Developmental physiology of cestodes. IX. Cytological characteristics of the germinative region of Hymenolepis diminuta.
Developmental physiology of cestodes. IX. Cytological characteristics of the germinative region of Hymenolepis diminuta.
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绦虫的发育生理学。
DOI：
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发表时间：
1971
影响因子：
1.3
通讯作者：
L. S. Roberts
中科院分区：
文献类型：
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作者：
R. Bolla;L. S. Roberts
根据细胞学特征和DNA合成活性，将小膜壳发育的萌发区定义为从200开始，在头节顶端之后，向后延伸到生发原基形成的点。描述了该区域萌发细胞类型的结构。对萌发区细胞分裂动力学进行了研究，估算出萌发期G1、S和G2的持续时间分别为3.0h、2.3h和3.2h。完成有丝分裂的时间估计为8.5小时以上。尽管大多数环叶绦虫的程序化被归因于从紧邻头节后面的未分割区域发芽(Wardle和McLeod，1952；Roberts，1961)，但所谓的发育区没有细胞学的定义或确切的位置。Roberts(1961)间接地将微小膜壳绦虫的萌发区定义为头节后没有观察到生殖器原基的区域。桑德曼(1959)定义的发芽区的衣壳，一种寄生虫的lapponica，位于后面的扩散区域的种球，其余的球果起源。如果萌发区确实负责孕节的产生，该区域的细胞活动应该与胚珠的其余部分显著不同，细胞分裂指数和参与DNA合成的细胞指数可以作为定义的标准。这个区域还应该包含一种细胞学上可识别的细胞类型，它可以作为产生孕节的“干细胞”。Bell和Smyth(1958)和Wikgren(1964)使用有丝分裂指数的标准来估计吸虫和吸虫的生长。巧妙(1967)讨论了将氚胸苷掺入DNA中作为DNA合成的指标，并因此将其用作细胞复制的指标。1970年7月30日收到待出版。*这项调查得到了马里兰州贝塞斯达美国国立卫生研究院的研究拨款AI-06153和培训补助金5 TOI AI226的支持，邮编：20014。现地址：印第安纳州圣母院，圣母大学生物系，46556。在本研究中，我们研究了微小隐孢子虫萌发区的细胞学特征以及该区域内一种可能的萌发细胞类型。已经估计了拟议的萌发细胞的细胞周期的持续时间。材料与方法将雄性Holtzman大鼠(体重120~180g)接种100个或10个囊虫，分别于感染后2、4、6~14d取回虫体。感染2天和4天后，用玻璃显微镜刮取肠道粘膜，放入磷酸盐缓冲(pH 7.4)平衡盐溶液(BSS)(Roberts和Fairbairn，1965)或加入0.1%葡萄糖的三马来酸缓冲林格液(TMR+G；pH 7.4)中(Read等，1963)。肠道刮片在解剖显微镜下进行检查，并用巴斯德吸管将蠕虫从碎片中分离出来。感染后6~14天，用少量盐溶液冲洗大鼠小肠内的老虫。每毫升含链霉素0.4 mg和青霉素G钾500单位的BSS或TMR+G液中孵育15min，然后在含有实验底物的盐溶液中孵育15min。所有的培养都在25毫升的Erlenmeyer培养瓶中进行，组织与培养液的比例为60 mg虫体鲜重/毫升培养液。孵化温度为37℃，氮气：二氧化碳(95：5)。孵化后，用盐溶液清洗虫体，固定进行细胞学检查。将测定有丝分裂指数的准备材料放在秋水仙素浓度中孵育一段时间，以获得最大数量的停滞中期图形。将整个2日龄和4日龄幼虫以及6~14日龄幼虫的前2厘米立即固定在3：1(v：v)70%乙醇：中性溶液中
The germinative region of developing Hymenolepis diminuta is defined based on cytological characteristics and DNA synthetic activity as beginning 200 ,u posterior to the apex of the scolex, and extending posteriorly to the point of germinal primordia formation. The structure of the germinative cell type in this region is described. The kinetics of cell division in the germinative region are studied, and estimates of the duration of G1, S, and G2 of prophase are 3.0, 2.3, and 3.2 hr, respectively. The time for a complete mitotic division is estimated to be 8.5 + hr. Although proglottisation in most cyclophyllidean cestodes has been attributed to budding from the unsegmented region immediately posterior to the scolex (Wardle and McLeod, 1952; Roberts, 1961), no cytological definition or exact location has been given to the so-called germinative area. Roberts (1961) indirectly defined the germinative area of Hymenolepis diminuta as the area behind the scolex in which genital primordia are not observed. Sandeman (1959) defined the germinative area of Capsulata edenesis, a parasite of Limosa lapponica, as a posteriorly located diffuse area of the strobila from which the rest of the strobila originates. If the germinative region is indeed responsible for proglottid production, the cellular activity in this region should be significantly different from the rest of the strobila, and cell division indices and an index of cells involved in DNA synthesis could be used as criteria of definition. This region should also contain a cytologically identifiable cell type which could act as a "stem cell" for production of proglottids. Bell and Smyth (1958) and Wikgren (1964) have used the criterion of a mitotic index to estimate growth in trematodes and cestodes. The use of the incorporation of tritiated thymidine into DNA as an index of DNA synthesis and, therefore, as an index of cell replication was discussed by Clever (1967). Received for publication 30 July 1970. * This investigation was supported by Research Grant AI-06153 and Training Grant 5 TOI AI226 from the NIH, U. S. Public Health Service, Bethesda, Maryland 20014. tPresent address: Department of Biology, Notre Dame University, Notre Dame, Indiana 46556. In the present study we have investigated the cytological characteristics of the germinative region of H. diminuta and a possible germinative cell type within this region. The duration of the cell cycle of the proposed germinative cell has been estimated. MATERIALS AND METHODS Recovery and incubation of H. diminuta Male Holtzman rats (120 to 180 g) were inoculated with either 100 or 10 cysticercoids and worms were recovered 2, 4, and 6 to 14 days postinfection, respectively. Worms were recovered 2 and 4 days postinfection from the hosts' small intestine by scraping the intestinal mucosa with a glass microscope slide into phosphate-buffered (pH 7.4) balanced salt solution (BSS) (Roberts and Fairbairn, 1965) or into tris-maleate-buffered Ringer's solution (Read et al., 1963) with 0.1% added glucose (TMR + G; pH 7.4). The gut scrapings were examined under a dissecting microscope, and the worms were separated from the debris with a Pasteur pipette. Older worms, 6 to 14 days postinfection, were flushed from the rat small intestine with a small amount of salt solution. The worms were washed, incubated 15 min in BSS or TMR + G containing 0.4 mg streptomycin and 500 units potassium penicillin-G per ml, and then incubated in the salt solution containing an experimental substrate. All incubations were done in 25-ml Erlenmeyer flasks with a tissue-to-medium ratio of 60 mg worm fresh weight/ml incubation fluid. Incubations were at 37 C in an atmosphere of N2:CO2 (95:5). Following the incubation, the worms were washed with salt solution and fixed for cytological studies. Preparation for determination of mitotic index Worms were incubated in a concentration of colchicine for a length of time necessary to obtain the maximum number of arrested metaphase figures. Entire 2and 4-day-old worms and the ant rior 2 cm of 6to 14-day-old worms were fixed immediately in 3:1 (v:v) 70% ethanol:neutral