重组水蛭素经蟾蜍肺泡膜的转运研究

 目的考察重组水蛭素跨膜转运的基本特点和影响因素,对转运的机制进行初步的探讨,为重组水蛭素肺部给药的体内评价打下理论基础。方法将蟾蜍肺泡膜作为肺液上皮体外模型,装在水平扩散池上,向给药池中加入荧光标记的重组水蛭素(FTTC-rHV₂),在不同的温度、不同的给药池初始浓度、不同的转运方向的条件下测定表观渗透系数,同时考察给药池溶液中高渗透压、酸性pH、加入渗透促进剂或者代谢抑制剂时的转运情况。同时监测膜电阻的变化。结果FTTC-rHV₂的表观渗透系数具有浓度非依赖性、温度非依赖性和方向非依赖性。酸性pH环境、高渗透压和吸收促进剂使表现渗透系数明显增加,而代谢抑制剂对袁观渗透系数没有明显的影响。3h内膜电阻没有明显的变化,说明膜的完整性得以保持。结论重组水蛭素经蟾蜍肺泡膜的转运是一个非能量依赖、非载体依赖的被动扩散过程。     

Detection of spontaneous CD4+ T-cell responses in melanoma patients against a tyrosinase-related protein-2-derived epitope identified in HLA-DRB1*0301 transgenic mice.

PURPOSE: The frequently expressed differentiation antigen tyrosinase-related protein-2 (TRP-2) has repeatedly been described as a target of spontaneous cytotoxic T-cell responses in melanoma patients, suggesting that it might be an ideal candidate antigen for T cell-based immunotherapy. As a prerequisite for immunization, T-cell epitopes have to be identified. Whereas a number of HLA class I-presented TRP-2-derived epitopes are known, information about HLA class II-presented antigenic ligands recognized by CD4+ T helper (Th) cells is limited. EXPERIMENTAL DESIGN: The search for TRP-2-derived Th epitopes was carried out by competitive in vitro peptide binding studies with predicted HLA-DRB1*0301 ligands in combination with peptide and protein immunizations of HLA-DRB1*0301 transgenic mice. In vivo selected candidate epitopes were subsequently verified for their immunogenicity in human T-cell cultures. RESULTS: This strategy led to the characterization of TRP-2(60-74) as an HLA-DRB1*0301-restricted Th epitope. Importantly, TRP-2(60-74)-reactive human CD4+ Th cell lines, specifically recognizing target cells loaded with recombinant TRP-2 protein, could be established by repeated peptide stimulation of peripheral blood lymphocytes from several HLA-DRB1*03+ melanoma patients. Even short-term peptide stimulation of patients' peripheral blood lymphocytes showed the presence of TRP-2(60-74)-reactive T cells, suggesting that these T cells were already activated in vivo. CONCLUSION: Peptide TRP-2(60-74) might be a useful tool for the improvement of immunotherapy and immune monitoring of melanoma patients.     

成人及脐血甲氨喋呤载体红细胞天然免疫粘附活性研究

[目的]研究成人及脐血甲氨喋呤(MTX)载体红细胞天然免疫粘附活性的变化情况。[方法]以改良低渗预膨胀法制备成人及脐血MTX载体红细胞,采用新鲜血天然免疫功能快速测定法,检测载体制备过程中洗涤、预膨胀和载药各阶段红细胞的天然免疫粘附活性的变化情况。[结果]低渗预膨胀处理的成人及脐血红细胞,天然免疫粘附能力(粘附率)分别为61.45%±8.51%、17.00%±2.16%,明显高于各自的对照组(P<0.05)。[结论]载药处理对红细胞天然免疫粘附活性无明显影响。     

乙酰丙酮钒在大鼠胃肠道吸收的初步研究

目的:研究乙酰丙酮钒在大鼠胃、小肠和大肠的吸收特性.方法:采用大鼠在体灌流实验,用石墨炉原子吸收法测定大鼠血浆中钒(V)浓度.结果:乙酰丙酮钒在10mg·kg-1剂量下在胃和大肠的吸收速率常数分别为2.853h-1和0.587h-1,AUC分别为158.6ng·mL-1·h和642.7ng·mL-1·h;在不同给药剂量(以钒计)10,25和50mg·kg-1时小肠的吸收速率常数分别为1.414,1.664和17.118h-1,AUC分别为1 714.0,4 641.2和5 554.7 ng·mL-1·h.结论:大鼠胃和大肠是乙酰丙酮钒的不良吸收部位,而小肠有较好吸收,吸收方式可能是被动扩散.     

学报类科研论文中有关实验动物学的文献统计

目的:分析实验动物学方法在中医药学科科研论文中的应用情况.方法:应用文献计量统计方法.结果:在21所中医药高等院校学报2003年发表的3893篇论文中有456篇与实验动物学有关,占论文总数的11.71%;在456篇论著中,接受厅局级以上资助的课题有235篇,占总数的51.54%;在人员构成上,标明第一作者具有副高以上职称和硕士以上学位水平者达到37.72%和47.15%.能清楚描述实验动物的种名品系、来源、性别、数目和体重(或年龄)的论著分别为91.89%、90.13%、89.25%、95.61%和94.96%;只有71篇论著使用的实验动物是清洁级或SPF级,49篇论著提及了实验动物的应用环境,分别占有关论文总数的15.57%和10.45%;饲料来源清楚的有5.26%.456篇论著共使用525批实验动物,涉及大鼠、小鼠和家兔的论文为276篇、163篇和52篇,分别占52.57%、31.05%和9.90%.结论:在继承和加快中医药发展的科学研究中,实验动物和动物实验已经占据了重要位置;广大科研工作者在设计科研课题时,既要考虑实验动物的遗传学控制,也要重视微生物学控制;在撰写论文时,应详细、全面、准确地记述实验动物和动物实验内容,以提高论著的研究水平和科学性.     

沙苑子黄酮对DMN诱导的大鼠肝纤维化形成的影响

目的　观察沙苑子黄酮对二甲基亚硝胺 (DMN)诱导的大鼠肝纤维化形成的影响。方法　采用DMN诱导大鼠肝纤维化 ,期间给予灌服沙苑子黄酮 (30 ,6 0 ,12 0mg·kg-1) ,设秋水仙碱组作对照 ,给药 4wk后 ,测定血清谷丙氨转氨酶(ALT)、谷草转氨酶 (AST)活性、总蛋白 (TP)和白蛋白 (Alb)含量 ,放射免疫分析法测定血清透明质酸 (HA)、层粘连蛋白(LN)含量。光镜和电镜观察肝细胞结构和肝纤维化程度。结果　沙苑子黄酮预防给药 ,明显降低DMN诱导肝纤维化大鼠血清ALT、AST活性 (P <0 0 1,P <0 0 1) ,提高血清TP和Alb含量 (P <0 0 5 ,P <0 0 1) ,降低血清HA和LN含量 (P <0 0 1)。病理学观察结果 ,沙苑子黄酮预防组大鼠胶原纤维沉积明显减轻 ,假小叶结构明显减少 ,电镜观察显示 ,沙苑子黄酮组肝细胞结构接近正常 ,disse间隙、肝细胞内胶原纤维明显减少。结论　沙苑子黄酮预防给药 ,可抑制DMN诱导的大鼠肝纤维化形成     

栀子酸调节胆汁淤积大鼠SHP-LRH-1信号通路的实验研究

目的：研究栀子酸(geniposidic acid,GPA)对α-萘异硫氰酸酯(α-naphthyl isothiocyanate,ANIT)诱导胆汁淤积 大鼠胆汁酸代谢小分子异源二聚体伴侣受体(small heterodimer partner,SHP)与肝受体同源物1(liver receptor homologue 1,LRH-1)信号通路的调节。方法：将50只SD大鼠随机分为5组,分别为空白组、ANIT组、ANIT+GPA 100 mg/kg组 (AG100组)、ANIT+GPA 50 mg/kg组(AG50组)、ANIT+GPA 25 mg/kg组(AG25组),每组10只,连续给药10 d,空白组和 ANIT组用生理盐水灌胃,ANIT+GPA各组给予GPA,给药的第8天,除空白组外,其余各组用ANIT(65 mg/kg)灌胃1 次造模,继续给药至第10天,测血清总胆固醇(total cholesterol,TC)、三酰甘油(triglyceride,TG)和总胆汁酸(total bile acids,TBA)的含量;分离并培养大鼠原代肝细胞(rat primary hepatocytes,RPH),将RPH分为空白组、40 μmol/L ANIT 组、40 μmol/L ANIT+GPA 4.00,1.00,0.25 mmol/L组(A4G,A1G和A0.25G组),real-time RT-PCR检测RPH中SHP和胆汁 酸合成关键酶胆固醇7α羟化酶(cytochrome P450 family 7 subfamily A member 1,CYP7a1) mRNA转录水平,蛋白印迹法检 测SHP和CYP7a1蛋白表达;沉默实验中,将RPH分为空白对照组、阴性转染组、siRNA-LRH-1组(ZR组)、siRNA-LRH- 1+GPA 4.00,1.00,0.25 mmol/L组(ZR4G、ZR1G、ZR0.25G组),检测SHP,CYP7a1和LRH-1蛋白和基因的表达;过表 达实验中,将RPH又分为空白对照组、阴性转染组(空白转染siRNA-阴性组)、LRH-1过表达组(LRH-1过表达质粒,OE 组)、LRH-1过表达质粒+GPA 4.00,1.00,0.25 mmol/L组(OE4G,OE1G,OE0.25G组),检测SHP,CYP7a1和LRH-1蛋 白和基因的表达。结果：在体实验中,与空白对照组比较,ANIT组的TC和TBA均显著升高(均P<0.01)、TG无差别; 与ANIT组比较,AG100组和AG50组的TC和TBA均显著降低(均P<0.01)。RPH实验中,与空白对照组比较,ANIT组中 SHP的蛋白和基因水平均显著降低(均P<0.01),CYP7a1的蛋白和基因水平均显著升高(均P<0.01);与ANIT组比较, A4G和A1G组的SHP基因和蛋白水平均显著升高(均P<0.01),A0.25G组SHP基因和蛋白水平均升高(P<0.05),A4G和A1G 组的CYP7a1基因和蛋白水平均显著降低(均P<0.01)。LRH-1沉默实验中,与阴性转染组比较,ZR组中CYP7a1和LRH-1 蛋白和基因均显著抑制(均P<0.01),SHP无变化;与ZR组比较,ZR4G,ZR1G和ZR0.25G组SHP基因和蛋白水平均显 著升高(均P<0.01),LRH-1和CYP7a1无显著变化。LRH-1过表达实验中,与阴性转染组比较,OE组的CYP7a1和LRH-1 蛋白和基因均显著抑制(均P<0.01),SHP无变化;与OE组比较,OE4G和OE1G组SHP基因和蛋白水平均显著升高(均 P<0.01),OE0.25G组SHP基因显著升高(P<0.05),LRH-1和CYP7a1无显著变化。结论：RPH中LRH-1的过表达能上调 CYP7a1的活性,GPA可以改善ANIT诱导的胆汁淤积大鼠的生物化学水平和肝脏病理,其机制可能与GPA激活大鼠 RPH中SHP造成LRH-1的消耗来降低CYP7a1的表达有关。     

miR-152-3p靶向IGF1基因对高糖诱导的ARPE-19细胞活性和凋亡的影响

目的：探究microRNA-152-3p(miR-152-3p)靶向胰岛素样生长因子1(IGF1)基因对高糖诱导的视网膜色素上皮ARPE-19细胞活性和凋亡的影响,并探讨其作用机制。

方法：高糖诱导ARPE-19细胞并转染miR-152-3p mimics,噻唑蓝(MTT)法检测细胞增殖活性,流式细胞术检测细胞凋亡情况,荧光定量PCR(RT-PCR)检测细胞中miR-152-3p水平,蛋白印迹(Western blot)法检测细胞中IGF1和VEGF表达水平,双荧光素酶报告基因检测IGF1和miR-152-3p靶向结合关系。

结果：高糖能够降低ARPE-19细胞活性,提高细胞凋亡率,抑制细胞中miR-152-3p的表达,提高IGF1和VEGF的表达; 而过表达miR-152-3p能够回调高糖诱导的细胞活性抑制及凋亡增加,抑制IGF1和VEGF的表达。双荧光素酶报告基因实验验证了IGF1是miR-152-3p的靶基因。

结论：miR-152-3p可通过靶向IGF1基因调节VEGF的表达抑制高糖诱导的ARPE-19细胞活性抑制和凋亡增加。     

Wnt通路中散乱蛋白在儿童急性淋巴细胞白血病中的表达及意义

目的 探讨Wnt信号通路中散乱蛋白（DVL）对儿童急性淋巴细胞白血病（ALL）发病及预后的意义。方法 选取33例新发ALL患儿为病例组,根据危险度将ALL患儿分为低危组（n=14）、中危组（n=5）和高危组（n=14）;29例免疫性血小板减少症（ITP）患儿为对照组。采集病例组患儿初发及诱导治疗第33天时,以及对照组骨髓血各2 mL,采用实时荧光定量聚合酶链反应（qRT-PCR）检测骨髓血细胞DVL1、DVL2、DVL3 mRNA表达。结果 病例组初发期DVL1、DVL2、DVL3 mRNA表达水平高于缓解期及对照组（P < 0.05）,与对照组比较,病例组缓解期DVL2 mRNA水平升高（P < 0.05）。DVL2 mRNA表达水平在病例组初发期及缓解期均高于DVL 1、DVL 3 mRNA （P < 0.05）。高、中危组DVL1、DVL2 mRNA表达水平高于低危组（P < 0.05）。DVL2 mRNA表达水平在低、中、高危组均高于DVL1、DVL3 mRNA （P < 0.05）。结论 Wnt通路中DVL,尤其是DVL2的表达变化可能对儿童ALL的发病及预后有一定意义。     

A New Treatment for Epiphora Secondary to Submandibular Gland Transplantation: Transcutaneous Atropine Gel

PurposeTo explore whether topical application of atropine gel controls epiphora secondary to submandibular gland (SMG) transplantion for severe keratoconjunctivitis sicca.MethodsEighteen patients with epiphora after SMG transplantation participated in a double-blind, controlled, crossover study. Patients were treated with topical smear common atropine (CA) and modified atropine (MA) gels (intervals ≥1 day). The control effect of atropine gels was observed for 5 hours. Tear flow was quantified in resting and stimulated (after exercise) states using the Schirmer I test.ResultsWith the CA gel, tear flow decreased significantly at 10, 30, and 120 minutes. Average epiphora reduction rates (AERRs) were 22.37%, 18.14%, and 13.27%, respectively (P<.05). With the MA gel, tear flow decreased significantly from 5 minutes to 5 hours; AERRs increased from 24.06% to 42.67% (P<.05); Maximum efficacy was maintained from 15 minutes to 3 hours (P<.01); Tear flow gradually increased from 4 to 5 hours but was still lower than that before atropine use (P<.05). According to results of the Schirmer I test and AERRs, the control efficacy of the MA gel was significantly better than that of the CA gel (P<.05).ConclusionsTopical application of atropine gel could effectively control mild epiphora for patients with SMG transplantation for severe keratoconjunctivitis sicca. The efficacy of MA gel was much better than that of the CA gel.