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.

Objectie: to evaluate the effects on fertility by immunization with anti-idiotypic antibodies to porcine zona pellucida (PZP) antigen. Method: anti-idiotypic antibodies (Ab2) were produced in New Zealand rabbits immunized with 17D3 monoclonal antibodies (mAbs) (IgG, Ab1) to PZP antigen. The antisera were first passed through immuno-affinity chromatography column linked to normal mouse IgG so as to remove the antibody bound to normal mouse IgG The passing elute was then purified by immuno-affinity chromatography using 17D3 mAbs to get the Ab2. Female BALB/c mice, 5-week-old, were grouped and immunized with the Ab2, PZP antigen, target antigen of the Ab1 and normal rabbit IgG, respectively. The treated female mice were mated with male BALB/c mice and sacrificed at gestation day 10. Analyses included ELISA measurement of anti-ZP antibody titer, fetal number determination and evaluation of ovarian histomorphology. Results: the Ab2 appeared as a single protein band by SDS-PAGE. Shown by a competitive inhibition ELISA, the Ab2 specifically bound to the variable region of the 17D3 Ab1. Compared with controls, the female mice immunized with Ab2 showed a decreased pregnancy rate and a statistically significant reduction in fetal numbers. Histological examination of ovaries demonstrated that Ab2 exposure interfered less with follicular development than did exposure to PZP. Conclusion: immunization of female mice with Ab2 to PZP mAbs suppresses fertility and fetal numbers with minimal ovarian pathology.

Objective: To express the hCGβ-C3d3 fusion protein in a CHO cell continual expression system to investigate further the adjuvant effects of C3d on contraceptive vaccination. Method: We constructed a plasmid pcDNA3-hCGβ-C3d3 which contains three copies of murine C3d cDNA and the hCGβ gene by cloning the chimerical hCGβ-C3d3 cDNA into the eukaryotic vector pcDNA3 downstream of the CMV promoter. The plasmid was transfected into a COS-7 cell transient expression system and a CHO cell continual expression system. RIA was used to detect hCGβ in the culture supernatant. Western blot and Raji cell immunohistochemical assays were performed to evaluate the expressed protein. Then, 6-/8-week-old female BALB/c mice were inoculated intramuscularly with pcDNA3-hCGβ and pcDNA3-hCGβ-C3d3, and ELISA was used to assess anti-hCGβ IgG antibody in serum. Results: In 72 h after COS-7 cells were transfected with the plasmid pcDNA3-hCGβ-C3d3, 1.0/105 cells could secrete 152 ng of the recombinant protein (calculated by hCGβ contained). The transfected CHO cells, which were then screened by G418, could continuously secrete the fusion protein at 660 ng/106 cells/48 h. The hCGβ-C3d3 protein was purified by anti-hCGβ immunoaffinity chromatography. Raji cell immunohistochemical assay demonstrated that both the hCGβ and C3d3 were successfully fused. After DNA immunization intramuscularly, the anti-hCGβ IgG antibody titer in the pcDNA3-hCGβ-C3d3 immunized group was 243-fold higher than that of the pcDNA3-hCGβ immunized group. Conclusion: We have expressed the hCGβ-C3d3 protein successfully, both in a transient expression system (COS-7 cells) and in a stable expression system (CHO cells). The C3d3 molecular adjuvant can enhance significantly the immunogenecity of hCGβ antigen in DNA immunization.

Objective: To compare the effects of treatment of tubal pregnancy (TP)and its following second pregnancy by intratubal methotrexate injection (IMI) alone and combination of IMI with Chinese herbal medicine. Metlmds: Thirty-five patients sudferiug from unruptured TP were divided into two groups at random, to the 19 patients in the treated group, the treatment of combined IMI with Ectopic Pregnancy decoction No.2 (EP2, a traditional Chinese medical decoction) was applied, and to the other 16 patients in the control group, IMI alone was applied for control. Sexum concentrations of h-man chorionic gonadotro-pin (HCG), size of the gestational sac, existent time of fetal cardiac beat and peritoneal fluid were measured before and after treatment. And hysterosalpingography were performed 6 months after pnding the treatment to verify the presence of tubal obstruction and the condition of relapse. Results: The treat-ment of all the 35 women was suceessful. The recovery duration of serum β-HCG, disappeerance duration of TP sac and existent time of peritoneal fluid in the treated group were 20.04-7.8 days, 1.24-0.7 months and 10.74-2.9 days respectively, which were significantly different from those in the control group (24.4 4-8.1 days, 3.64-1.7 months and 19.14-3.2 days respectively(P＜0.05, P＜0.01 and P＜0.05 respec-β-tivdy), but the existent time of fetal cardiac beat in the two groups (8.84-1.9 days vs 9.0 4-1.3 days) was not significantly different (P~0.05). The post-treatment oviduct obstructive rate in the two groups was 10.5% and 43.8% respectively, that in the treatment group was less significant (P＜0.05). The relapse rate of EP in the treatment group was insignificantly different from that in the control group (5.3% vs 18.8%,P＜0.05).usion: The two therapies (IMI alone and IMI combined with EP2) could obtain e-qual efficacy in curing TP. Compared with IMI alone, the combined therapy appears to have the effects of accelerating the resorption of gestational sac and peritoneal fluid, improving the patency of fallopian tube and ameliorating the cirolm~tance of pregnancy, which is hvorable to improvement of the re-pregnancy rate and reduction of the re-occurrence of ectopic pregnancy as well as to the enhancement of the effect of IMI in killing trophocytes. But there is not enough proof to show the potency of EP2 in killing embryo.

CD28/CTLA-4 interactions with their specific B7-ligands (CD80 and CD86) play a decisive role in antigenic and allogenic responses. Recently, experimental transplant studies demonstrated that donor-specific tolerance was achieved by blocking these interactions. However, the role of blockade of CD28/B7 costimulatory pathway in the maintenance of materno-fetal tolerance has received little attention. In the present study, abortion-prone CBA/J females mated with DBA/2 males were administered with anti-CD80 and anti-CD86 monoclonal antibodies (mAbs) on day 4 of gestation (time of murine implantation). We demonstrated that the combined use of anti-CD80 and anti-CD86 mAbs induced maternal tolerance to the fetus in the abortion-prone CBA/J mice, and displayed expansion of the maternal CD4+CD25+ regulatory T cell population and up-regulated expression of CTLA-4, suggesting an active mechanism of regulatory T cells in suppressing maternal rejection to the fetus. In addition, the anti-CD80/86 mAbs treatment enhanced Th2 and reduced Th1 cytokine production in mice, implying that the development of Th2 cells might contribute to maternal tolerance to her fetus. Together, these findings indicated that blocking CD80 and CD86 enhanced maternal tolerance to her fetus in mice by increasing regulatory T cell function and skewing toward a Th2 response. Our data might provide an enhanced understanding of the maternal-fetal immune relationship and be helpful in clinical trials for immunotherapy of recurrent spontaneous abortion.

More than 70% of decidual lymphocytes are NK cells characterized by CD56brightCD16- phenotype, but the mechanisms by which these NK cells are recruited in the decidua are still almost unrevealed. In this study, we first analyzed the transcription of 18 chemokine receptors in the first-trimester decidual CD56brightCD16- NK cells. Among these receptors, CXCR4 and CXCR3 were found highly transcribed, and the expression of CXCR4 was verified in most of the decidual CD56brightCD16-NK cells by flow cytometry. The first-trimester human trophoblasts were found expressing CXCL12/stromal cell-derived factor 1, the specific ligand of CXCR4, by way of in situ hybridization and immunohistochemistry. The primary cultured trophoblast cells were also found to secrete stromal cell-derived factor 1-spontaneously, and its concentration was 384.6-90.7pg/ml after the trophoblast cells had been cultured for 60 h. All of the ligands for CXCR3 were below the minimal detectable concentration when trophoblast cells were cultured for up to 48 h. Both recombinant human SDF-1-and supernatants of the cultured trophoblast cells exhibited chemotactic activity on decidual CD56brightCD16- NK cells. Our findings suggest that human first-trimester trophoblast cells produce CXCL12, which in turn chemoattracts decidual CD56brightCD16- NK cells. This activity could contribute to the recruitment mechanism of decidual lymphocytes, especially CD56brightCD16-NK cells, in decidua, and may be used at a local level to modulate the immune milieu at the materno-fetal interface.