.85 ± 8.14) μm in CECs,(12.51±1.40)μm and (10.93 ±1.69) μm in uninduced iPSCs,and(36.12±10.29) μm and(31.53±9.65)μm in CECs-induced iPSCs.Both the length and width values of CECs-induced iPSCs were statistically bigger than those uninduced iPSCs,with significant differences between them (P＜0.05),but no significant difference was seen in the width valne of CECs-induced iPSCs in comparison with CECs(P＞0.05).The convex structure of CECs cytomembrane surface showed the digitation in shape with the size and height(2.11 ± 1.03) μm and (115.68±92.08) nm respectively,and the concave structure of cytomembrane surface of CECs was fenestrae-like depression and the size was (1.49 ± 0.65) μm.The numerical valuc of mean square root roughness (Rq)and average roughness (Ra)of cytomembrane surface of CECs were(39.20±7.82)nm and (30.37±5.32)nm respectively.The convex surface of cytomembrane of iPSCs was granular-like in shape with size and height(0.39±0.22)μm and(13.11±9.18)nm respectively.The concave surface of cytomembrane of iPSCs was worm-eaten-like concave with the size(0.34±0.18)μm.The numerical value of Rq and Ra of geometrical parameters of cytomembrane surface of iPSCs were (26.60 ± 4.93)nm and (9.97 ± 3.78) nm respectively.The convex surface of cytomembrane of induced iPSCs was digital-like in shape with the size and height (1.91±0.76) μm and(106.55±77.27) nm respectively.The concave surface of cytomembrane of induced iPSCs was fenestrae-like depression and the size of concave was(1.6l±1.25) μm.The numerical value of Rq and Ra on surface of cytomembrane of induced iPSCs was (57.33± 12.80) nm and (43.63± 11.17) nm respectively.The numerical values of the size and height of convex,the size of concave,Rq and Ra on surface of cytomembrane in induced iPSCs were statistically bigger than in iPSCs(P＜0.05)and were not significant differences in comparison with CECs (P＞0.05).Conclusions Morphology of iPSCs translate toward the CECs after induce for 7 days under the AFM.This outcome lays the foundation for further study on iPSCs.

Objective To observe the effects of casein and excessive iodine on histomorphalogY and ultrastructure of mouse thymicL Methods Based on 2 × 3 factorial design,the experimental mice were divided into 6 groupg Animal models were estabhshed by feeding the mice with different levels of iodine water and casein food.The levels of iodine were 50,600 μg/L in drinking water and 0(Ⅰ),10%(Ⅱ),20%(Ⅲ)of casein in food respectively.After 12 months,the thyroid weight was measured and the morphology of thyroid was observed under optical and electron microscope.Results Factorial analysis showed that iodine factors obviously affected the thyroid absolute and relative weiights of mice(F=16.23,9.47,P＜0.01),and there was interaction between casein and iodine(F=5.29,4.68,P＜0.01 or＜0.05).Compared wiht 150Ⅰ[(5.91±0.82)rag,(117.0±22.2)mg/kg]and 50Ⅲ[(4.90±0.63)rag,(106.1±13.3)mg/kg]groups.thyroid absolute and relative weights of the mice increased in 600 Ⅰ[(7.60±2.40)mg,(143.3±43.2)mg/kg]and 600Ⅲ[(8.63±1.88)mg,(166.2±39.4)mg/kg]groups(P＜0.05 or＜0.01),respectively.But compared with 600 Ⅰ and 600Ⅲ groups.they were reduced obviously in 600Ⅱ[(5.76±1.13)mg,(109.8±16.5)mg/kg]group(P＜0.05 or＜O.01).Colloid goiter,lymphocyte infiltration were found,some of the follicles epithelial cells appeared active under light and electron microscope in iodine excels group,which,however,decreased obviously along with the increase of casein dose.Conclusions Long-term excessive iodine may cause colloid goiter and inflammation injury of mice,possibly leading the development of thyroiditis in mice,which may be partly reduced by casein.

Background The human transforming growth factor beta-induced gene (TGFBI) is the first determined pathogenic gene to corneal dystrophy.But the molecular genetic mechanism is completely unknown.The study of concerning role of TGFBI is very important for us understand the physiological function of cornea,and the pathogenesis of corneal dystrophy.Objective The vector of human transforming growth factor beta-induced gene (TGFBI) in eukaryotic expression was constructed and transfected into the human corneal epithelial cells in order to explore its influence on the growth of human corneal epithelial cells.Methods Total RNA was extracted from normal donor cornea tissue and cDNA was obtained by reverse transcription.TGFBI cDNA was synthesized by reverse transcription-PCR and cloned into pCMV-N-HA vector and identified by sequencing with PCR and EcoRV,XhoI double restriction endonuclease.The cells were grouped into recombinant pCMV-N-HA-TGFBI plasmid group,pCMVN-HA plasmid group,non-transfected group and pGFP-C2 transfected group.The recombinant pCMV-N-HA-TGFBI plasmid was transfected to human corneal epithelial cells and identified by observing the expression of enhanced green fluorescence protein(EGFP) in the cells.The TGFBI mRNA and proteins were harvested from the cells for real-time PCR analysis and Western blot assay respectively in 58 hours after transfection.The growth of the transfected cells was assessed by Cell Counting Kit-8.The expressions of matrix metalloproteinase(MMP) and tissue inhibitors of matrix metalloproteinase (TIMP) proteins and their mRNA in transfected cells were detected using SYBR fluorescence realtime PCR analysis and Western blot assay.Results The sequencing result of pCMV-N-HA-TGFBI positive clone plasmid showed that amplified TGFBI eDNA inserted into the vector at the correct sequence.EGFP was expressed in transfected cells in 48 hours after transfer of pGFP-C2 with the transfer efficacy 70％.The expression intensity of TGFBI mRNA was significantly higher in recombinant pCMV-N-HA-TGFBI plasmid group compared with pCMV-N-HA plasmid group and non-transfected group,and TGFBI protein was expressed in recombinant pCMV-N-HA-TGFBI plasmid group.No significant difference was found in the A450value among recombinant pCMV-N-HA-TGFBI plasmid group,pCMV-N-HA plasmid group and non-transfected group ( F=3.34,P＞0.05 ).The mRNA level of MMP1,MMP3in the transfected cells was significant elevated but that of TIMP1 was declined in the recombinant pCMV-N-HA-TGFBI plasmid group compared with pCMV-N-HA plasmid group and non-transfected group (all P ＜ 0.05 ).Meanwhile,the expressions of MMP1,MMP3 and TIMP1 proteins appeared the same tendency( all P＜0.05).Conclusions Eukaryotic expression vector harboring human TGFBI eDNA can be successfully constructed and efficiently overexpressed in human corneal epithelial cells.TGFBI gene is involved in the physical and pathological conditions of human corneal epithelial cells by regulating the activity of MMP1,MMP3 and TIMP1.The results offer a new approach for the study of the role of TGFBI in pathogenesis of corneal transparency.

Objective:To stablish of chemiluminescent Southern blot detection system for examining HBV DNA replication intermediates in HepG2.2.15,and analyse the inhibition of HBV replication with three kind of drugs with different targets.Methods:The HBV DNA replication intermediates were extracted and analyzed by Southern blot with HBV probe,which(pTHBV1047) was labelling with digoxigenin.The results of the hybridization were detected by chemiluminescent,and the condition of hybridization was optimized.After treated with lamivudine,Bay41-4109,?-Galcer in different concentration,the HBV DNA from the HepG2215 cells were detected with the system.Results:the sensitivity of the system was 1pg of pTHBV1047,and HBV specific positive signals was detected with the DNA from HepG2.2.15.The three kinds of drugs can inhibit the HBV replication obviously with chemiluminescent Southern blot detection system,the IC50 were 1.53?mol/L,0.41?mol/L,0.01?mol/L.Conclusion:The HBV replication intermediates from the cell of HepG2.2.15 can reflect the antiviral effect accurately with different targets drugs and this mothod would be used in the study of Chinese-midicine.

Objective:To develop an HPLC method for determining matrine , oxymatrine and oxysophocarpine in wine-processed Sophora flavescens.Methods:An HPLC method was used with a Dalian Elite HYP-ODS column (250 mm ×4.6 mm, 5 μm).The mobile phase consisted of methanol-phosphate buffer (12:88) with the flow rate of 1.0 ml· min-1 .The detection wavelength was 205 nm, the column temperature was 25℃and the injection volume was 20 μl.Results:The calibration curve was linear within the range of 0.105-0.420 μg for matrine,0.760-3.040μg for oxysophocarpine and 1.615-6.460μg for oxymatrine.The average recovery of ma-trine, oxysophocarpine and oxymatrine was 99.70％(RSD=1.92％), 99.41％( RSD=1.93％) and 98.60％(RSD=1.79％)(n=6), respectively.Conclusion:The method is accurate and reliable with good reproducibility , promising specificity and high sensitivi-ty, which can be used for the quality control of wine-processed Sophora flavescens.