条件性子宫细胞剔除的研究

为了研究体内细胞的功能和各种细胞之间的相互作用,试验研究了可以特异性消除特定细胞的条件性细胞剔除新方法。通过PR~(Cre/+)工具鼠与pCAG-loxp-STOP-loxp-DTR-2A-EGFP转基因小鼠(DTR小鼠)交配获得子宫特异表达白喉毒素受体(DTR)的转基因小鼠,经过PCR和琼脂糖凝胶电泳鉴定基因后,筛选出PR~(Cre/+)/pCAG-loxp-STOP-loxp-DTR-2A-EGFP雌性小鼠(PR-DTR小鼠)。以DTR小鼠和野生型小鼠(WT小鼠)作为试验对照组,利用免疫组织化学和Western blotting检测子宫中绿色荧光蛋白(GFP)的表达。腹腔注射白喉毒素1 ng/d,连续2 d,观察PR-DTR小鼠、DTR小鼠和WT小鼠子宫的外部形态差异,并通过HE染色的方法观察子宫的内部形态。使用免疫组织化学方法检测注射白喉毒素后PR-DTR小鼠、DTR小鼠和WT小鼠子宫中叉头框蛋白A2(FOXA2)的表达。试验发现在小鼠子宫上皮、基质和肌层细胞中都有GFP的表达,注射白喉毒素后PR-DTR小鼠子宫红肿膨胀,DTR小鼠和WT小鼠子宫没有显著变化。HE染色试验结果发现,PR-DTR小鼠子宫中出现异常空腔,而DTR小鼠和WT小鼠子宫没有明显变化,并且对注射白喉毒素的PR-DTR小鼠子宫中FOXA2表达的检测结果表明,这些异常的空腔不属于腺体,说明白喉毒素使细胞死亡,证实在小鼠子宫中表达孕酮受体的细胞表达了DTR。试验结果表明,白喉毒素在PR-DTR小鼠子宫中可以条件性剔除子宫中表达孕酮受体的细胞,为研究子宫细胞的功能以及子宫细胞相互之间的作用提供了新方法。     

Tgfβ信号通路介导孕酮注射导致的初生仔鼠子宫腺体缺失

为探索小鼠子宫内膜上皮细胞中的Tgfβ信号通路是否能够介导孕酮(P_4)注射导致的新生仔鼠子宫腺体缺失,本研究检测了P_4处理的仔鼠子宫上皮细胞中Tgfβ信号通路受体基因Tgfbr1、Tgfbr2、Tgfbr3及靶基因的mRNA表达水平。结果显示,P_4处理的仔鼠子宫内膜上皮细胞中Tgfbr2和Tgfbr3及靶基因Ctgf和Mmp9的表达水平明显上升,说明Tgfβ信号通路在仔鼠子宫上皮中被P_4激活。此外,在注射P_4的同时给仔鼠注射Tgfβ受体抑制剂,结果发现该抑制剂可以部分挽救因P_4注射导致的小鼠子宫腺体缺失。这些结果说明Tgfβ信号通路部分介导了P_4注射导致的初生仔鼠子宫腺体缺失。     

囊胚注射转基因ES细胞制作嵌合体的研究

在小鼠胚胎成纤维细胞制作的饲养层上培养并成功的维持了携带LacZ基因的胚胎干细胞系（S8），在此基础上，以S8为供体细胞，以远交系昆明白小鼠3．5d胚胎为受体，通过显微注射法将供体细胞转移到受体的囊胚腔内，经过恢复培养，移植到代孕鼠昆明白雌鼠的子宫中；后代在嵌合体出生一周后进行判定。本试验用8～13代的S8细胞共注射胚胎597枚，经1～3h恢复培养，有585枚胚胎重新具有膨大的囊胚腔，细胞轮廓分明，滋养层细胞间连接也清晰可见，胚胎成活率为97％；胚胎移植后，代孕母鼠共移植胚胎228枚，经17～19d的妊娠期后，产仔37只（2只死胎），产仔率为16％；有35只仔鼠（雄鼠18只，雌鼠17只）存活到可以判断毛色，共获得8只S8细胞毛色嵌合体小鼠，嵌合体的产生率为21．6％。结果表明用S8细胞经囊胚注射后能够获得嵌合体，并且嵌合体明显发生了性偏离现象。本试验为国内利用囊胚注射携带LacZ基因的胚胎干细胞获得嵌合体小鼠的首例报道。     

带有GFP的小鼠Sox2基因逆转录病毒载体的构建及其检测

为了建立体细胞重编程过程中检测Sox2基因表达变化的方法,本研究利用克隆获得的小鼠Sox2基因与逆转录病毒载体pMX—IRES—GFP连接,构建带有报告基因GFP的逆转录病毒载体,将其转染293GP细胞后获得假病毒上清。将获得假病毒上清侵染小鼠成纤维细胞（NIH3T3细胞）后观察GFP的表达和检测Sox2转录量的变化。其结果,成功构建带有GFP的Sox2基因逆转录病毒载体pMX—Sox2-IRES—GFP;将构建的载体转染后293细胞后获得能侵染NIH3T3细胞的假病毒上清,且与未侵染的NIH3T3细胞相比,侵染后的NIH3T3细胞的Sox2表达量提高近10倍。本研究为开展在体细胞重编程过程中Sox2表达与重编程效率的相关性提供依据．     

NO在小鼠早期胚胎吸收过程中的作用

给妊娠 7d小鼠尾静脉注射细菌脂多糖 (L PS)诱导早期胚胎吸收。注射 L PS后 12、2 4、36 h,以 EL ISA、比色法检测血清和子宫匀浆中 Th1型细胞因子 IFN- γ、IL- 12与 NO的含量变化 ;免疫组织化学法观察 3种一氧化氮合酶(n NOS,e NOS,i NOS)和 Th2型细胞在小鼠子宫表达的变化。此外 ,还观察了 NO供体硝普钠 (SNP)诱导孕鼠流产效果及氨基胍 (AG)对抗 L PS诱导孕鼠流产的效果。结果显示 ,相对于正常妊娠组 ,L PS处理组孕鼠子宫匀浆 NO含量及 IFN- γ、IL- 12水平极显著升高 (P<0 .0 1) ,血清 NO含量也极显著升高 (P<0 .0 1) ;n NOS、i NOS在 L PS处理组小鼠子宫可见阳性标记 ,而 e NOS未见阳性标记 ;大量 Th2型阳性细胞标记仅在正常妊娠组小鼠子宫内膜基质可见 ,L PS处理组未见阳性细胞。腹腔注射 SNP致使孕鼠早期胚胎吸收 ,然而妊娠 6～ 9d腹腔注射 AG却未能降低 L PS诱导的孕鼠早期胚胎吸收。上述结果提示 ,L PS处理后 ,Th1型免疫反应增强 ;源自升高表达的 i NOS的子宫局部高浓度 NO可能作为一种效应分子介导小鼠早期胚胎吸收。     

“益母散”对去卵巢小鼠发情和雌激素受体表达的影响

旨在研究纯中药制剂"益母散"是否对小鼠子宫有直接作用并影响小鼠的发情和雌激素受体的表达。以性成熟雌性小鼠为研究对象,通过手术去除小鼠卵巢,用"益母散"进行灌胃试验,试验期间每天进行阴道涂片,检测小鼠的发情情况,试验结束后取出子宫,测子宫质量,同时对子宫组织HE染色观察子宫的组织形态,通过PCR检测子宫雌激素受体表达,并测定发情期血清雌激素水平。结果表明,"益母散"可明显抑制去卵巢小鼠子宫的萎缩(P0.01),并能促进去卵巢小鼠子宫发育、维持子宫正常组织形态、促进雌激素受体ERαmRNA表达,使去卵巢小鼠出现发情表现。这表明"益母散"具有一定的类雌激素样作用,可通过上调雌激素受体表达,使去卵巢小鼠在内源性雌激素水平较低的情况下出现发情表现。     

金黄色葡菌球菌脂蛋白对M1型小鼠骨髓源巨噬细胞免疫作用的影响

本研究旨在阐明金黄色葡萄球菌脂蛋白对M1型小鼠骨髓源巨噬细胞免疫作用的影响，为金黄色葡萄球菌致病性研究提供理论参考。以金黄色葡萄球菌野生株SA113（WT SA113）和SA113 lgt：：ermB脂蛋白表达缺失菌株（SA113Δlgt株）体外感染M1型小鼠骨髓源巨噬细胞，分为3组：空白对照组、WT SA113感染组（MOI：3：1）、SA113Δlgt感染组（MOI：3：1）。采用ELISA法检测M1型小鼠骨髓源巨噬细胞中肿瘤坏死因子-α（TNF-α）、白细胞介素1β（IL-1β）、趋化因子（RANTES）和白细胞介素10（IL-10）的水平，实时荧光定量PCR检测Toll样受体2（TLR2）、Toll样受体4（TLR4）和含NLR家族Pyrin域蛋白3（NLRP3）基因的表达，免疫荧光法检测脂蛋白对M1型小鼠骨髓源巨噬细胞吞噬金黄色葡萄球菌作用的影响。结果显示，与空白对照组相比，WT SA113感染组和SA113Δlgt感染组均可显著上调M1型小鼠骨髓源巨噬细胞中TNF-α、RANTES、IL-10分泌量以及WT SA113感染组TLR2、NLRP3基因表达水平（P<0.05），而TLR4基因表达量显著降低（P<0.05）；与WT SA113感染组相比，SA113Δlgt感染组M1型小鼠骨髓源巨噬细胞中TNF-α、IL-1β、RANTES、IL-10分泌量以及TLR2（12 h除外）、NLRP3基因表达水平显著降低（P<0.05）。免疫荧光结果显示，M1型巨噬细胞对SA113Δlgt株的吞噬作用显著低于对WT SA113株的吞噬作用（P<0.05）。综上，金黄色葡萄球菌的脂蛋白在M1型小鼠骨髓源巨噬细胞中主要通过激活TLR2和NLRP3受体，诱导细胞因子TNF-α、IL-1β、RANTES和IL-10的产生和释放。     

人类α-actin 启动子真核表达载体的构建及应用

利用PCR技术克隆了人类α-actin基因的启动子（约450bp），尝试用去掉启动子的pEGFP—N1作为框架结构，成功构建了真核表达载体pEGFP-N1-α-actin—P。应用Lipofectamine 2000将所构建的pEGFP-N1-α-actin-P转染入小鼠ES细胞。通过比较，发现在本实验室条件下，质粒DNA浓度以3．0～5．0μg／mL，转染时间约在2～3h最佳。200μg／mL的G418较适宜于靶细胞的转染与筛选。得到表达心肌a—actin和GFP的小鼠ES细胞，基本维持小鼠ES细胞的形态。PCNA染色结果表明，转染后的小鼠ES细胞具有增殖能力。α—actin抗体免疫组化染色结果表明，转染后细胞表达α-actin和GFP，揭示构建的真核表达载体pEGFP-N1-α-actin-P转染小鼠ES细胞，可能促进其向心肌细胞分化并对其筛选。     

川楝素对昆明小鼠的胚胎毒性研究

取体重18~25 g雌鼠,超数排卵后与公鼠合笼。获得的孕鼠随机分为 6 组,分别用于研究川楝素对孕鼠2 细胞胚、桑椹胚、囊胚3个时期胚胎的毒性。每个时期均设对照组,以小鼠腹腔注射1/30半数致死量(LD50)的川楝素剂量对以上3个时期试验组孕鼠染毒2次,对照组孕鼠用等量PBS注射。研究其着床时期胚胎毒性时不对小鼠进行超数排卵,但试验组孕鼠染毒3次,对照组孕鼠用等量PBS注射。最后计数、观察4个时期孕鼠子宫内的胚胎数量和形态。结果表明:2 细胞胚、桑椹胚、囊胚时期试验组胚胎总数与对照组相比,没有统计学上差异(P>0 05); 2 细胞期试验组正常胚胎、异常胚胎数量与对照组相比,也无统计学上的差异(P> 0 05);桑椹胚、囊胚时期试验组正常胚胎、异常胚胎数量与对照组相比,具有极显著差异(P<0 01)。3 次染毒对着床后的胚胎毒性最大,子宫内胚胎几乎全部溶解,无法计数。母体组织病理切片观察发现川楝素对主要器官心、肝、肺、肾、子宫仅有轻微损害,提示腹腔注射小剂量川楝素对怀孕小鼠具有特定的胚胎毒性。     

小鼠感染猪圆环病毒2型的超微结构病变观察

为探讨猪圆环病毒2型（PCV2）对小鼠超微结构的病理损伤及其病毒在细胞内的复制,20只6周龄的昆明小鼠随机平均分成2组（即A组和B组）,A组小鼠经腹腔注射PCV2细胞培养物0.1mL/只（含病毒1 000TCID50）,B组以同样的方式和剂量注射无菌细胞培养液作为对照。于PCV2感染后14d,处死所有小鼠,取其组织做电镜观察和PCV2PCR检测。结果显示,在电镜下,所有PCV2感染鼠的超微结构病变基本一致,主要表现为淋巴器官、心脏、肝脏、肺脏、肾脏、脑、肠等脏器实质细胞凋亡或坏死,细胞内线粒体水肿、内质网扩张,间质毛细血管淤血、炎症细胞浸润,并在脾脏、胸腺、淋巴结的淋巴细、巨噬细胞,以及肝细胞,肾足细胞,脑神经细胞的胞浆或胞核内发现病毒包涵体。同时通过PCR检测,所有PCV2感染鼠的组织均可检出PCV2DNA。B组（对照组）小鼠除在淋巴结可见极少数淋巴细胞凋亡外,其他组织均无任何超微结构病变;同时,所有组织也未检出PCV2DNA。由此说明PCV2可在昆明小鼠多脏器实质细胞内复制,并诱导细胞凋亡。     

重组白喉毒素活性的研究

为获得高活性、副作用小的重组白喉毒素,在免疫毒素药物ONTAK的基础上,从免疫毒素构建、纯化筛选、对动物及人类的活性研究方面出发,对近年来的重组白喉毒素的研究内容进行了汇总分析。重组白喉毒素的构建方式简捷,临床应用效果良好,通过动物实验可用于多种癌症的治疗,因此重组白喉毒素对肿瘤细胞具有靶向特异性、高效性的优点,是靶向治疗癌症的重要研究方向。     

精氨酸和维拉帕米对缓解胎鼠宫内发育迟缓的影响

本研究在日粮中添加精氨酸(L-Arg)和钙阻滞剂维拉帕米,旨在探讨L-Arg通过钙信号对小鼠宫内发育迟缓(IUGR)的影响。将160只雌性小白鼠随机分为4组,对照组饲喂基础日粮,L-Arg组、钙阻滞剂组、L-Arg+维拉帕米组分别在基础日粮中添加0.5%L-Arg、8 mg/kg维拉帕米、0.5%L-Arg+8 mg/kg维拉帕米。在妊娠第10、12、14、16天颈部放血处死母鼠,剖腹取胎鼠、子宫和胎盘等样品,测定相关指标,取14 d母鼠子宫固定于Carnoy’s和多聚甲醛中,分别用作肥大细胞(MC)、HE和免疫组化染色。结果表明:Arg组胎鼠数量、胎鼠体重等IUGR指标显著高于对照组(P0.05),维拉帕米组显著或极显著低于Arg组(P0.05或P0.01);L-Arg组子宫MC数显著低于对照组(P0.05),极显著低于维拉帕米组(P0.01);Arg组子宫巨噬细胞数较对照组显著增多(P0.05),维拉帕米组较对照组显著减少(P0.05),Arg组与维拉帕米组差异极显著(P0.01);L-Arg组子宫微血管(MVD)数较对照组显著增多(P0.05),较维拉帕米组显著减少(P0.05)。综上,L-Arg通过调控MC释放介质来提高母鼠的繁殖能力,维拉帕米能阻止Arg该作用,表明L-Arg通过钙信号发挥生物学作用,是其缓解IUGR的机理之一。     

Oxytocin receptors in dioestrous and anoestrous canine uteri

The aim of the study was to localize oxytocin receptors (OTR) and measure mRNA expression of OTR in the canine uterus with and without the influence of progesterone. Uterine samples were taken from nine anoestrous and eight dioestrous bitches during ovariohysterectomy. Histological changes were evaluated in haematoxylin and eosin (HE)‐stained samples. Purified polyclonal antibody for OTR was used in immunohistochemistry to localize receptors in uterine layers. Relative mRNA concentration of OTR was evaluated with real‐time PCR from full‐thickness uterine samples taken from the middle horn and the body. Myometrial smooth muscle cells, endometrial luminal epithelium (LE) and deep and superficial glandular epithelium were positively stained for oxytocin receptors in non‐pregnant animals. No significant difference in staining intensity was detected between uterine middle horn and body. However, the staining intensity of LE was significantly higher in dioestrous than in anoestrous uteri (p < .05). Leucocytes and endothelium of blood vessels were also positively stained for OTR. Real‐time PCR showed no significant differences in OTR mRNA expression between the middle horn and the body of the uterus, or between anoestrous and dioestrous uterus. No correlation was noted between OTR mRNA expression and blood progesterone concentration. In conclusion, despite the apparent inactivity, the uterus of the non‐pregnant bitch expresses OTR. The distribution or relative expression of OTR does not differ between uterine horn and body in dioestrus or anoestrus except in LE. LE may have more oxytocin‐dependent activity during dioestrus than anoestrus.     

Expression of interleukin-18 receptor mRNA in the mouse endometrium

Interleukin-18 (IL-18) is a proinflammatory cytokine involved in chronic inflammation, autoimmune diseases, and a variety of cancers, and is expressed in mouse uteri. Our previous study suggested that IL-18 acts as a paracrine factor, regulating endometrial function. To elucidate the physiological roles of IL-18 in the mouse endometrium, the expression of the IL-18 receptor (IL-18R) alpha subunit was analyzed. IL-18Ralpha mRNA was expressed in several mouse organs in addition to the endometrium. In situ hybridization analysis using a biotin-labeled mouse IL-18Ralpha riboprobe demonstrated that IL-18Ralpha mRNA expression was detected in glandular epithelial cells, stromal cells around uterine glands, and myometrial cells in the mouse uterus, suggesting that these cells are targets for IL-18. The uterine IL-18Ralpha mRNA expression level changed with the estrous cycle. The uterine IL-18Ralpha mRNA levels of estrous mice were higher than those of diestrous mice. In addition, the IL-18Ralpha mRNA levels in uteri at 3 and 14 days after ovariectomy were higher than those at diestrus and decreased following treatment with estradiol-17beta or progesterone. These findings suggest that IL-18Ralpha gene expression is regulated by estrogen and progesterone and that the uterine IL-18 system is involved in the regulation of uterine functions in a paracrine manner.     

Differentiation and elimination of uterine natural killer cells in delayed implantation and parturition mice

To clarify the roles of uterine natural killer (uNK) cells in implantation and parturition, differentiation and elimination of uNK cells in the pregnant uterus was examined using artificial delayed implantation (DI) and delayed parturition (DP) mice. To prepare DI mice, pregnant mice were ovariectomized on the third day of pregnancy (D3) and treated with 2 mg progesterone daily. The same amount of progesterone was administered on D15 or D17 of normal pregnant mice at 24 h intervals until sampling to prepare DP mice. The uNK cells contained PAS-positive granules on D8 in DI mice. The uNK cells in DI mice were smaller in size, and differentiation of these cells was delayed compared to those of the control mice. From D19 to D21 in DP mice, the metrial gland was well developed and uNK cells were present. The number of uNK cell granules decreased on D21, and there were no uNK cells in the normal pregnant mice. This result suggests that differentiation of uNK cells is not directly related to implantation, but elimination of these cells is closely involved in parturition.     

Corynebacterium ulcerans from diseased wild boars

Two Corynebacterium strains were isolated from lymph nodes of wild boars showing severe alterations caused by caseous lymphadenitis. The wild boars came from different districts in southern Germany; one was found dead, the other had been shot. The two Corynebacterium strains obtained were both positive for phospholipase D. Further analysis of biochemical profiles did not allow unambiguous differentiation between Corynebacterium ulcerans and Corynebacterium pseudotuberculosis. Fourier-transformed infrared spectroscopy as well as partial sequencing of the genes for 16S rRNA and RNA polymerase beta subunit (rpoB) clearly identified both strains as Corynebacterium ulcerans. The tox gene for diphtheria toxin (DT) could be detected in both porcine isolates by PCR. Partial DNA sequencing of this tox gene showed significant differences from sequences described for other Corynebacterium ulcerans strains and a higher degree of similarity to that of Corynebacterium diphtheria. Production of diphtheria toxin could not be detected. These results indicate that wild game could be a reservoir for zoonotic Corynebacterium ulcerans.     

葛根素对去卵巢小鼠乳腺及子宫发育的影响

目的：观察葛根素对去卵巢小鼠乳腺及子宫的影响,研究其雌激素样作用。方法：将21日龄昆明系雌性小白鼠50只随机分成5组,每组10只。①正常对照组,正常＋蒸馏水;②阴性对照组,去势＋蒸馏水;③阳性对照组Ⅰ,去势＋己烯雌酚;④阳性对照组Ⅱ,去势＋己烯雌酚＋黄体酮;⑤试验组,去势＋葛根素。做乳腺标本和乳腺及子宫的常规切片,光镜观察。结果：摘除卵巢后的小鼠乳腺及子宫发育停止,乳腺导管和腺泡数（P〈0.01）、子宫系数（P〈0.01）、子宫壁腺体数（P〈0.05）与正常对照组比较差异显著或极显著。试验组的各项指标与阴性对照组比较差异不显著（P〉0.05）,但高于阴性对照组。结论：葛根素有微弱的雌激素样作用,对去卵巢小鼠乳腺和子宫发育有显著的影响。     

薄荷油对雌性小鼠的抗生育作用

为探讨薄荷油对雌性小鼠的抗生育作用,将80只健康的雌性性成熟昆明小鼠随机分成3个试验组和1个对照组,各试验组小鼠分别灌胃含薄荷油0.135,0.270,0.540g/（kg·d）的0.5%羧甲基纤维素钠（sodium carboxymethylcellulose,CMC）混悬液,对照组灌胃0.5%CMC溶液,1周后按雌雄比2∶1合笼,继续对雌鼠灌胃总计19d,剖检灌胃雌鼠,统计雌鼠的怀孕率,采集血清进行血液生化指标检测,并采集脾脏和肾脏,计算其脏器系数。采集卵巢、子宫进行固定,并进行病理学观察。同时进行抗着床、抗早孕、抗中期和抗晚期妊娠试验。结果表明,薄荷油具有明显的抗生育作用,并对妊娠各阶段有显著影响。对脾脏和肾脏无明显副作用,其脏器系数均无统计学意义。病理学观察结果显示,卵泡间质组织毛细血管充血,卵泡细胞发生灶性坏死,可见炎性细胞浸润。子宫肌层、固有层和黏膜层发生不同程度的充血、出血、水肿和炎症,严重者,可见固有层细胞发生坏死。这表明薄荷油对雌性小鼠的抗生育作用主要是通过破坏子宫和卵巢等生殖器官而实现的,其作用机制需进一步研究。     

Histopathologic features, environmental factors, and serum estrogen, progesterone, and prolactin values associated with ovarian phase and inflammatory uterine disease in cats.

Forty-four female American Shorthair cats with inflammatory uterine disease or infertility were evaluated. Data collected included age, month of diagnosis, housing, reproductive history, results of bacteriologic culture of uterine specimens, serum concentrations of estrogen, progesterone, and prolactin and histopathologic features of the ovaries and uterus. Histologically, the ovaries of 19 cats were dominated by active or cystic follicles, whereas 25 cats had luteal-phase ovaries. Of the 25 cats with active corpora lutea, 20 had either recently weaned litters (n = 11) without subsequent exposure to a male cat, or had been housed individually for lengthy periods (n = 9). The finding of active corpora lutea under these circumstances indicates that in queens, ovulation may occur by mechanisms not involving coitus. Prominent, active corpora lutea on the ovaries were associated with adenomatotic proliferative changes in the superficial and glandular epithelium of the uterus and with myometrial hyperplasia, compared with the uterus of cats with follicular ovaries (P less than 0.01). Serum progesterone concentration greater than or equal to 1.87 ng/ml was consistently associated with luteal-phase ovaries. Serum progesterone values less than or equal to 0.15 ng/ml were consistently associated with follicular-phase ovaries. Escherichia coli was the organism most commonly isolated from uterine contents.