T-2毒素对小鼠睾丸间质细胞相关功能基因mRNA表达的影响

目的探讨T-2毒素对小鼠睾丸间质细胞相关功能基因mRNA表达的影响。方法对分离出的4~5周龄BABL/c小鼠睾丸间质细胞进行体外培养,分别暴露于含10 ng/ml hCG(对照)和10-9、10-8、10-7mol/L T-2毒素+10 ng/ml hCG的培养液中24h。采用RT-PCR方法检测相关功能基因(3β-HSD-1,P450scc和StAR)mRNA的表达水平。结果与对照组相比,10 ng/ml hCG+10-8mol/L T-2毒素和10 ng/ml hCG+10-7mol/L T-2毒素染毒组小鼠睾丸间质细胞3β-HSD-1 mRNA的表达水平及10 ng/ml hCG+各剂量T-2毒素染毒组小鼠睾丸间质细胞P450scc/HPRT、StAR/HPRT mRNA的表达水平均下降,差异有统计学意义(P0.05,P0.01);且随着T-2毒素染毒剂量的升高,10 ng/ml hCG诱导小鼠睾丸间质细胞3β-HSD-1、P450scc和StAR mRNA的表达水平呈下降趋势。结论 T-2毒素可通过抑制小鼠睾丸间质细胞3β-HSD-1、P450scc和StAR mRNA表达而对雄性小鼠产生生殖毒性。     

双酚A对大鼠睾丸支持细胞的功能影响及机制

目的 探讨双酚A(BPA)对雄性SD大鼠睾丸支持细胞功能的影响及损伤机制.方法 将16～22日龄SPF级雄性SD大鼠的睾丸支持细胞制成3.0×10~6～3.5×10~6个/ml的细胞悬液,设立空白对照组、溶剂对照组和1×10~(-7)、1×10~(-6)、1×10~(-5)、1×10~(-4)mol/L BPA染毒组,观察睾丸支持细胞的增殖、细胞周期以及PCNA、Vimentin蛋白表达的情况.结果 各染毒组睾丸支持细胞的存活率明显低于对照组(P<0.05),G_0/G_1期细胞构成比增加,而S期和M期细胞构成比降低,PI下降.免疫细胞化学结果显示,10~(-4)mol/L、10~(-5) mol/L BPA实验组显著抑制大鼠睾丸支持细胞PCNA的表达.BPA在10~(-4) mol/L、10~(-5)mol/L、10~(-6) mol/L剂量组显著抑制大鼠睾丸支持细胞内Vimentin的表达.结论 双酚A可通过影响睾丸支持细胞的细胞增殖、细胞周期和抑制PCNA、Vimentin蛋白表达而表现出生殖毒性.

Abstract：

Objective To investigate the effects of bisphenol A on SD rat Sertoli cell function and the injury mechanism. Methods SD rat Sertoli cells were treated with bisphenol A at different doses,and the control group,solvent control group were set. Sertoli cell proliferation,cell cycle and PCNA.Vimentin protein expression were observed. Results The survival rate of Sertoli cells in the treated groups was significantly lower than the control group (P<0.05), constitution ratio of G_0/G_1 phase cells increased,while for S phase and M-phase, it decreased,and PI decreased. Immunocytochemistry showed PCNA expression in Sertoli cells was significantly inhibited by 10~(-4),10~(-5) mol/L BPA. Vimentin expression in Sertoli cells was significantly inhibited by 10~(-4), 10~(-5),10~(-6) mol/L BPA. Conclusion Bisphenol A can affect Sertoli cell proliferation,cell cycle and inhibit PCNA.Vimentin protein expression in Sertoli cells.     

兴奋性氨基酸递质系统在乐果诱导星形胶质细胞活化中的作用

Objective To study the involvement of excitatory amino acid system in astrocytes activation caused by dimethoate. Methods Pure-cultured astrocytes were gained by three passages from primary cultured rat nerve cells, then treated with 10-6,10-5,10-4 mol/L dimethoate for 48 h, 50 μmol/L and 100μmol/L MK801, a NMDA receptor blocker, was used to intervene the effects induced by 10-4 mol/L dimethoate.HPLC-FLD was utilized to measure the concentrations of excitatory amino acid (EAA), RT-PCR was used to detect the expression levels of NR2B, GLT-1, GLAST, GFAP and S100β mRNA, and immunofluresence staining method was applied to measure the expression levels of GFAP and S100β proteins. Results The expression levels of GLAST mRNA in all exposure groups were 67.8% ,68.6% and 76.2% of control level,respectively, which were significantly lower than that of control group (P＜0.05); The concentrations of EAA significantly decreased in 10-4 mol/L dimethoate group, as compared with control group (P＜0.01); the expression levels of GFAP mRNA in 10-4 mol/L dimethoate group, of S100β mRNA in 10-5 mol/L dimethoate group, of GFAP protein in 10-4 mol/L and 10-5 mol/L dimethoate groups and S100β protein in 10-4 mol/L dimethoate group were significantly higher than those in control group (P＜0.01). The expression levels of GLT-1 and GLAST mRNA in 10-4 mol/L dimethoate plus 50 μ mol/L or 100 μ mol/L MK801 groups increased significantly, as compared with 10-4 mol/L dimethoate group (P＜0.01), the expression levels of NR2B mRNA in 10-4 mol/L dimethoate plus 50 μ mol/L or 100 μmol/L MK801 groups increased significantly, as compared with control group (P＜0.05 or P＜0.01); the concentration of Glu in 10-4 mol/L dimethoate plus 100 μ mol/L MK801 group increased significantly, as compared with 10-4 mol/L dimethoate group (P＜0.01); the expression levels of GFAP mRNA and protein in10-4 mol/L dimethoate plus 50 μ mol/L or 100 μ mol/L MK801 groups decreased significantly (P＜0.01); S100β protein expression level in 50 μ mol/L MK801 intervention group was significantly higher than thatl in control group (P＜0.01). Conclusion Excitatory amino acid system involved in astrocytes activation caused by dimethoate. MK801 was useful to control astrocytes gliosis.     

叔丁基对苯二酚对百草枯神经细胞毒性和氧化应激的拮抗作用

Objective To investigate the protective effects of the tert-butylhydroquinone (tBHQ)pretreatment on neurotoxicity and oxidative stress induced by paraquat (PQ) in PC12 cells. Methods Cytoyoxicity of PC12 cells was measured by MTT assay, following the PC12 cells treatment with different concentrations of 100, 300 μmol/L PQ for 24 h and 48 h. PC12 cells were pretreated with or without 40 μmol/L tBHQ for 4 h, PC 12 cells were exposed to PQ at the doses of 0, 100, 300 μmol/L for 24 h and 48 h, respectively.The viability of PC 12 cells was measured by MTT assay, the apoptosis rates of PC 12 cells were detected by flow cytometry (FCM) and the malondialdehyde (MDA) levels of PC 12 cells were examine by thiobarbituric acid (TBA) method. Results When the exposure doses of PQ were 100 and 300 μmol/L for 24 h, the viability of PC 12 cells pretreated with tBHQ was significantly higher than that of PC 12 cells only exposed to PQ (P＜0.05 or P＜0.01). When the exposure dose of PQ was 100μmol/L for 48 h, the viability of PC12 cells pretreated with tBHQ was significantly higher than that of PC12 cells only exposed to PQ (P＜0.01). When the exposure doses of PQ were 100 and 300 μmol/L for 24 h, the apoptosis rates and MDA levels of PC12 cells pretreated with tBHQ were significantly lower than those of PC12 cells only exposed to PQ (P＜0.05 or P＜0.01). Conclusions tBHQ preteatment can reduce the cytotoxicity, apoptosis and oxidative stress induced by PQ in PC12 cells.     

牛磺酸锌对铅暴露雄性大鼠血脑屏障通透性的影响

目的 探讨牛磺酸锌(taurine-zinc,TZC)对生长发育期铅暴露雄性大鼠血铅含量和血脑屏障(blood-brain barrier,BBB)通透性的影响.方法 选取3周龄雄性SD大鼠.随机分为对照组、乙酸铅组、二巯丁二酸(dimercaptosuccinic acid,DMSA)组和TZC组,每组8只.TZC组大鼠饮用0.46 g/L的TZC,其余各组大鼠均饮用自来水.乙酸铅组、DMSA组和TZC组大鼠腹腔注射0.12%乙酸铅生理盐水溶液(每100 g体重0.2 ml),DMSA组大鼠灌胃5 mg/ml的DMSA(每100 g体重0.2 ml).染毒3周后,以原子吸收法测血铅含量,以硝酸镧示踪法观察血脑屏障通透性变化.结果 与乙酸铅组相比,TZC组和DMSA组血铅含量显著降低(P＜0.01);透射电镜观察显示,对照组未见镧盐颗粒渗透出血脑屏障,乙酸铅组大鼠普遍出现镧盐颗粒穿过血脑屏障进入神经网络,TZC组和DMSA组均有部分毛细血管存在镧盐颗粒透过内皮细胞到达基膜,但未穿过基膜进入神经网络.结论 TZC能显著降低铅暴露大鼠血铅含量,对血脑屏障有良好的保护作用.

Abstract：

Objective To explore the effects of taurine-zinc (TZC)on blood lead content and blood-brain barrier(BBB)permeability of growing rats exposed to lead.Methods Male SD rats aged 3 weeks were randomly divided into four groups(8 in each group):control group,lead exposure group,dimercaptosuccinic acid (DMSA)group and TZC group.The exposure group treated with 0.12%lead acetate(0.2 ml/100 g body weight)by intraperitoneal injection,DMSA group and TZC group were treated with the same dose of lead acetate but were respectively supplemented with 5 mg/ml DMSA(2 ml/100 g body weight) by ig and 0.46g/L TZC solution through drinking.Three weeks later,blood lead content was measured by atomic absorption spectroscopy(AAS)and changes of BBB permeability were observed using lanthanum nitrate tracer method.Results Compared with the lead exposure group,the blood lead level in TZC group and DMSA group was significantly reduced(P＜0.01).Under transmission electron microscope.it was found that the lanthanum granules generally seeped into neural network through BBB in lead exposure group,but those only to basement membrane through some capillary endothelial cell in TZC group and DMSA group.Conclusion TZC can significantly reduce the blood lead level and protect BBB from injury induced by lead exposure.     

利尿剂在三聚氰胺及三聚氰酸致大鼠急性肾损伤中的治疗作用

Objective To investigate the effect of diuretic (furosemide) therapy on kidney injury induced by melamine and cyanuric acid in rats. Methods 36 male Spragne Dawley rats were random disided into 3 groups. Group A was treated with 2mL of water daily, group B was treated with melamine and cyanuric acid ( each 100 mg/kg) daily for 4 days and then 2ml of water daily, group C was treated with the same as group B at the first 4 days and then treatment with furosemide (20mg/kg) daily. Samples of blood and 24h urine were collected to detective biochemical indexes, and kidney sections were performed on days 4 and 11 ( each end point, n = 6). The kidneys were observed with histopathology and renal crystal deposition scores were determined. Results On the 4th day, group B and group C were resulted in acute kidney injury such as oliguria [ ( 3. 39 ± 1.02 ) ml, ( 3. 20 ± 0. 86 ) ml ] and high serum creatinine [ ( 153.54 ±27. 08)μmol/L, (160. 11 ± 19. 55)μmol/L] and renal melamine cyanurate crystal were found in the renal tissues. On the 11th day, the renal crystal deposition score in the rats was reduced by 9. 52% ( P >0. 05). Compared with those of the 4th day in group B, it reduced by 63.63%( P <0.05) in group C. Urine volume were increased significantly compared with those of the 4th day( P < 0. 05 ) in group C [ from (3.20±0. 86)ml to (25.96 ±5.97)ml] and group B [ from(3. 39 ± 1.02)ml to (8. 57 ± 1.66)ml] , and Urine volume in group C was increased significantly more than that in group B ( P < 0. 05 ). The serum creatinine was obviously reduced as compared with those of the 4th day in group B and C( P <0.05), from[ (153. 54±27.08) μmol/L] to [ ( 106. 10 ±5.53) μmol/L] in group B and from [ ( 160. 11 ± 19. 55) μmol/L] to [ (67. 17 ± 12. 80 ) μmol/L] in group C, but the serum creatinine in group B was still higher than that in group A and C ( P < 0. 05). Conclusions Furosemide can attenuate the damage of acute kidney injury induced by melamine and cyanuric acid.     

氟铝单独及联合染毒对MC3T3-E1细胞增殖能力及细胞周期的影响

目的 观察不同浓度的氟、铝对体外培养的小鼠颅顶前成骨细胞亚克隆14(MC3T3-E subclone 14)增殖及细胞周期的影响,以进一步阐明地方性氟中毒机制提供实验依据.方法 以10~(-9)～10~(-3)mol/LNaF染毒MC3T3-E1细胞,同时以50μmoI/L NaF及5μmol/L AICl3单独或者联合染毒MC3T3-E1细胞,培养72 h.应用CCK-8(cell counting kit-8)观察MC3T3-E1细胞增殖能力的影响;采用流式细胞术检测MC3T3-E1细胞周期变化情况.结果 氟无显著促进MC3T3-E1细胞增殖的作用,较高浓度的氟(1 mmol/L)抑制MC3T3-E1细胞增殖(P<0.01).氟铝联合染毒显著刺激MC3T3-E1细胞增殖(P<0.01):G_2/M期细胞明显增多,增殖指数(PI)升高(P<0.05),DNA相对含量增高(P<0.05).结论 氟对MC3T3-E1细胞无显著促增殖作用,氟铝联合能显著提高MC3T3-E1的增殖能力,使G_2/M期细胞明显增多,促进成骨细胞的增殖分裂,细胞处于活跃生长状态.

Abstract：

Objective To explore the effects of different concentrations of fluoride, aluminum alone and in combination exposure on mice parietal bone cell subclone 14 (MC3T3-E subclone 14), and to elucidate the pathogenesis of endemic fluorosis. Methods The proliferation of MC3T3-E1 cells exposed to 10~(-9)-10~(-3)mol/L NaF alone, 50 μmol/L NaF and 5 (μmol/L AlCl_3 aloneand in combination ,was measured by CCK-8, and the change of cell cycle was measured by flow cytometry after treatment with various concentrations of fluoride and aluminum. Results Fluoride alone did not promote osteoblast MC3T3-E1 cells proliferation, higher concentration fluoride inhibited MC3T3-E1 cells proliferation. Fluoride and aluminum combined exposure (50 μmol/L NaF +5 μmol/L AlCl_3) stimulated proliferation of MC3T3-E1 cells (P<0.01 ).and significantly induced increase of G_2/M phase, PI (proliferation index) and DNA relative content. Conclusion Fluoride does not promote the MC3T3-cells proliferation, aluminium plus fluoride may increase MC3T3-E1 cells proliferation and affect the cell cycle, which can significantly increase the number of G_2/M phase cells.     

三氧化二砷联用华蟾素对K562细胞增殖和凋亡的影响

Objective To investigate the effect of As2 O3 in combination with cinobufacini on proliferation and apoptosis of the K562 cells and provide theoretical basis for clinical application.Methods Cell proliferation was assayed by analyzing the growth and viability of the cells.Apoptosis was assayed by performing cell morphology,Annexin-V/PI staining,DNA-PI staining,and DNA gel electrophoresis.Results After exposure to As2O3 and cinobufacini,the growth of K562 cells was inhibited and the viability of K562 cells was decreased. After treated with 1.0μmol/L As2O3,0.125μg/ml cinobufacini,0.25μg/ml cinobufacini,1.0μmol/L As2O3 + 0.125 μg/ml cinobufacini,1.0μmol/L As2O3 + 0.25μg/ml cinobufacini for 24 and 48 hours,the proliferation inhibition rate were(24 ± 1.3)%,(21 ± 1.5)%,(38 ± 3.1)%,(57 ±2.7)%,(66 ±3.3)% and(49 ±2.9)%,(48 ±2.7)%,(61 ±2.1)%,(77 ±3.8)%,(82 ±4.2)%,the apoptosis rate of K562 cells were(4.8 ± 0.5)%,(5.6 ± 0.7)%,(9.8 ± 0.6)%,(11.9 ± 1.2)%,(15.2±1.5)% and(11.0 ±0.9)%,(12.9 ±1.1)%,(18.4 ±1.5)%,(21.0 ±2.0)%,(28.0 ±1.9)%.The percentage of apoptotic cells was a time- and dose-dependent manner.Typical DNA ladder was shown by DNA gel electrophoresis.Conclusions As2O3 combined with cinobufacini inhibited the proliferation of K562 cells and induced apoptosis of the K562 cells,the combination of the two drugs had better effect.     

叔丁基对苯二酚对亚砷酸钠致Chang liver细胞毒性的影响

目的 探讨叔丁基对苯二酚(tert-butylhydroquinone,tBHQ)对亚砷酸钠(NaAsO_2)致Chang liver细胞毒性的影响.方法 Chang liver细胞培养48 h后分别以20、40、60和80 μmo/L的NaAsO_2染毒24和48 h,作为NaAsO_2单独作用组.以5和20μmol/L的tBHQ预处理Chang liver细胞24h,以40和60μmol/L的NaAsO_2染毒24和48h,作为tBHQ预处理组;对照组处理同NaAsO_2:单独作用组.每个浓度设3个复孔.用Alamar Blue法检测细胞活力.结果 NaAsO_2单独作用24和48 h组Alamar Blue还原率显著下降,与对照组比较,差异有统计学意义(P<0.05);且NaAsO_2单独作用48 h组的Alamar Blue还原率均显著低于24 h组,差异有统计学意义(P＜0.01).5 μmol/L的tBHQ预处理组与对应的60 μmol/L NaAsO_2单独作用24h组相比,显著提高了Alamar Blue还原率(P＜0.01);20 μmol/L的tBHQ预处理组的Alamar Blue还原率均显著高于相对应NaAsO_2单独作用24 h组(P＜0.05).5 μmol/L的tBHQ预处理组的Alaraar Blue还原率均显著高于相对应NaAsO_2单独作用48h组(P＜0.01);20μmol/L的tBHQ预处理组与对应的40μmol/L NaAsO_2单独作用48h组相比,显著提高了Alamar Blue还原率(P<0.01).结论 tBHQ能够降低NaAsO_2致Chang liver细胞的毒性,增强细胞对NaAsO_2毒性的抵抗能力.

Abstract：

Objective To study the antagonism of text-butylhydroquinone (tBHQ)to NaAsO_2 induced cytotoxicity in Chang liver cells in vitro.Methods Chang liver cells were exposed to NaAsO_2(0,20,40,60 and 80μmol/L)for 24 and 48 hours,or Chang liver cells were treated with tBHQ(5 and 20 μmol/L),then exposed to NaAsO_2(40 and 60 μmol/L)for 24 and 48 hours.The conditions of control group was the same as NaAsO_2 group.Alamar Blue was used to evaluate the viabifity of cells.Results Chang liver cells were exposed to NaAsO_2 for 24 and 48 hours,Alamar Blue reduction rates decreased significantly and Alamar Blue reduction rates of 48 hours group were lower than 24 hours group (P<0.01).Alamar Blue reduction rate of 5 μmol/L tBHQ pretreatment group was higher than 60 μmol/L NaAsO_2 group for 24 h(P<0.01);Alamar Blue reduction rate of 20μmol/L tBHQ pretreatment group were higher than respective NaAsO_2 group for 24 h(P＜0.05).Alamar Blue reduction rate of 5 μmol/L tBHQ pretreatment group were higher than respective NaAsO_2 group for 48 h(P＜0.01);Alamar Blue reduction rate of 20 μmol/L tBHQ pretreatment group was higher than 40 μmol/L NaAsO_2 group for 48 h(P<0.01).Conclusion tBHQ can decrease the cytotoxieity induced by NaAsO_2 in Chang liver cells,increase the resistance to the cytotoxieity induced by NaAsO_2 in Chang liver cells.     

微波诱导小胶质细胞的促炎症反应

Objective To explore the relationship between microglia] proinflammatory and electro-magnetic radiation and unveil the role of microglia in microwave radiation induced central nervous system in-jury. Methods N9 microglia cells cultured in vitro were exposed to microwave at 90 mW/cm2. Cell flow cy-tometry was used to observe the expression of CD1 lb at different time points after exposure; ELISA was used to detect the concentration of TNF-α in N9 cell culture supernatant; RT-PCR analysis confirmed iNOS mRNA ex-pression in N9 microglia cells; and Nitrate Reductase Method was used to test NO amount in culture super-natant. Results The CD11b positive microglial cells increased significantly at 3 h after microwave exposure (P<0.05), continued to increase until 24 h and peaked at 6 h after exposure. The amount of TNF-α rose dra-matically from 1 h to 24 h after exposure (P<0.01) and peaked at 3 h [(762.1±61.5) pg/ml] after exposure (P< 0.01). The level of NO started to increase at 1 h [(4.48±0.59) μmol/L] and lasted for 24 h after exposure. The expression of iNOS mRNA increased significantly at 1 h(P<0.05), and tripled the original expression at 6 h af-ter exposure, hereafter, it decreased slightly, but all were higher than the control group within 24 h after exposure. Conclusion Microwave radiation could induce the activation of microglia cells. The activated microglia cells could induce microglial proinflammatory by producing large amounts of TNF-α, NO, etc.     

双酚A对体外培养小鼠睾丸睾酮合成的影响及机制

目的探讨睾丸体外培养模型下双酚A(BPA)暴露对小鼠睾丸睾酮合成及基因表达的影响。方法采用睾丸器官体外培养方法,将睾丸随机分为DMSO溶剂对照组和4个BPA剂量染毒组(终浓度为10-7、10-6、10-5和10-4mol/L)。分别培养24、48和72h,每24h通气一次。HE染色光镜下观察睾丸组织形态学变化,放射免疫法检测培养液睾酮浓度,免疫组化法检测睾丸组织相关蛋白表达。实时荧光定量PCR检测睾丸组织相关基因表达水平。结果 10-4mol/L剂量组支持细胞数目减少,部分间质细胞核固缩或呈絮状改变;随着时间的延长睾酮分泌逐渐减少,与DMSO对照组相比,10-5mol/L剂量组在48h时睾酮合成增加(P<0.05),其余各剂量组睾酮分泌降低,以10-4mol/L剂量组较明显(P<0.01);睾酮合成相关酶3β-羟基类固醇脱氢酶(3βHSD)、胆固醇侧链裂解酶(P450scc)、细胞色素P45017a羟化酶(P450c17)及波形蛋白(Vimentin)的基因及蛋白表达水平下降。结论 BPA能通过抑制3βHSD、P450scc、P450c17及Vimentin等相关酶的表达,干扰间质细胞睾酮的合成。     

氯乙烯对雄性大鼠生殖内分泌激素的影响

目的 探讨氯乙烯对雄性大鼠生殖内分泌激素的影响.方法 雄性SD大鼠60只,随机分为对照组和10、100和1000mg/kg氯乙烯染毒组,每组15只,腹腔注射染毒14和28d.测定大鼠血清中睾酮(T)、卵泡刺激素(FSH)、黄体生成素(LH)、雌二醇(E2)、抑制素B(InhB)和睾丸中E2、T、InhB的水平,并观察睾丸支持细胞和间质细胞超微结构的改变.结果 与对照组比较,染毒14 d各染毒组大鼠血清中T和E2水平降低,InhB和LH水平升高,仅100 mg/kg染毒组LH水平差异有统计学意义(P<0.05).染毒28 d,100、1000 mg/kg染毒组大鼠血清中T分别为(10.90±1.56)和(8.52±2.85)ng/ml,InhB水平分别为(31.40±6.21)和(28.39±5.67)pg/ml,均明显低于对照组[T为(15.89±4.03)ng/ml,lnhB为(46.87±8.74)pg/ml],差异有统计学意义(P<0.05);各染毒组大鼠血清中FSH水平均明显高于对照组.差异有统计学意义(P<0.05).与染毒14d比较,染毒28d各染毒组大鼠血清中InhB和LH水平均明显降低,差异有统计学意义(P<0.05).染毒28 d时100、1000mg/kg染毒组大鼠睾丸组织中T和InhB水平分别为(8.05±2.19)、(6.75±1.94)ng/mg pro和(39.32±5.55)、(35.53±8.71)pg/mg pro,与对照组[T为(11.90±2.33)ng/mg pro、InhB为(49.45±7.01)pg/mg pro]比较,差异有统计学意义(P<0.05).超微结构观察可见,染毒组的睾丸间质细胞和支持细胞出现了细胞核畸形、线粒体肿胀等改变.结论 氯乙烯对雄性大鼠生殖内分泌系统有损害作用,可引起生殖内分泌激素水平改变和睾丸间质细胞、支持细胞超微结构变化.     

Regulation of Steroidogenesis by Cordyceps sinensis Mycelium Extracted Fractions with (hCG) Treatment in Mouse Leydig Cells

The in vitro effect of extracted fractions of Cordyceps sinensis (CS) mycelium on hCG-treated testosterone production from purified normal mouse Leydig cells was examined. Different fractions extracted from CS (F1-water soluble polysaccharide, F2- water soluble protein and F3- poorly water soluble polysaccharide, and protein) were added to Leydig cells with hCG, and the production of testosterone was determined by radioimmunoassay (RIA). Testosterone productions stimulated by hCG in mouse Leydig cells were suppressed by F2 at 10 mg/ml and F3 at doses from 3 to 10 mg/ml, respectively. F2 and F3 at 10 mg/ml did inhibit dbcAMP-stimulated testosterone productions which indicated that F2 and F3 might affect steroidogenesis at the site after the formation of cyclic AMP. Finally, cycloheximide inhibited F2- and F3-treated mouse Leydig cell testosterone production.     

大骨节病患者踝关节软骨IGF-1、IGFBP2基因表达的特征及意义

目的探讨大骨节病(KBD)患者踝关节软骨胰岛素样生长因子1(IGF-1)、胰岛素样生长因子结合蛋白2(IGFBP2)基因表达的特征及意义。方法采用病例-对照研究方法,选择2010年1月至2016年12月在陕西省人民医院骨科住院的KBD患者10例为KBD组,并以同期因外伤致踝关节骨折但无距骨损伤的患者10例为对照组,收集两组患者软骨组织。分别采用免疫组织化学、实时荧光定量PCR及蛋白免疫印迹法检测患者软骨组织中IGF-1、IGFBP2阳性细胞、mRNA和蛋白表达情况。根据KBD患者踝关节软骨IGF-1、IGFBP2基因表达情况,在陕西省人民医院选择1例创伤致截肢患者,取踝关节软骨制备软骨细胞进行体外细胞验证实验;将软骨细胞分为对照组(0 ng/ml T-2毒素)、T-2处理组(20 ng/ml T-2毒素)、T-2+IGFBP2沉默组(20 ng/ml T-2毒素+50 nmol/L IGFBP2 siRNA),采用MTT法和二甲基亚甲基蓝染色法检测3组软骨细胞活性及硫酸糖胺多糖(sGAG)分泌情况。结果对照组与KBD组患者软骨组织IGF-1[(47.26±8.97)、(68.15±7.42)个]、IGFBP2阳性细胞数[(27.56±5.40)、(71.85±7.62)个]比较,差异均有统计学意义(t=4.487、9.402,P均<0.01);与对照组比较,KBD组患者软骨组织IGF-1、IGFBP2 mRNA和蛋白表达水平均较高,差异均有统计学意义(t=3.340、20.700,4.684、8.699,P<0.05或<0.01)。细胞实验中,对照组、T-2处理组、T-2+IGFBP2沉默组软骨细胞活性和sGAG含量比较,差异均有统计学意义(F=226.70、80.66,P均<0.01);其中,T-2处理组、T-2+IGFBP2沉默组细胞活性、sGAG含量均低于对照组(P均<0.05),且T-2+IGFBP2沉默组均高于T-2处理组(P均<0.05)。结论KBD患者踝关节软骨中IGF-1、IGFBP2基因表达明显较高。沉默IGFBP2基因能够降低T-2毒素对软骨细胞活性及sGAG分泌的抑制作用。     

氟虫腈对PC12细胞凋亡及Bcl-2蛋白表达的影响

目的 研究氟虫腈对神经细胞凋亡的影响并初步探讨其神经毒作用机制.方法 设3.13×10~(-6)、1.25×10~(-5)、5.00×10~(-5)mol/L 3个氟虫腈染毒组和1个对照组,用荧光显微镜检测各组PC12细胞凋亡荧光强度并用流式细胞仪对凋亡率进行测定,并对3个染毒组Bcl-2蛋白表达进行荧光强度检测.结果 细胞凋亡荧光强度检测发现,3.13×10~(-6)mol/L染毒组细胞凋亡率为17.00%±0.57%,5.00×10~(-5)mol/L染毒组细胞坏死率为13.95%±1.95%,均明显高于对照组(分别为9.80%±0.30%、3.10%±0.40%),差异均有统计学意义(P<0.05).对Bcl-2蛋白表达含量进行定性检测,结果显示,3.13×10~(-6)mol/L染毒组Bcl-2蛋白表达明显低于对照组,其他染毒组Bcl-2蛋白表达与对照组比较无明显差异.结论 氟虫腈在较低剂量时可能是通过影响Bcl-2蛋白表达来诱导PCI2细胞凋亡,而在较高剂量时可能通过其他非细胞凋亡途径直接致细胞坏死发生.     

双酚A雄性生殖毒性的体内外实验研究

目的探讨双酚A对雄性动物生殖机能的影响。方法将双酚A混入饲料(0、1和5g／kg)连续饲喂成年32只SD大鼠14d,放免法测定睾酮和雌二醇并进行右睾组织形态分析;对原代培养的支持细胞染毒(0、10^-7、10^-6、10^-5、10^-4moL／L)。结果5g／kg的双酚A组的右睾平均重量1．53g显著低于对照组1．62g,但1g／kg组与对照组差异无统计学意义。形态观察发现,2个双酚A组中的曲细精管基底膜均与生精细胞分离;部分生精细胞和支持细胞发生核固缩和空泡变性;同时,双酚A处理使黏附于支持细胞的生精细胞平均数量由对照组的7．94个分别减少为4．13和3．04个。此外,双酚A在体内外实验中均抑制睾丸支持细胞的波形蛋白表达,阻碍细胞骨架和胞间联结的形成,使支持细胞在体外培养中的形态变得异常细长。但双酚A对血清雌二醇和睾酮浓度的影响没有统计学意义。结论双酚A可能通过破坏支持细胞骨架和改变支持细胞形态而损害雄性生殖功能。     

EFFECTS OF TREMELLA MESENTERICA ON STEROIDOGENESIS IN MA-10 MOUSE LEYDIG TUMOR CELLS

Tremella mesenterica (TM), a yellow jelly mushroom, has been traditionally used as food and crude medicine to improve several kinds of symptoms in Chinese society for a long time. Recent studies have illustrated that the fractions of fruiting bodies of TM exhibit a significant hypoglycemic activity in diabetic mouse models, which usually suffer from sexual dysfunction. In a previous study, we showed that TM reduced plasma testosterone production in normal rats without any positive effect in diabetic rats. It evolved a question of TM directly regulating Leydig cell steroidogenesis. In this study, MA-10 mouse Leydig tumor cells were treated with vehicle, different dosages of TM with or without human chorionic gonadotropin (hCG 50 ng/ml) to clarify the effects. Results showed that TM at different dosages (0.01–10 mg/ml) did not have any effect on MA-10 cell steroidogenesis (p > 0.05). In the presence of hCG, there was an inhibitory trend that TA suppressed MA-10 cell progesterone production at 3 hr treatment with a statistically significant difference by the 10 mg/ml TM (p < 0.05). In time course effect, TM alone did not have any effect on MA-10 cell steroidogenesis from at 1, 2, 3, 6 and 12 hr (p > 0.05). However, TM did reduce hCG-treated MA-10 cell progesterone production at 1, 2 and 3 hr (p < 0.05), respectively. To determine whether TM would have adverse effects on MA-10 cell steroidogenesis in the presence of hCG, MTT assay and recovery studies were conducted. MTT assay indicated that TM had no effect on surviving cells. In addition, with the removal of TM, and then the addition of hCG (2 and 4 hr), progesterone levels were restored within 4 hr. Taken together, present studies suggested that TM suppressed hCG-treated steroidogenesis in MA-10 cells without any toxicity effect.     

姜黄素抑制人肝癌细胞BEL-7402中VEGF和HIF-1α表达通过PI3K／AKT／FRAP信号传导通路

目的探讨MAPK和PI3K信号传导通路在姜黄素调节VEGF和HIF-1α表达中的作用。方法分别加入LY29400225μmol／L、50μmol／L，U012610μmol／L、20μmol／L，rapamycin 5ng／ml、10ng／ml处理人肝癌细胞BEL-7402，30min后加入姜黄素10μmol／L，对照组单独加入0、10μmol／L姜黄素，缺氧环境中培养6h后，行RT—PCR和Western Blot检测VEGF蛋白、mRNA和HIF-1α蛋白表达；分别加入不同浓度的的姜黄素以及LY294002 25 μmol／L处理人肝癌细胞BEL-7402，缺氧环境中培养6h后，行Western Blot检测磷酸化AKT和总AKT蛋白表达。结果姜黄素10μmol／L＋LY29400225μmol／L组、姜黄素10μmol／L＋LY294002 50 μmol／L组、姜黄素10μmol／L＋rapamycin 5 ng／ml组、姜黄素10μmol／L＋rapamycin 10 ng／na组HIF-1α蛋白、VEGF蛋白、mRNA表达分别与姜黄素10μmol／L组相比降低，差异有统计学意义（P〈0．01）；而姜黄素10μmol／L＋U012610μmol／L组、姜黄素10μmol／L＋U0126 20 μmol／L组HIF—1α蛋白、VEGF蛋白、mRNA表达分别与姜黄素10μmol／L组相比差异无统计学意义（P〉0.05）；不同浓度的姜黄素、LY294002 25 μmol／L处理的人肝癌细胞BEL-7402缺氧6h后磷酸化AKT蛋白表达逐渐降低，LY294002 25 μmol／L可以基本阻断磷酸化AKT蛋白的表达，而对总AKT蛋白表达无明显变化。结论姜黄素对人肝癌细胞BEL-7402中HIF-1α蛋白和VEGF的表达通过P13K／AKT／FRAP信号传导通路。     

Effects of monobutyl and di(n-butyl) phthalate in vitro on steroidogenesis and Leydig cell aggregation in fetal testis explants from the rat: comparison with effects in vivo in the fetal rat and neonatal marmoset and in vitro in the human

BACKGROUND: Certain phthalates can impair Leydig cell distribution and steroidogenesis in the fetal rat in utero, but it is unknown whether similar effects might occur in the human. OBJECTIVES: Our aim in this study was to investigate the effects of di(n-butyl) phthalate (DBP), or its metabolite monobutyl phthalate (MBP), on testosterone production and Leydig cell aggregation (LCA) in fetal testis explants from the rat and human, and to compare the results with in vivo findings for DBP-exposed rats. We also wanted to determine if DBP/MBP affects testosterone production in vivo in the neonatal male marmoset. METHODS: Fetal testis explants obtained from the rat [gestation day (GD)19.5] and from the human (15-19 weeks of gestation) were cultured for 24-48 hr with or without human chorionic gonadotropin (hCG) or 22R-hydroxycholesterol (22R-OH), and with or without DBP/MBP. Pregnant rats and neonatal male marmosets were dosed with 500 mg/kg/day DBP or MBP. RESULTS: Exposure of rats in utero to DBP (500 mg/kg/day) for 48 hr before GD21.5 induced major suppression of intratesticular testosterone levels and cytochrome P450 side chain cleavage enzyme (P450scc) expression; this short-term treatment induced LCA, but was less marked than longer term (GD13.5-20.5) DBP treatment. In vitro, MBP (10(-3) M) did not affect basal or 22R-OH-stimulated testosterone production by fetal rat testis explants but slightly attenuated hCG-stimulated steroidogenesis; MBP induced minor LCA in vitro. None of these parameters were affected in human fetal testis explants cultured with 10(-3) M MBP for up to 48 hr. Because the in vivo effects of DBP/MBP were not reproduced in vitro in the rat, the absence of MBP effects in vitro on fetal human testes is inconclusive. In newborn (Day 2-7) marmosets, administration of a single dose of 500 mg/kg MBP significantly (p = 0.019) suppressed blood testosterone levels 5 hr later. Similar treatment of newborn co-twin male marmosets for 14 days resulted in increased Leydig cell volume per testis (p = 0.011), compared with co-twin controls; this is consistent with MBP-induced inhibition of steroidogenesis followed by compensatory Leydig cell hyperplasia/hypertrophy. CONCLUSIONS: These findings suggest that MBP/DBP suppresses steroidogenesis by fetal-type Leydig cells in primates as in rodents, but this cannot be studied in vitro.