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Background The incidence of dry eye is gradually increased,and researches showed that inflammation participated in the pathogenesis and development of dry eye.The current therapy for dry eye can only relieve symptom but not achieve final cure.Stem cell therapy has been used in the treatment of limbal stem cell deficiency.However,whether it is feasible for the stem cell treating dry eye is still unclear.Objective This study attempted to investigate a new approach to treat dry eye syndrom by using human umbilical cord mesenchymal stem cells (HUCMSCs).Methods The use and care of experimental animals complied with the Tsinghua University School of Medicine Laboratory Animal Care Details.HUCMSCs were cultured and cell suspension was prepared with the cell density of 5×105/ml.Twenty 24-week-old male NOD/Ltj mice were randomized into 4 groups.0.1 ml PBSHUCMSCs suspension was injected via tail vein or lacrimal respectively in the caudal vein injection group and lacrimal injection group,and 10 μ1 PBS-HUCMSCs suspension was topically administed in the eye drops group.The NOD/Ltj mice without any treatment served as the model group.Five male ICR mice were used as the normal control group.Tear secretion was quantitatively detected with phenol red cotton thread in 1,2,3 weeks after injection,and corneal epithelial defect was scored by fluorescein staining.The serum levels of interleukin (IL)-6,IL-17a,tumor necrosis factor-α (TNF-αt) and interferon-γ (IFN-γ) were assayed by ELISA.The relative expression levels of p65,Stat3,Stat5 and extracellular signal-regulated kinases (Erk)-1 in lacrimal gland were detected by Western blot.Results The tear secretion amount was significantly different among the model group,caudal vein injection group,lacrimal injection group and eye drops group in various time points (1 week:F =3.700,P =0.040;2 weeks:F =5.150,P =0.008;3 weeks:F=10.130,P＜0.001).The tear secretion amount was increased in the caudal vein injection group and lacrimal injection group compared with the model group in different time points (all at P＜0.05),and no significant difference was seen in tear secretion amount between eye drops group and model group among various time points (all at P＞0.05).The fluorescein staining score was 3.00±0.63,9.40±1.62,5.20±1.17,4.20±1.17 and 7.20±0.98 in the ICR mouse control group,model group,caudal vein injection group,lacrimal injection group and eye drops group 1 week after injection respectively,and the scores were significantly lower in the caudal vein injection group,lacrimal injection group and eye drops group than those in the model group (P =0.001,0.000,0.033).The serum levels of IL-6,IL-17a and TNF-α in the caudal vein injection group were evidently lower than those in the model group (t =4.70,3.46,11.0,all at P＜0.01),but no significant difference was displayed in the serum IFN-γ level among the five groups (F=1.740,P=0.170).The expressions of STAT5 were significantly decreased in the mice treated with tail vein injection and lacrimal injection compared with mice without treatment (both at P＜0.05).Conclusions Administration of HUCMSCs via intravenous and lacrimal injection can alleviate the inflammatory response during progression of dry eye syndrome by down-regulating the serum level and expression of inflammation-related factors in NOD/Ltj mice.The topical administration of HUCMSCs eye drops can attenuate the symptom of dry eyes by lubricating the cornea and suppling nutrition.
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Background Recently, the morbidity of orbital lymphoma increased gradually, and we have made deeper research in pathology,therapy and pathogenesis of the disease.There were few reports of mice model of orbital lymphoma up to now for its lower morbidity and culture difficulty.Objective This study was to establish a mouse model of orbital diffuse large B cell lymphoma (DLBCL) by injection of systemic DLBCL cell line pfeiffer.Methods Ten SPF BALB/c mice and 5 nod-SCID mice were radiated firstly using 137Cs,with the absorption dose 3.5 Gy in the BALB/c mice and 2.6 Gy in the nod-SCID mice,and then pfeiffer cells were intraobitally injected in 4eyes of BALB/c mice (orbital injection group) and suncutaneously injectied in 4 eyes of BALB/c mice and 4 eyes of nod-SCID mice (subcutaneous injection group) at the concentration of 1.5×l08/ml.The developing status of tumors were examined once per day and the growth curve was drawn.The tumors and nearby lymph nodes were obtained 54 days after injection for the preparation of 4 μm thickness of serial sections.Hemotoxylin-eosin staining was used to examine the histopathology of the tumors, and immunochemistry was employed to detect the expressions of CD20,CD79α and CD45RO proteins.The tumors were typed based on the expressions of CD10, BCL-6 and mum-1 in the specimens,and the expressions of Ki-67 and survivin were assyed to assess the prognosis of the tumor.All the results were compared with 3 diagnosed human orbital DLBCL sections.The use and care of the mice complied with Chinese Administration Rule of Laboratory Animal.Results The tumor formation rates were 100％ in both the orbital injection group and subcutaneous injection group, and the tumors grew much faster in nod-SCID mice than BALB/c mice.Infiltration of tumor cells in lymph nodes were found in the subcutaneous injection group rather than the orbital injection group.The pathological features were accordant among the orbital injection group, subcutaneous injection group and human orbital DLBCL sections.The number of ＜50％ and ≥50％ CD20+ specimens was 3 and 5,CD79αwas 2 and 6,CD45RO was 8 and 0 in the BALB/c mice;while that in the nod-SCID mice was 1 and 3 in CD20,0 and 4 in CD79α,4 and 0 in CD45RO;the number of human orbital DLBCL specimens was 1 and 2 in CD20,1 and 2 in CD79α,2 and 1 in CD45RO,without significant differences among them (all at P=1.00).No significant differences were seen in Ki-67+ number and survivin+ number among the BALB/c mice, nod-SCID mice and human orbital DLBCL specimens (all at P=1.00).The detection of CD10,BCL-6 and mum-1 expressions indicated that the tumors of BALB/c mice,nod-SCID mice and human orbital DLBCL specimens all were the non-germinal center B cell-like types.Conclusions The orbital and subcutaneous DLBCL mouse models are successfully established by injection of pfeiffer cell line.There are the same findings and features in biological behavior, pathology and immunohistochemistry in orbital,subcutaneous models with human orbitl DLBCL.Nod-SCID mice appear to be more suitable for the growth of lymphoma cells.
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Objective To investigate the mechanism of pioglitazone preventing diabetes and the role of nuclear factor of actived T cells (NFAT) on non-obese diabetic(NOD) mice .Methods (1)Female NOD mice at 4 weeks of age were randomly divided into pioglitazone group(n=21) and control group(n=21) .The accumulative diabetes incidence was followed-up to 30 weeks of age in each group of NOD mice .(2)Pancreas were removed from NOD mice at 12 weeks of age in each group(n=15) to score insulitis se-verity by routine HE staining .IL-4 ,IFN-γand peroxisome proliferator-activated receptor γ(PPARγ) mRNA levels in spleens were tested by RT-PCR .IL-4 and IFN-γlevels in sera ,the activity of PPARγand NFATc1 nuclear protein in spleens were measured by enzyme linked immunosorbent assay (ELISA) .Results (1) At 15 weeks of age ,the diabetes incidence was 4 .76% in pioglitazone group ,and 33 .33% in control group(P0 .05) .(2) At 12 weeks of age ,the insulitis score in pioglitazone group was lower than that in control group[(1 .79 ± 0 .75) vs .(2 .38 ± 0 .66) ,P<0 .05] .(3) IFN-γ mRNA level in pioglitazone group was lower than that in control group[(0 .16 ± 0 .07) vs .(0 .53 ± 0 .26) ,P<0 .05] ,and PPARγmRNA level in pioglitazone group was higher than that in control group(0 .91 vs .0 .25 ,P<0 .05) .(4)IFN-γ level in pioglitazone group was lower than that in control group [(561 .05 ± 78 .61)pg/mL vs .(666 .43 ± 28 .42)pg/mL ,P<0 .05] .(5)At 12 weeks of age ,the spleen PPARγnuclear protein activity in pioglitazone group was higher than that in control group [(0 .05 ± 0 .01) vs .(0 .02 ± 0 .01) ,P<0 .05)] ,and NFATc1 nuclear protein activity was low-er than that in control group[(0 .23 ± 0 .04) vs .(0 .33 ± 0 .04) ,P<0 .05] .Conclusion Pioglitazone could activate PPARγ nuclear protein ,inhibit activity of NFATc1 nuclear protein ,downregulate IFN-γ,diminish Th cells deviating to Th1 ,and sequently prevents insulitis and diabetes onset in NOD mice .
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CONTEXTO: A terapia a laser de baixa intensidade (LLLT) tem sido relatada como importante moduladora da cicatrização de feridas cutâneas aumentando a proliferação fibroblástica associada ao aumento da expressão da citocina fator transformador de crescimento- β2 (TGF-βB2). OBJETIVO: No presente estudo foram avaliados os efeitos da LLLT sobre a expressão da enzima ciclooxigenase 2 (COX2) no sítio do reparo tecidual utilizando o modelo experimental com camundongos diabéticos não obesos (NOD) para estudar a cicatrização de feridas cutâneas. MÉTODOS: Foram utilizados 30 camundongos NOD, destes 14 ficaram diabéticos e foram divididos em dois grupos: o grupo I (n=7) foi submetido a um procedimento cirúrgico de feridas cutâneas e o grupo II (n=7) foi submetido a um procedimento cirúrgico de feridas cutâneas e tratados com LLLT. O grupo II foi submetido à LLLT nos seguintes parâmetros: 15 mW de potência, dose de 3,8 J/cm² e tempo de aplicação de 20 segundos. Após sete dias do ato cirúrgico e após aplicação do laser, os animais foram eutanasiados com sobredose de anestesia e amostras das feridas foram colhidas para posterior análise histopatológica, histomorfométrica e imuno-histoquímica. RESULTADOS: A LLLT promoveu a inibição da expressão da COX2 em feridas cutâneas de camundongos diabéticos. CONCLUSÃO: Em conjunto, os resultados sugeriram que a LLLT é capaz de modular negativamente a expressão da enzima COX2 contribuindo para o controle da resposta inflamatória em feridas cutâneas de camundongos NOD.
BACKGROUND: Low-level laser therapy (LLLT) has been reported to modulate the healing of wounds by inducing an increase in fibroblast number associated with increased expression of the cytokine transforming growth factor-β2 (TGF-β2). OBJECTIVE: In the present study, the effect of LLLT on expression of COX2 at the site of tissue repair was evaluated, using an experimental model with non obese diabetic mice (NOD) to study cutaneous wound healing. METHODS: Thirty NOD mice were used, of which 14 were diabetic and were divided into two groups: group I (n=7) underwent a surgical procedure of skin wounds and group II (n=7) underwent a surgical procedure of skin wounds and treated with LLLT. Group II was submitted to LLLT in the following parameters: 15 mW of power, dose of 3.8 J/cm² and exposure time of 20 seconds. Seven days after surgery and after laser application, animals were euthanized with an overdose of anesthesia and tissue samples were collected for subsequent histological analysis, histomorphometry and immunohistochemistry. RESULTS: The LLLT has promoted the inhibition of COX2 expression in skin wounds in mice diabetic. Taken together the results suggest that LLLT modulate the expression of COX2 improved the control of inflammatory reaction in cutaneous wound lesions in NOD mice. CONCLUSION: Taken together, the results suggested that LLLT is able to negatively modulate the expression of COX2 enzyme contributing to the inflammatory response in cutaneous wounds in NOD mice.
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Animals , Mice , Diabetes Mellitus, Experimental/blood , Low-Level Light Therapy , Mice, Inbred NOD , Nitric Oxide/adverse effects , Animal Experimentation/ethics , Wounds and Injuries/veterinary , Blood Glucose/analysisABSTRACT
Objective To investigate the mechanism of preventing islet β-cell apoptosis in NOD mice with pioglitazone.Methods Female NOD mice at 4 weeks of age were divided into pioglitazone group ( n =21,0.02％pioglitazone was added into the feed ) and control group ( n =21,fed with regular diet).The accumulative incidence of diabetes was followed-up to 52 weeks of age in each group of NOD mice.Pancreas was removed from NOD mice at 12 weeks of age in each group ( n =15 ) to score severity of insulitis by routine H-E staining.The apoptotic β-cells in islets were observed with double-labeling technique of TUNEL in situ combined with standard sensitive avidin-biotin complex (sABC) immunohistochemical method.The spleens were taken for cell culture; IL-4 and IFN-γ levels in sera and supernatants of cultured splenocyte,the activity of PPARγ and NF-κB nuclear proteins in cultured splenocyte were measured by ELISA.Results (1)At 30 and 52 weeks of age,the respective incidences of diabetes were 57.1％ and 76.2％ in pioglitazone group,and 76.2％ and 90.5％ in control group ( all P＞0.05 ).At 15 weeks of age,the incidence became 4.8％ in pioglitazone group,and 33.3 ％ in control group ( P =0.045 ).( 2 ) At 12 weeks of age,the percentages of non infiltrated islet and peri-insulitis islet in pioglitazone group were higher than those in control group ( 14.73％ vs 5.69％,P＜0.01 ; and 26.02％ vs 15.72％,P＜0.01 ),and that of intraislet insulitis was lower than that in control group ( 59.25％ vs 78.59％,P＜0.01 ).The percentage of apoptotic β-cell in pioglitazone group was lower than that in control group( 6.17％ ±3.62％ vs 10.62％ ±4.43％,P=0.008 ).(3) In sera,IFN-γ level in pioglitazone group was lower than that in control group [( 561.05±78.61 ) vs ( 666.43 ± 28.42 ) pg/ml,P =0.045].In cultured splenocyte supernatant,the level of IFN-γ in pioglitazone group was lower than that in control group[(605.84+65.60) vs (692.20+44.98) pg/ml,P=0.041].(4) In cultured splenocyte,PPARγ nuclear protein activity in pioglitazone group was higher than that in control group ( 0.06 ± 0.01 vs 0.03 ± 0.01,P =0.013 ),and NF-κB nuclear protein activity was lower than that in control group ( 0.03 ± 0.01 vs 0.08± 0.01,P =0.001 ).Conclusions Pioglitazone activates PPARγ nuclear protein,inhibits activity of NF-κB nuclear protein,downregulates IFN-γ,diminishes differeutiation of Th cells to Th1,and subsequently prevents insulitis and β-cell apoptosis in NOD mice.
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Type 1 diabetes mellitus is caused by the autoimmune destruction of beta cells within the islets. In recent years, innate immunity has been proposed to play a key role in this process. High-mobility group box 1 (HMGB1), an inflammatory trigger in a number of autoimmune diseases, activates proinflammatory responses following its release from necrotic cells. Our aim was to determine the significance of HMGB1 in the natural history of diabetes in non-obese diabetic (NOD) mice. We observed that the rate of HMGB1 expression in the cytoplasm of islets was much greater in diabetic mice compared with non-diabetic mice. The majority of cells positively stained for toll-like receptor 4 (TLR4) were beta cells; few alpha cells were stained for TLR4. Thus, we examined the effects of anti-TLR4 antibodies on HMGB1 cell surface binding, which confirmed that HMGB1 interacts with TLR4 in isolated islets. Expression changes in HMGB1 and TLR4 were detected throughout the course of diabetes. Our findings indicate that TLR4 is the main receptor on beta cells and that HMGB1 may signal via TLR4 to selectively damage beta cells rather than alpha cells during the development of type 1 diabetes mellitus.
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Animals , Female , Humans , Mice , Diabetes Mellitus, Type 1/immunology , Gene Expression Regulation , Glucagon-Secreting Cells/immunology , HMGB1 Protein/genetics , Immunity, Innate , Insulin-Secreting Cells/immunology , Macrophages/immunology , Mice, Inbred C57BL , Mice, Inbred NOD , Necrosis , Protein Binding , Signal Transduction , Toll-Like Receptor 4/antagonists & inhibitorsABSTRACT
To explore the protective effect of adenovirus mediated IGF-Ⅰgene(Ad-IGF-Ⅰ)transfer on non-obese diabetic(NOD)mice. The results showed that the incidence of diabetes and degree of insulitis were significantly reduced in mice receiving Ad-IGF- Ⅰ . Treatment with Ad-IGF- Ⅰ significantly decreased apoptosis rate,expression of Fas and caspase-3, and increased expression of Bcl-xl. This study indicates the potential of IGF- Ⅰ gene therapy in protecting NOD mice from insulitis and apoptosis.
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Objective To investigate the anti-inflammatory effect of peroxisome proliferator-activated receptor-γ(PPAR-γ)in non-obese diabetic mice(NOD mice)with Sj(o)gren's syndrome(SS)．Methods Twenty 8-weeks-old female NOD mice were randomly divided into 2 groups．Rosiglitazone and normal saline were administered for the rosiglitazone group and control group respectively．At age of 12 weeks and 15 weeks，one mouse in the rosiglitazone group and the control group were killed respectively,and the others were sacrificed at the age of 17 weeks．Blood were obtained by cardiac puncture，and minor salivary glands (MSG) were resected．The histopathological changes waere examined by H&E staining．The level of IL-1β，IL-4，IL-6 and TNF-α in serum were measured by ELISA．Real-time PCR waft used to evaluate the mRNA expression level of IL-1β，IL-4，IL-6 and TNF-α in MSG．Student's t-test was used to assess the differences．Results Compared with the control group，the mice in the rosiglitazone group showed that：①histopatho-logical change was significantly ameliorated．②At the age of 17 weeks，IL-6[(26±7)vs(37±11)，t=-2．298] and TNF-α[(57±22)vs(79±21)，t=-2．188] were expressed significantly lower and IL-4[(26±13)vs(12±4)，t=2．438 ] was expressed significantly higher in serum(P<0．05)．③The expression of TNF-αwas significantly decreased and the expression of IL-4 was significantly increased in MSG (P<0．05)．Conclusion PPAR-γ can ameliorate SS on NOD mice effectively．The mechanism may be related to reduction of Th1 cytokines，and the Th1/Th2 balance is changed into Th2 predominant．
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Objective To investigate the preventive effect of complete Freund's adjuvant induced thymocyte apoptosis on NOD mice with type 1 diabetes.Methods Forty-two female NOD mice of 3 weeks old were randomly divided into complete Freund's adjuvant group(CFA)and phosphate buffer saline group(PBS)(21 each).Animals of CFA group received injection of 50?l CFA at hind foot-pad,and those in PBS group received 50?l PBS at the same location.Five mice of each group were sacrificed at the 6th and 12th week,respectively,and the remainders of each group were sacrificed at the 30th week for the examination of insular ? cell apoptosis,thymocytes apoptosis,insulitis severity and diabetes incidence.Results Both the insulitis severity score and the ? cell apoptosis declined(P
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Objective To study the effect of phosphodiesterase inhibitor pentoxifylline (PTX) on type 1 diabetes in NOD mice and its possible mechanism.Methods Blood glucose, urinary glucose, insulitis and incidence of diabetes were investigated in NOD mice treated with PTX, insulitis was observed by HE staining and the expressions of IFN ?, TNF ?, IL 10 in pancreas were measured by RT PCR technique. Results The incidence of diabetes in the PTX group was 30.0% which was significantly lower than 67.9% in the control group (P
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Objective To investigate the role of antigen-specific regulatory T cells in spontaneous diabetes of NOD mice. Methods Glutamic acid decarboxylase (GAD)-peptide specific T cells with fluorescein-labelled Class Ⅱ MHC tetramers were isolated by flow cytometer from NOD mice with spontaneous diabetes and diabetes resistant BALB/c mice. The cytokine profiles of these T cells were detected by antigen stimulated assay, ELISA and intracellular cytokine staining. Adoptively transferred diabetes was determined by intravenously injecting these T cells to NOD/scid mice. Results With different peptides working on the same strain, it showed that NOD mice-derived T cells secreted different amounts of interforn-? but comparable interlenkin (IL)-4 or IL-10, however, similar cytokine profiles were shown in BALB/c mice-derived T cells. With the same peptide working on different strains, NOD mice-derived T cells secreted less IL-2 but more IL-4 and IL-10 than BALB/c mice-derived T cells did. Interestingly, these NOD mice-derived T cells effectively inhibited diabetes development in adoptively transferred NOD/scid mice. Conclusion NOD mice-derived T cells inhibit development of diabetes, however, different cytokine profiles are expressed by these T cells induced by two adjacent GAD peptides. It suggests that these T cells are diabetes-inhibiting regulatory T cells displaying unique cytokine profiles.
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Due to their high immunostimulatory ability as well as the critical role they play in the maintenance of self-tolerance, dendritic cells have been implicated in the pathogenesis of autoimmune diseases. The non-obese diabetic (NOD) mouse is an animal model of autoimmune type 1 diabetes, in which pancreatic beta cells are selectively destroyed mainly by T cell-mediated immune responses. To elucidate initiation mechanisms of beta cell-specific autoimmunity, we attempted to generate bone marrow-derived dendritic cells from NOD mice. However, our results showed low proliferative response of NOD bone marrow cells and some defects in the differentiation into the myeloid dendritic cells. NOD dendritic cells showed lower expressions of MHC class II, B7-1, B7-2 and CD40, compared with C57BL/6 dendritic cells. In mixed lymphocyte reactions, stimulatory activities of NOD dendritic cells were also weak. Treatment with LPS, INF-gamma and anti-CD40 stimulated NOD dendritic cells to produce IL-12p70. The amount of IL-12, however, appeared to be lower than that of C57BL/6. Results of the present study indicated that there may be some defects in the development of NOD dendritic cells in the bone marrow, which might have an impact on the breakdown of self tolerance.
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Mice , Animals , Autoimmune Diseases/pathology , Autoimmune Diseases/immunology , Bone Marrow Cells/pathology , Bone Marrow Cells/immunology , Bone Marrow Cells/chemistry , Cell Differentiation/immunology , Cell Differentiation/drug effects , Dendritic Cells/pathology , Dendritic Cells/immunology , Dendritic Cells/chemistry , Diabetes Mellitus, Type 1/pathology , Diabetes Mellitus, Type 1/immunology , Enzyme-Linked Immunosorbent Assay , Granulocyte-Macrophage Colony-Stimulating Factor/pharmacology , Interleukin-12/analysis , Interleukin-4/pharmacology , Lipopolysaccharides/pharmacology , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Inbred NOD , ObesityABSTRACT
NOD mice were treated intraperitoneally with tripterygium wilfordii ployglycosidium (TWP) for 4 weeks to observe the incidence of cyclophosphamide accelerated diabetes.The apoptosis of?-cells was detected by TUNEL,the expression of caspase-3 in islets of the NOD mice was detected by immunohistochemistry and the expression of caspase-8 mRNA in pancreas by RT-PCR.The results revealed that the incidence of diabetes in TWP group was lower than that in control group.The apoptosis index of?-cells was decreased in TWP group. The expression of caspase-3 in islets and the expression of caspase-8 mRNA in pancreas in TWP group were lower than those in control group.