稳定转染HSP72奶牛乳腺上皮细胞系建立与表达鉴定

为了构建HSP72慢病毒表达载体,建立稳定表达HSP72的牛乳腺上皮细胞系。以牛HSP72基因序列为模板,设计并合成引物,PCR扩增目的基因,连接到慢病毒表达质粒中,用包装获得的慢病毒感染牛乳腺上皮细胞,经一定浓度的嘌呤霉素筛选,Hochest33342法对细胞核定位,倒置荧光显微镜下观察转染效率,用免疫组化和Western Blot方法验证感染细胞是否稳定表达HSP72。结果显示,携带HSP72基因的慢病毒滴度约为1. 0×109TU/m L;确定嘌呤霉素的最佳筛选浓度为2μg/m L;牛HSP72慢病毒感染细胞表达HSP72,感染的奶牛乳腺上皮细胞在倒置荧光显微镜下可看到绿色荧光,转染效率可达90%以上;免疫组化结果表明,正常细胞和空载体感染细胞,HSP72呈现低表达,转染携带HSP72目的基因载体细胞,HSP72高表达,呈现颜色较深的棕色; Western Blot方法检测证实与正常转染细胞相比,慢病毒空载体转染的奶牛乳腺上皮细胞HSP72表达稍有降低,但无统计学差异,而携带HSP72基因的慢病毒载体转染的细胞HSP72呈现高表达,且差异极显著(P 0. 01),与慢病毒空载体转染的奶牛乳腺上皮细胞相比,携带HSP72基因的慢病毒载体转染的细胞HSP72呈现高表达,且差异极显著(P 0. 01)。成功建立稳定表达HSP72的牛乳腺上皮细胞系,可为研究奶牛乳腺炎分子调控机制以及抗性分子药物筛选提供新的理论依据和工作基础。     

钙/钙调素依赖性蛋白激酶Ⅱ对热休克小鼠胚胎成纤维细胞HSP70表达的影响

为证实热休克小鼠胎儿成纤维细胞中钙/钙调素依赖性蛋白激酶Ⅱ(Ca2+/calmodulin-dependent protein kinaseⅡ,CaMKⅡ)对热休克蛋白70(Heat shock protein70,HSP70)基因表达的影响及其作用机理,将体外培养的小鼠胎儿成纤维细胞(Mouse Embryonic Fibroblasts,MEF)随机分为39℃和41℃热处理组(分别处理0.5,1,1.5,2 h)和常温对照组(37℃),测定各组细胞CaMKⅡ和HSF1 mRNA的量。此外,将培养的细胞分为37℃和39℃CaMKⅡ特异性抑制剂(myr-AIP)处理组和相应的空白对照组,分别测定各组细胞HSP70、HSF1及其mRNA的量。结果表明,39℃热休克1 h后HSF1和CaMKⅡ的mRNA表达量极显著高于常温对照组,经过myr-AIP处理的39℃组细胞HSP70及其mR-NA含量显著低于39℃空白对照组,而37℃myr-AIP处理组与37℃对照组没有显著差异;myr-AIP对37℃组和39℃组的HSF1 mRNA和蛋白含量影响均不显著,却使p-HSF1的含量显著降低。热休克能增加CaMKⅡ和HSF1 mRNA的转录,且CaMKⅡ通过磷酸化HSF1促进HSP70基因表达。     

不同浓度赖氨酸对猪肠黏膜上皮细胞碱性氨基酸转运载体mRNA表达的影响

研究通过建立猪肠黏膜上皮细胞(Intestinal epithelial cells,IEC)原代培养的方法,研究了赖氨酸对体外培养的猪小肠黏膜上皮细胞碱性氨基酸转运载体mRNA表达的影响。用机械刮取初生仔猪小肠黏膜加胶原酶Ⅱ消化的方法,成功地在体外培养了新生仔猪IEC;分别用浓度为0、2、8mmol/L赖氨酸处理细胞96h,提取细胞总RNA,采用实时荧光定量PCR方法,检测了碱性氨基酸转运载体在不同处理细胞中的表达丰度。结果表明:高浓度的赖氨酸对CAT-1 mRNA的表达有上调作用,差异极显著(P0.01),并且呈剂量依赖效应;不同浓度的赖氨酸对CAT-2 mRNA的表达影响不大,8mmol/L和2mmol/L赖氨酸组CAT-2 mRNA的表达丰度均有低于0mmol/L的趋势,但二者之间差异不显著(P0.05);不同浓度的赖氨酸对y+LAT1 mRNA表达影响差异不显著(P0.05);高浓度的赖氨酸可提高4F2hc mRNA的表达;0mmol/L和2mmol/L赖氨酸组4F2hc mRNA的表达丰度均低于8mmol/L,且差异显著(P0.05);0mmol/L和2mmol/L赖氨酸组之间差异不显著(P0.05)。     

奶牛HSP 70表达量与温湿指数的相关性研究

利用免疫学和分子生物学手段研究荷斯坦牛和娟-荷F1牛,在环境温湿指数(THI)影响下,血液淋巴细胞热应激蛋白70(HSP 70)的表达情况,旨在探索HSP 70表达量与THI相关性以及不同品种差异性。结果表明：(1)荷斯坦成乳牛在热应激和非热应激条件下均可表达HSP 70,严重热应激期(THI 93.5±2.1)HSP 70表达量分别是中度热应激期(THI 76.7±2.6)1.9倍(P<0.01)和非热应激期(THI 56.5±1.7)6.9倍(P<0.01),中度热应激期HSP 70表达量是非热应激期3.6倍(P<0.05),HSP 70表达量与THI呈强度正相关(R=0.91)(P<0.01),THI每上升1个单位,HSP 70表达量增加2172 OD/mm2;(2)在严重热应(THI 95.3)条件下,娟-荷F1成乳牛HSP 70表达量极显著高于荷斯坦牛(P<0.01)。 关键词：奶牛;热应激蛋白;温湿指数;相关性     

金黄色葡萄球菌对小尾寒羊乳腺组织TLR2表达的影响

Toll样受体能够识别细菌等病原微生物及介导炎症反应信号通路,在先天免疫和获得性免疫中发挥着重要作用。为了解Toll样受体2(TLR2)在小尾寒羊正常乳腺组织及由金黄色葡萄球菌引起的乳腺炎乳腺组织中的表达情况,探讨TLR2在金黄色葡萄球菌乳腺炎致病过程中的作用机制及乳腺上皮细胞在乳腺免疫防御中所起的作用,采用qRT-PCR技术和免疫组织化学染色(IHC)法检测了正常小尾寒羊及金黄色葡萄球菌乳腺炎病理模型小尾寒羊乳腺组织中TLR2基因mRNA及其蛋白的表达情况。结果显示,小尾寒羊正常乳腺组织、金黄色葡萄球菌感染乳腺组织均有TLR2 mRNA及其蛋白表达;但金黄色葡萄球菌感染后乳腺组织中TLR2基因及TLR2蛋白显著高于正常乳腺组织,且感染后48 h TLR2 mRNA和TLR2蛋白相对表达量最高(P0.05),96 h TLR2 mRNA和蛋白相对表达量较48 h显著降低(P0.05),正常对照组TLR2的相对表达量最低。通过免疫组织化学染色发现,正常乳腺组织中TLR2蛋白主要定位于乳腺腺泡上皮,而感染金黄色葡萄球菌后乳腺组织中TLR2蛋白主要表达在乳腺腺泡腔中脱落的乳腺上皮细胞和以淋巴细胞为主的炎性细胞上。结果表明,乳腺感染金黄色葡萄球菌后,乳腺上皮细胞是病原菌作用的靶标,通过上调表达TLR2识别病原菌,激发先天免疫。     

SD大鼠热应激模型的制作及评价

为了对热应激SD大鼠模型的制作及生理生化指标进行检测。将20只SD大鼠随机分成两组,一组为对照组,二组为模型组,模型组在霉菌培养箱内以40℃高温、50%相对湿度热攻击2 h,使热应激发生,对照组正常饲养,2 h后观察大鼠行为变化、死亡个体剖检、眼底静脉丛采血、检测各种理化指标和肝脏Hsp70表达量。结果表明：与对照组相比,模型组的pH升高,差异不显著(P＞0.05),Na⁺、K⁺浓度降低,其中K⁺达到差异极显著(P＜0.01);血浆蛋白TP、ALB、HB浓度下降,达到差异极显著(P＜0.01);血清酶ALT、GOT、ALP、LDH、CK活性均表现为升高,达到差异显著或极显著（P＜0.05或(P＜0.01);SOD浓度降低,差异极显著(P＜0.01),MDA浓度升高,差异极显著(P＜0.01);肝脏Hsp70相对表达量升高,差异极显著(P＜0.01)。说明模型组符合热应激指征,SD大鼠热应激模型制作成功。     

不同浓度赖氨酸对猪IEC碱性氨基酸转运载体mRNA表达的影响

本研究通过建立猪肠粘膜上皮细胞（Intestinal epithelial cells, IEC）原代培养的方法,研究了赖氨酸对体外培养的猪小肠粘膜上皮细胞碱性氨基酸转运载体mRNA表达的影响。用机械刮取初生仔猪小肠粘膜加胶原酶Ⅱ消化的方法,成功地在体外培养了新生仔猪IEC;分别用浓度为0.5 mM、2 mM、8 mM赖氨酸处理细胞96 h,提取细胞总RNA,采用实时荧光定量PCR方法,检测了碱性氨基酸转运载体在不同处理细胞中的表达丰度。结果表明：高浓度的赖氨酸对CAT-1 mRNA的表达有上调作用,差异极显著(P<0.01),并且呈剂量依赖效应;不同浓度的赖氨酸对CAT-2 mRNA的表达影响不大,8 mM和2 mM赖氨酸组CAT-2 mRNA的表达丰度均有低于0.5 mM的趋势,但二者之间差异不显著(P>0.05);不同浓度的赖氨酸对y+LAT1 mRNA表达影响差异不显著(P>0.05);高浓度的赖氨酸可提高4F2hc mRNA的表达;0.5 mM和2 mM赖氨酸组4F2hc mRNA的表达丰度均低于8 mM,且差异显著(P<0.05);0.5 mM和2 mM赖氨酸组之间差异不显著(P>0.05)。     

脂多糖诱导奶牛子宫内膜上皮细胞炎性损伤的研究

为了初步探讨大肠杆菌型奶牛子宫内膜炎发生机制,进而为后续筛选出作用于关键信号通路靶点的药物奠定基础。试验通过组织块培养法和传代培养分离纯化得到奶牛子宫上皮细胞(Bovine endometrial epithelial cell,bEEC)。30μg/mL的脂多糖(Lipopolysaccharide,LPS)刺激bEEC 12 h后,收集细胞用于奶牛子宫内膜上皮细胞体外炎性损伤模型的研究。通过荧光定量RT-PCR检测LPS作用于bEEC 2、6、9、12 h后细胞内IL-1β、IL-8、IL-6、TNF-α mRNA表达,然后通过Western-blot检测LPS诱导bEEC NF-кB和MAPKs相关蛋白的表达。结果表明：30μg/mL的LPS作用b EEC细胞2、6、9、12 h可极显著(P<0.01)诱导IL-1β、IL-8、IL-6、TNF-α mRNA表达。LPS可上调MAPKs相关蛋白P38、ERK、JNK的磷酸化水平(P<0.01),促使NF-κB抑制蛋白IκBα的降解(P<0.05)。说明LPS可激活MAPKs和NF-кB信号通路,诱导大量炎症介质的...     

中草药方剂对鲫鱼热应激蛋白HSP 70表达的影响

选用4种中草药方剂AH、CD、Ⅳ和APS,每日按体质量的3%混饲鲫鱼30 d。采用实时荧光定量RT-PCR方法,监测适温(18℃)及热刺激(28℃)1 h后的鲫鱼脑及肝脏中HSP 70表达水平变化。试验结果表明,对照组鲫鱼体内2种组织中HSP 70的表达量在热刺激条件下均有不同程度的升高,且适温和热应激条件下,鲫鱼肝脏组织中HSP 70表达量均明显高于脑组织。而AH组、IV组和APS组热刺激后HSP 70 mRNA含量升高,但CD组热刺激后HSP 70 mRNA转录水平降低,且在肝脏中表达水平降低极为显著。4种方剂中尤以补血养气为主要功能的AH方剂在鲫鱼肝脏中对HSP 70表达水平有极大的促进作用,在适温及热应激条件下,分别是对照组的4.86,3.43倍。因此,中草药方剂AH对促进鲫鱼HSP 70基因高水平表达有较好的作用,有助于鱼体抵抗热刺激防止热损伤,可作为有效的鲫鱼抗热应激备选药物。     

半定量RT-PCR方法检测热应激对鲫鱼肝脏中HSP70 mRNA含量的影响

依据GenBank已公布的鲫鱼热激蛋白70(Heat shock protein70,HSP70)和beta-actin mRNA序列,分别设计两对引物,应用半定量RT-PCR方法检测应激处理后不同恢复时期鲫鱼(Carassius auratus)肝脏中HSP70 mRNA的表达情况。结果表明,诱导后肝脏组织中HSP70 mRNA的表达量呈现时间依赖性,存在先升高后回落的趋势。在诱导后1h即有所升高,在4h时达到最高值,之后逐渐降低,至48h,表达量降至最初值。研究结果为鲫鱼HSP70的研究提供了基础数据,也为将HSP70用作鱼类生理状态的诊断提供了理论依据。     

Heat Transfer Model and Application of Buried Tube Heat Exchanger for Ground Source Heat Pump

This paper introduces the theoretical basis and application method for heat transfer model of buried tube (pipe in pipe and U- type) heat exchanger for ground source heat pump, and a practical example is calculated. Then, the calculation results are analyzed and discussed. In particular, the correlation coefficient and calculation method of tube group exchanger is discussed; the method for determining known parameter and some basic data for main procedure GSHP.HTM is analyzed. The calculation formula and data sampling for JSW.HTM subroutine is introduced. At last, the problems in application of calculation procedure, to which should pay attention, are discussed.     

Experimental Study of Ground-source Heat Pump Systems with Horizontal Heat Exchanger

This paper describes the double layer horizontal ground source heat pump systems constructed in 1998 with the buried depth of 0.9 m and 1.8 m. The program of study includes experimental equipments, heat exchanger size, testing meter and methods for data analysis. In addition, the heating and cooling performance at typical days are also illustrated. Among the influencing factors on the performance of the ground source heat pump systems, the parameter of heat exchanger and operation patterns of heat pump systems are analyzed.A heat transfer model has been set up for horizontal heat exchanger.     

Experiment and Simulation on Heat Transfer Efficiency of Double pipe Heat Exchanger for River Water source Heat Pump

The river water source heat pump system is an important technique to utilize renewable energy in Chongqing and heat exchanger can influence overall energy efficiency of system obviously. A test was carried out to study the heat transfer efficiency of double pipe heat exchanger with semicircular reentrant tube. It was found that the heat transfer efficiency would increase while curvature of semicircular reentrant tube rose. And when the ratio of R to r was five, heat transfer efficiency for semicircular reentrant tube would be increased 2.5% than that of straight double pipe heat exchanger. Flow, temperature field and heat transfer process of semicircular reentrant tube were simulated with computational fluid dynamics(CFD) tool to study the heat transfer mechanism. The modified RNG κ ε Turbulence Model was adopted to ensure the accuracy of simulation analysis under the situation of rotary flow and great curvature. Thus, it was indicated that there was obvious secondary flow vertex in reentrant tube. And disturbance of vertex on boundary layer was the main reason for which heat transfer efficiency of double pipe heat exchanger would increase as curvature of semi toroidal reentrant tube rose. Based on the experiment and simulation, the recommended curvature for reentrant tube was proposed with the consideration of sediment in river water and heat transfer efficiency of heat exchanger.     

小麦耐热性获得和耐热性表现关系的研究

以Seui82×Siete Cerros重组近交系为供试材料,采用电解质渗漏测定细胞膜热稳定性的方法,对耐热性获得与大田生产条件下的耐热性表现的关系进行了研究,发现供试材料幼苗相当热损伤率的变幅为39.0%～75.8%,基因型间在该性状上表现出显著的差异.在幼苗相对热损伤率和大田环境条件下耐热性表现的相关分析中,发现相对热损伤率与大棚升温及第2播期的千粒重、穗粒重热感指数呈显著的正相关,并和第2播期产量性状表现显著的负相关,和升温处理的产量性状也表现出较高的负相关.研究认为细胞膜热稳定性是一个耐热性评价的理想生理指标.     

小麦耐热性鉴定和热处理对品质的影响

为鉴定育成小麦品种的耐热性,同时也为小麦耐热育种提供种质资源,用千粒重热感指数和产量热感指数法鉴定河北、山西和山东省13个推广品种的耐热性。结果表明,晋麦47、石优20、泰山5366和金禾9123耐热性好;石麦22和石4185的产量在正常和热处理环境中差异不大,耐热性较好;衡136的产量和千粒重在正常环境下较高,但在热处理下产量和千粒重较低,耐热性较差。尧麦16的湿面筋、蛋白质、吸水率和延展性的差异极显著。石麦19、邢麦6、沧麦028、石麦22和金禾9123的湿面筋和蛋白质差异显著。     

Coupled Conduction-convective Heat Transfer in the Louvered Fin Heat Exchanger

The heat transfer process in the louvered fin heat exchanger is analyzed,and the corresponding physical and mathematical models on the coupled conduction-convective heat transfer for the louver fin geometry are proposed.The control volume-based finite element method(CVFEM) is employed to solve numerically the problem.The computed(results) reveal the flow structure and heat transfer in the geometry in detail.Compared with the empirical correlation of heat transfer and friction coefficients previously proposed,the computed results show better consistency with the experimental results.     

Research on Neural Network Applied in Heat Load Control of Heat Pump System

In recent years, lots of heat pump systems have been applied for district heating. In order to insure safe and efficient running of these systems, perfect load control methods are necessary. In this paper, the load matching problem between heat pump syst     

Experimental Study of Flow and Heat Transfer in Heat Exchanger with Small Channels

The experimental study of the flow resistance and heat transfer characteristics are conducted for water and ethylene glycol solution (66% Wt) flowing in the heat exchanger with small rectangular microchannels . The heat exchanger having the channels of 0.4 mm in width, 2.0 mm in height, and 20 mm in length is heated by a hearing rod at the bottom surface, the upper and two side surfaces are adiabatic. During experiments, the Reynolds number are ranged from 2 to 2 500. The experimental results show that the flow friction factor decreases and Nusselt number increases with increasing Reynolds number for water and ethylene glycol solution. At a fixed Reynolds number, the Nusselt number for ethylene glycol solution with larger Prandtl number is greater than that for water. Meanwhile, the correlations of flow resistance and heat transfer in the heat exchanger with small channels are obtained for engineering application.