器官培養におけるヒト胎盤絨毛のviabilityに関する研究

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The method and technique of organ culture of human chorionic villi was elaborated in our laboratory. In this report, placental specimens obtained at 15 to 18 weeks of gestation were studied in organ culture for 7 days in terms of the maintenance of morphological integrity and the preservation of functions. The morphological aspect of the viability of the various vinous elements with special emphasis on the trophoblast cells was described histologically and ultrastructurally. The functional aspect of the viability was discussed by analysis of the suppressive effect of the cultivated villi on plasminogen activator (PA) secretion by OK-432 elicited mouse peritoneal macrophages, by analyses of the activity of Δ5 -3β-hydroxysteroid dehydrogenase coupled with Δ5 -Δ4-isomerase (HSD) and of the radioactivity of [125I] -Iododeoxyuridine ([125I] - IUdR) retained in the DNA of the trophoblast cells. Specimens of normal placenta were obtained at the time of induced abortion. The gestational ages were 15, 16 and 18 weeks. Specimens for organ culture were prepared under sterile conditions within one hour after expulsion of the placenta. Through gross dissection, the villi were isolated and minced into fragments of approximately 1 to 2 mm3. Incubation was carried out at 37°C in a conventional static chamber with a gas mixture of 95% air, 5% CO2. The culture dishes and culture media were renewed every day. Data are the mean values of the duplicate incubations. In the first series of organ culture, placental fragments were removed at the end of each day of incubation, washed thoroughly and transferred to the new dishes with a culture medium freed from fetal bovine serum, where further incubation was performed for 24 hours. After this, placental fragments were fixed in 4% neutral buffered formalin, processed through paraffin embedding, serially sectioned at 5 μm and stained by periodic-acid Schiff (PAS) procedure. Culture medium obtained in this series at each end of incubation was put into a “macrophage plate” with the addition of plasminogen in the concentration of 2 IU/ ml, in which OK-432 elicited mouse peritoneal macrophages with the capacity to secrete PA were cultured. Reaction was terminated at 24 hours unless otherwise indicated. Ten μl of the medium in the “macrophage plate” was applied to the fibrin plate, and reaction was performed for 18 hours. PA activity was expressed by plasmin activity converted from plasminogen measured by the single radial immuno-diffusion method. The suppressive effect of the villi on PA secretion was examined and expressed as per cent of the PA activity against the control, which was measured by adding only bovine serum free medium to the “macrophage plate” by the same procedure as previously described. In the second series, placental fragments were removed at the end of each day of incubation, washed thoroughly and transferred to the new dishes with a culture medium containing 1.75 n moles (0.1 μCi) of [4-14C] -5-pregnen-3β-20-one ([4-14C] -P5; S.A. 57.2 mCi/m mole) as the substrate for HSD. Incubation was terminated at 20 hours unless otherwise indicated, and extraction of steroids was carried out with dichloromethane from the medium. The extracts were chromatographed on a silica gel thin layer plate, and radioactive regions were detected by a β-chromatogram camera. Placental fragments obtained in this series were homogenized by sonication for protein determination by the Lowry method with bovine serum albumin as standard. The activity of HSD was expressed as × 10-11 moles/mg protein/hour. In the third series, 1.0 μCi of [125I] -IUdR (S.A. 2,000 Ci/m mole) was added to the culture medium prior to the cultivation,
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器官培養におけるヒト胎盤絨毛のviabilityに関する研究

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