Standaert F E, Chew B P, Wong T S, Michal J J
Department of Animal Sciences, Washington State University, Pullman, Washington 99164-6320.
Am J Reprod Immunol. 1991 May;25(4):175-80. doi: 10.1111/j.1600-0897.1991.tb01090.x.
Lymphocyte conditioned medium (CM) was prepared by incubating 0 (cell-free control) or 4 x 10(6) lymphocytes/ml in serum-supplemented RPMI containing 0, 10(-9), 10(-7), and 10(-5) M luteinizing hormone releasing hormone (LHRH), 10(-5) M LHRH antagonist (LHRHA), or 10(-7) M LHRH + 10(-5) M LHRHA. Treatments were applied with and without 10 micrograms/ml concanavalin A (con A), and media were analyzed for LH. Aliquots of the CM from cultures incubated for 48 h were later applied to porcine granulosa cell cultures (suspended to 1.25 x 10(5) cells in 450 mul). Thereafter, 50 mul of CM were added to granulosa cell cultures. Media were collected after 12, 24, and 48 h and progesterone determined. Immunoreactive LH increased with time of incubation in lymphocyte CM but not cell-free CM. LH content of lymphocyte CM increased as LHRH concentration increased. LHRHA significantly reduced the amount of LH measured. The presence of con A in the medium resulted in maximal concentrations of LH, irrespective of dose of LHRH or LHRHA. Cell-free CM containing LHRH, LHRHA, and/or con A did not affect progesterone production by granulosa cells at any of the time periods. Lymphocyte CM containing LHRH caused a dose-dependent increase in progesterone production at 48 h. This stimulation was blocked by lymphocyte CM containing LHRHA. Lymphocyte CM containing con A also stimulated progesterone production at all of the LHRH concentrations studied. This response was not inhibited by lymphocyte CM containing the LHRHA.(ABSTRACT TRUNCATED AT 250 WORDS)
淋巴细胞条件培养基（CM）的制备方法如下：将0（无细胞对照）或4×10⁶个淋巴细胞/毫升接种于添加血清的RPMI中，该培养基含有0、10⁻⁹、10⁻⁷和10⁻⁵M促黄体生成素释放激素（LHRH）、10⁻⁵M LHRH拮抗剂（LHRHA）或10⁻⁷M LHRH + 10⁻⁵M LHRHA。在有和没有10微克/毫升伴刀豆球蛋白A（cona）的情况下进行处理，并分析培养基中的促黄体生成素（LH）。将培养48小时的CM等分试样随后应用于猪颗粒细胞培养物（悬浮于450微升中，细胞浓度为1.25×10⁵个细胞）。此后，向颗粒细胞培养物中加入50微升CM。在12、24和48小时后收集培养基并测定孕酮。在淋巴细胞CM中，免疫反应性LH随孵育时间增加，但在无细胞CM中则不然。淋巴细胞CM中LH含量随LHRH浓度增加而增加。LHRHA显著降低了测得的LH量。培养基中cona的存在导致LH达到最大浓度，与LHRH或LHRHA的剂量无关。含有LHRH、LHRHA和/或cona的无细胞CM在任何时间段均不影响颗粒细胞的孕酮产生。含有LHRH的淋巴细胞CM在48小时时导致孕酮产生呈剂量依赖性增加。这种刺激被含有LHRHA的淋巴细胞CM阻断。含有cona的淋巴细胞CM在所有研究的LHRH浓度下也刺激了孕酮产生。含有LHRHA的淋巴细胞CM并未抑制这种反应。（摘要截断于250字）
