抗小鼠精子特有乳酸脱氢酶C4的与分泌片装配或未装配的单克隆抗体IgA的生物学功能

为了测定抗精子ＩｇＡ在免疫不育和抗精子避孕疫苗研制方面的生物学作用，用肠道内免疫的方法制备了一组抗乳酸脱氢酶Ｃ４（ＬＤＨ－Ｃ４）的单克隆ＩｇＡ抗体（ｍｏＩｇＡ）。以免疫印迹证实了它们的异质同形体。大部分ｍｏＩｇＡ（ＰＡ１－ＰＡ５）是用肠道内免疫和以派依尔氏淋巴细胞作为亲本细胞进行融合来获得的。在豚鼠血清补体存在的情况下，小鼠精子可以被ｍｏＩｇＡＰＡ１、ＰＡ２和ＰＡ４所制动。高浓度ＰＡ４和ＰＡ５可凝集小鼠精子。小鼠体外受精率可被３个ｍｏＩｇＡ（ＰＡ２、ＰＡ３和ＰＡ４）显著降低，但用ＰＡ１、ＰＡ２和ＰＡ５被动免疫之后，小鼠体内受精无明显变化。纯化的小鼠胆汁分泌片可同纯化的ｍｏＩｇＡ或腹水中的ｍｏＩｇＡ在体外组装起来。同分泌片结合之后，ｍｏＩｇＡ对精子的制动、凝集和体外受精无明显变化。这些研究结果提供了抗ＬＤＨ－Ｃ４的ｍｏＩｇＡ和分泌性ＩｇＡ对精子功能和体外受精的生物学作用的直接证据，在免疫不育的防治，避孕疫苗的研制以及性传播疾病的防治方面均有一定的指导意义。

IgA抗体的结构、功能与调控

IgA抗体是介导机体粘膜免疫的主要因子,其在粘膜分泌物中能有效地阻止外界抗原(包括可溶性抗原、病原微生物以及存在于食物中的致癌物质)附着或侵入粘膜上皮,从而避免这些外来物给机体造成损伤,保持机体粘膜完整并避免胃肠道疾病、呼吸道疾病及泌尿生殖道各种疾病的发生。为了能有效地利用IgA抗体的免疫保护功能,解决诸多困扰人类的粘膜性疾病,前人从很多方面对IgA抗体做了大量的研究,得到很多有意义的结果。本文试图总结这些研究成果,结合最新的研究进展,对IgA抗体的结构、功能及调控等进行评述。

人精子抗原决定簇的末端单糖参与小鼠IgA单抗与抗原的结合

对消化道免疫获得的33个抗精子IgA单抗, 12个IgG和35个IgM单抗靶抗原的生化性质及末端单糖做了鉴定。免疫印迹的结果显示, IgA、IgG和IgM类单抗靶抗原的分子量范围分别为10-89、11-75和12-94KDa。有12个单抗的靶抗原为非蛋白类的糖复合物, 一个IgA单抗(A22)的靶抗原为不含糖的蛋白。凝集素封闭和糖苷酶消化试验的结果显示, 98.7%单抗的靶抗原分子末端含一种或几种糖。五种凝集素对IgA类单抗靶抗原的抗原的封闭效应均较强, 表明IgA类单抗靶抗原的抗原决定簇含有岩藻糖、乙酰氨基葡萄糖、乙酰氨基半乳糖、半乳糖和甘露糖等末端单糖者较多。IgG类单抗靶抗原的抗原决定簇则含有带岩藻糖、乙酰氨基半乳糖和#alpha#-甘露糖等末端单糖者较多。内切-#beta#-半乳糖苷酶消化试验的结果表明, 54.4%IgA类单抗的靶抗原为依赖半乳糖苷连接的糖肽化合物。这些结果表明, 经消化道免疫能产生IgA及其它类别抗体的绝大多数人精子抗原为含多种类型末端单糖的膜表面分子。

SECRETORY MONOCLONAL IGA ANTIBODY TO HUMAN SPERM PRODUCED BY GASTROINTESTINAL IMMUNIZATION INHIBITS HUMAN SPERM ACTIVITY AND MOUSE INVITRO FERTILIZATION

BALB/c mice were immunized intragastrically with human sperm. Cells from the Peyer's patches and spleens of the immunized mice were for the preparation of hybridomas secreting antisperm monoclonal IgA (mcIgA). The specific ratio of IgA-secreting cells in Peyer's patches was much higher than that in spleen. The binding site on human sperm of 9 of 19 mcIgA was in the post-acrosomal region using an immunofluorescent assay. Two of eight selected mcIgA caused strong human sperm agglutination and three of them produced significant inhibition of mouse in vitro fertilization. No mcIgA tested caused obvious human sperm immobilization or inhibited mouse in vivo fertilization. In vitro assembly of selected mcIgA in ascites with mouse secretory component (SC) caused no significant changes in effects on sperm function and in vitro fertilization. By use of Western blotting, dimer or higher polymers were demonstrated in all selected mcIgAs and corresponding protein antigens in 6 of 8 selected mcIgAs. These results suggest that human sperm function may be inhibited and fertilization rate reduced by specific secretory IgA to human sperm and that secretory immunity to protein antigens of human sperm could be induced by intragastrointestinal immunization.

Transport of anti-sperm monoclonal IGA and IGC into murine male and female genital tracts from blood - Effect of sex hormones

Ab levels in the genital tract may be important in fertility and in preventing sexually transmitted diseases, In this study, I-125-labeled polymer or monomer mAb IgA (C4pIgA or C4mIgA) and IgC2b (C4IgC) to murine lactate dehydrogenase C4 and a polymer mAb IgA (npIgA) not cross-reacting with mouse sperm were intravenously injected into BALB/c mice, and the relative distribution of these Abs was determined. Polymer IgA was transported much more efficiently into the genital tract, trachea, and duodenum of both sexes than C4IgG and C4 mIgA (p < 0.01), The transport of polymer IgA (C4pIgA and npIgA) into the male genital tract greatly increased following orchiectomy (p < 0.01); this change was not affected by testosterone, suggesting that the unknown regulatory factor(s) from the testis may suppress polymer IgA transport, However, the transport of polymer IgA into female genital tissues was significantly decreased by ovariectomy (p < 0.01); this decline can be rectified by P-estradiol but not progesterone treatment, suggesting that estradiol may stimulate polymer IgA transport, Furthermore, the transport of C4IgG into tissues of the Fallopian tubes and the uterus was significantly decreased by treatment with progesterone (p < 0.01). Together, these findings indicate that serum polymer IgA can be transported selectively into the genital tracts of both sexes, that this transport is strongly under the control of gonads, and that transport of Ige into the Fallopian tubes and uterus is downregulated by progesterone.

抗 LDH－C4 IgA 和IgG 抗体在粘膜免疫系统的分布及其抗生育作用的研究

已有的研究表明，小鼠背部携带能分泌抗原特异的IgA 单克隆抗体的杂交细 胞瘤，可以保护小鼠抵抗微生物和病毒等多种病原体经粘膜途径感染机体。我们 利用背部携带能分泌抗精子特异抗原（LDH-C4）的IgA 和IgG 杂交细胞瘤、以 及抗DNP 的IgA 骨髓细胞瘤的小鼠为动物模型，采用定量ELISA 法研究了抗 LDH－C4 IgA 与抗DNP IgA 单克隆抗体在呼吸道、肠道及生殖道内转运和分布， 抗LDH-C4 IgG2b 在肠道内转运与分布，以及抗LDH-C4 IgA 和IgG 单克隆抗体 在体内抗生育作用。 研究结果表明，带瘤小鼠血液中含有较高水平抗原特异的 IgA 和IgG 单克 隆抗体。PA4 和MOPC IgA 单克隆抗体在呼吸道、肠道以及雌性生殖道分泌物 内有较高的分布水平。在肠道，PA4 和MOPC IgA 单克隆抗体的分布水平显著 高于IgG（p<0.01 和p<0.05）。在肠道和生殖道的不同部位，IgA 抗体的分布水 平不同。在肠道，结肠分泌物中的IgA 单克隆抗体显著高于其它肠道部位 （p<0.01）。在生殖道，IgA 单克隆抗体分布水平以子宫角分泌物中最高。雄性 的前列腺也有较高的IgA 抗体水平。在呼吸道、肠道以及雌性生殖道相应部位的 分泌物内，PA4 IgA 单克隆抗体的水平显著高于MOPC IgA 单克隆抗体的分布水 平（p<0.05）。PA4 和MOPC IgA 单克隆抗体在粘膜分泌物内的分布水平差异可 能与其IgA 聚合形式的不同有关。另外，除气管外，在两时间点间分泌物中的 IgA 抗体水平没有显著差异。 检测背部带瘤小鼠交配后的两细胞胚胎期，发现携带PA4 和G2b 杂交细瘤 的雌性小鼠的受精率与对照组并没有显著的差异，这表明抗LDH-C4 IgA 和IgG 单克隆抗体在体内不能明显抑制小鼠的精子和卵子的结合或受精过程。注射细 胞后的27 天，检测鼠着床胚胎时，发现带瘤两性小鼠均携带PA4 时或者只有 雌性携带PA4 杂交瘤时，以及雌雄两性小鼠均携带G2b 杂交瘤细胞时，交配后的怀孕率与能分泌抗DNP 抗体的MOPC 的骨髓瘤细胞瘤的相应组别相比，显 著降低（p<0.01）。但PA4 各组与G2b 各组之间无显著差异(p>0.05）。然而，雌 雄的小鼠均带瘤时，最高怀孕减少率未能达100%。这些结果提示，抗LDHC4 IgA 和IgG 单克隆抗体在小鼠体内不能有效地抑制小鼠的精子与卵子的结 合，但能显著地抑制小鼠受精后胚胎的发育。抗LDH-C4 的IgA 和IgG 单克隆 抗体单独存在时，在体内均具有抗生育作用，但不能完全抑制生育。

抗LDH-C4 IgA和IgG抗体在带瘤小鼠生殖道和其它粘膜分泌物内分布特性的研究

中文摘要 　 已有的研究表明，小鼠背部携带能分泌抗原特异的IgA单克隆抗体的杂交细胞瘤，可以保护小鼠抵抗微生物和病毒等多种病原体经粘膜途径感染机体。我们利用背部携带能分泌抗精子特异抗原（LDH-C4）的IgA和IgG杂交细胞瘤、以及抗DNP的IgA骨髓细胞瘤的小鼠为动物模型，采用定量ELISA法研究了抗LDH-C4 IgA与抗DNP IgA单克隆抗体在呼吸道、肠道及生殖道内转运和分布，抗LDH-C4 IgG2b在肠道内转运和分布，以及抗LDH-C4 IgA和IgG单克隆抗体与体内抗生育作用的关系。研究结果表明，带瘤小鼠血液中含有较高水平抗原特异的IgA和IgG单克隆抗体。PA4和MOPC IgA单克隆抗体在呼吸道、肠道以及雌性生殖道分泌物内有较高的分布水平。在肠道，PA4和MOPC IgA 单克隆抗体的分布水平显著高于IgG（p < 0.01和p < 0.05）。在肠道和生殖道的不同部位，IgA抗体的分布水平不同。在肠道，结肠分泌物中的IgA单克隆抗体显著高于其它肠道部位（p < 0.01）。在生殖道，IgA单克隆抗体分布水平以子宫角分泌物中最高。雄性的前列腺也有较高的IgA抗体水平。在呼吸道、肠道以及雌性生殖道相应部位的分泌物内，PA4 IgA单克隆抗体的水平显著高于MOPC IgA单克隆抗体的分布水平（<0.05）。PA4和MOPC IgA单克隆抗体在粘膜分泌物内的分布水平差异可能与其IgA聚合形式的不同有关。另外，除气管外，在两时间点间分泌物中的IgA抗体水平没有显著差异。检测背部带瘤小鼠交配后的两细胞胚胎期，发现携带PA4或G2b杂交细胞瘤的雌雄小鼠的受精率与对照组并没有显著性差异，这表明抗LDH-C4 IgA和IgG单克隆抗体在体内不能显著抑制小鼠的精子和卵子的结合或受精过程。注射细胞后的27天，检测着床胚胎时，发现带瘤两性小鼠均携带PA4时或者只有雌性携带PA4杂交瘤时，以及雌雄性小鼠均携带G2b杂交瘤时，交配后的怀孕率与带能分泌抗DNP抗体的MOPC骨髓瘤细胞瘤的相应组别相比，显著降低（p < 0.01）。但PA4各组与G2b各组之间无显著差异（p < 0.05）。然而，雌雄小鼠均带瘤时，最高怀孕减少率也未达到100%。这些结果提示，抗LDH-C4 IgA和IgG单克隆抗体在小鼠体内不能有效地抑制小鼠的精子和卵子的结合，但能显著地抑制小鼠受精后胚胎的发育。抗LDH-C4 的IgA或者IgG单克隆抗体单独存在时，在小鼠体内均具有抗生育作用，但不能完全抑制生育。

抗LDH-C4的IgA单克隆抗体在生殖道局部的分布

为了弄清生殖道内抗体，特别是IgA抗体的准确来源和它的调控因子，同时也为了弄清生殖的局部免疫与典型的粘腊免疫之间的关系，以同位素标记的针对精子特有抗原乳酸脱氢酶C4(LDH-C4)的多聚IgA单抗及其单体，与小鼠精子发生反应的IgA单抗，以及LDH-C4特异的IgG抗体，尾静脉注射给雌雄Balb/c小鼠，4小时后测定小鼠的生殖道及其分汾物，肠道、呼吸道及其分泌物，各相关淋巴组织以及其它器官内这些抗体的分布。还研究了特异抗原刺激、性激素等对这些抗体分布状况的影响。

抗小鼠乳酸脱氢酶C4（LDH-C4）IgA单克隆抗体的制备及其对精子功能的影响

乳酸脱氢酶C4 （LDH-C4）是一种人和哺乳动物精子特有的乳酸脱氢酶同功酶。用纯化的小鼠LDH-C4 免疫动物，有一定的避孕效果。这种避孕效果与血清特异性抗体水平并不完全一致。这可能是由于受精过程是在生殖道内进行的，而生殖道内又有粘膜免疫因素存在。IgA抗体是生殖道内的主要抗体成分，研究抗LDH-C4 IgA抗体的抗精子作用有助于了解局部分泌性免疫系统在抗精子免疫避孕中的作用。由于足够量的特异性IgA抗体难于从动物或人粘膜分泌液中分离到，为了获得供体内外试验用的该种抗体，直接证明它在抗生育方面的作用，我们采用一种特殊的免疫方法制备了一系列的抗LDH-C4的单克隆抗体，包括6株IgA和9株IgM。这种免疫方法的主要特点是将抗原直接注射到派伊尔氏淋巴小结（PP）或小肠腔内。ELISA检测表明，按这种方法免疫后，分泌IgA的克隆出现的比例明显高于常规免疫的结果。这是因为PP是粘膜免疫的中枢，其中含有大量的IgA前体细胞，直接将抗原注射到PP内有助于刺激IgA前体细胞的分化和增殖，诱导局部分泌性免疫反应。我们用所得到的单克隆抗体研究了LDH-C4在人，小鼠和树鼩精子表面的定位。结果表明，大多数单克隆抗体可以结合到这些精子的表面；不同的单抗在同一物种的精子表面呈现不同的结合区域。这一方面说明来源于人，小鼠和树鼩的LDH-C4的抗原决定簇有很高的同源性；另一方面提示LDH-C4在精子表面不同的区域所暴露的抗原决定簇不同。初步的功能试验表明，某些抗LDH-C4的IgA单克隆抗体可以凝集或制动精子，说明生殖道内的IgA抗体可以通过凝集和制动作用来影响精子的功能，从而影响精子的受精力。

Circulating immunoglobulin A- and immunoglobulin G-secreting hybridoma cells in peripheral blood preferably migrate to female genital tracts. The role of sex hormones

Antigen-specific circulating immunoglobulin-secreting cells (ISC) migrate to various secondary and tertiary lymphoid tissues. To understand the migration of the cells into the genital tract and its regulation by sex hormones, spleen-derived SG2 hybridoma cells secreting immunoglobulin G2b (IgG2b) and Peyer's patch-derived PA4 hybridoma cells secreting polymer IgA were labelled with (3) H-TdR, and intravenously injected into syngeneic mice of both sexes. Using flow cytometry, surface molecular markers of plasma cells, CD38 and CD138, and adhesion molecules, CD49d, CD162, and CD11a were found to be positive in SG2 and PA4 cells, but CD62L, alpha4beta7 and CD44 were not expressed on these cells. The relative distribution indexes (RDIs) of the cells in genital tract and other tissues were measured. The means of RDIs of SG2 and PA4 cells in female genital tissues were 6.5 and 4.5 times as many as the means in male genital tissues, respectively. The treatment of ovariectomized mice with beta-oestradiol significantly increased the RDIs of PA4 cells in cervix and vagina, but decreased the RDIs of SG2 cells in vagina, horn of uterus, uterus and rectum (P <0.05). Progesterone treatment increased the RDIs of PA4 cells in vagina and rectum (P <0.05). The treatment with testosterone significantly increased the RDIs of SG2 and PA4 cells in epididymis and accessory sex glands (P <0.05). These results demonstrate that the female genital tract is the preferable site for the migration of circulating hybridoma cells to the male genital tract, and sex hormones play an important role in regulation of the migration of circulating ISC to genital tracts.

Effects of testosterone undecanoate as a male contraceptive candidate on rat immunological features

Testosterone undecanoate (TU) is under phase III clinical trial as a hormonal male contraceptive in China. Sex hormones can modulate the immune system. Female hormonal contraceptives may affect SIV/HIV-1 transmission. To evaluate the safety of TU and to understand whether long-term use of TU for a male contraceptive affects users' immunological features, adult male rats were treated for a 32-week TU-treated phase at the dose of 20 mg TU/kg body weight and a 24-week recovery phase. The reproductive and immunological parameters of 4-6 rats in each subgroup were examined at the stated time point. The mean sperm count and viability in the treated rats were significantly suppressed (p < 0.01). In the TU-treated group: the mean blood leukocyte and lymphocyte counts; the proliferation indexes of T cells from peripheral blood mononuclear cells (PBMC) and spleen; and, of B cells from spleen, as well as the mean counts of blood T, NK, and B cells decreased in comparison with those of control group. These decreases were not significant (p > 0.01). Similarly, the mean serum IgM, IgG, and IgA levels and complement activity in TU-treated rats were lower than those in control group (p > 0.01), and the changes in the antibody levels of the examined genital secretions were not significant (p > 0.01). The changes in the thickness of urethra epithelium, and in secretory component (SC) expression in genitals were not observed in the treated group. These results demonstrated that long-term supraphysiological TU injection did not obviously affect the examined rat immunological parameters.

Migration of mouse antibody-secreting hybridoma cells from blood to genital tract and its regulation by sex hormones are associated with the differential expression patterns of adhesion molecules and chemokines in the tract rather than in the antibody-secreting cells

To understand better the molecular mechanisms of differential migration of antibody-secreting cells (ASCs) into mouse genital tracts, and regulation by sex hormones, surface markers, hormone receptors and adhesion molecules in mouse SG2 and PA4 hybridoma cells, respectively, secreting IgG2b and polymeric IgA antibody were detected by flow cytometry or RT-PCR. Semiquantitative RT-PCR was also used for measuring mRNA expression of adhesion molecules and chemokines (VCAM-1, ICAM-1, P-selectin, JAM-1 and CXCL12) in genital tracts of various adult mouse groups. The mRNAs of androgen receptor, estrogen receptor beta and CXCR4 were expressed in the ASCs. Sex hormones had no effect on expression of these molecules in ASCs. Except for VCAM-1, mRNA of all examined genes was expressed in normal mouse genital tracts. The mean of relative amounts of ICAM-1 and CXCL12 mRNA in all examined organs of females were higher (2.1- and 1.9-fold) than those in males. After orchiectomy or ovariectomy, the expression of ICAM-1, CXCL12 and P-selectin mRNA in the examined organs increased, except JAM-1 in male and CXCL12 in female. Sex hormone treatment recovered the changes to normal levels of mRNA expression in many examined genital tissues. In combination with our previous work, preferential migration of ASCs into female genital tract and regulation of migration by sex hormones are associated with expression patterns of adhesion molecules and chemokines in genital tract rather than in ASCs. (C) 2006 Elsevier Ireland Ltd. All rights reserved.

IgM, IgD and IgY and their expression pattern in the Chinese soft-shelled turtle Pelodiscus sinensis

Three Ig isotypes, IgM, IgD, and IgA, were previously known in reptiles. Here, in this report we describe IgM, IgD and a novel immunoglobulin heavy-chain isotype upsilon (IgY) in Chinese soft-shelled turtle (Pelodiscus sinensis). The IgM and IgY constant domains are characteristically similar to their counterparts described in other vertebrates. The expression of IgM and IgD were detected at mRNA level early during embryonic development, and their expression increased during further development. However, the IgY expression was not detected in larval turtles until 90 days after hatching-out. The increase in the transcription of these three Ig molecules was analyzed by using real-time PCR in spleen, kidney and blood following the injection of inactivated Aeromonas hydrophila. The primary increase in the expression of these three Igs was observed I week after the first injection, although not statistically significant, and the second injection 2 weeks after the first injection provoked a significant increase in the expression of these Igs, revealing a pattern of primary and secondary antibody response in the turtle. The present study represents the first report on reptile IgY and the pattern of IgM, IgD and IgY transcription in reptiles. (C) 2009 Elsevier Ltd. All rights reserved.

BaLiF_3∶Ce~(3+)纳米粒子的制备及其光谱特性

BaLiF3属立方钙钛矿型复合氟化物,作为高效闪烁晶体可用于热中子检测[1].由于其能带隙宽,易于实现各种不同价态稀土离子掺杂,可以获得许多可调谐性质,因此它也是比较理想的光学功能材料的基质[2].Ce3+激活的BaLiF3晶体作为紫外发射的短波固体激光材料和光放大材料的研究多有报道[3~5].Ce3+的发射特性强烈依赖于基质晶体结构[6,7].氟化物基质中氧杂质的含量是影响光谱性质的重要因素[8].为了寻找氧含量低、发射波长更短、可调谐范围更宽的激光材料,本工作制备了BaLiF3∶Ce3+纳米晶,获得了一些新的有意义的实验结果.如果将其引入合适的聚合物基体制备出纳米复合体系,有可能为新一代杂化激光材料提供更理想的光学活性组分[9].1实验部分1.1仪器用日本R igaκu D/maxⅡB型X射线衍射仪(XRD)测量样品的晶体结构,辐射源CuKα1(λ=0·154 1 nm);用日本H itachi S-570环境扫描电子显微镜(ESEM)观察样品的形貌和粒径大小;用日立F-4500荧光光谱仪测试样品的激发和发射光谱;用英国VG公司ESCALAB MKⅡX射线电子能谱仪(XPS)检测样品的含氧量.1.2实验过...