目的：探讨环孢素A（cyclosporin A, CsA）作用于人滋养层细胞差异表达的功能基因。方法：应用抑制性消减杂交筛选110μmol·L-1 CsA作用于JAR细胞系前后差异表达的功能基因，经RT2PCR及蛋白质印迹进一步验证CsA作用前后原代分离的人早孕期滋养层细胞及JAR细胞株titin 表达的变化。结果：CsA作用于人JAR细胞系前后出现6个具有差异表达的基因，其中1 个为已知基因titin，2个为功能未知的mRNA，3个为位于16号染色体上的EST。RT2PCR显示，CsA作用于人滋养层细胞24h后出现titin mRNA的表达，72h达高峰，并呈现明显的剂量依赖性，110μmol·L-1 CsA作用最明显。蛋白质印迹显示，CsA可诱导titin的表达。结论：CsA可能通过诱导滋养层细胞titin的高表达，改变其生物学行为,从而有利于胚胎着床及早期发育。

Objectives: To test the possibility of vaccination with lactobacillus expressing hCGβ antigen administered by vaginal mucosal immunization. Methods: A plasmid pIlac-hCGβ was constructed and then transfected into Lactobacillus casei CECT5276, which stably expressed hCGβ protein. RIA was used to detect hCGβ in the culture supernatant and cell lysate.Western blotting was performed to evaluate the expressed protein of interest. Female BALB/c mice aged 6–8 weeks received inoculations in the vagina of the recombinant L. casei CECT5276. ELISA was used to determine the anti-hCGβ IgA antibody in vaginal lavage fluid from the BALB/c mice after vaginal mucosal immunization. Results: The pIlac alone appeared to have a higher efficiency than pIlac-hCGβ, and the highest transfection efficiency of both plasmids was at pulse voltages of 1200V and 1500V. About 78.5% of the hCGβ protein was excreted into the culture supernatant. Excretion of hCGβ was most efficient when the pH of the culture medium was adjusted to around 7.0 and the concentration of lactose was around 1%. The hCG protein in the vaginal lavage fluid of these BALB/c mice was positive on the third day after vaginal inoculation. Anti-hCGβ IgA antibody continued to be found in the vaginal lavage fluid for 2 weeks following a booster vaginal inoculation. The splenic lymphocytes of the mice immunized with hCGβthrough the vagina underwent a proliferative reaction to hCGβ antigen restimulation in vitro. Interferon gamma (IFN-γ) and interleukin (IL)-4 were secreted at higher levels after vaginal mucosal immunization of L. casei expressing hCG than after vaginal mucosal immunization of L. casei alone. Conclusions: Vaginal immunization of lactobacillus expressing hCG induced an antihCGβ antibody response in the murine vaginal mucosa. Induction of the antigen-specific antibodies in the reproductive tract following vaginal inoculation of recombinant lactobacillus will lead to the development of a safe, efficient, and easy-to-use form of immunocontraception.

To show that an anti-human chorionic gonadotrophin-β (hCGβ) antibody response can be induced by inoculating Lb. Expressing hCGβ through diVerent mucosal pathways in mice of two strains, female BALB/c and C57BL/6 mice were immunized via vaginal, oral or nasal routes with 108, 109, and 1010 Lb.hCGβ (a recombinant Lactobacillus expressing hCGβ). The mice were immunized twice with a booster in study week 3. An indirect ELISA was used to determine anti-hCGβ IgG and IgA antibodies in vaginal lavage and serum, obtained from the 2nd to 8th week after the primary immunization. Flow cytometry was used to analyze the lymphocyte proliferation from these tissues, 1 week after the primary immunization. The hCGβ antigen-speciWc antibody-secreting cells of spleen, uterus, and vagina were evaluated by enzyme-linked immunospot assay (ELISpot), 2 weeks after the booster. The analysis showed that 109 and 1010 Lb.hCGβ inoculations induced similar anti-hCGβ antibody responses, while the three mucosal pathways induced similar antibody responses. The antiserum obtained after boosters with 109 and 1010 Lb. hCGβ was able to neutralize more than 100 ng/ml hCGβantigen, both in BALB/c and C57BL/6 mice. The highest antibody titer induced by vaginal mucosal immunization was stronger than that obtained via the other mucosal pathways. The B cells in the vagina appeared to proliferate after vaginal immunization (P <0.05). The numbers of anti-hCGβ IgG and IgA antibody-secreting cells in the uterus and vagina were greater than in the spleen. Therefore, the vaginal mucosal route appears to be a better immunization pathway to induce higher anti-hCGβ antibody levels in the reproductive tract.

目的探讨脱氢表雄酮（DHEA）对成骨细胞（oste0blasts，OB）及CD4+T细胞表达协同刺激分子的调控作用。方法颅骨酶解法培养鼠oB，体外模拟雌激素撤退；免疫磁珠细胞分选（rnag-c cell 9ort，MAC. S）法分离CD4+T细胞；将oB或CD4 T细胞分为对照组、E2及DHEA处理组，并以LPS刺激；以流式细胞术分析OB表面CDS0、CD86以及CD4+T细胞表面CD28、CⅡA4的表达。结果经E2及DHEA处理后，OB表达CDS0、CD86显著增加（P<0.05，P<0.01）；CsA可降低对照组及DHEA处理组OB CD80、CD86的表达（P<0.01）。除DHEA组CD28 T细胞百分比增加外（P<0.05），其余各组CD4 T细胞CD28和CIIA4的表达无显著改变（P>0.05）。结论DHEA可上调鼠OB协同刺激分子CDS0、O386表达，该作用可被CsA阻滞；DHEA还上调CD4 T细胞cD28的表达，提示可改善骨。免疫调节网络。

The embryo expresses paternal Ags foreign to the mother and therefore has been viewed as an allograft. It has been shown that anergic T cells generated by blocking of the CD28/B7 costimulatory pathway with anti B7-1 and anti B7-2 mAbs can be transferred as suppresser cells to prevent allograft rejection. Little is known, however, about the in vivo function of anti-B7-treated T cells after their transfer into abortion-prone mice in the maintenance of materno-fetal tolerance. In the present study, abortion-prone CBA/J females mated with DBA/2 males were administered anti-B7-1 and anti-B7-2 mAbs on day 4 of gestation (murine implantation window). The anti-B7-treated T cells subsequently were adoptively transferred into abortion-prone CBA/J mice. We demonstrated that costimulation blockade with anti-B7 mAbs at the time of implantation resulted in altered allogeneic T cell response and overcame increased maternal rejection to the fetus in the CBA/J DBA/2 system. The transferred anti-B7-treated T cells appeared to be regulatory, decreasing responsiveness and generating clonal deviation in maternal recipient T cells. The transferred CFSE-labeled T cells were found to reside in the spleen and uterine draining lymph nodes, and a few were localized to the materno-fetal interface of the maternal recipient. Our findings suggest that the anti-B7-treated T cells not only function as potent suppresser cells, but also exert an immunoregulatory effect on the maternal recipient T cells, which cosuppresses maternal rejection to the fetus. This procedure might be considered potentially useful for fetal survival when used as an immunotherapy for human recurrent spontaneous abortion.