小鼠皮肤局部缺血模型的建立

目的研究建立稳定的昆明小鼠皮肤局部缺血模型。方法取40只雌性昆明小鼠，随机分为A／B两组，每组20只。分别在小鼠背部建立深达皮下筋膜层的u型皮瓣。A组为3．3×1．5cm皮瓣组．B组为2．5×1．5CITI皮瓣组。皮瓣游离缘垫以医用橡胶，缝合切口，造成皮肤缺血模型，术后观察皮瓣发绀水肿及坏死情况，并用散斑全景实时血流成像仪（MoorFLH）检测皮瓣血流。结果肉眼观察，3．3×1．5cm皮瓣组在术后第2天，50％小鼠皮瓣末端坏死，坏死面积达皮瓣面积的（37．20±4．83）％。术后第3天100％小鼠皮瓣末端坏死。并随时间的延长，坏死面积逐渐扩大。2．5×1．5皮瓣组在术后第1天皮瓣出现发绀，水肿，发绀水肿面积占（77．46±4．51）％，术后第3，第5天发绀水肿区面积逐渐减小，至术后第7天，发绀、水肿完全消退。散斑全景实时血流成像仪（MoorFLPI）检测皮肤血流显示，术后第1天皮瓣显著性缺血（P〈0．05），术后第7天血流基本恢复到术前水平。结论成功建立了昆明小鼠皮肤局部缺血模型，为进一步研究组织微血管再生的机理和策略提供简单而稳定的动物模型。     

刚地弓形虫RH株感染对BALB／c小鼠情绪行为的影响

目的研究刚地弓形虫RH株感染对BALB／c小鼠学习记忆行为的影响及可能机制。方法将72只周龄、大小相近的雄性BALB／c小鼠采用随机数字表分为生理盐水对照组与不同数量（3×10^3／ml、3×10^4／ml、3×10^5／m1）弓形虫RH株感染组,每组18只。于感染第5周从每组各随机抽取6只分别进行高架十字迷宫实验、旷场实验及强迫游泳实验,观察各组小鼠在实验中情绪行为变化。并记录各项指标进行统计分析。结果在高架迷宫试验与旷场实验中,3×10^3／ml弓形虫感染组小鼠与生理盐水对照组小鼠比较,各项行为学指标无明显差异。在3×10^4／ml、3×10^5／ml弓形虫感染组小鼠与对照组小鼠比较出现明显降低（P〈0．05）．以3×10^5／ml感染组最为明显,其小鼠运动活力（0E＋cE）、进入开放臂次数比例（OE％）、开放臂停留时间比例（OT％）小鼠爬行总格数、中央格在总格数中比例分别为11．08±2．12、28．73±0．59％、25．62±2．33％、32．30±17．26、2．42±0．65％。在强迫游泳试验中,各弓形虫感染组小鼠游泳的静止时间均高于对照组小鼠（P〈0．05）,3×10^5／ml感染组静止时间最长,达226．6±1．9S。结论刚地弓形虫RH株感染可引起小鼠情绪行为改变,具有焦虑抑郁倾向。     

肉毒毒素A对大鼠慢性神经源性疼痛镇痛作用的研究

目的观察肉毒毒素A(BoNT-A)对L5、L6脊神经结扎引起慢性神经源性疼痛模型大鼠行为学的影响。方法建立Wistar大鼠左侧L5、L6脊神经结扎模型(SNL模型)。SNL模型大鼠随机分为3组(n=8)。1)实验组:SNL术后第3天同侧足底皮下注射BoNT-A30U/kg;2)对照组:SNL术后同侧足底皮下注射相同容积生理盐水;3)假手术组:仅暴露而不结扎L5和L6脊神经。均分别测定术前、给药前、给药后1d、3d、5d、7d、14d、21d的机械缩足反射阈值(MWT)和热缩足反射潜伏期(TWL)。结果实验组手术同侧足底皮下注射BoNT-A可以增加大鼠的MWT和TWL,实验组手术对侧、对照组、假手术组应用BoNT-A对MWT和TWL无影响。结论足底皮下注射BoNTA能够改善SNL模型大鼠手术同侧肢体的MWT和TWL,有镇痛作用。     

金丝桃素抑制慢性病毒性心肌炎小鼠心肌细胞凋亡及Fas／FasL蛋白表达

目的观察金丝桃素对慢性病毒性心肌炎（VMC）小鼠心肌细胞凋亡及Fas／FasL蛋白表达的影响。方法Balb／C小鼠多次接种心肌柯萨奇段病毒复制慢性VMC模型,首次感染病毒45天后将存活小鼠分为模型组、金丝桃素组及氯沙坦组,同时设正常对照组．分别给予相应药物干预30天,采用原位末端标记法检测心肌细胞凋亡及免疫组化方法检测Fas／FasL蛋白表达。结果模型组心肌细胞凋亡率较正常组显著增加（P〈0．05）,金丝桃素组和氯沙坦组凋亡率较模型组显著降低（P〈0．05）。模型组Fas／FasL蛋白表达较正常组显著增多（P〈0．01）,金丝桃素组和氯沙坦组较模型组显著降低（P〈0．01）。结论金丝桃素保护心肌细胞与抑制VMC心肌细胞的凋亡．下调Fas／FasL蛋白表达有关。     

Roux-en-Y胃旁路术对2型糖尿病大鼠肾脏非对称性二甲基精氨酸代谢的影响

目的研究Roux-en-Y胃旁路术对2型糖尿病大鼠肾脏非对称性二甲基精氨酸(ADMA)代谢的影响。方法 40只SD大鼠,随机分为正常对照组(NC组,n=8)和糖尿病模型组(32只)。糖尿病模型组成模30只,随机分为糖尿病对照组(DC组,n=8)、糖尿病假手术组(DSO组,n=8)及糖尿病手术组(DO,n=14),DO组行胃旁路术,DSO组行胃窦十二指肠离断原位吻合。检测术前、术后1、2、4、8周血胰高血糖素样肽(GLP-1)水平,测定术前、术后8周空腹血糖(FBG),肾脏ADMA、血尿素氮(BUN)、肌酐(Cr)、一氧化氮(NO)水平,一氧化氮合酶(NOS)活性;检测蛋白质精氨酸甲基转移酶1(PRMT1)mRNA及二甲基精氨酸二甲胺水解酶1(DDAH1)mRNA,PRMT1蛋白表达水平,PAS染色观察肾脏组织学变化。结果随着手术时间延长,D0组GLP-1水平增加,其余各组无明显变化(P0.05),术后8周,DC和DSO组较DO组和NC组FBG、BUN、Cr、ADMA水平,PRMT1mRNA和蛋白表达水平均明显升高(P0.05),NO、NOS、水平、DDAH1的mRNA表达明显降低(P0.05);PAS染色示DC组和DSO组肾小球基质沉积,阳性染色物增多,余无明显变化。结论 2型糖尿病肾脏组织中的ADMA升高;胃旁路术促进ADMA代谢,改善了肾功能。     

外源性细胞色素C对脓毒症小鼠心肌TGF-β1和Smad1/5/8影响的实验研究

目的 探讨外源性细胞色素c (Cytc)对脓毒症小鼠心肌TGF-β1和Smad1/5/8的作用及影响.方法 雄性昆明小鼠78只,随机分为假手术组( Sham)、脓毒症组(CLP)、细胞色素C处理组(T).CLP组和T组用盲肠结扎穿孔法造模,Sham组仅开腹翻动盲肠.24 h造模成功后,T组经尾静脉注射外源性Cyt c(20 mg/kg)、Sham组和CLP组分别经尾静脉注射150(1生理盐水.30 min后以0、6h、12 h点取材,每个时间点5只小鼠,测左心室收缩压(LVSP)、左心室舒张末期压(LVEDP)、左室内压力变化最大上升和下降速率(LV±dp/dt-max);取心肌组织分别做组织学检查、RT-PCR和Western-Blot检测TGF-β1和Smad1/5/8蛋白的表达;24h点留取电镜标本,余下的小鼠观察生存率.结果 与CLP组比较,T组一般情况好转,生存率提高,病理学改善;LVSP、LVEDP和LV±dp/dt-max提高(P＜0.05);各组TGF-β1和Smad1/5/8蛋白均有表达,CLP组表达明显增加,经外源性Cytc干预后蛋白表达减少,CLP组与其它组比较差别有统计学意义(P＜0.05).结论 外源性Cytc逆转脓毒症小鼠心肌线粒体功能抑制和心肌抑制,改善心功能.     

解聚复肾宁对糖尿病肾病大鼠TNF—OL、MCP-1的影响

目的观察解聚复肾宁（Jiejufushenning,JJFSN）对糖尿病（Diabetes mellitus,DM）大鼠肾组织肿瘤坏死因子-α（Tumorneerosisfactor-alpha,TNF-α）、单核细胞趋化蛋白-1（Monocyte chemoattractant protein-1,MCP-1）和血清TNF-α的影响,探讨其肾保护作用机制。方法腹腔注射链脲佐菌素（Streptozotocin,STZ）建立SD大鼠DM模型,将成模DM大鼠随机分为3组：DM模型组（B组）、JJFSN组（C组）、厄贝沙坦（Irbesartan）组（D组）,另设正常对照组（A组）。采用相应干预措施处理12周。常规方法测定12周末各组大鼠空腹血糖（FBG）、血尿素氮（BUN）、血肌酐（Scr）、肾重／体重（KW／BW）、24h尿蛋白（24huFro）;放免法测血清TNF-α含量：免疫组织化学方法测肾组织TNF—Ot、MCP-1的表达;PAS染色评估细胞外基质;电镜观察肾组织超微结构改变。结果与A组相比．B组大鼠FBG、BUN、Scr、KW／BW、24huPro及血清TNF-α升高（P〈0．05）,肾组织TNF—Ot、MCP-1表达明显增高（P〈0．05）;肾组织超微结构明显异常;C组和D组上述指标显著改善（P〈0．05）,肾组织超微结构异常改善。结论竹FSN能延缓DM大鼠肾损害进程,其机制可能与抑制TNF-α、MCP-1上调有关。     

甘草、红景天及黄芪粗提取物对UVB诱导小鼠皮肤慢性光损伤的保护作用

目的研究甘草、红景天和黄芪三种粗提取物对皮肤慢性光损伤的保护作用。方法甘草、红景天和黄芪三种粗提取物局部外用于小鼠背部皮肤后给予UVB辐射,每天1次,连续30天,分别采用TUNEL、ELISA和免疫印迹检测小鼠背部皮肤组织的细胞凋亡指数、TNF-α含量和caspase-8蛋白水平。结果与空白对照组相比,UVB照射组(单纯给予UVB照射)小鼠皮肤中的细胞凋亡指数、TNF-α含量及caspase-8水平均显著增高(P0.05);不同浓度甘草等粗提取物处理后可下调TNF-α、caspase-8表达水平及细胞凋亡指数(P0.05),尤以甘草组明显。结论UVB慢性辐射可诱导皮肤光老化,而甘草、红景天和黄芪三种粗提取物可缓解紫外线引起的皮肤慢性光损伤,其作用机制与抑制炎症因子TNF-α释放、caspase-8表达和细胞凋亡有关。     

单纯及丙泊酚联合电休克对抑郁大鼠海马TNF—α表达的影响

目的探讨单纯电休克及丙泊酚联合电休克对抑郁大鼠电休克治疗效果及海马内TNF-α表达的影响。方法50只成年Wistar大鼠随机分为5组（n=10）：正常对照组（C组）、抑郁组（D组）、单纯丙泊酚组（DP组）、单纯电休克组（E组）、丙泊酚联合电休克组（M组）。C组正常饲养,其余采用经典的慢性不可预见性应激法建立抑郁模型。建模成功后,E组直接行电休克治疗,M组腹腔注射丙泊酚90mg／kg,麻醉产生效应后行电休克治疗,每日进行以上处理1次,连续7日。以Open—field法和糖水消耗实验进行行为学评分。实验完成后处死大鼠,用免疫组化染色法检测TNF—α受体蛋白的表达;ELISA法海马TNF-α的含量,RT-PCR法测海马TNF—α mRNA表达。结果E组和M组大鼠的行为学评分高于D组和DP组,且M组高于E组（P〈0．05）;D组和DP组海马内TNF-α含量以及mRNA的表达高于其它各组（P〈0．05）,M组海马内TNF—α含量以及mRNA的表达低于E组（P〈0．05）,与C组差异无统计学意义（P〉0．05）;D组和DP组海马区TNF-α受体表达量低于C组（P〈0．05）,M组的表达量接近于C组,但高于E组（P〈0．05）。结论慢性应激使大鼠海马内TNF—α含量升高及其受体表达下调,从而产生抑郁症。丙泊酚可以改善电休克抑郁症状可能与进一步下调抑郁大鼠海马内TNF—α的含量和上调其受体表达有关。     

分枝杆菌噬菌体 D29的生物学特性研究及其抗耐药结核潜力的探讨

目的研究分枝杆菌噬菌体 D29的生物学特性,为 D29抗耐药结核治疗奠定基础.方法观察噬菌体电镜结构和噬菌斑形态;测定 D29最佳感染复数(MOI);一步生长实验;检测 pH值对 D29活力的影响;斑点法测定裂解谱;中和实验检测抗原性.结果 D29噬菌斑圆形透明,边界清楚;D29尾长129nm,最佳 MOI为10-4;D29感染宿主菌的潜伏期约为50min,裂解量为10;pH值对 D29存活率影响大,酸性环境不影响 D29裂解能力;D29能裂解分枝杆菌临床耐药株;D29K值为1069.50.结论 D29属于长尾噬菌体科(siphoviridae),裂解谱广,抗原性较高,具有抗耐药结核潜力     

Evidence for a role of IFN gamma in control of Listeria monocytogenes in T cell deficient mice.

The role of interferon (IFN) gamma in controlling chronic infections of Listeria monocytogenes (Listeria) was studied in athymic C57BL/6 nu/nu mice, and by treating thymectomized C57BL/6 +/+ mice with monoclonal rat CD4 and CD8-specific monoclonal antibodies (Mab). Mice treated with a combination of the two T cell subset antibodies were similar to athymic, nude mice in being able to control Listeria infection, keeping the titers below 3-5 log10 bacteria per organ, but they could not eliminate them completely. Treatment with antibodies to IFN gamma of nude or CD4+ + CD8+ - T cell-depleted mice suffering from chronic Listeria infection caused a marked increase of Listeria titers in liver and spleen. This result implies a role of IFN gamma in maintaining anti-Listeria resistance in mice lacking mature T cells.     

microRNA-451对人结肠癌细胞SW620裸鼠皮下种植瘤生长的抑制作用及机制探讨

目的 探讨microRNA-451(miR-451)对人结肠癌细胞系SW620裸鼠皮下种植瘤生长的影响,并探讨其可能机制.方法 18只裸鼠随机分为3组,实验组(SW-620-451组)、阴性对照组(SW-620-NC)、空白对照组(SW-620组),每组6只,分别皮下接种转染miRNA-451 agomir的SW620细胞、转染了阴性片段agomir Negative Control(NC)的SW620细胞和不经处理的结肠癌SW620细胞,并每周两次于瘤体内分别注射miRNA-451 agomir、miRNA-451 agomir NC片段及生理盐水.比较各组裸鼠皮下形成瘤体的大小并计算抑瘤率,接种四周后处死裸鼠取组织,用免疫组化法和Western-blot法定性定量分析和比较肿瘤相关基因c-myc的表达.结果 各组裸鼠皮下接种肿瘤细胞6天后均有瘤体形成,成瘤率100％,实验组(SW-620-451组)瘤体生长速度明显低于其他两组(p＜0.05);时间终点为第30天时,实验组(SW-620-451组)裸鼠皮下种植瘤的体积为(0.95±0.13)cm3,阴性对照组(SW-620-NC组)为(2.25±0.50) cm3,空白对照组(SW-620组)为(2.46±0.59)cm3;实验组(SW-620-451组)瘤体体积显著小于其他两组体积(p＜0.05);实验组(SW-620-451组)瘤体质量为(1.15±0.13)g,明显低于SW-620-NC组(2.59±0.46)g及SW-620组(2.76±0.44)g(p ＜0.05).实验组种植瘤中c-myc的表达量较另外两组下降(p＜0.05).结论 转染miR-451后可以抑制结肠癌细胞SW620裸鼠皮下移植瘤的生长,其机制可能与下调c-myc表达有关.     

Effect of light on variations in blood testosterone in the ram: demonstration of a photosensitive phase in diurnal rhythm]

6 groups each of 5 adult rams were subjected in light controlled pens to a pretreatment of normal variations in daylength during May and June. Then one group followed the normal daylength and the other five different "skeleton photoperiods" of 8 hrs of light per day consisting of: 7 hrs continuous light (7 L) + 1 hr light (1 L) situated at different times of the dark period (D) according to groups. Blood samples were taken once each week and hourly during one 24 hr period in June, July and September. Peripheral plasma testosterone measured by radio-immunoassay was used as an index of testicular activity. There was increase in the level of testosterone resuting from a rise in the number of peak release during the 24 hrs. In the different light treatments tested, only the treatment (7L+9D+1L)+7D) stimulated testosterone secretion with the pattern of testosterone of this group being similar to that of the control group. This indicates that there exists a photosensitive phase at 16 to 17 hrs after the beginning of the principal light (the subjective dawn).     

2-Carboxyethylgermanium sesquioxide,a synthetic organogermanium compound,as an inducer of contrasuppressor T cells

2-Carboxyethylgermanium sesquioxide (Ge-132), a synthesized organogermanium compound with immunomodulaing activities, was shown to be an inducer of anti-suppressor T cells in normal mice. The suppressor cell activity of T6S cells, a clone of burn-induced CD8⁺ IL-4-producing suppressor T cells, was clearly inhibited when a mixed lymphocyte-tumor cell reaction of the clone was conducted with splenic mononuclear cells from mice treated orally with a 100 mg/kg dose of Ge-132. The activity of anti-suppressor cells was demonstrated in spleens of mice 2 days after treatment with Ge-132 and reached its peak on day 3. The anti-suppressor cells induced by the compound were of a contrasuppressor T cell-linage, because they were characterized as CD4⁺ CD28⁺ TCR/⁺ Vicia villosa lectin-adherent T cells. These cells produced IFN- but did not produce IL-2, IL-4, IL-6 or IL-10 in their culture fluids. CD4⁺ anti-suppressor T cells induced by Ge-132 may be different from other subsets of CD4⁺ T cells because Th1 and Th2 cells generated in our laboratory did not adhere toVicia villosa lectin-coated petri dishes, and each produced specific cytokines. Th1 cells produced IFN- and IL-2 while Th2 cells produce IL-4 and IL-10 in vitro. These results suggest that Ge-132 may be useful as an inducer of contrasuppressor T cells in immunocompromised individuals bearing suppressor T cells. To eliminate suppressor T cells from immunocompromised hosts may result in improved resistance from various opportunistic infections.     

Effets de l'AET sur la spermatogenèse de souris irradiées

Summary The testes of hybrid mice (CBA × C57Bl) were irradiated with 1368 R of X-rays. Half of the animals received an intraperitoneal injection of 8 mg of AET before irradiation. Each week, 5 males of each group were sacrificed. Histological examination of the testes and the epididymus showed that AET is able to protect, to some extent, against testicle damage induced by X-rays.     

Evidence for a role of IFN γ in control ofListeria monocytogenes in T cell deficient mice

Summary The role of interferon (IFN) in controlling chronic infections ofListeria monocytogenes (Listeria) was studied in athymic C57BL/6nu/nu mice, and by treating thymectomized C57BL/6 +/+ mice with monoclonal rat CD4 and CD8-specific monoclonal antibodies (Mab). Mice treated with a combination of the two T cell subset antibodies were similar to athymic, nude mice in being able to contol Listeria infection, keeping the titers below 3–5 log₁₀ bacteria per organ, but they could not eliminate them completely. Treatment with antibodies to IFN of nude or CD4⁺ + CD8⁺-T cell-depleted mice suffering from chronic Listeria infection caused a marked increase of Listeria titers, in liver and spleen. This result implies a role of IFN in maintaining anti-Listeria resistance in mice lacking mature T cells.     

Blockage of the NLRP3 inflammasome by MCC950 improves anti-tumor immune responses in head and neck squamous cell carcinoma

The NLRP3 inflammasome is a critical innate immune pathway responsible for producing active interleukin (IL)-1β, which is associated with tumor development and immunity. However, the mechanisms regulating the inflammatory microenvironment, tumorigenesis and tumor immunity are unclear. Herein, we show that the NLRP3 inflammasome was over-expressed in human HNSCC tissues and that the IL-1β concentration was increased in the peripheral blood of HNSCC patients. Additionally, elevated NLRP3 inflammasome levels were detected in tumor tissues of Tgfbr1/Pten 2cKO HNSCC mice, and elevated IL-1β levels were detected in the peripheral blood serum, spleen, draining lymph nodes and tumor tissues. Blocking NLRP3 inflammasome activation using MCC950 remarkably reduced IL-1β production in an HNSCC mouse model and reduced the numbers of myeloid-derived suppressor cells (MDSCs), regulatory T cells (Tregs) and tumor-associated macrophages (TAMs). Moreover, inhibiting NLRP3 inflammasome activation increased the numbers of CD4⁺ and CD8⁺ T cells in HNSCC mice. Notably, the numbers of exhausted PD-1⁺ and Tim3⁺ T cells were significantly reduced. A human HNSCC tissue microarray showed that NLRP3 inflammasome expression was correlated with the expression of CD8 and CD4, the Treg marker Foxp3, the MDSC markers CD11b and CD33, and the TAM markers CD68 and CD163, PD-1 and Tim3. Overall, our results demonstrate that the NLRP3 inflammasome/IL-1β pathway promotes tumorigenesis in HNSCC and inactivation of this pathway delays tumor growth, accompanied by decreased immunosuppressive cell accumulation and an increased number of effector T cells. Thus, inhibition of the tumor microenvironment through the NLRP3 inflammasome/IL-1β pathway may provide a novel approach for HNSCC therapy.     

The use of cyclophosphamide as an enhancer of the vaccine against foot-and-mouth disease

The immunization of biungulate animals with killed foot-and-mouth disease virus (FMDV) requires periodic vaccinations due to a low vaccine immunogenicity. Therefore, FMDV antigens need to be combined with adjuvants such as aluminum hydroxide, saponin or oil emulsions. Animal handling for periodic inoculations, and the repeated doses of vaccines that have to be administered increase the commercialization costs. Moreover, the use of adjuvants may induce adverse effects.In the present work we show that it is possible to increase the life span of neutralizing antibodies in serum when a single dose of cyclophosphamide (Cy) is administered four days before vaccination with aluminum hydroxidesaponin FMDV vaccine.     

Impact of biofilm matrix components on interaction of commensal Escherichia coli with the gastrointestinal cell line HT-29

Commensal Escherichia coli form biofilms at body temperature by expressing the extracellular matrix components curli fimbriae and cellulose. The role of curli fimbriae and cellulose in the interaction of commensal E. coli with the intestinal epithelial cell line HT-29 was investigated. Expression of curli fimbriae by the typical commensal isolate E. coli TOB1 caused adherence and internalization of the bacteria and triggered IL-8 production in HT-29 cells. In particular, induction of IL-8 production was complex and involved curli-bound flagellin. While cellulose alone had no effect on the interaction of TOB1 with HT-29 cells, co-expression of cellulose with curli fimbriae decreased adherence to, internalization and IL-8 induction of HT-29 cells. Investigation of a panel of commensal isolates showed a partial correlation between expression of curli fimbriae and enhanced internalization and IL-8 production. In addition, a high immunostimulatory flagellin was identified. Thus, the consequences of expression of extracellular matrix components on commensal bacterial-host interactions are complex.