Immunohistochemical study of osteopontin in the spinal cords of rats with clip compression injury

Expression of osteopontin (OPN) was investigated in the spinal cords of rats with clip compression injury. Western blot analysis demonstrated that OPN protein increased significantly in the spinal cord during the early stages after injury. The increased expression of OPN was partially paralleled by that of proliferating cell nuclear antigen (PCNA). Immunohistochemical staining showed that OPN was expressed in proliferating activated microglia/macrophages in core lesions and in some astrocytes at the periphery of lesions. These results indicate that expression of OPN protein increases mainly in activated microglia/macrophages after spinal cord injury, suggesting that OPN is related to cell proliferation during the early stages after injury, probably leading to tissue remodeling.     

Coccidia-induced mucogenesis promotes the onset of necrotic enteritis by supporting Clostridium perfringens growth

This study tested the hypothesis that a host mucogenic response to an intestinal coccidial infection promotes the onset of necrotic enteritis (NE). A chick NE model was used in which birds were inoculated with Eimeria acervulina and E. maxima and subsequently with Clostridium perfringens (EAM/CP). A second group of EAM/CP-infected birds was treated with the ionophore narasin (NAR/EAM/CP). These groups were compared to birds that were either non-infected (NIF), or infected only with E. acervulina and E. maxima (EAM), or C. perfringens (CP). The impact of intestinal coccidial infection and anti-coccidial treatment on host immune responses and microbial community structure were evaluated with histochemical-, cultivation- and molecular-based techniques. Barrier function was compromised in EAM/CP-infected birds as indicated by elevated CFUs for anaerobic bacteria and C. perfringens in the spleen when compared to NIF controls at day 20, with a subsequent increase in intestinal NE lesions and mortality at day 22. These results correlate positively with a host inflammatory response as evidenced by increased ileal interleukin (IL)-4, IL-10 and IFN-gamma RNA expression. Concurrent increases in chicken intestinal mucin RNA expression, and goblet cell number and theca size indicate that EAM/CP induced an intestinal mucogenic response. Correspondingly, the growth of mucolytic bacteria and C. perfringens as well as alpha toxin production was greatest in EAM/CP-infected birds. The ionophore narasin, which directly eliminates coccidia, reduced goblet cell theca size, IL-10 and IFN-gamma expression, the growth of mucolytic bacteria including C. perfringens, coccidial and NE lesions and mortality in birds that were co-infected with coccidia and C. perfringens. Collectively the data support the hypothesis that coccidial infection induces a host mucogenic response providing a growth advantage to C. perfringens, the causative agent of NE.     

Protective effect of Pasteurella multocida cell-free antigen and toxoid against challenge with toxigenic strains of pasteurella multocida in mice

The cell-free antigen (CFA) obtained from the culture supernatant of Pasteurella multocida (P. multocida) and the toxin (PMT) purified from CFA were inactivated and mixed with oil adjuvant to prepare a trial vaccine. Both of the mice immunized with CFA and PMT toxoid vaccine were noticeably protected against intratracheal challenge with toxigenic strains of P. multocida. Nevertheless, the protective indices of the mice immunized with CFA vaccine indicate that it is more protective and clears away the bacteria more promptly than in the mice immunized with PMT vaccine. The results suggested that CFA would possibly be good as an effective antigen to toxigenic strains of P. multocida infection.     

小鼠骨桥蛋白的原核表达及多克隆抗体的制备与鉴定

为探索骨桥蛋白(OPN)调控骨代谢及与肿瘤的关系,本试验利用DNAMAN与Mega 5.02软件分析OPN蛋白系统进化关系;采用RT-PCR与分子克隆方法制备OPN蛋白表达载体;利用SDS-PAGE电泳切胶纯化、尿素浓度梯度复性获得OPN蛋白,免疫小鼠制备OPN蛋白多克隆抗体;采用ELISA法检测抗体滴度,Western blotting检测抗血清特异性。OPN序列的同源性比对分析发现,在不同进化层次动物中均存在Arg-Gly-Asp(RGD)短肽重复序列;OPN序列系统进化分析显示,OPN在动物间具有明显的进化趋势;提取小鼠肝脏RNA,OPN重组载体双酶切、DNA测序鉴定结果及蛋白表达、纯化条带大小均与预测一致;ELISA法确认OPN抗血清滴度达1:1 600;Western blotting证实抗血清具有很好的特异性。结果表明本试验对OPN序列进行了遗传进化分析,成功克隆、表达、纯化及复性OPN蛋白,并制备、鉴定了小鼠多克隆抗体。     

Prokaryotic Expression,Polyclonal Antibody Preparation and Identification of Mouse Osteopontin Protein

In order to explore the function of osteopontin (OPN) regulated bone metabolism and the relationship between OPN and tumor, DNAMAN and Mega 5.02 softwares were used to analyze phylogenetic relationships of OPN protein, the OPN expression vector was prepared by RT-PCR and molecular clone; OPN was purified by SDS-PAGE gel slices, renatured by urea concentration gradient, and immunized mice to prepare the polyclonal antibody; The antibody titer and specificity were detected by ELISA and Western blotting, respectively.The homology comparison result of OPN sequence showed that it existed short repeat sequenc Arg-Gly-Asp (RGD) in different animals with evolution levels.Phylogenetic tree of OPN sequence showed that OPN had obvious evolution trend among animals.RNA was extracted, OPN recombinant vector had been digested and sequenced to confirm the correct construction, and strip lengths of OPN expression and purification were agreed with the prediction.The OPN antiserum titer was 1:1 600 detected by ELISA, and Western blotting proved that the antiserum had good specificity.We had successfully analyzed genetic evolution relationship of OPN sequence, gotten OPN expression vector, purified and renatured OPN, and prepared and identified the polyclonal antibody against OPN.     

Effect of active immunization against LHRH or LH in boars: reproductive consequences and performance traits

Forty crossbred boars were equally divided into eight groups at birth. Four groups were immunized (200 micrograms/boar) at 12 wk of age against either luteinizing hormone-releasing hormone (LHRH) conjugated to human serum globulin (LHRH-hSG) in complete Freund's adjuvant (CFA), LHRH-hSG in muramyldipeptide adjuvant (PEP), procine luteinizing hormone (LH) conjugated to hSG (pLH-hSG) in CFA or ovine LH (oLH) in CFA. Equal doses of boosters were given in either PEP or incomplete Freund's adjuvant (IFA) at 16 and 18 wk of age. Two groups of boars were immunized with either hSG + CFA or hSG + PEP (adjuvant controls). Two groups were castrated either at the time of weaning (castrate weaning) or at 16 wk when immunized boars were given their first booster injections (castrate booster). All pigs were slaughtered at 24 wk of age. Serum levels of LH and testosterone (T), LHRH or LH antibody titers, as well as testicular and accessory sex gland weights and histology were determined. By wk 16, LHRH antibody titers began to rise in those boars immunized against LHRH-hSG. Luteinizing hormone-releasing hormone antibody titers on wk 18, 20 and 22 were greater than those at wk 16. By 22 wk of age, LHRH-hSG boars had non-detectable plasma LH and T and reduced weights of testes and acessory sex glands. Boars immunized against oLH did not respond to treatment, whereas pLH-hSG boars showed a reduction in serum T levels and accessory sex gland weights. Immunization had no effect on average daily gain, hot carcass weights or loin eye area.(ABSTRACT TRUNCATED AT 250 WORDS)     

饲粮添加包被蛋氨酸和包被叶酸对杜寒杂交二代公羔生长性能、养分消化代谢和瘤胃微生物蛋白质合成的影响

本试验旨在研究饲粮添加包被蛋氨酸（coated methionine,CM）和包被叶酸（coated folic acid,CFA）对杜寒杂交二代公羔生长性能、养分消化代谢及瘤胃微生物蛋白质合成的影响。采用2×2完全随机试验设计,试验因素为按照试验动物体重添加CFA（0 mg,CFA-或4 mg·（kg·d）^-1,CFA+）和CM（0 g,CM-或0.1 g·（kg·d）^-1,CM+）。将40只体重相近（（30.08±1.34）kg）的4月龄杜寒杂交二代公羔随机分为4组,分别饲喂基础日粮（CM-&CFA-）、基础日粮+CFA 4 mg·（kg·d）^-1（CM-&CFA+）、基础日粮+CM 0.1 g·（kg·d）^-1（CM+&CFA-）、基础日粮+CFA 4 mg·（kg·d）^-1+CM 0.1 g·（kg·d）^-1（CM+&CFA+）。试验期85 d,其中预试期15 d,正试期70 d,在试验期结束前10 d开展消化代谢试验。试验结果表明：1）饲粮添加CFA显著提高了1~30 d平均日增重和饲料转化率（P<0.05）,而添加CM仅显著提高了1~30 d饲料转化率（P=0.036）,对1~30 d平均日增重无显著影响（P>0.05）;饲粮中添加CFA、CM显著提高了杜寒杂交二代公羔31~60 d和1~60 d平均日增重及相应阶段饲料转化率（P<0.05）,二者对1~60 d饲料转化率具有显著互作效应（P=0.007）;2）饲粮添加CFA显著提高了干物质、有机物、中性洗涤纤维和酸性洗涤纤维的表观消化率（P<0.05）,饲粮添加CM有提高有机物表观消化率的趋势（P=0.066）及增加干物质消化率的趋势（P=0.095）,二者对养分消化率无显著互作效应（P>0.05）;3）饲粮添加CFA、CM显著降低了尿能排出量,显著提高了能量消化率和代谢率（P<0.05）;通过显著降低粪氮、尿氮及总排泄氮（P<0.05）,从而显著提高了氮的表观消化率和沉积氮水平（P<0.05）,且二者同时添加对减少氮排泄、提高氮消化率有显著互作效应（P<0.05）;4）饲粮添加CM、CFA显著提高嘌呤衍生物的尿排泄量,提高瘤胃微生物蛋白质合成量（P<0.05）。饲粮添加CM、CFA提高干物质和有机物表观消化率,改善总能消化率和代谢率,增加氮表观消化率和氮沉积量,促进杜寒杂交二代公羔的生长。     

Identification of anti-idiotypic antibody in dogs

There is general agreement that idiotype/anti-idiotype (id/anti-id) networks are important mechanisms of immune regulation, based primarily on studies conducted using inbred laboratory animals. To determine if anti-idiotypic antibody (anti-id) could be induced during an immune response in outbred dogs, the dogs were immunized to the hapten-carrier combination dinitrophenol-ascaris (DNP-ASC) and subsequently immunized with autologous antibody in complete Freund's adjuvant (CFA). Auto-anti-id was detected in three of five dogs during the DNP-ASC response. A cross-reactive anti-id was detected in dogs immunized with autologous antibody when a mouse monoclonal antibody was used as the id. These experiments further suggest that the regulation of the immune response via network interaction, as first illucidated in inbred animals, may occur in dogs.     

松果菊苷对大鼠成骨细胞胃桥素基因和蛋白质的影响

为了探讨松果菊苷对体外培养大鼠成骨细胞骨桥素(OPN)mRNA和蛋白质表达的影响,选取出生24 h内的SD大鼠颅盖骨,依次用胰酶和胶原酶消化分离培养成骨细胞,用不同浓度的松果菊苷作用于第3代细胞,选取雌二醇为阳性对照,分别在药物作用24、48和72 h后提取细胞总RNA的表达量,用荧光定量PCR(FQ-PCR)法检测细胞中OPN mRNA的表达量;用Western blotting检测细胞中OPN蛋白的表达量。结果显示,各浓度松果菊苷在各个时间段均增加OPN基因的表达,尤其是5×10^-5和5×10^-6 mg/mL松果菊苷在各时间段均具有显著或极显著地促进作用(P＜0.05或P＜0.01);各浓度组在作用48 h时蛋白质的表达量均高于对照组。结果表明,松果菊苷对成骨细胞OPN基因表达有显著的促进作用,同时对OPN蛋白的分泌有促进的趋势。     

Comparative cardiac pathological changes of Atlantic salmon (Salmo salar L.) affected with heart and skeletal muscle inflammation (HSMI), cardiomyopathy syndrome (CMS) and pancreas disease (PD)

The heart is considered the powerhouse of the cardiovascular system. Heart and skeletal muscle inflammation (HSMI), cardiomyopathy syndrome (CMS) and pancreas disease (PD) are cardiac diseases of marine farmed Atlantic salmon (Salmo salar) which commonly affect the heart in addition to the skeletal muscle, liver and pancreas. The main findings of these diseases are necrosis and inflammatory cells infiltrates affecting different regions of the heart. In order to better characterize the cardiac pathology, study of the inflammatory cell characteristics and cell cycle protein expression was undertaken by immunohistochemistry. Immunohistochemistry was performed on paraffin embedded hearts from confirmed diseased cases applying specific antibodies. The inflammatory cells were predominantly CD3⁺ T lymphocytes. The PD diseased hearts exhibited moderate hypoxia inducible factor-1α (HIF1α) immuno-reaction that suggested tissue hypoxia while recombinant tumor necrosis factor-α (rTNFα) antibody identified putative macrophages and eosinophilic granulocytes (EGCs) in addition to endocardial cells around lesions. There were strong to low levels of major histocompatibility complex (MHC) class II immunostaining in the diseased hearts associated with macrophage-like and lymphocyte-like cells. The diseased hearts expressed strong to low levels of apoptotic cells identified by caspase 3 and terminal deoxynucleotidyl transferase nick-end labeling (TUNEL) staining. The strong signals for proliferative cell nuclear antigen (PCNA) and TUNEL, and moderate levels of caspase 3 immuno-reactivity suggested a high cell turnover where DNA damage/repair might be occurring in the diseased hearts. Interestingly, the apparently similar cardiac diseases exhibited differences in the immunopathological responses in Atlantic salmon.     

Chronic lung inflammation affects plasma amino acid concentrations in pigs

Metabolic changes associated with inflammatory processes and immune response can modify protein and AA requirements. Improved knowledge of these processes will provide opportunities for nutritional intervention to sustain growth and animal defense at the same time. The objective of the study was to identify AA whose metabolism is affected by chronic lung inflammation. Six pairs of littermate piglets were selected at 28 d of age on the basis of their BW. After catheterization of the jugular vein, one littermate received complete Freund's adjuvant (CFA) intravenously, whereas its littermate was injected with a sterile saline solution (CON). Piglets within a litter were pair-fed in order to avoid confounding effects of feed intake and inflammation on plasma AA concentrations. Blood samples were taken after an overnight fast and 2 h after the morning meal for 9 d. Rectal temperature, food consumption, weight gain, plasma haptoglobin, and AA concentrations were measured. The CFA injection decreased food intake, and increased body temperature and plasma haptoglobin concentration. Plasma tryptophan, glutamine, proline, glycine, tyrosine, ornithine, total AA concentrations, and the ratio of tryptophan to large neutral AA were less in CFA than in CON (P < 0.05), independent of time and meal. In contrast, plasma histidine concentration was higher (P < 0.05) in CFA than in CON pigs. Plasma serine, arginine, alanine, asparagine, and total AA concentrations were lower in CFA than in CON pigs only in the fed state (P < 0.05). Among essential AA, only plasma tryptophan concentration was lower (P < 0.01) in CFA than in CON pigs in both fasted and fed state. These results show that chronic lung inflammation affects individual AA differently and suggest that the utilization of some AA increased during chronic lung inflammation in pigs. Activation of tryptophan catabolism enzyme indoleamine 2,3-dioxygenase seems a relevant hypothesis to explain the increased tryptophan utilization, although its incorporation in acute-phase proteins and the existence of other catabolic pathways cannot be excluded.     

Type-I hypersensitivity as a component of eosinophilic myositis (muscular sarcocystosis) in cattle

Eight bovine hearts with lesions of eosinophilic myositis (EM) and 2 bovine hearts without EM lesions were collected at slaughter. Blood samples from these 10 hearts, and the heart of a newborn calf also were collected. Histologically, Sarcocystis cruzi was identified in the 8 hearts with EM lesions and the 2 hearts without EM lesions, but not in the heart of the newborn calf. Serum was harvested from the 10 blood samples and was used in homologous, modified, passive cutaneous anaphylaxis tests. Antigen was prepared from S cruzi bradyzoites isolated from the 2 hearts without EM lesions. Serum samples from the 8 cattle with EM lesions reacted positively to S cruzi antigen. When heat-inactivated IgE in serum (56 C for 4 hours) was used, all passive cutaneous anaphylaxis responses were considered negative. Using ELISA, serum IgE concentrations from the 10 cattle with and without EM lesions were 2.2 to 9 U/ml. As determined by radial immunodiffusion, IgM concentrations were 80 to 215 mg/dl. Immunoglobulin G concentrations were 420 to 2,050 mg/dl, but most were less than or equal to 1,700 mg/dl. Immunoglobulin A concentrations were 0 to 62 mg/dl; 1 steer with EM lesions had 0 mg/dl. Double-gel immunodiffusion confirmed the presence of Sarcocystis-specific precipitating antibodies. Sera from the 10 cattle with and without EM lesions formed at least 1 precipitin band.     

The Effects of Adjuvants on Autoimmune Responses Against Testicular Antigens in Mice

Experimental autoimmune orchitis (EAO) is a model of immunologic male infertility and pathologically characterized by lymphocytic inflammation, which causes breakdown of the testicular immune privilege with spermatogenic disturbance. Generally, murine EAO is induced by immunization with testicular homogenate (TH) from the testes of donor mice + complete Freund''s adjuvant (CFA) + Bordetella pertussigens (BP), and it has been considered that treatment with these two adjuvants is required to enhance the immune response against testicular antigens. However, there remains a possibility that CFA and BP may affect autoimmune responses against the testicular antigens without TH. In the present study, we examined this possibility using real-time RT-PCR, Western blotting and immunohistochemical staining. The results demonstrated that immunization with TH in combination with CFA and BP evoked more severe EAO than that with only TH. Real-time RT-PCR analyses revealed that Fas mRNA expression in TH+CFA+BP-induced EAO was significantly higher than that in TH-induced EAO. Interestingly, IL-6 mRNA expression dramatically increased in TH+CFA+BP-induced EAO; however, no apparent change in IL-6 mRNA expression occurred in TH-induced EAO. It was also noted that treatment with CFA and BP alone augmented autoimmune reactions against some testicular autoantigens. These results indicates that these adjuvants are helpful in evoking severe EAO, and treatment with the adjuvants alone can evoke autoimmune reactions against some testicular autoantigens despite the use of no TH.     

Active immunization of heifers against luteinizing hormone-releasing hormone, human chorionic gonadotropin and bovine luteinizing hormone

Seventy crossbred heifers were allotted randomly to 10 treatment groups. Treatments consisted of active immunization against ovalbumin (OV) conjugates of luteinizing hormone-releasing hormone (LHRH), human chorionic gonadotropin (hCG) and bovine luteinizing hormone (bLH) with each of three adjuvants. The adjuvants were complete Freund's adjuvant (CFA), M103(6) and 6VR6. Control animals were immunized against OV alone using CFA. Bulls were placed with the heifers following immunization to allow comparison of pregnancy rates between groups. Blood samples were collected weekly for 14 wk to determine antibody concentrations. Significant levels of circulating LH or LHRH antibodies were detected in heifers immunized with each of the hormone conjugates. Complete Freund's adjuvant was the most effective for stimulating antibody response to these antigens; however, M103 was equally effective when used with bLH or hCG conjugates. None of the heifers in the bLH-OV-CFA, bLH-OV-M103 or LHRH-OV-CFA immunization groups was pregnant at slaughter, whereas 71% of the OV-CFA control heifers were pregnant. Fertility suppression may be achieved in the bovine by active immunization against any of these three hormone conjugates. However, the duration of this study (8 wk after immunization) does not allow evaluation of the duration of effectiveness of each of the treatments.     

Construction of a Salmonella Gallinarum ghost as a novel inactivated vaccine candidate and its protective efficacy against fowl typhoid in chickens

In order to develop a novel, safe and immunogenic fowl typhoid (FT) vaccine candidate, a Salmonella Gallinarum ghost with controlled expression of the bacteriophage PhiX174 lysis gene E was constructed using pMMP99 plasmid in this study. The formation of the Salmonella Gallinarum ghost with tunnel formation and loss of cytoplasmic contents was observed by scanning electron microscopy and transmission electron microscopy. No viable cells were detectable 24 h after the induction of gene E expression by an increase in temperature from 37 °C to 42 °C. The safety and protective efficacy of the Salmonella Gallinarum ghost vaccine was tested in chickens that were divided into four groups: group A (non-immunized control), group B (orally immunized), group C (subcutaneously immunized) and group D (intramuscularly immunized). The birds were immunized at day 7 of age. None of the immunized animals showed any adverse reactions such as abnormal behavior, mortality, or signs of FT such as anorexia, depression, or diarrhea. These birds were subsequently challenged with a virulent Salmonella Gallinarum strain at 3 weeks post-immunization (wpi). Significant protection against the virulent challenge was observed in all immunized groups based on mortality and post-mortem lesions compared to the non-immunized control group. In addition, immunization with the Salmonella Gallinarum ghosts induced significantly high systemic IgG response in all immunized groups. Among the groups, orally-vaccinated group B showed significantly higher levels of secreted IgA. A potent antigen-specific lymphocyte activation response along with significantly increased percentages of CD4⁺ and CD8⁺ T lymphocytes found in all immunized groups clearly indicate the induction of cellular immune responses. Overall, these findings suggest that the newly constructed Salmonella Gallinarum ghost appears to be a safe, highly immunogenic, and efficient non-living bacterial vaccine candidate that protects against FT.     

Activin A as a novel biomarker of equine inflammatory abdominal disease: preliminary findings

This study evaluated the inflammatory mediator activin A in horses with acute abdominal disease and compared this putative novel biomarker with serum amyloid A (SAA). Thirty-three adult horses referred for evaluation of acute abdominal disease were grouped into three lesion categories, non-strangulating, strangulating or inflammatory. Eleven healthy adult horses served as controls. Serum activin-A was significantly increased in animals with inflammatory or strangulating lesions compared with controls. Horses with non-strangulating, strangulating or inflammatory lesions had significantly elevated SAA concentrations. Activin A, along with other biomarkers, may be useful in monitoring inflammation in cases of acute abdominal disease in horses. Further validation is warranted to determine the utility of this biomarker in evaluating the effectiveness of novel anti-inflammatory treatments for equine colic and endotoxaemia.     

Fibrinolytic Activity in Plasma From Horses With Gastrointestinal Diseases: Changes Associated With Diagnosis, Surgery, and Outcome

Plasma fibrinolytic activity was evaluated over 5 consecutive days in 59 horses admitted to the Large Animal Teaching Hospital with acute gastrointestinal diseases. Only horses hospitalized for at least 5 days were included in the study. Tissue plasminogen activator (tPA) and plasminogen activator inhibitor type-1 (PAI-1) were quantitated using standard chromogenic activity assays. Statistical analyses were performed using analysis of variance; differences were considered significant when P < .05. Activity of PAI-1, the primary endogenous inhibitor of fibrinolysis, was significantly increased on hospital days 2, 4, and 5 in horses that died, when compared with those that were discharged from the hospital. Plasma PAI-1 activity was not different at admission, but was significantly increased on hospital days 2 and 3 in horses that underwent surgery, when compared with those that did not, suggesting an acute phase response to surgical intervention. Horses with strangulating intestinal lesions had significantly increased PAI-1 activity on day 3, while PAI-1 activity was significantly greater in horses with inflammatory conditions at the time of admission, when compared with horses with strangulating or nonstrangulating/noninflammatory lesions. Among all horses, PAI-1 activity was significantly higher and tPA activity was significantly lower on day 2 when compared with other hospital days. These results suggest that fibrinolysis is inhibited early in the course of inflammatory gastrointestinal diseases and in response to surgery. In addition, among all horses, the prognosis for survival was poor for those with persistently increased PAI-1 activity, reflecting treatment failure and the loss of hemostatic regulation.     

The expressions of HSP70 and ��B-crystallin in myocarditis associated with foot-and-mouth disease virus in lambs

This study describes the expression of heat shock protein70 (HSP70) and alpha-basic-crystallin (α-BC) and their association with apoptosis and some related adaptor proteins in the pathogenesis of foot-and-mouth disease virus (FMDV)-induced myocarditis in lambs. HSP70 was generally overexpressed in the myocardial tissues and inflammatory cells of FMDV-induced myocarditis with differential accumulation and localization in same hearts when compared to non-foot-and-mouth disease control hearts. α-BC immunolabeling showed coarse aggregations in the Z line of the cardiomyocytes in FMDV-infected hearts in contrast to control hearts. Overall, the results of this study show that the anti-apoptotic proteins, HSP70 and α-BC, were overexpressed with increased apoptosis in FMDV-infected heart tissues. Both proteins failed to protect the cardiomyocytes from apoptosis as defense mechanisms to the FMDV during the infection, suggesting that the virus is able to increase apoptosis via both downregulation and/or upregulation of these anti-apoptotic proteins.     

OPN5对鸭颗粒细胞凋亡、增殖及类固醇激素生成的影响

旨在研究视神经蛋白（neuropsin,OPN5）对鸭颗粒细胞凋亡、增殖及类固醇激素生成的影响。本研究分别对鸭颗粒细胞进行OPN5过表达质粒和OPN5 siRNA处理72 h （n=6）,应用EdU细胞增殖检测技术、流式细胞技术、Annexin V-FITC、RT-PCR、Western blot及ELISA技术系统地检测颗粒细胞的增殖、凋亡情况及生殖相关基因的mRNA、蛋白水平及激素水平的变化规律。结果显示,OPN5过表达能促进鸭卵泡颗粒细胞增殖,抑制鸭卵泡颗粒细胞凋亡;能极显著地促进GnRHR、FSHR、LHR的表达 ,并抑制GnIH、GnIHR的表达（P<0.01）;能显著或极显著上调StAR、CYP11A1、3β-HSD、CYP17A1、CYP19A1的mRNA水平（P<0.05或P<0.01）;显著升高OPN5、3β-HSD及CYP19A1的蛋白表达水平（P<0.05）和极显著升高E2、P4分泌水平（P<0.01）,并极显著降低INHβ的水平（P<0.01）。OPN5 siRNA能够显著降低OPN5的表达水平（P<0.01）,抑制卵泡颗粒细胞增殖并促进凋亡,极显著下调GnRH、GnRHR、FSHR、LHR（P<0.01）,促进GnIH、GnIHR的表达（P<0.01）;并极显著抑制StAR、CYP11A1、CYP17A1的表达（P<0.01）;显著降低OPN5、3β-HSD及CYP19A1蛋白表达水平（P<0.05）及极显著降低E2、P4的分泌水平（P<0.01）,并能极显著升高INHβ的水平（P<0.01）。研究表明,OPN5能促进鸭卵泡颗粒细胞的增殖,抑制其凋亡,促进颗粒细胞中类固醇激素的生成和分泌。