Immunoregulatory effects of miR-31-5p overexpression on peripheral blood Th17 cell in a rabbit model of autoimmune dry eye / 中华实验眼科杂志

Objective:To investigate the immunoregulatory effects of lentivirus-mediated microRNA (miR)-31-5p overexpression on peripheral blood T helper cell 17 (Th17) in a rabbit model of autoimmune dry eye.Methods:The miR-31-5p recombinant lentiviral vector was constructed.Lentivirus overexpressing miR-31-5p and its control virus were packaged.The concentration measurement and lentiviral titer determination were carried out.A rabbit model of autoimmune dry eye was established and the peripheral blood mononuclear cells (PBMC) of the rabbits were isolated.PBMC infected with miR-31-5p and negative control lentivirus particles were assigned as the miR-31-5p overexpression group and control group, respectively.The miR-31-5p expression level was detected using quantitative real-time PCR (qRT-PCR). Then PBMC in the two groups were co-cultured with γ-ray irradiated lacrimal gland epithelial cells.The expressions of Th17 cell related transcription factor retinoic acid-receptor-related orphan receptor C (RORC) and interleukin-17 (IL-17) mRNA, IL-1β, IL-6 and IL-23 were determined by qRT-PCR.The IL-17 protein expression level was detected by Western blot.The use and care of animals complied with Regulation for the Administration of Affair Concerning Experiment Animals by State Science and Technology Commission.The study protocol was approved by the Ethics Committee of Tianjin Medical University Eye Hospital (No.TJYY20201221036).Results:The construction of the miR-31-5p recombinant lentiviral vector was verified by DNA sequencing.The lentiviral titer of lentivirus overexpressing miR-31-5p and control lentivirus particles was 3.82×10 7 TU/ml and 3.50×10 7 TU/ml, respectively.The miR-31-5p relative expression level of PBMC was significantly increased in miR-31-5p overexpression group in comparison with control group, showing a statistically significant difference ( t=-9.696, P<0.001). When PBMC were co-cultured with lacrimal gland epithelial cells in vitro, the relative expression levels of RORC and IL-17 mRNA in miR-31-5p overexpression group were 0.33±0.03 and 0.28±0.09, which were significantly decreased in comparison with 1.00±0.00 and 1.00±0.00 in control group, with statistically significant differences between them ( t=46.256, 13.810; both at P<0.05). The relative expression level of IL-17 protein in miR-31-5p overexpression group was significantly reduced than control group ( t=4.977, P=0.008). The relative expression levels of IL-1β, IL-6 and IL-23 mRNA were significantly lower in miR-31-5p overexpression group than control group ( t=220.076, 6.641, 13.271; all at P<0.05). Conclusions:The overexpression of miR-31-5p can inhibit the Th17-immune response via down-regulating the expression of IL-6, IL-1β and IL-23.

Effect of interleukin-23R overexpression on Th17/Treg balance in experimental autoimmune uveitis mice / 中华眼底病杂志

Objective:To investigate the effect of interleukin (IL)-23 receptor (IL-23R) overexpression on the balance of T helper 17 (Th17 cells)/regulatory T cells (Treg cells) in experimental autoimmune uveitis (EAU) mice.Methods:Twelve 8-week-old female C57BL/6J mice were randomly divided into LV-Ctrl group and LV-IL-23R group, with 6 mice in each group. Two groups of mice were injected with LV-Ctrl and LV-IL-23R lentiviruses through the tail vein, respectively; 7 days after injection, the EAU mouse model was established by active immunization with vitamin A-binding protein 1-20 between photoreceptors. Starting from 13 days after immunization, the fundus of the mice was observed by indirect ophthalmoscopy every 2 days and clinical scores were performed; 30 days after immunization, hematoxylin-eosin staining was used to observe the histopathological changes of mouse retina. The levels of IL-17 in serum of the two groups of mice were detected by enzyme-linked immunosorbent assay; the proportion of Th17 cells and Treg cells was detected by flow cytometry. The relative mRNA expression of IL-23R, IL-17, retinoic acid-related orphan receptor γt (RORγt), IL-10 and forkhead transcripyion factor p3 (Foxp3) were detected by real-time quantitative polymerase chain reaction. Comparisons between groups were performed using repeated measures analysis of variance, independent samples Mann-Whitney U test, and independent samples t test. Results:Compared with the LV-Ctrl group, the retinal inflammatory reaction of the LV-IL-23R group was more severe. At 13 days after immunization, there was no significant difference in fundus inflammation scores between LV-IL-23R group and LV-Ctrl group ( t=-2.001, P=0.058); 15-29 days after immunization. The fundus inflammation scores of LV-IL-23R group were higher than those of LV-Ctrl group, and the difference was statistically significant ( t=-4.429,-6.578, -7.768, -10.183, -6.325, -7.304, -4.841, -6.872; P<0.001). Histopathological examination showed that the infiltration of inflammatory cells in the fundus increased, the retinal structure was damaged more seriously, and the histopathological score was significantly increased, and the difference was statistically significant ( t=-4.339, P=0.001). Compared with the LV-Ctrl group, the relative expression of IL-23R mRNA in the spleen of the LV-IL-23R group was significantly increased, and the difference was statistically significant ( Z=2.087, P=0.037). The relative expression of IL-17 and RORγt mRNA increased, while the relative expression of IL-10 and Foxp3 mRNA decreased, and the differences were statistically significant ( t=-6.313,-5.922, 4.844, 7.572; P=0.003, 0.004, 0.008, 0.002). Compared with the LV-Ctrl group, the level of IL-17 in the serum of the mice in the LV-IL-23R group was significantly increased, and the difference was statistically significant ( t=-5.423, P=0.002); the proportion of Th17 cells in the spleen and lymph nodes was significantly increased, whereas, the proportion of Treg cells was significantly reduced, and the difference was statistically significant ( t=-4.290, 3.700; P=0.002, 0.006). Conclusion:IL-23R overexpression can promote Th17/Treg imbalance in EAU mice, and aggravate the clinical and pathological manifestations of EAU.

Correlation analysis between corneal biomechanical parameters and high-order aberrations in myopia children / 国际眼科杂志(Guoji Yanke Zazhi)

AIM:To investigate the correlation between corneal biomechanical parameters measured by the corneal visualization Scheimpflug Technology(Corvis ST)and corneal high-order aberrations(HOAs)in children with mild to moderate myopia.METHODS:A cross-sectional study. A total of 255 pediatric patients with myopia enrolled from April to July 2021 in Tianjin Medical University Eye Hospital were continuously collected, and all the right eyes were taken for analysis. Corneal biomechanical parameters were obtained from Corvis ST. Pentacam three-dimensional anterior segment analyzer was used to measure total corneal higher-order aberrations(RMSh), third order aberrations(RMS3)and fourth order aberrations(RMS4).RESULTS:RMS3 was positively correlated with the second applanation time(A2T)(r=0.175, P=0.009)and negatively correlated with the axis length(AL)(r=-0.155, P=0.014). RMS4 was negatively correlated with the highest concavity radius(HCR)(r=-0.165, P=0.009). RMSh was negatively correlated with HCR and AL(r=-0.152, P=0.037; r=-0.175, P=0.005).CONCLUSION:There is a correlation between corneal biomechanical parameters and HOAs in children with myopia. Cornea with higher stiffness and stronger deformation resistance has smaller RMS3, RMS4 and RMSh.

Enhanced effect of microRNA-338-3p in bone marrow-derived dendritic cells on IRBP 1-20-specific T helper 17 cells activation in experimental autoimmune uveitis / 中华实验眼科杂志

Objective:To investigate the role of microRNA-338-3p (miR-338-3p) in regulating the generation and function of interphotoreceptor retinoid-binding protein (IRBP) 1-20-specific T helper 17 (Th17) cells in experimental autoimmune uveitis (EAU). Methods:Bone marrow cells were flushed from the femurs and tibiae of wild-type C57BL/6 mice and cultured in the presence of granulocyte macrophage-colony stimulating factor (GM-CSF) and interleukin-4 (IL-4)to differentiate into bone marrow-derived dendritic cells (BMDCs). On day 5 after induction, immature BMDCs were collected and divided into miR-338-3p mimics transfection group and mimics negative control transfection group, then transfected with miR-338-3p mimics or negative mimics according to grouping.Twenty-four hours after transfection, the BMDCs were stimulated with 100 ng/ml of lipopolysaccharide to mature.Relative expression levels of miR-338-3p, IL-6, IL-23 and IL-1β mRNA in BMDCs of the two groups were detected by quantitative real-time polymerase chain reaction (qRT-PCR). The EAU model was established with IRBP 1-20, incomplete Freund adjuvant and mycobacterium tuberculosis (H37Ra) in mice.On day 13 after modeling, T cells were isolated from the mice spleen or draining lymph nodes and co-cultured with miR-338-3p mimics or negative control mimics-transfected BMDCs under Th17-polarizing conditions.Concentration of IL-17 in the supernatant was detected by ELISA.Relative expression levels of retinoic acid receptor-related orphan nuclear receptor γt (RORγt) and IL-17 mRNA were analyzed by qRT-PCR.The proportion of IL-17 + cells among T cells co-cultured with BMDCs was assessed by flow cytometry.To further verify the role of miR-338-3p in dendritic cells on Th17 cells, BMDCs transfected with miR-338-3p inhibitor or control inhibitor were co-cultured with T cells isolated from spleen or draining lymph nodes of EAU mice.Concentration of IL-17 in the supernatant was detected by ELISA.The use and care of the animals complied with Regulations for the Administration of Affairs Concerning Experiment Animals by State Science and Technology Commission.The study protocol was approved by the Institutional Animal Care and Use Committee of Tianjin Medical University (No.TJYY2019110117). Results:Relative expression level of miR-338-3p in BMDCs was significantly increased in the miR-338-3p mimics transfection group than the mimics negative control group ( t=6.861, P=0.002). In T cells co-cultured with miR-338-3p mimics-transfected BMDCs, the relative expression levels of RORγt and IL-17 mRNA were 1.34±0.16 and 1.33±0.16, which were significantly higher than 1.00±0.01 and 1.00±0.01 in the mimics negative control group ( t=3.632, P=0.022; t=3.681, P=0.021). ELISA showed that the concentration of IL-17 in the supernatant was (5 941.00±452.40)pg/ml in the miR-338-3p mimics transfection group, which was significantly higher than (4 299.00±348.30)pg/ml in the mimics negative control group ( t=4.979, P=0.008), and IL-17 concentration in the supernatant was (3 092.00±200.90)pg/ml in the miR-338-3p inhibitor transfection group, which was lower than (4 063.00±131.50)pg/ml in the inhibitor negative control group ( t=7.005, P=0.002). The proportion of IL-17 + cells among T cells was (8.03±1.35)% in the miR-338-3p mimics transfection group, which was significantly higher than (4.52±0.73)% in the mimics negative control group ( t=3.968, P=0.017). The relative expression levels of IL-6, IL-23, and IL-1β mRNA were 2.23±0.21, 2.21±0.56, 2.32±0.43, respectively in the miR-338-3p mimics transfection group, which were significantly higher than 1.00±0.06, 1.00±0.07, 1.01±0.15 in the mimics negative control group ( t=10.290, P=0.001; t=3.747, P=0.020; t=5.280, P=0.006). Conclusions:Overexpression of miR-338-3p in BMDCs can promote the IRBP 1-20-specific Th17 cell response by increasing the expression of Th17-polarizing cytokines including IL-6, IL-1β and IL-23.

Regulation of human umbilical cord mesenchymal stem cells derived exosomes on peripheral blood macrophages from rabbit autoimmune dry eye / 中华实验眼科杂志

Objective@#To investigate the effect of human umbilical cord mesenchymal stem cells derived exosomes (hUC-MSC-exo) on the phenotype of peripheral blood macrophages from rabbit autoimmune dry eye and the expression of related cytokines.@*Methods@#The hUC-MSCs were isolated and characterized.Exosomes derived from hUC-MSCs were extracted by ultracentrifugation and observed directly using electronic microscopy.Specific markers of exosomes were analyzed by Western blot.Six rabbits were randomly divided into the normal control group and the dry eye group by using the random number table method, 3 rabbits for each group.Rabbit model of autoimmune dry eye was established in the dry eye group, and the lacrimal glands were collected for quantitative real-time PCR (qRT-PCR) at the 8th week.In vitro, activated peripheral blood mononuclear cells (PBMCs) from rabbit autoimmune dry eye model were incubated with hUC-MSC-exo or phosphate buffered saline (PBS). After 48 hours, cells from the hUC-MSC-exo group and the PBS control group were collected.The mRNA expression levels of related cytokine genes and subpopulation-related marker genes in macrophages were quantified by qRT-PCR.The use and care of the animals complied with Regulations for the Administration of Affair Concerning Experimental Animals by State Science and Technology Commission.This study protocol was approved by Ethic Committee of Tianjin Medical University Eye Hospital (No.TJYY20181217001). Written informed consent was obtained from each family before obtaining umbilical cord.@*Results@#Exosomes derived from hUC-MSCs had typical morphology and specific markers.qRT-PCR results showed that, the relative expression quantity of M1 macrophages phenotypic molecular nitric oxide synthase 2 (NOS2) mRNA and inflammatory factor tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) mRNA in the lachrymal organization in the dry eye model group was significantly higher than those in the normal control group (3.06±1.00 vs. 1.00±0.03, 2.77±0.72 vs. 1.01±0.02 and 1.30±0.08 vs. 1.01±0.01, respectively), the relative expression quantity of M2 macrophages phenotypic molecular arginase 1 (Arg1), CD206 and IL-10 mRNA in the lachrymal organization in the dry eye model group was significantly lower than those in the normal control group (0.55±0.07 vs. 1.00±0.00, 0.60±0.13 vs.1.00±0.00, 0.65±0.14 vs. 1.01±0.01, respectively), with significant differences between them (all at P<0.05). In vitro, the relative expression quantity of M1 macrophages phenotypic molecular NOS2 mRNA and inflammatory factor TNF-α, IL-1β mRNA in PBMCs in the hUC-MSC-exo group was significantly lower than those in the PBS control group (0.59±0.08 vs.0.98±0.03, 0.56±0.07 vs. 1.03±0.11, 0.47±0.04 vs.1.00±0.08)(all at P<0.05); the relative expression quantity of M2 macrophages phenotypic molecular Arg1, CD206 mRNA and anti-inflammatory cytokine TGF-β, IL-10 and IL-4 mRNA in PBMCs in the hUC-MSC-exo group was significantly higher than those in the PBS control group (2.13±0.28 vs. 1.10±0.17, 1.32±0.03 vs. 1.01±0.06, 1.53±0.20 vs. 1.05±0.10, 1.47±0.08 vs.0.98±0.03, 1.51±0.16 vs. 1.01±0.03), with significant differences between them (all at P<0.05).@*Conclusions@#hUC-MSC-exo can polarize peripheral blood macrophages toward immune-suppressive M2-like phenotype, inhibit the production of pro-inflammatory cytokines TNF-α and IL-1β, and meanwhile increase the expression of anti-inflammatory factors IL-10 and TGF-β.

Establishment and application of chemical fingerprint and pattern recognition for Panacis Japonici Rhizoma / 中草药

Objective To establish and identify HPLC fingerprints of Panacis Japonici Rhizoma from different regions, and provide a scientific method for quality its control and standardization. Methods The fingerprints of Panacis Japonici Rhizoma were established based on HPLC method. The similarity evaluation, cluster analysis (CA), and principal component analysis (PCA) were applied for the experimental data, in order to find out the similarities and differences among the 10 batches of Panacis Japonici Rhizoma from different regions. Meanwhile, the qualitatively and quantitatively analyze were adopted in the common peaks of Panacis Japonici Rhizoma by total statistical moment method. Results The HPLC fingerprint with 16 common peaks was established, for the common chemical components, six were identified as ginsenoside Rg1, ginsenoside Re, ginsenoside Rb1, chikusetsusaponin V, chikusetsusaponin IV, and chikusetsusaponin IVa, respectively. Among them, the contents of chikusetsusaponin V and IV in different batches of Panacis JaponiciRhizoma samples showed little differences. The similarities of samples from various regions except S7 were over 0.90. The samples were classified into three categories by CA and PCA methods. The parameters of total statistical moment for five species of Panax genus showed significantly differences. Conclusion The developed HPLC fingerprint of Panacis Japonici Rhizoma combined with chemical pattern recognition can provide a scientific instruction for the quality control of Panacis Japonici Rhizoma and the differentiation of five herb medicines from Panax genus.

Research progression on mesenchymal stem cells in dry eye / 眼科新进展

Mesenchymal stem cells (MSCs) are multipotent stromal cells derived from the mesoderm that can differentiate into a variety of cell types.Recently,as the study of MSCs in the treatment of various autoimmune diseases is maturing,more and more researchers have advocated the immune function of MSCs and the treatment for dry eye.The rate of dry eye is higher.The duration of therapy in dry eye,especially immune related dry eye,is long,and the therapeutic effect is poor.Dry eye has affected people's quality of life,so it is important to find new methods to treat it.This article reviews MSCs function in dry eye.

Effects of mesenchymal stem cells on pathological changes and cytokines expression in rabbit model of autoimmune dry eye / 眼科新进展

Objective To establish the model of autoimmune dry eye and analyze the level of cytokines and the pathological changes of autoimmune dry eye rabbits treated with human umbilical cord mesenchymal stem cells (MSCs).Methods Twentyfour female New Zealand rabbits were randomly divided into three groups:Normal control group,dry eye group and MSCs treatment group,8 cases in each group.After treated with MSCs for six weeks,the pathological changes were observed,and the expression of Th1 and Th17 cell differentiation related cytokines mRNA in lacrimal gland tissue was detected by real-time quantitative PCR.The percentage of regulatory T cells (CD4 +Foxp3 + cells) in the lacrimal gland and spleen tissues of the dry eye model group and MSCs treatment group was measured by flow cytometry.Results After treatment with MSCs for 6 weeks,HE staining from each rabbit lacrimal gland showed that,the normal control group showed no or only a few lymphocytes;The lacrimal gland cells were atrophied in the dry eye group,the distribution of lymphocyte scattered around the gland and small blood vessels;Compared with dry eye group,the lacrimal gland lymphocyte infiltration reduced in MSCs treatment group,and the cell morphology were better.Compared with dry eye group,lymphocyte infiltration and aggregation in the conjunctival conjunctiva in MSCs treatment group were significantly reduced,the epithelial structure was intact,and the degeneration and atrophy cells were rare.The expression of inflammatory factors including interferon-γ(IFN-γ),Interleukin-17 (IL-17) and transcription factor T-bet mRNA were decreased in MSCs treatment group,there were significant difference compared with dry eye group (all P ＜ 0.05);Thl7 cell associated cytokines IL-17 expression levels were decreased,but no significant difference was found between two groups (P ＞ 0.05);The expression of transcription factor RORrt mRNA in dry eye group was significantly decreased,the difference was statistically significant (P ＜ 0.05).The expression of inflammatory factors TNF-α in the lacrimal gland tissue of MSCs treatment group was significantly lower than that in the dry eye model group,while the expression of the anti-inflammatory factor TGF-β was significantly higher than that in the dry eye model group,and the differences were statistically significant (all P＜0.05).The regulatory T cells (CD4 + Foxp3 + cells) in the lacrimal gland of dry eye model group accounted for 10％ of lymphocytes was,while the treatment group accounted for 27.8％,MSCs group was significantly higher than dry eye group,the difference was statistically significant (P ＜ 0.05).Conclusion MSCs can reduce the histopathological changes of the immune dry eye,and they may have immunoregulatory effects on autoimmune dry eye.The mechanisms of immunomodulatory effects may be related to the balance of inflammatory cytokines and anti-inflammatory cytokines.

Recovery of anterior corneal biometry after discontinuation of orthokeratology / 眼科新进展

Objectivc To observe the short-term changes of anterior corneal biometry after discontinuation of orthokeratology in patients with 2-year wearing.Methods Retrospective study.Sixty myopic patients aged from 8-14 years old during October 2012 and October 2014 were wearing orthokeratology for 2 years in Tianjin Medical University Eye Hospital.According to the degree of myopia,they are divided into three groups(SE≤-2.00 D for group A,-2.00 D ＜ SE ≤-4.00 D for group B and -4.00 D ＜ SE ≤-6.00 D for group C).The recovery of anterior corneal curvature,including flat K(FK),steep K(SK),average K (AVEK),changes of axial length and central corneal thickness (CCT) at 1 week,2 weeks,1 month after discontinuation of orthokeratology were observed.Results There was no statistical differences in FK,SK,AVEK before and 2 weeks after wearing orthokeratology in group A (all P ＞ 0.05).While in group B,there was no significant difference in FK,SK,AVEK before and 1 month after wearing orthokeratology;There were statistically differences in FK,SK,AVEK at 1 month after discontinuation in group C compared with the baseline (all P ＜0.05).As for CCT,there was no statistical differences among group A,B,C after discontinuation of orthokeratology for 2 weeks (all P ＞ 0.05).There were statistical differences in the axial length between 1 week and 2 weeks after discontinuation of orthokeratology in group B and C (all P ＜ 0.05);There were statistical differences in the axial length between 1 month and 2 weeks after discontinuation of orthokeratology in three groups (all P ＜ 0.05);Compared with the state before wearing orthokeratology,the increase of axial length in group A,B,C were (0.43 ± 0.36) mm,(0.35 ± 0.21)mm and (0.36 ± 0.29) ram,respectively.Conclusion The time course of returning to the original corneal parameter varies among different degree of myopia,and the axial length has no significant growth after short-term discontinuation.

Recent advances in immune regulation of mesenchymal stem cells on autoimmune dry eve / 眼科新进展

Mesenchymal stem cells (MSCs) have good immune regulatory function,can inhibit many immune cell proliferation,direct effects on activation and proliferation of T cell,play a role in immune regulation by Treg cells or by the secretion of soluble factors regulating Thl/Th2 secretion and reaction equilibrium,inhibit the inflammation through the anti-inflammatory,regulation of cytokines expression at the same time,so reduce the expression levels of matrix metalloproteinase-2 and-9,which may promote lacrimal gland tissue damage,and thus play a role in immune regulation.MSCs can reduce the autoimmune dry eye clinical index,recovery secretion function of part lacrimal gland.This article reviews the research advances in inmmmne regulation of MSCs on autoimmune dry eye.

Comparison of ocular biometry by two kinds of optical biometers in Chinese adolescents / 眼科新进展

Objective To evaluate the repeatability and agreement of two optical biometers (Lenstar LS900 (R) and SW-9000) for ocular biometry in Chinese adolescents.Methods A prospective study was conducted which included 65 ametropic patients,with an average age of (11.45 ± 2.67) years (age ranging from 8 to 18 years).The ocular biometry for right eyeball was performed with Lenstar LS900 (R) and SW-9000 respectively,followed by evaluation of the repeatability of the two biometers using one-way analysis of variance,and the agreement of the two instruments using the Bland-Altman plot.Results The repeatability of parameters measured by Lenstar LS900 (R),including axial length (AL),K value in the flattest meridian (K1),K value in the steepest meridian (K2),central corneal thickness (CCT),anterior depth (AD),lens thickness (LT),pupil diameter (PD),was well,and all intraclass correlation coefficient (ICC) ＞ 0.9;the repeatability of white to white (WTW) was inferior to other parameters,but it was still ＞0.88.The repeatability of AL,K1,K2,CCT measured by SW-9000 was good,with their ICC ＞ 0.9,but the repeatability of other parameters was poor.The parameters with good repeatability including AL,K1,K2,CCT measured by SW-9000 and Lenstar LS900 (R) were compared respectively,and the results showed that AL and CCT examined by SW-9000 were slightly longer than those measured by Lenstar LS900 (R),and the difference was statistically significant (all P ＜ 0.05).However,there was no significant difference about K1,K2 (all P＞0.05).Moreover,the AL,K1,K2 and CCT measured by the two instruments had close linear correlation (all r ＞0.97,all P ＜0.01).The BlandAltman plot showed that 95％ LoA (limits of agreement) of AL was (-0.057 to 0.133) mm,K1 was (-0.456 to 0.369) D,K2 was (-0.388 to 0.549) D and CCT was (-3.483 to 8.016) μm.Conclusion Biometric parameters including AL,K1,K2,CCT measured by Lenstar LS900 (R) and SW-9000 have good repeatability in the adolescents aged 8-18 years and they are highly correlated;meanwhile,the agreement of AL,K1,K2,CCT measured by SW-9000 with Lenstar LS900 (R) is acceptable in clinical practices.

Ocular inflammation and pathological characteristics of recurrent experimental autoimmune uveitis in rat / 中华实验眼科杂志

Background Most animal models of experimental autoimmune uveitis (EAU) are single attacked procedure,with a different feature from the natural course of human recurrent autoimmune uveitis.So establishing a recurrent EAU model is necessary for the clinical study on EAU.Objective This study was to establish the recurrent EAU model in rat and investigate the ocular inflammation and pathological manifestation and interleukin-17 (IL-17)expression in the eye.Methods T cells isolated from the spleen and draining lymph nodes of Lewis rats immunized with interphotoreceptor retinoid-binding protein (IRBP) 1177-1191 peptide fragments (R16) 10 days earlier were re-stimulated with R16 in vitro and injected into naive syngeneic rats to establish the recurrent EAU models,and the normal Lewis rats were used as controls.The eyes of model rats were then examined daily for clinical signs of uveitis by slit-lamp biomicroscopy and scored Caspi's criteria.The rats were sacrificed 1 month,2,3months after injection respectively,and the retinal sections were prepared for the pathological examination by hemotoxylin & eosin staining.Immunohistochemistry was performed to detect the expression of IL-17 in the retina.Results Adoptive transfer of R16-specific T cells to Lewis rats induced recurrent uveitis.The inflammatory scores on the fourth day,the sixth day,and the inflammatory response disappeared on the tenth day after injection.However,the inflammatory reaction occurred repeatedly 4 or 5 times in the 2-month duration after that,and the right and left eyes of a single recipient showed a different pattern of relapse,and the clinical manifestations of EAU was similar to the natural course to those of human autoimmune uveitis.In the retinal specimens of 1-,2-and 3-month group,the number of inflammatory cells was gradually decreased as the time lapse.Compared with the normal group,the thicknesses of the entire retina,outer nuclear layer and inner nuclear layer decreased with a significant difference among the 4 groups (F=20.46,288.40,4.43,all P=0.00).The number of RGCs in the normal group,1-,2-and 3-month group was 231.27 ± 15.36,225.36 ± 17.79,132.18 ±9.39 and 67.45 ± 11.90,respectively,showing a significant difference among them (F=68.94,P=0.00).Immunohistochemistry showed that the scores of the IL-17 expression in the rat retina were 0.64 ± 0.17,1.92 ± 0.19,1.17 ± 0.23 and 0.83 ± 0.23,showing statistically significant difference (F=64.10,P=0.00).Conclusions The stimulation of R16-specific T cells can induce recurrent EAU in Lewis rat.Th17 is involved in the disease course.

Web Research Learning of Physiology in Rehabilitation Specialty / 中国康复理论与实践

@# This paper introduced the implementing process, characteristics and effects of the web research learning of physiology. Research learning based on Web promoted reform of physiology teaching, enhanced ability of self-study, integration and innovative of students

Involvement of inhibition of nucleus GAPDH over-expression in erythropoietin's reduction of neuronal apoptosis induced by brain ischemia/reperfusion in rats / 生理学报

To study whether recombinant human erythropoietin (rhEPO) reduces neuronal apoptosis through inhibiting over-expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in nucleus induced by brain ischemia/reperfusion in rats, 48 adult Sprague-Dawley rats were randomly divided into 3 groups: sham, saline and EPO groups. Animal models of brain ischemia/reperfusion were established by middle cerebral artery occlusion in rats. The effects of EPO on the sizes of ischemia tissue were observed by TTC staining. The over-expression of GAPDH in nucleus was detected by Hoechst-33258 and anti-GAPDH antibody double staining. The neuronal apoptosis in penumbral was detected by Nissl's staining and Hoechst-33258 immunofluorescence, respectively. The results showed that rhEPO treatment (3 000 U/kg, three times daily, i.p.) apparently reduced the sizes of infarct brain tissue in ischemia/reperfusion rats. rhEPO inhibited over-expression of GAPDH in nucleus of apoptotic neurons. In the meantime rhEPO decreased the number of apoptotic neurons in ischemia/reperfusion rats. These results suggest that rhEPO may induced reduction of neuronal apoptosis in penumbra may be through inhibiting over-expression of GAPDH in nucleus of apoptotic neurons induced by ischemia/reperfusion. Reduction of GAPDH over-expression in nucleus may play a pivotal role in EPO inhibiting neuronal apoptosis in cerebral ischemia/reperfusion rats, providing experimental evidence for EPO neuro-protecting effects against ischemia/reperfusion.

Problem-based Learning Based on Web for Rehabilitation Therapy Teaching / 中国康复理论与实践

@# Objective To apply the problem-based learning (PBL) based on the web in the rehabilitation therapy teaching. Methods The 64 students of rehabilitation therapy in grade 2009 and 2010 were taught with selected 3 chapters of physiology, using traditional teaching and PBL based on the web respectively. They were investigated through questionnaire after learning. Results Compared with he traditional teaching, PBL based on the web could improve the interesting of learning, self-study ability, and scope of knowledge. Conclusion PBL based on the web could improve the quality of physiology teaching for students of rehabilitation therapy.

Effect of valsartan on postprandial plasma inflammatory factors in patients with essential hypertension / 中南大学学报(医学版)

OBJECTIVE@#To explore the effect of valsartan on the concentrations of plasma inflammatory factors after a high-fat meal in patients with essential hypertension in very short time.@*METHODS@#Fifty hypertensive patients and 25 healthy controls were studied. Patients randomly accepted lacidipine 4 mg/d (lacidipine group) or valsartan 80 mg/d (valsartan group) for 1 week. The concentrations of plasma lipid profiles, high-sensitivity C-reactive protein (hsCRP) and soluble P-selectin were measured in fasting state and at 4 h after a single high-fat meal in all subjects at baseline and in patients after 1 week.@*RESULTS@#The concentrations of postprandial plasma hsCRP and soluble P-selectin significantly increased after a high-fat meal in patients (P < 0.05), as compared with those at fasting levels, but not in the controls. The postprandial plasma triglyceride concentrations significantly increased in the healthy controls (P < 0.05), but were lower than those in hypertensive patients (P < 0.01). Postprandial change in plasma concentration of triglyceride was significantly correlated with those of log (hsCRP) (r = 0.344)and soluble P-selectin (r = 0.432), respectively (n = 75, both P < 0.01). Lipids profiles did not change significantly after 1 week. There was no significant difference between the fasting and postprandial plasma concentrations of either hsCRP or soluble P-selectin in valsartan group, while the postprandial increments of inflammatory factors were still significant in the lacidipine group.@*CONCLUSION@#High-fat meal can induce postprandial inflammation response in patients with essential hypertension. Valsartan effectively attenuates this postprandial inflammation response within a very short time.

Effects of puerarin on ADRP gene expression in fatty tissue of type 2 diabetes mellitus rats / 中国中药杂志

<p><b>OBJECTIVE</b>To observe the effects of puerarin on ADRP gene mRNA expression in fatty tissue of type 2 diabetes mellitus rats (T2DM).</p><p><b>METHOD</b>Wiastar rats of T2DM model were made by feeding with high glucose and fat diet and injecting with small dose of streptozocin (25 mg x kg(-1)). 40 model rats were randomly divided into model control group and three puerarin groups (40, 80, 160 mg x kg(-1)), another 10 rats were selected as normal control group. FBG and FINS were measured to calculate IR after rats were injected consecutively for 6 weeks. The level of ADRP gene mRNA in fatty tissue was determined by RT-PCR after rats were injected eight weeks.</p><p><b>RESULT</b>Compared with model control group, high and middle dosage of puerarin can decreased ADRP gene mRNA expression in fatty tissue obviously, FBG, IR level in each puerarin group and FINS in high and middle dosage puerarin groups decreased obviously.</p><p><b>CONCLUSION</b>Puerarin can decrease the blood glucose level of T2DM by downregulating ADRP mRNA expression and depressing the insulin resistance.</p>

Construction of selectable marker-removable plant expression vectors / 生物工程学报

The commonly used plant constitutive expression vector pBI121 was modified by insertion of two directly orientated lox sites each at one end of the selectable marker gene NPTII and by replacing the GUS gene with a sequence composed of multiple cloning sites (MCS). The resulting plant expression vector pBI121-lox-MCS is widely usable to accommodate various target genes through the MCS, and more importantly to allow the NPTII gene removed from transformed plants upon the action of the Cre recombinase. In addition, the CaMV 35S promoter located upstream of the MCS can be substituted with any other promoters to form plant vectors with expression features specified by the introduced promoters. Provided in this paper is an example that an enhanced phloem-specific promoter of the pumpkin PP2 gene (named dENP) was used to construct an NPTII-removable phloem-specific expression vector pBdENP-lox-MCS. Moreover, to facilitate screening of selectable marker-removed gene and the composite sequence is flanked by lox sites. Thus the selectable marker-free plants can be visually identified by loss of GFP fluorescence. The above newly created plant expression vectors can be used to develop selectable marker-removable transgenic plants for a variety of purposes.

Effect of niacin on adiponectin levels in the adipocytes secretion in rabbits / 中南大学学报(医学版)

OBJECTIVE@#To explore the effect of niacin on the serum adiponectin concentration in hypercholesterolemia rabbit and the adiponectin concentration secreted by adipocytes in normal rabbits.@*METHODS@#Ten male New Zealand white rabbits fed with high cholesterol diet for 8 weeks were randomly divided into 2 groups: (1) The high cholesterol group maintained a high cholesterol diet for 8 weeks. (2) The same cholesterol diet plus niacin (0.4g/kg*d ) were administrated for 6 weeks in the niacin group. A control group was fed with normal diet for 14 weeks. Subcutaneous adipose from the control group was collected for adipocyte culture. Matured adipocytes were incubated with various concentrations of niacin (0, 0.25, 0.5, 1.0, and 2.0micromol/L). Adiponectin concentrations in the serum and adipocyte culture supernatant were measured by enzyme-linked-immunosorbent assay.@*RESULTS@#Compared with the control group, rabbits in the high cholesterol group showed higher serum levels of total cholesterol, and low density lipoprotein cholesterol (LDL-C), all of which were significantly reduced by niacin treatment (P<0.01),and serum high density lipoprotein-cholesterol (HDL-C) significantly increased (P<0.01). At 8th week, the mean adiponectin concentration of rabbits fed with high cholesterol diet was significantly lower than that of the control group[(1.268+/-0.039)mg/L vs.(1.449+/-0.107)mg/L,P<0.01]. Niacin treatment significantly elevated the serum adiponectin level which was positively related to HDL-C,and negatively related to TC and LDL-C. Cell experiment in vitro indicated that niacin could significantly induce the adiponectin secretion of adipocytes in a dose-dependent manner.@*CONCLUSION@#Niacin can significantly promote the adiponectin secretion of adipocytes, suggesting that niacin probably has an ability of elevating the serum adiponectin level in addition to lipid-lowering effect.

Practice and exploration on physiology interactive learning website / 中华医学教育探索杂志

The network-based course is a teaching resource based on modern multimedia technology and network-based teaching environment.In order to improve the quality of teaching,we designed and developed the physiology interactive learning website,including online class,study column,online test and network resource database.The teaching practice indicates teaching by the website has many merits such as rich content,giving prominence to emphasis,combination of word and picture and good mutuality.