胞质转导肽-HBcAg18-27-Tapasin体外诱导HBV转基因小鼠髓源性树突状细胞成熟及在T淋巴细胞增殖中的作用

目的:观察融合蛋白胞质转导肽(cytoplasmic transduction peptide,CTP)-HBcAg18-27-Tapasin体外诱导HBV转基因小鼠髓源性树突状细胞(dendritic cell,DC)成熟和对T淋巴细胞增殖的作用.方法:体外分离、培养HBV转基因小鼠及近交系C57BL/6小鼠髓源性DC,加入重组粒细胞-巨噬细胞集落刺激因子和白介素(interleukin,IL)-4培养5d,再加入实验组10μg/mL CTP-HBcAg18-27-Tapasin、对照组10μg/mL CTP-HBcAg18-27、10μg/mL HBcAg18-27-Tapasin及空白组RPMI1640完全培养液.流式细胞术测定DC表面分子CD80、CD83、MHC-1的表达,ELISA法测定DC培养上清液中的IL-12p70的水平,细胞计数试剂盒(CCK-8)检测T淋巴细胞增殖反应,流式细胞仪检测增殖的T淋巴细胞内的细胞因子.结果:体外成功诱导小鼠髓源性DC;CTP-HBcAg18-27-Tapasin能明显上调DC表面分子CD80、CD83、MHC-1的表达;并且CTP-HBcAg18-27-Tapasin组诱导DC分泌的IL-12p70水平及诱导DC增殖T淋巴细胞增殖能力明显高于对照组及空白组[IL-12p70转基因小鼠(F=205.85,P=0.000);C57BL/6小鼠(F=406.20,P=0.000)];流式细胞仪检测实验组融合蛋白诱导的CTL水平也高于对照组[转基因小鼠(F=155.45,P=0.000);C57BL/6小鼠(F=392.90,P=0.000)],同时HBV转基因小鼠DC表面分子及在T淋巴细胞增殖中的作用要比C57BL/6小鼠低.结论:分子伴侣Tapasin修饰胞内化抗原肽能促进HBV转基因小鼠髓源性DC的分化、成熟,并能增强DC刺激T淋巴细胞增殖能力及诱导CTL的产生.     

真核表达载体pcDNA3-CTLA4Ig转染小鼠对移植胰岛功能的影响

目的:探讨真核表达载体pcDNA3-CTLA4Ig转染受体小鼠肌细胞后对胰岛移植后排斥反应的影响.方法:受体C57BL/6小鼠随机分为对照组、空白转染组和实验组(转染pcDNA3-CTLA4Ig).Western blot检测受体小鼠血清CTLA4-Ig表达,观察移植物存活时间,术后7 d取受体肾脏做HE染色及胰岛素免疫组化染色,采用微量全血~3H-胸腺嘧啶掺入法检测单个核细胞在刀豆蛋白A刺激下的增殖程度,流式细胞仪检测外周血T细胞亚群表达情况.结果:受体小鼠转染5 d后,Western blot检测到血清内有CTLA4Ig表达,转染效率为27.50%;实验组小鼠血糖维持正常时间明显延长(P<0.05):术后7 d,转染组单个核细胞在刀豆蛋白刺激下的增殖程度明显降低(P<0.05),CD4~ 、CD8~ T淋巴细胞表达与对照组和空白转染组相比有显著差异差异(CD4~ :14.38%±0.84% vs 20.56%±0.68%,21.04%±1.14%,P<0.05:CD8~ :14.77%±0.92% vs 24.63%±1.30%,23.84%±1.21%,P<0.05),小鼠肾被膜下胰岛素免疫组化染色强度明显增高.结论:利用脂质体法可将pcDNA3-CTLA4Ig转染到活体小鼠肌细胞内并在小鼠体内表达CTLA4Ig,从而阻断B7/CD28共刺激信号途径,抑制胰岛移植术后排斥反应.     

日本血吸虫磷酸丙糖异构酶Th1型表位的免疫学鉴定

目的筛选和鉴定日本血吸虫磷酸丙糖异构酶（SjTPI）的Th1型细胞表位,为构建短肽疫苗奠定基础。方法用BLAST比对并预测SjTPI的T细胞表位SjTPI-P18及其对照表位SjTPI-P9。设计并合成其编码DNA,重组入原核表达载体pET-32c（＋）后进行表达,获得纯化的重组融合蛋白rSjTPI-P18和rSjTPI-P9。用rSjTPI、rSjTPI-P18及rSjT-PI-P9刺激经照射致弱尾蚴免疫、rSjTPI加强免疫的C57BL/6和C3H/HeJ小鼠淋巴细胞,^3H-TdR掺入法检测淋巴细胞的增殖效果及细胞培养上清中IL-2水平;分别用重组rSjTPI-P18和合成SjTPI-P18刺激rSjTPI-P18、SjTPI-P18或PBS加弗氏佐剂两次免疫的C57BL/6小鼠淋巴细胞,并检测其细胞培养上清中IFN-γ及IL-4水平。结果参照曼氏血吸虫TPI T细胞表位预测的SjTPI-P18及SjTPI-P9,获得了与Trx融合表达的重组肽rSjTPI-P18及rSjTPI-P9。与rSjTPI-P9相比,rSjTPI及rSjTPI-P18均可刺激辐照致弱尾蚴免疫、rSjTPI加强免疫的C57BL/6或C3H/HeJ小鼠的淋巴细胞增殖且IL-2分泌量增加（P均〈0.05）;SjTPI-P18及rSjTPI-P18刺激经rSjTPI-P18或SjTPI-P18免疫两次的C57BL/6小鼠淋巴细胞分泌IFN-γ水平升高（P均〈0.05）,而IL-4水平较低。结论筛选和鉴定出的SjTPI-P18是C57BL/6小鼠特异的Th1型表位。     

NKT细胞在诱导小鼠皮肤移植免疫耐受中的作用

目的探讨NKT细胞在诱导小鼠皮肤移植免疫耐受中的作用机制。方法 C57BL/6小鼠作为供体,BALB/c小鼠作为受体,建立皮肤移植模型。对照组:单纯行皮肤移植,无其他处理;NKT组:受鼠于术前1 d尾静脉注射供体来源NKT细胞(5×106个/只);CsA组:受鼠于手术当日开始腹腔注射CsA(4mg.kg-1.d-1);NKT+CsA组:受鼠于手术前1 d尾静脉注射供体鼠NKT细胞(5×106个/只),手术当天开始腹腔注射CsA(4mg.kg-1.d-1)。术后每日观察移植皮肤存活情况;术后第10天行混合淋巴细胞反应(Mixed lymphocyte reaction,MLR),确定耐受的状态;并通过过继转移实验,进一步探讨NKT细胞在耐受形成中的作用机制。结果术前输注供体NKT细胞,可延长移植皮片存活时间,并且此种耐受可被过继转移。结论 NKT在诱导小鼠皮肤移植免疫耐受过程中起重要作用。     

N-3多不饱和脂肪酸的抗炎效应对C57BL/6-PD-1~(-/-)基因敲除小鼠心房电重构的影响

目的:观察N-3多不饱和脂肪酸(PUFAs)饲养对C57BL/6-PD-1~(-/-)小鼠体内炎症和心房电重构的影响。方法:观察和比较C57BL/6、PD-1~(-/-)和PUFAs组小鼠的血清炎性细胞因子表达水平和心房有效不应期。结果:与C57BL/6组小鼠相比,PD-1~(-/-)组小鼠血清白细胞介素(IL)-2、IL-4、IL-6、IL-10、IL-17A、肿瘤坏死因子(TNF)和γ干扰素(IFN-γ)水平明显升高,同时PD-1~(-/-)小鼠右心耳、右房低侧壁、右房高侧壁、右房游离壁心房肌有效不应期明显缩短;PUFAs组小鼠血清炎性细胞因子水平较C57BL/6小鼠组大部分仍升高,但升高趋势较PD-1~(-/-)组得到一定程度的遏制,PUFAs组心房有效不应期和C57BL/6组相比无统计学差异。与PD-1~(-/-)组相比,PUFAs组血清炎性细胞因子水平均明显降低,心房有效不应期明显延长。结论:PD-1基因敲除后C57BL/6小鼠体内炎性细胞因子水平明显升高,并出现心房有效不应期缩短,N-3多不饱和脂肪酸饲养能够遏制这种变化趋势。     

自然调节性T细胞对脑型疟疾易感鼠和抵抗鼠感染结局的影响

目的探讨自然调节性T细胞(nTregs)在伯氏疟原虫感染过程中的活化特点及其与疾病进展的相关性。方法用伯氏疟原虫ANKA分别感染C57BL/6、BALB/c和DBA/2小鼠,计数红细胞感染率;感染前和感染后3、5、8d制备脾细胞悬液,流式细胞术检测脾细胞悬液中nTregs百分含量;ELISA和Griess方法检测脾细胞培养上清IFN-γ、IL-10和NO水平。结果C57BL/6小鼠感染后8～11d死于脑疟,BALB/c和DBA/2小鼠感染后3～4w死于贫血和过度虫体血症。3种小鼠于感染后3d,nTregs百分数达到峰值后逐渐下降,于感染后8d,C57BL/6小鼠nTregs百分数下降最为显著并明显低于正常水平,整个感染过程中,C57BL/6小鼠的nTregs百分含量显著低于BALB/c和DBA/2小鼠。3种小鼠脾细胞培养上清IFN-γ、NO和IL-10水平于感染后开始升高,感染后5d达到峰值,感染后8d与感染后5d相比,C57BL/6小鼠IFN-γ、NO水平轻微下降,IL-10水平显著下降;BALB/c和DBA/2小鼠IFN-γ、NO水平显著下降,IL-10水平轻微下降。并且C57BL/6小鼠IFN-γ、NO水平高于BALB/c和DBA/2小鼠,而IL-10水平低于BALB/c和DBA/2小鼠。3种小鼠感染后脾脏nTregs数量与IFN-γ和NO水平呈负相关,IL-10水平与感染率呈正相关。结论伯氏疟原虫感染过程中,nTregs数量的动态变化与宿主感染结局密切相关。     

带有HPV-16 E6/E7基因的DC疫苗抑制裸鼠宫颈癌生长的机制

目的 观察由携带HPV-16 E6/E7基因的树突细胞(dendritic cell,DC)疫苗免疫得到的细胞毒性T淋巴细胞(CTL)在裸鼠体内对官颈癌的抑制作用及相关机制.方法 诱导培养C57B L/6小鼠骨髓的未成熟DC(imDC),将已经成功构建的携带人乳头状瘤病毒(HPV)-16 E6/E7基因的重组腺病毒感染imDC,Western印迹检测E7蛋白的表达;构建裸鼠官颈癌细胞移植瘤模型,应用DC疫苗诱导的CTL处理后,观察裸鼠肿瘤的体积变化;CCK8(cell counting kit8)法检测经DC疫苗免疫的BALB/c小鼠脾脏T淋巴细胞的增殖;免疫组化法检测裸鼠脾脏组织CD4+T淋巴细胞的表达.结果 成功培养出DC并制备出DC疫苗;第28天各组裸鼠肿瘤体积pAd-E6E7-DC-CTL-CaSki组与对照组pAd-mock-DC-CTL-CaSki组、DC-CTL-CaSki组、CaSki组相比较,pAd-E6E7-DC-CTL-CaSki组裸鼠肿瘤体积最小(P＜0.05);CCK8法检测经DC疫苗免疫的BALB/c小鼠脾脏T淋巴细胞的增殖,结果pAd-E6/E7-DC组淋巴细胞OD值明显高于pAd-mock-DC组、DC组和PBS组(P＜0.01);免疫组化检测各组裸鼠脾脏组织CD4 T淋巴细胞的表达结果显示,pAd-E6E7-DC-CTL-CaSki组CD4表达量最高,pAd-mock-DC-CTL-CaSki组和DC-CTL-CaSki组CD4表达量次之,CaSki组CD4表达量最低.结论 带有HPV-16 E6/E7基因修饰的DC疫苗,能够促进小鼠体内T淋巴细胞的增殖,诱导CTL抑制裸鼠宫颈癌移植瘤的生长.     

动脉粥样硬化斑块内FIZZ1表达促进平滑肌细胞清道夫受体-A上调

目的探讨ApoE基因敲除鼠动脉粥样硬化斑块内FIZZ1表达情况及其对平滑肌细胞清道夫受体A(SR-A)表达的影响.方法C57BL/6J ApoE基因敲除鼠及C57BL/6J野生型小鼠各9只,分别喂养高脂饲料及普通饲料,24周后处死小鼠,石蜡包埋血管后做连续切片,行HE染色及FIZZ1免疫组化.用氧化型低密度脂蛋白(ox-LDL)以及终浓度分别为3×10-6mmol/L、9×10-6mmol/L、2.7×10-5mmol/L的FIZZ1刺激培养的平滑肌细胞,激光共聚焦显微镜确认SR-A表达后,流式细胞术检测FIZZ1对ox-LDL诱导的平滑肌细胞SR-A表达的影响.结果ApoE基因敲除鼠高脂饲养24周后,主动脉根部明显形成动脉粥样硬化,可见FIZZ1在动脉粥样硬化斑块内明显表达,同龄野生型C57BL/6J鼠正常血管壁内,未见FIZZ1表达,重组FIZZ1能明显促进ox-LDL诱导的平滑肌细胞SR-A表达(与对照组比较,P<0.01).结论C57BL/6J野生型小鼠正常血管不表达FIZZ1,C57BL/6JApoE基因敲除鼠动脉粥样斑块表达FIZZ1,FIZZ1促进ox-LDL诱导的平滑肌细胞SR-A表达,提示FIZZ1可能在ApoE基因敲除鼠动脉粥样硬化进展中起一定的促进作用.     

诱骗受体3和谷氨酸脱羧酶65重组腺病毒对糖尿病小鼠的作用研究

构建诱骗受体3(DcR3)和谷氨酸脱羧酶65( GAD65)重组腺病毒载体,转染树突状细胞,干预1型糖尿病小鼠,观察小鼠体内CD+8T细胞的活化抑制情况和血糖水平.结果显示转染重组腺病毒的树突状细胞能有效抑制GAD65特异性T细胞的增殖和细胞因子的分泌,γ-干扰素[(50.5±7.2)对(95.4±6.9)、(91.2±6.5)pg/ml]和白细胞介素2[(46.3±5.1)对(86.1±5.2)、(80.3±7.3) pg/ml]低于阴性和空白对照组(均P＜0.05),同时延缓1型糖尿病的发生.提示DcR3和GAD65重组腺病毒可用于1型糖尿病的基因治疗.     

小鼠巨噬细胞Ipr1基因真核表达载体的构建及表达

目的构建小鼠Ipr1(intracellular pathogen resistance 1)基因真核表达载体。方法从C57BL/6J小鼠胸腺组织提取总RNA,以RT-PCR法调取Ipr1基因编码序列,克隆至pMD19-Tsimple载体中,转化大肠杆菌JM109,筛选的阳性克隆作PCR及酶切鉴定后测序分析。其中的突变碱基经点突变修复后将Ipr1基因亚克隆于原核真核双系统表达质粒pQE-TriSystem中得到pQE-TriSystem-Ipr1,用脂质体瞬时转染小鼠巨噬细胞株RAW264.7细胞,RT-PCR鉴定Ipr1基因的表达。结果从C57BL/6J小鼠胸腺组织内扩增出大小为1338bp片段。阳性克隆测序鉴定发现一个点突变,经点突变修复后与GenBank报道的Ipr1基因序列一致。亚克隆获得重组质粒pQE-TriSystem-Ipr1,转染RAW264.7细胞经RT-PCR扩增出Ipr1基因相应大小的DNA片段。结论成功调取小鼠Ipr1基因并成功构建含Ipr1基因的真核表达载体,为进一步研究Ipr1基因的功能奠定基础。     

Interaction of mouse prolactin with mouse hepatic receptors

Lactogenic receptors are usually studied in heterologous systems where prolactin is derived from one species and receptors prepared from another. In such systems the foreign prolactin could be seen as a growth hormone by the host tissue. We have therefore developed a homologous radioreceptor assay using secreted mouse prolactin (smPRL) and mouse hepatic receptors. In this system, monovalent anions augment the smPRL-receptor interaction in the order F- greater than Cl- greater than Br- greater than I-. Divalent cations (Mg2+, Ca2+, Sr2+), phosphate and acetate also increase smPRL binding. Temperature and pH optima are at 8 degrees C and pH 8.3, respectively. Under optimum conditions, the percent total, specific and nonspecific binding are 55%, 45% and 10%, respectively. At infinite receptor concentration the maximum specific bindability of labeled smPRL is 50%. The effects of ions on binding of smPRL to the receptor show that hydrophobic forces participate in smPRL-receptor coupling. The biphasic dissociation kinetics show initial and final rate constants of 1.56 X 10(-4)/s and 7.62 X 10(-6)/s, respectively. The lactogenic receptor does not bind mouse growth hormone; however, it binds both mouse placental lactogen (mPL) and smPRL with equilibrium association constants of 3.90 X 10(8) M-1 and 2.25 X 10(8) M-1, respectively, suggesting that smPRL and mPL share biological roles by acting through the same receptor.     

Studies on dissociation of mouse prolactin from mouse hepatic receptors

The influence of pH, temperature, ethylene glycol, urea, chaotropic anions and excess unlabelled secreted mouse prolactin (smPRL) on the dissociation kinetics of 125I-iodosmPRL from mouse hepatic receptors was investigated. The destabilization of smPRL-receptor complexes by chaotropic anions followed the typical trend of the Hofmeister series: I- greater than Br- greater than Cl- greater than F-. Increasing the temperature of the dissociation reaction from 8 degrees C to 23 degrees C and 30 degrees C caused partial dissociation of 125I-iodosmPRL-receptor complexes. Dissociation of 125I-iodosmPRL from mouse hepatic receptors was pH dependent, with the slowest rate of dissociation occurring at pH 8 and the fastest rate of dissociation occurring at pH 5 and 6. Both ethylene glycol and urea accelerated the rate of dissociation of 125I-iodosmPRL from mouse hepatic receptors in a concentration-dependent manner. Dissociation of 125I-iodosmPRL from mouse hepatic receptors was 6-fold faster in the presence of excess unlabelled smPRL than in its absence. The results of these investigations suggest that both protonation/de-protonation reactions and hydrophobic interactions play important roles in stabilizing the smPRL-receptor complex. In addition, they suggest that cooperative interactions may be involved in the binding of smPRL to mouse hepatic receptors.     

Hybridization of mouse leukemia virus c-DNA to mouse repeated DNA sequences.

Experiments of hybridization between mouse leukemia virus synthetic 3H-DNA probe and mouse main band and satellite DNAs indicate that there is not a higher concentration of viral sequences in the satellite DNA. On the contrary, viral sequences appear to be enriched in the fast renaturing intermediate main band DNA.     

Lactogenic response of cultured mouse mammary epithelial cells to mouse placental lactogen

The ability of mouse placental lactogen (mPL), mouse prolactin (mPRL), mouse GH (mGH) and ovine prolactin (oPRL) to stimulate synthesis of alpha-lactalbumin was tested in a primary culture of mouse mammary gland epithelial cells. Mammary tissue was obtained from 10-day pregnant Swiss Webster mice, enzymatically dissociated and the cells were cultured on floating collagen gels for 5 days. The basic culture medium consisted of Nutrient Mixture F12/Dulbecco's Modified Eagle's Medium (1:1, v/v), containing 10 mg insulin/1, 5 mg cortisol/l, 10 micrograms epidermal growth factor/l, 5 g bovine serum albumin/l and 50 mg gentamycin/l. Mouse PL, mPRL, mGH and oPRL were added to the basic medium in concentrations from 1 microgram/l to 1 mg/l. Accumulation of alpha-lactalbumin in the culture medium was measured. For that purpose, mouse alpha-lactalbumin was purified from mammary tissue obtained from lactating Swiss Webster mice and a radioimmunoassay was developed. Mouse PL, mPRL and oPRL stimulated a dose-dependent increase in alpha-lactalbumin secretion. Mouse GH also caused a significant, but dose-independent, increase in alpha-lactalbumin secretion. Mouse PL showed the greatest activity in stimulating alpha-lactalbumin secretion. It was concluded that mPL is an important lactogenic hormone in the latter half of pregnancy in the mouse, when circulating mPRL concentrations are low.     

Echocardiography in mouse

Assessing cardiac phenotype requires invasive or noninvasive techniques in mouse. Echocardiography is a noninvasive technique for evaluating cardiac function. The purpose of this paper is to underline echocardiography modalities and new tools Doppler applications like tissue Doppler imaging.     

Immunosurgery of mouse blastocyst.

Mouse blastocysts with and without zonae pellucidae are susceptible to complement-dependent antibody cytotoxicity. Exposure of blastocysts to rabbit anti-mouse serum together with complement results in the death of all cells; however, when blastocysts are exposed to antiserum alone and then transferred to guinea pig complement, only the trophoblastic cells are killed. These results suggest that the mouse blastocyst is not permeable for certain antibodies. The inner cell masses can easily be separated from the remnants of trophoblastic cells and are then able to grow and differentiate in vitro. This method of immunosurgery can be used to obtain large quantities of pure inner cell masses in a relatively short period of time.