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。

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.

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.

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.

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.