摘要
用常规电生理细胞内记录技术了解了影响蝾螈胚胎非典型表皮细胞兴奋性的一些因素。1.小牛血清、蝾螈胚胎内胚层和肌节中胚层、非洲爪蟾胚胎外胚层及嫁接于受体胚胎都提高非典型表皮显示动作电位样品的百分比。血清琼脂包饺子的作用较培养在含血清溶液明显。内、外胚层的作用更为明显。嫁接于受体胚胎的非典型表皮百分之百地显示兴奋性。2.血清、内、中、外胚层及嫁接于受体胚胎都降低表皮细胞的刺激阈值。各处理组的平均阈值均低于对照组,其巾嫁接于受体胚胎的非典型表皮细胞最低,接近于正常胚胎的表皮细胞。3.血清琼脂块和内胚层包饺子的实验表明,影响兴奋性的因素必须始终与表皮细胞保持接触,才‘能产生作用。一旦脱离接触,其作用就逐渐消失。4.胚胎表皮细胞兴奋性的产生不依赖于外来因素,是一种自主的功能分化。但是外来因素能使培养的表皮细胞的兴奋性增强,对低强度的刺激产生兴奋。这种外来因素可能是一些非专一性的与代谢有关的物质。
It has been reported that in Cynopsorientalis the cells in the atypical epidermisresulted from isolation of the ectoderm fromthe early gastrula and cultured in vitro pos-sesses very low excitability.The great ma-jority of the explants didn't display actionpotential (AP) with electric stimulus up to6 V duration 1 ms).If,however,the epi-dermis was taken from the flank region of aneurula with the underlying mesoderm,insuch explant the epidermis cells bacame exci-table at a lower threshold of stimulus.Themesoderm seemed to exert some influence uponthe epidermis.In order to study whethertissues other than mesoderm possess similareffect on the excitability of epidermis cells,different combinations of explants were madewith the experimental embryological techni-que and their excitability was examined elec-trophysiologically. In all experiments the explants were sti-mulated extracellularly and the action po-tential was recorded intracellularly.The spe-cimens were stimulated with electric stimulusat 6 V at first,and,if they displayed APthe strength was lowered to determine thethreshold of stimulus to evoke AP.The du-ration of stimulus was fixed at 1 ms. For the control of all series,the ecto-derm or epidermis was isolated at the samestage and cultured for the same length ofperiod as the experimantal explants but with-out any treatment. 1).The effect of calf serum. In this group the effect of calf serumwas examined and following series were car-ried out:a) Gastrula ectoderm cultured inHoltfreter solution containing 10% calfserum;b) calf serum+agar as implant:calfserum was mixed with agar (1:1) and cutinto small pieces when cooled,and the serum+agar pieces were wrapped with ectoderm;c) heated serum+agar,heated serum was mix-ed 1:1 with agar and the small pieces werewrapped with ectoderm. In all the experiments the excitability ofthe epidermis cells was raised more or lessbut the agar pieces containing fresh serumseemed to be more effective,perhaps due tothe higher concentration of the serum andthe intimate contact with the epidermis cells(Tab.1) In the series with serum+agar as im-plant and in the series with heated serum+agar as implant,it occurred frequently thatduring cultivation the implants will be ex-truded and after the extrusion of the implantthe remaining epidermis became less sensitiveto stimulation.For example in the serieswith serum+agar as implant,the percentages ??of explants revealed excitability were reducedto 21.7% (n=23) at stage 27,to 10.5%(n=19) at stage 33—34.and to 28.0%(n=25) at stage 36,that is,approximately tothose in the control series.It seems that,when the epidermis lost the contact with theimplant,the influence of the later graduallydisappeared.2) The effect of mesoderm and somite. The somitic mesoderm of the mid neural(stage 18—19) or the somite of the swim-ming larva (stage 40—42) was removed andwrapped with the ectoderm of late gastrulawhich had lost the competence to form neu-ral tissue upon induction.The results aresummerized in Tab.2 from which it can beseen that in both series the percentage ofexplants with ability of generation of AP wasincreased and the threshold of stimulus waslowered. The effect of larval somite is worthy ofmention,because at stgae 40—42 at whichthe somite was removed,the epidermis hadalready lost the excitability,but the somiteis still effective to influence the excitabilityof the epidermis. ??3) The effect of endoderm. In the normal embryo there is no directcontact between endoderm and ectoderm,theyare separated by the mesoderm.In order tostudy whether the endoderm may influencethe excitability of the cultured epidermiscells,the yolk endoderm was removed fromthe early gastrula and wrapped with the ec-toderm of the same embryo.After certainperiod of cultivation the excitability was exa-mined and the result is shown in Tab.3.Comparing this table with the table 2,it canbe seen that the endoderm is no less effec-tive than the mesoderm. Due to the affinity change between theendoderm and the ectoderm during develop-ment,if the ectoderm healed up incompletelyafter the operation,the endoderm will beextruded during the cultivation.The remain-ing epidermis was tested electrophysiologi-cally 1—2 days after the extrusion of theendoderm.At stage 31,the percentage of ex-plants showing excitability was 8.3 (n=12)they behaved like the control.4) The effect of epidermis of Xenopus laevis. The atypical epidermis of Xenopus laevisbehaves quite defferently from that of Cynopsorientalis.Isolated at stage 10 (Xenopus stageaccording to Nieuwkoop and Faber) all ofthe explants revealed excitability when exa-mined eletrophysiologically at stage 33,35,42,45 and 46 (unpublished result).Sincethe influence of different tissues altered theexcitability of the Cynops-epidermis,it is in-teresting to know whether the epidermis ofXenopus may possess such ability.Chimericexplants consisted of gastrula ectoderm fromboth species were made,and the excitabilityof the Cynops cells was examined when theydeveloped to stages 27,29,32 and 37 )the ??Xenopus epidermis reached the stages 43,45,47 and 48 respectively). From Tab.4 it can be seen that in com-parison with the Cynops control,in thechimeric explants the percentage of the ex-citable Cynops-part was increased considera-bly and the threshold cf stimulus was ob-viously lowered.Besides it should be notedthat the Xenopus-epidermis possesses no moreexcitability when it is older than stage 47,but the Cynops-epidermis in the chimericexplants when developed to stages 34 and 37(the Xenopus-epidermis already reached stages47 and 48 respectively) still showed muchhigher percentage of excitability than thecontrol series.This means,although theXenopus-epidermis itself is no more excitable,but the influence on excitability still persis-ted.5) The effect of grafting the epidermis tohost embryo. If the epidermis was cultured for 2 daysafter removal from early gastrula and thengrafted to a normal embryo as we have men-tioned in a preliminary report,the tran-splant became more excitable than those cul-tured in isolated condition.As shown inTab.5,after the operation all the grafts re-vealed excitability and the threshold of sti-mulus was obviously lowered,approximatelyto the level of the in situ epidermis. If,however,the graft was amputatedafter it has acquired the excitability change andcultured further,its excitability lowered againto the level of the control after 1—2 daysin isolated condition,just like the cases ingroups 1) and 3) after the extrusion of theimplants. In the above mentioned experiments theexcitability of the cultured epidermis cellswas influenced to different degree.Since theexcitability can be raised by various means,it can be concluded that:1) The appearanceof excitability represents an autonomous func-tional differentiation of the embryonic epi-dermis cells.Even the explants which didn'trespond to stimulus were potentially excita-ble,perhaps the mechanism to produce exci-tability was impaired or interfered by thein vitro culture condition,and the excitabi-lity could be restored by external factor(s).2) Factor(s) influencing the excitability ofthe epidermis cells is non-specific. Since the excitability of the treated epi-dermis became lowered to the level of thecontrol under the following conditions:a.after the extrusion of the implant (eitherserum+agar or endoderm);b.after ampu-tation from the host embryo,it seems plau-sible that,for the maintenance of a moresensitive excitability a continous supply ofthe effective factor(s) is required.Once thesupply is interrupted,during a certain period ??of time the effective factor(s) in the epider-mis cells will be completely metabolized andexcitability become lowered to the originallevel of cultured epidermis without treatment
出处
《实验生物学报》
CSCD
1989年第1期111-122,共12页
Acta Biologiae Experimentalis Sinica
基金
国家自然科学基金资助项目
关键词
东方蝾螈
胚胎
表皮细胞
兴奋性
Embryo of Cynops orientalis.Epidermis cells.Excitability.
