小鼠动情周期内雌孕激素波动与皮层及海马δ-亚基的突触外γ-氨基丁酸A型受体表达的相关性

目的 探讨雌性小鼠动情周期不同阶段血清孕酮浓度与大脑皮质和海马中含δ-亚基的突触外γ-氨基丁酸A型受体(δGABAARs)表达的相关性.方法 动情间期和动情期成熟卵巢周期雌性小鼠(4～6周,n=12)血清雌激素和孕酮浓度采用ELISA试剂盒检测;动情间期和动情期大脑皮层和海马中δGABAARs表达采用免疫组织化学方法检测.结果 在动情间期,皮层和海马δGABAARs表达明显高于动情期,血清孕酮的浓度与δGABAARs的积分吸光度值成线性关系.结论 动情期不同阶段孕酮的生理波动会影响δGABAARs在海马及皮层等特定脑区的表达,而海马神经元δGABAARs表达的差异会影响脑区神经元的兴奋度,从而可能影响雌性动物的行为特性.     

表达hIFNα-2b基因重组乳杆菌治疗HSV-2感染的研究

目的 探讨表达人干扰素α-2b的重组乳杆菌应用于阴道HSV-2感染的治疗作用。方法 建立阴道感染HSV-2的小鼠模型，使用重组菌菌液于感染前24h进行预防，或感染后48h进行治疗，记录病损程度并检测小鼠阴道分泌物中HSV-2滴度。结果使用重组菌预防或治疗的小鼠症状轻且病程缩短，病灶中病毒滴度下降很快。结论表达IFNα-2b的重组乳杆菌具有较强的局部抗病毒作用，可用于预防及治疗由HSV-2感染引起的生殖器疱疹。     

一氧化氮和前列腺素F2α对大鼠颗粒细胞雌二醇及孕酮分泌的影响

为探讨一氧化氮（NO）和前列腺素（PGF2α）对大鼠卵巢颗粒细胞分泌雌、孕激素的影响，给未成年雌性大鼠注射孕马血清促性腺激素（PMSG），48h后取卵巢，收集颗粒细胞并分组培养。24h后，分别加入NO供体硝普钠（SNP）、PGF2α和SNP＋PGF2α，48h后收集细胞培养液，用放免法测定雌、孕激素的含量。结果发现SNP可增加颗粒细胞孕酮分泌，减少雌二醇分泌，且呈量效关系；PGF2α可使雌二醇和孕酮含量均降低；SNP和PGF2α联合可使孕酮含量升高，雌二醇含量降低。结果提示SNP和PGF2α都参与大鼠卵巢颗粒细胞雌、孕激素合成的调节。     

小鼠动情各期子宫内膜的显微结构及动情期凋亡因子在生殖器官的表达

目的:探讨动情期小鼠卵巢、输卵管、子宫中Bcl-2、Bax、caspase-9的表达及定位。方法:取各期小鼠卵巢、输卵管和子宫,石蜡切片、H-E染色,显微镜观察;免疫组织化学法检测Bcl-2、Bax、caspase-9在动情期小鼠的卵巢、输卵管、子宫中的表达。结果:动情各期子宫内膜结构各有特点,动情期及前后的子宫内膜厚度较厚,与动情间期比较差异有统计学意义;Bcl-2、Pax、caspase-9在动情期小鼠生殖器官均有强弱不等的表达。结论:本研究提供了小鼠动情各期子宫内膜的结构变化,明确了Bcl-2、Bax和caspase-9在动情期卵巢、输卵管、子宫中的定位和表达。     

Effect of estrogen and progestogen on the expression of interleukin-8 in mouse endometrium and blastocyst during preimplantation

目的:研究雌、孕激素对着床前小鼠子宫内膜及胚泡白细胞介素8(IL-8)表达的影响.方法:利用经典小鼠胚泡延迟着床模型,应用免疫组织化学显色、免疫印迹及图像分析技术,对着床前小鼠子宫内膜及胚泡IL-8的蛋白表达进行定位及半定量分析.结果:IL-8主要表达于妊娠小鼠子宫内膜腔上皮细胞及胚泡内细胞群和滋养层细胞的细胞质内.在子宫内膜组织中,切除双侧卵巢后单独给予孕酮组(P4组),IL-8表达低于联合应用雌二醇和孕酮组(E2+P4组)和对照组;E2+P4组IL-8表达上调,但低于对照组.在各组胚泡中,单独给予孕酮获得的静止胚泡、IL-8蛋白含量低于正常胚泡;联合应用雌二醇、孕酮获得的激活胚泡、IL-8的蛋白表达高于静止胚泡.结论:切除卵巢后,联合应用雌、孕激素可上调着床前小鼠子宫内膜及胚泡IL-8蛋白的表达.     

HSV-2感染人外周血淋巴细胞后TCRVβ基因片段的选择性扩增

T细胞受体（TCR）Vβ基因 1—20亚家族在识别和杀伤病毒及肿瘤抗原方面各自有着重要的作用。本研究采用单纯性痢疾病毒（HSV-2）感染正常人的外用血淋巴细胞（PBLs）后发现TCR Vβ2,6,7,8基因在体外选择性扩增,而用 HSV-2攻击生殖器单纯疱疹性皮肤病患者不同病程时期的 PBLs时发现 TCRVβ基因的表达水平随病程不同而改变。结果提示Vβ7,8在识别HSV-2时呈选择性扩增而限制 HSV-2的增殖。     

动情期和动情间期小鼠输卵管中氧化应激和抗氧化基因表达差异

目的观察昆明(Kunming,KM)小鼠输卵管中氧化应激和抗氧化基因的表达是否随动情周期而改变。方法分别取动情期和动情间期小鼠输卵管进行RNA提取,用PCR芯片检测氧化应激和抗氧化基因表达的差别,然后用Real Time PCR对差异表达基因进一步验证。结果与动情间期比较,动情期小鼠输卵管中NADPH氧化酶1(NADPH oxidase1,NOX1)、谷胱甘肽过氧化物酶2(glutathion peroxidase 2,GPX2)、超氧化物歧化酶3(superoxide dismutase 3,SOD3)、一氧化氮合酶2(nitric oxide synthase 2,NOS2)等4个基因表达上调。结论动情期小鼠输卵管中NOX1、GPX2、SOD3和NOS2等4个基因表达上调,提示其可能参与维持动情期小鼠输卵管腔内适度的活性氧自由基(reactive oxygen species,ROS)和一氧化氮(nitric oxide,NO)水平,为卵子受精和早胚发育提供必要第二信号分子。     

雌、孕激素对小鼠uNK细胞在子宫壁内分布的影响

目的:探讨甾体激素对小鼠子宫自然杀伤(uterine Natural Killer,uNK)细胞在子宫壁内分布的调节作用。方法:用uNK细胞特异性的DBA-lectin抗体,对去卵巢以及卵巢甾体激素处理的小鼠子宫进行免疫组织化学标记。结果:在去卵巢小鼠给予雌二醇后,uNK细胞主要分布于子宫的基质,为一些小的、圆形细胞;给予孕酮后,DAB-lectin染色主要见于一些血管内皮细胞;联合给予雌二醇和孕酮后,DBA-lectin染色分布于子宫的基质,既可见于一些小的、圆形uNK细胞,也见于一些血管内皮细胞。雌、孕激素作用均可被其相应受体的拮抗剂所阻断。结论:雌、孕激素对小鼠uNK细胞在子宫壁内分布的具有协同性的调节作用,共同参与妊娠子宫对半同种异体胚胎的保护性免疫反应。     

Expression of Maspin in mouse endometrium during estrus cycle and early pregnancy

目的:初步探讨Maspin基因在动情周期及早孕小鼠子宫内膜的表达规律.方法:采用实时荧光定量PCR和免疫组织化学分别检测动情前期、动情期、动情后期、动情间期及孕2、4、5、7d小鼠子宫内膜Maspin基因mRNA及蛋白的时空表达规律.结果:动情周期中动情期MaspinmRNA和蛋白表达较强,与其他3期相比差异有统计学意义,其他3期表达较弱,无差异.早孕小鼠子宫内膜组织Maspin基因mRNA和蛋白的表达高于未妊娠小鼠,且随着妊娠天数的增加呈逐渐增强的趋势,到妊娠第5天达到最高.结论:Maspin在动情周期及早孕小鼠子宫内膜呈规律性表达,说明其可能在胚泡着床过程中发挥着重要的作用.     

小鼠动情周期与脂多糖诱导肝、肺组织病理变化的关系

目的探讨小鼠动情周期与脂多糖(LPS)所致肝、肺组织病理损伤的关系及可能的机制。方法小鼠分别于动情期、动情后期、动情间期等不同阶段腹腔注射亚致死量(12.5 mg/kg)LPS,观察LPS注射后肝、肺病理的损伤,并于LPS注射后0、5、10、20 h同步测定血浆E2、P及血浆和卵巢TNF-α、EGF含量,分析它们之间的关系。结果小鼠于动情间期血浆E2和P含量最低,LPS组与对照组比较血浆E2和P含量均于10 h明显下降(P<0.05和P<0.01),并持续至20 h(两者P<0.01);血浆和卵巢TNF-α于5 h明显升高(P<0.05),并持续至20 h(两者P<0.01);血浆和卵巢EGF于5 h明显下降(P<0.05)并分别持续至20 h(P<0.01)和10 h(P<0.05)。与动情期和动情后期LPS组比较,动情间期肝、肺组织病理损伤程度较重,血浆E2、P含量明显下降(P<0.01),血浆和卵巢中TNF-α含量升高(P<0.01),而EGF含量下降(P<0.05)。结论动情期和动情后期高水平的E2和P对LPS造成的组织损害有保护作用,其机制可能与TNF-α产生减少和EGF产生升高有关。     

X-linked resistance of mice to high doses of herpes simplex virus type 2 correlates with early interferon production.

Mice inoculated intraperitoneally with herpes simplex virus type 2 develop focal necrotizing hepatitis and eventually die from ascending myelitis and encephalitis. The genetics of resistance to the infection were analyzed in crosses between resistant C57BL/10 mice and susceptible BALB/c mice. It was shown that the resistance of C57BL/10 mice to hepatitis induction was influenced by an X-linked dominant gene as previously shown for the GR mouse strain. The course of infection in the liver pointed to early, natural defense mechanisms as being responsible for the difference between the mouse strains, whereas the clearance of virus from the liver, probably mediated by specific immunity, was exerted at the same time and with equal efficiency for all groups of mice. In mortality experiments, resistance was shown to be an autointerference phenomenon in that a considerable number of C57BL/10 mice survived an intraperitoneal injection of 10(6) PFU, whereas all mice were killed by 10(5) PFU. This resistance of C57BL/10 mice to high doses of HSV-2 was retrieved in all groups of F1 mice in crosses between C57BL/10 and BALB/c mice except the (BALB/c female X C57 male) male group, in which the mice receive the X chromosome from the susceptible BALB/c female. Thus, the autointerference phenomenon also seems to be influenced by loci on the X chromosome. A similar pattern of inheritance was observed when early interferon induction (4 to 5 h after infection) in response to HSV-2 was measured. The possible relevance of this early interferon response in conjunction with other potential natural defense mechanisms is discussed.     

Virus specificity of the antiviral state induced by IFN gamma correlates with resistance to MHV 3 infection

Summary A comparative study was carried out to investigate the correlation between the antiviral effect induced in macrophages by IFN gamma and the resistance of A/J and BALB/c mice to an experimental infection of MHV 3, MHV 4, and MHVA 59. Both mouse strains were resistant to intraperitoneal infection with MHV 4 or MHVA 59 and only the A/J mice showed resistance to MHV 3, the BALB/c mice being fully susceptible to this virus infection. Comparable growth kinetics, for all three viruses, were observed in both mouse strains, except for the MHV 3 growth in BALB/c mice, where the virus titre increased to a peak on day 2, remaining high until day 4 when the mice died of acute hepatitis. The IFN gamma titres in the peritoneum of mice preceded and correlated with the virus growth, higher titres being found in MHV 3 infected BALB/c mice. The highest titre was always observed 24 to 48 h after infection. Among viral strains grown in cultured macrophages, higher titres were always observed in cultures infected with MHVA 59, followed by MHV 3 and the lowest those infected with MHV 4. The macrophage activation by IFN gamma-induced a partial restriction of virus growth only in MHV 3 infected A/J mouse macrophages. A virus specificity of the IFN gamma-induced antiviral state was shown to be in direct correlation with the resistance of mice to MHV 3 infection.     

Genetic susceptibility to vaginal candidiasis.

To enable future studies on host resistance factors and therapy, inbred and outbred mouse strains were tested for susceptibility to vaginal candidiasis. Groups of mice were given 0.5 mg estradiol 3 days before and 4 days after intravaginal challenge with a suspension of Candida albicans. On day 1 after challenge, a swab was used to quantitate infection in all groups and to assure equivalent infection levels. On day 6, this was repeated and the experiment was terminated. BALB/c, the reference strain in repeated experiments, was susceptible, showing persistent infection with levels of cfu at day 6 falling within a range between a twofold decrease and a fourfold increase in relation to day 1 levels. CD-1 outbred mice were markedly resistant, with day 6 cfu levels showing a 74- to 87-fold decrease with respect to day 1 levels, whereas other outbred strains (CF-1, SW, ICR) were susceptible. A BALB/c substrain (ByJ) was also susceptible. With exception of CBA/J, which showed modest resistance, all inbred strains were similarly susceptible, including DBA/2, AKR/J, C3H/HeN, A/J and C57BL/6. The differences between CD-1 and BALB/c mice were also seen with a second C. albicans isolate. Our results show susceptibility to vaginal candidiasis is independent of the major histocompatibility locus H2 haplotype and any effect ascribable to use of particular commercial mouse suppliers. Differences among mouse strains in susceptibility to C. albicans, as seen in previous studies involving nonvaginal challenge routes, are not reflected in this vaginal candidiasis model; in general, such resistance patterns appear specific to the route of challenge administration. The resistance seen in mouse strain CD-1 is of particular interest in that CD-1 is known to be resistant to endocrine disruption by estrogen. Our results suggest this estrogen insensitivity may have broad-ranging effects on processes other than gametogenesis, including vaginal susceptibility to candidiasis.     

Experimental Gonococcal Genital Tract Infection and Opacity Protein Expression in Estradiol-Treated Mice

The development of effective prophylactic agents against gonorrhea and the study of adaptation by Neisseria gonorrhoeae to the urogenital mucosa are hindered by the lack of a well-established animal model of gonococcal genital tract infection. Here, a murine model of long-term gonococcal genital tract infection is described. Female BALB/c mice were treated with 17-beta-estradiol and inoculated intravaginally with wild-type gonococcal strain FA1090 or MS11. N. gonorrhoeae was recovered from vaginal swabs for an average of 12 to 13 days following inoculation with 10(6) CFU of either strain. Inflammation occurred in over 80% of infected mice, and diplococci were associated with epithelial cells and neutrophils in stained vaginal smears. Ascended infection occurred in 17 to 20% of mice inoculated with strain FA1090. An outbred mouse strain (SLC:ddY) previously reported to be naturally susceptible to N. gonorrhoeae was also tested; however, as with BALB/c mice, estradiol was required for prolonged infection. Although piliation was not maintained during experimental murine infection, 46 to 100% of vaginal isolates from four of eight BALB/c mice and three of four SLC:ddY mice expressed one or more opacity (Opa) proteins within 4 days after inoculation with an Opa-negative variant of strain FA1090. The observed selection for and/or induction of gonococcal Opa protein expression during murine infection appears to parallel events that occur during experimental urethritis in volunteers.     

Central nervous system and genital infection with reactivation of latent herpes simplex virus type 2 in mice

To establish a reactivation model of genital and central nervous system infection, 3- to 12-week-old outbred or BALB/c mice were inoculated vaginally with the HG-52 strain of herpes simplex virus type 2 (HSV-2). Primary infection was confirmed by serially positive vaginal cultures. Mortality in 6- and 12-week old infected mice was about 20%. In survivors, clearance of infectious virus was confirmed in serially negative vaginal cultures. At 6 weeks, immunosuppression of survivors with cyclophosphamide and antilymphocyte serum was begun. Recurrent virus shedding, monitored by daily vaginal cultures, was detected in the majority of animals. All mice became moribund or died, usually during the third to fifth weeks of immunosuppression. Brains and spinal cords from which all sensory ganglia had been removed were homogenized and inoculated onto cultures. One or both central nervous system (CNS) samples were virus-positive in nearly half of these mice, and cell-free virus was isolated from most positive brain and cord supernatants tested. Three-fourths of mice had evidence of virus reactivation with immunosuppression, as indicated by vaginal or CNS isolations, and by failure to isolate virus by identical means in matched infected, non-immunosuppressed controls. Vaginal, spinal cord and brain isolates occurred independently of one another in many immunosuppressed mice, and could not be predicted from presence or absence of external genital lesions during primary infection. These experiments show that with immunosuppression, reactivations of latent HSV-2 infections in mice can be detected in the genital tract and CNS, and provide a model to study productive, recurrent CNS infection and disease.     

Primary respiratory syncytial virus infection in mice

A mouse model of respiratory syncytial virus (RSV) infection is described. A high-titered, large-volume inoculum results in replication of RSV to a high titer in lungs of BALB/c mice. Mice older than 15 weeks of age are more susceptible to RSV infection. Titers up to 10(6.9) plaque-forming units (pfu)/gram lung can be attained in 32-week-old mice. Older mice experience a clinical illness manifested by ruffled fur, reduced activity, and weight loss. Lung histology of older mice infected with RSV shows bronchiolitis and increased number of lymphocytes and macrophages in alveolar spaces compared with that of mice less than 8 weeks old. This model will serve as the basis for investigating immunodeterminants of recovery and protection from RSV infection.     

Herpetic keratitis in athymic (nude) mice.

The inflammatory response to herpes simplex virus infection of the cornea was studied in athymic nude (nu/nu) and heterozygote (nu/+) BALB/c mice. Although athymic mice were highly susceptible to HSV infection and died 13 to 17 days after corneal inoculation, they failed to develop necrotizing keratitis of the cornea. Heterozygote mice survived the initial virual infection, but many of these mice developed necrotizing keratitis and permanent corneal scarring. Light and electron microscopy showed numerous inflammatory cells (polymorphonuclear leukocytes and lymphocytes) in the corneas of heterozygote mice, but not in the athymic mice. These studies suggest that the immune system plays a dual role in herpes simplex virus infection of the cornea: protection against dissemination of the virus and immunopathogenesis of necrotizing keratitis in the cornea.     

Failure of Mycoplasma pneumoniae infection to confer protection against Mycoplasma genitalium: observations from a mouse model

Mycoplasma pneumoniae and M. genitalium are genomically distinct but share antigens that induce some serological cross-reactivity. Therefore, the possibility that M. pneumoniae infection of the human respiratory tract might provide immunity to M. genitalium infection of the genital tract was considered. Because of the difficulty of assessing this proposition in man, it was evaluated experimentally in a mouse model. Female BALB/c mice were susceptible to infection of the vagina with M. pneumoniae, whereas those infected previously in the oropharynx with M. pneumoniae were completely immune to infection of the vagina with this mycoplasma. However, all mice with such a respiratory tract infection were susceptible to infection of the vagina with M. genitalium. The findings suggest that an M. pneumoniae infection of the human respiratory tract is unlikely to influence infection of the genital tract by M. genitalium.     

Immunological studies of hsv-infection of resistant and susceptible inbred strains of mice

DBA/2 mice were found to be quite susceptible to infection with herpes simplex virus (HSV) while C57BL/6 mice and F1 hybrids between the 2 strains were relatively resistant. This difference was most marked after ip infection, but could also be demonstrated after intracerebral, intravenous or subcutaneous infection. In both strains the LD50 was considerably higher after ip infection than after iv infection, and a dose of X-irradiation was required to kill the mice by sc infection. A/J and BALB/c mice were equally susceptible after ip infection but differed significantly after iv infection. C57BL/6 were made susceptible to ip infection by immunosuppression with antilymphocyte serum or cyclophosphamide. LPS, when given simultaneously with HSV also markedly increased the susceptibility of C57BL/6 mice. Susceptible DBA/2 mice which surrvived a low dose of HSV ip were not immune but C57BL/6 mice surviving a high dose were immune against rechallenge. Both strains of mice could be protected by an apathogenic, tissue-culture-attenuated strain of HSV against infection with the virulent strain. They could also be protected by iv injection of a sublethal dose against a lethal ip infection.     

Recovery of mice from herpes simplex virus type 2 hepatitis: adoptive transfer of recovery with immune spleen cells.

Young BALB/c mice inoculated intraperitoneally with herpes simplex virus type 2 develop focal necrotizing hepatitis. After infection, the livers of these mice show increasing virus titers, which reach a maximum on day 3 after infection; this is followed by a dramatic decrease in the amount of virus recovered on days 4 and 5. This decrease in virus content is accompanied by a progressive infiltration of the lesions with mononuclear leukocytes and an apparent resolution of the lesions. Adoptive transfer of immune spleen cells from mice infected 6 days earlier accelerated this process. When 50 x 10(6) to 100 x 10(6) immune spleen cells were transferred 24 h after infection, the inflammatory response and the clearance of virus from the livers were advanced by almost 2 days. As few as 12 x 10(6) immune spleen cells accelerated the healing process, whereas fewer immune cells, disrupted immune cells, or normal spleen cells did not have an effect. The protection conferred by herpes simplex virus type 2-sensitized immune spleen cells was specific since mouse cytomegalovirus- or vaccinia virus-sensitized immune spleen cells had no effect on the course of infection with herpes simplex virus type 2, whereas some cross-reactivity was observed between herpes simplex virus types 1 and 2. This model seems to be suitable for examining the immunological mechanisms that are active during recovery from visceral herpes simplex virus infections.