一种改良宫颈鳞癌原代培养方法的建立

目的：探讨不同培养条件对宫颈鳞癌原代培养的可行性。方法：取确诊为宫颈鳞癌患者的活检或手术标本,通过对消化时间及重悬液量进行改良原代细胞培养,观察鳞癌细胞生长状况,并比较其与传统消化法的细胞生长率,免疫组化进行细胞鉴定。结果：消化时间为3小时,重悬液量为1ml时细胞生长率最高。改良法24小时细胞生长率为61．54％,传统消化法24小时细胞生长率为7．69％（x2=5．14,P〈0．05）。活检标本细胞生长率80％,手术标本3例无一例细胞生长。结论：采用消化时间为3小时,重悬液量为1ml成功建立了宫颈鳞癌原代培养方法,活检标本较手术标本更易培养成功。     

成人宫颈上皮细胞的原代培养及鉴定

目的:探索采用无血清培养基原代培养成人宫颈上皮细胞的方法。方法:以成人的宫颈上皮组织为研究对象,采用胰蛋白酶-EDTA消化法获得宫颈上皮细胞悬液,于上皮细胞专用无血清培养基中培养,采用免疫细胞化学法测定细胞中角蛋白及波形蛋白的表达,对细胞纯度进行鉴定。结果:原代培养10-15天细胞融合达60%,传代至4-6代,细胞出现生长衰退。早期细胞生长状态良好,细胞纯度在90%以上。结论:采用酶消化法及K-SFM无血清培养基培养可获得纯度高的成人宫颈上皮细胞。     

兔骨髓间充质干细胞的分离、培养与鉴定

目的:寻求家兔骨髓间充质干细胞(Bone Mesenchymal Stem cells,BMSCs)培养与分离简单易行的方法,为下一步骨髓间充质干细胞在呼吸系统疾病的应用打好基础。方法:取3个月龄家兔1只,经双侧髂前上棘抽取骨髓共5.0 ml,密度梯度离心法和贴壁筛选法相结合分离、纯化获得BMSCs,倒置差显微镜观察其形态学特性,流式细胞仪鉴定CD34、CD133表达。结果:接种细胞于24小时有少量细胞贴壁,72小时多数细胞可贴壁,贴壁细胞形态多为长梭型或多边形,原代细胞呈集落生长,12-16天可达90%融合,1%胰酶消化后传代培养,培养至第三代备用。流式细胞仪鉴定90%为骨髓间充质干细胞。结论:通过密度梯度离心法与贴壁筛选法可成功分离培养出骨髓间充质干细胞,此方法简单易行,成功率比较高。     

原代地鼠肾细胞及狂犬病毒不同培养方式的研究

目的通过比较原代地鼠肾细胞在转瓶、微载体、细胞工厂的3种培养方式的培养效果,为原代地鼠肾细胞选择一种易扩大规模、培养高质量细胞的培养方式,进而提高狂犬病毒的产量。方法消化取得的细胞悬液分别在转瓶、微载体、细胞工厂中培养,通过显微镜观察细胞形态、计数等结果比对培养的差别。结果细胞工厂可以很好地培养原代地鼠肾细胞和狂犬病毒;而细胞在微载体上贴附性差,生长不好。结论实验结果表明细胞工厂可以取代转瓶,用于大规模培养原代地鼠肾细胞扩大狂犬病毒的产量。     

CTX免疫抑制小鼠替代裸鼠用于成瘤实验的研究

目的:探讨小鼠注射环磷酰胺CTX形成暂时性免疫缺陷后,在其肾筋膜接种Hela细胞,建立免疫抑制小鼠肿瘤细胞模型的可行性。方法:复苏培养Hela细胞,做软琼脂克隆形成实验,挑取生长状态良好的克隆团,分离具有较强增殖活力的细胞,取对数生长期细胞,接种(1~3)×107/ml的单细胞悬液0.1 ml于CTX免疫抑制小鼠左侧肾上腺区肾筋膜内,接种BMEMs细胞作为阴性对照。经核磁共振成像,组织病理HE染色和免疫组化检测确认癌细胞在肾筋膜内成瘤情况。结果:39只CTX免疫抑制小鼠,荷瘤成功39只,总荷瘤率100%。结论:成功建立了CTX免疫抑制小鼠肾筋膜接种Hela细胞模型,为研究肿瘤细胞悬液在非免疫缺陷的免疫抑制机体内生长的生物学特性打下良好的基础。     

成年SD大鼠心肌原代成纤维细胞分离及培养的优化方案

目的:摸索及优选成年SD大鼠心肌原代成纤维细胞的体外分离、培养及鉴定的实验方法。方法:将成年SD大鼠心脏剪成小组织块,采用以下四种方案(A:0.08%胰酶+0.1%胶原酶II消化15 min,B:0.2%胶原酶II消化15 min,C:0.2%胶原酶II消化60min,D:0.2%胶原酶II消化90 min)提取成年大鼠心脏原代成纤维细胞,再通过差速贴壁分离方法培养原代成纤维细胞。采用倒置显微镜观察成纤维细胞的基本形态特征,并进行Vimentiin免疫荧光染色对培养的原代细胞进行荧光鉴定;采用台盼兰染色对培养的原代成纤维细胞存活率进行鉴定;采用细胞计数对培养的成纤维细胞生长趋势进行鉴定。结果:四种方法均能培养成纤维细胞,但单酶消化60 min可一次性提取较多细胞,并且细胞状态佳,3 d即可传代。72 h成纤维细胞Vimentin免疫荧光染色阳性率高达97%。台盼兰染色可见其细胞死亡率明显降低,并且细胞计数可见细胞生长状态极佳。结论:单酶消化60 min是提取成年SD大鼠心肌原代成纤维细胞的高效、快速、稳定的实验方法,为心脏疾病的基础及临床研究提供了较为理想的细胞学实验模型。     

一种原代肺上皮细胞体外培养方法

目的:建立一种操作简便、重复性好的体外培养BALB/c小鼠原代肺上皮细胞(AEC)的方法,探究不同发育阶段小鼠AEC中柯萨奇病毒和腺病毒受体(CAR)表达量的变化,及其对小鼠原代AEC贴壁的作用。方法:手术获取小鼠肺组织,机械法剪碎肺组织,PBS缓冲液清洗肺组织块数次去血,联合使用链霉蛋白酶和胶原酶Ⅰ消化、分离肺组织块获得单细胞悬液,差速离心逐步清除其他种类的细胞,达到纯化AEC的作用。细胞在Ⅰ型鼠尾胶原蛋白包被过的细胞板中培养,光学显微镜下观察不同发育阶段AEC贴壁、生长状态;免疫荧光法鉴定AEC,检测AEC中CAR的表达量。结果:体外获得不同发育阶段BALB/c小鼠的原代AEC;胎鼠、幼鼠AEC贴壁并开始增殖所需时间较成体鼠短;胎鼠及幼鼠AEC中CAR的表达量明显较成体鼠高。结论:建立了稳定可重复的分离、纯化、体外培养小鼠原代AEC的方法,证明了AEC中的CAR可以促进原代AEC贴壁,为完善原代细胞培养方法提供了科学依据。     

小鼠小脑颗粒神经元的原代培养与鉴定

本研究通过体外分离培养及鉴定原代小鼠小脑颗粒神经元(cerebellar granule neurons,CGNs),为神经科学研究提供原代神经元细胞模型。取生后第7天的ICR乳鼠小脑,在解剖体视镜下剥离脑膜及血管,经刀片切碎、0.25%胰酶消化、移液器吹打、70μm尼龙滤网制备单细胞悬液,差速贴壁后接种于新鲜配置的培养基。倒置相差显微镜观察不同时间CGNs的形态变化。采用神经元的标记蛋白微管相关蛋白-2(microtubule associated protein 2,MAP2),星形胶质细胞的标记蛋白胶质纤维酸性蛋白(glial fibrillary acidic protein,GFAP),小胶质细胞的标记蛋白(type 3 complement receptor,CR3/CD11b)进行免疫荧光检测培养的原代CGNs纯度。原代培养CGNs接种20 min后即贴壁良好,培养24 h后细胞伸出突起,培养第7天神经元成熟,形成丰富的轴突,树突和胞间突触连接。经免疫荧光鉴定,CGNs纯度可达95%以上。成功体外分离培养出高纯度的原代CGNs,可应用于CGNs的体外研究。     

胰蛋白酶消化强度优化获得高纯度体外培养的星形胶质细胞

本文旨在观察胰蛋白酶消化对体外培养的星形胶质细胞纯度的影响,优化星形胶质细胞培养方法。常规分离新生Sprague Dawley(SD)大鼠大脑皮质,分别用0.25%胰蛋白酶消化20、30和40 min制备单细胞悬液并接种细胞。细胞长满瓶底时进行恒温摇床振荡,前两个不同消化时间组再分为常规消化的对照组和二次胰蛋白酶消化组进行传代纯化。倒置相差显微镜观察细胞生长情况,MTT法检测细胞增殖,GFAP免疫荧光分析星形胶质细胞纯度并观察其形态,流式细胞术分析细胞凋亡。结果显示,胰蛋白酶消化20 min的细胞在原代培养9 d长满瓶底。而延长胰蛋白酶消化时间至30 min,细胞增殖更快,培养7 d即可铺满瓶底,且星形胶质细胞形态正常,纯度达(70.2±4.0)%,较20 min组有显著性提高(P0.05)。40 min组虽然星形胶质细胞纯度也较20 min组有所提高,但细胞增殖缓慢且损伤明显。在恒温摇床振荡结束后,进行二次胰蛋白酶消化可减少传代后杂细胞数量。二次胰蛋白酶消化组第一代(P1)的GFAP阳性率普遍高于各自对照组,其中30 min+二次胰蛋白酶消化组GFAP阳性率为(98.1±1.7)%,相当于20 min+对照组P3水平,且二者的凋亡率无显著性差异。以上结果表明,胰蛋白酶消化30 min+二次消化能有效提高星形胶质细胞纯度,缩短原代培养及纯化时间,是体外快速获得高纯度星形胶质细胞的有效方法。     

人肺癌细胞系HB-99的建立及其生物学特征

利用一例肺鳞癌手术标本通过原代培养建立了肺癌细胞系命名为HB-99。该细胞系呈单层贴壁生长,从相差显微镜和电镜分析具有细胞的多形性,细胞倍增时间为24小时,克隆形成率40%,染色体改变复杂,众数63-65。将细胞移植到裸鼠体内而生长的肿块具有与原始病人手术标本相似的组织形态。免疫组织化学分析,近100%的细胞表达角蛋白17（CK17）,10%的细胞表达波形蛋白（vimentin）。根据该细胞系的生物学特征提示HB-99是一新建立的肺鳞癌细胞系。 Abstract：We have established a human lung squamous carcinoma cell line ,designed HB-99, by culturing primary tumor sample. The cells of HB-99 derived from resected specimen of a male patient with lung squamous cancer. They grew in monolayers and showed cellular morphology by phase contrast and electronic microscopy. The HB-99 cells had a doubling time of 24 hours and a cloning efficiency of 40%. Chromosomal analysis showed complicated rearrangements with a modal number of 63～65. When hetero-transplanted to nude mice, HB-99 grew to form tumor with the same morphology as the original one from the patient. The results of immunohistochemistry suggested that CK17 expressed in almost all cells while only 5%～10% cells had Vimentin. HB-99 is really a newly established cell line of lung squamous carcinoma.     

六种藓类植物茎的比较解剖学研究

通过对6种藓类植物,即褶叶青藓(Brachythecium salebrosum(Web.et Mohr.)B.S.G.)、湿地匐灯藓(Plagiomnium acutum(Lindb.)Kop.)、侧枝匐灯藓(Plagiomnium maximoviczii(Lindb.)Kop.)、大凤尾藓(Fissidensnobilis Griff.)、大羽藓(Thuidium cymbifolium(Doz.et Molk.)B.S.G.)和大灰藓(Hypnum plumaeforme Wils.)嫩茎和老茎的石蜡切片和显微观察发现,同一藓类植株的嫩茎和老茎,茎结构稳定,不同种藓类植物茎横切面具有不同特征.植物体茎横切面形状、表层细胞的层数、细胞大小和细胞壁厚薄、皮层细胞大小和形状、中轴的有无以及比例等特征可以作为藓类植物的分科分类依据之一.     

Cause of Senescence of Nine Sorts of Flowers

The levels of endogenous phytohormones and respiratory rate in nine sorts of flowers such as Cymbidium faberi Rolfe, Nopalxochia ackermannii Kunth and others were investigated both at full bloom and senescence and meanwhile the effect of exogenous phytohormones on prolonging the blossoms and promoting ethylene production were tested. There is a high content of endogenous ethylene in all the long-lived flowere, about 3–16 folds higer than the short-lived ones. There is a high level of ABA at full blooming flowers of short-lived flowers, in which there is no or only some cytokinins in it, but the ratio of CTK (6BA+zeatin)/ABA is smaller(l.7). The endogenous ABA reached a much higher level at senescence in all nine sorts of flowers, so it is reasonable to consider that it is ABA which plays an important role of regulation in controlling flower's senescence. There is a much higher level of GA3 and zeatin in the long-lived flowers which is not demonstrated in the shortlived ones. The respiratory rate is one of the factors controtling the longevity of flowers, but it does not play a decided role. Application of 6BA and zeatin prolongs distinctly orchid’s longevity, however exogenous IAA through the promotive action on ethylene production, evidently extends the longevity of the flowers of the Nopalxochia ackermannii Kunth.     

龙胆科药用植物化学成分的研究现状

龙胆科植物在我国的分布范围很广,且多数为药用植物,其多数种属的药用植物,至今其化学成分尚未被系统研究。综述了目前龙胆科药用植物的化学成分的研究现状及一般提取方法,对近年来发现的环烯醚萜及裂环烯醚萜类化合物进行了总结,为本科药用植物的更深入研究提供了参考。     

The effects of glutamine of the maintenance of embryogenic cultures of Cryptomeria japonica

Summary Embryogenic tissues of sugi (Cryptomeria japonica) were induced on a modified Campbell and Durzan (CD) medium containing 1 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 600 mg l⁻¹ glutamine, and subcultured in the medium of the same composition for over 1 yr. This resulted in a mixed culture of embryogenic and non-embryogenic cells. When embryogenic cells were isolated and cultured independently, their capacity to form embryogenic aggregates was lost. Thus, the non-embryogenic cells present within a mixed culture system were essential to the formation of embryogenic aggregates. When embryogenic tissues were isolated and cultured independently on a high glutamine-containing (2400 mg l⁻¹) medium, dry weights and endogenous levels of glutamine increased, and the tissue could generate a large number of embryogenic aggregates. Amino acid analysis of embryogenic and non-embryogenic cells from the maintenance culture indicated a higher level of glutamine was present in the latter. The high endogenous level of glutamine in the non-embryogenic portion of mixed cell masses may be the supplier of glutamine for maintaining the embryogenic property of the tissues.     

Specificity of action of piperidylcholine derivatives as substrates of cholinesterases of various origin

The review deals with study of enzymologic properties of a novel highly specific acetylcholinesterase substrate, N-(β-acetoxyethyl) piperidinium iodomethylate (“piperidylcholine”), and its 30 derivatives that were tested as effectors of cholinesterases of mammals and various species of Pacific squids. It was proven for the first time that responsible for specificity of action was structure of cyclic ammonium grouping of the alcohol part of molecule of the ester substrate. Analysis of specificity is performed based on enzymatic hydrolysis parameters—activity of catalytic center of cholinesterases and bimolecular constant of the reaction rate that are determined at optimal and low substrate concentrations. Among the specially synthesized group of thioester compounds there is revealed one more highly specific acetylcholinesterase substrate—N-(β-acetoxyethyl) piperidinium.     

真菌类遗传学分析的知识结构教学

本文以认知结构理论为指导,讨论了真菌类遗传分析与高等动植物遗传分析的内在联系,认为利用这种内在联系进行教学可收到好的效果并说明了作者的具体教学过程。 Abstract:In the paper, the relationship between genetic analysis of Fungi and genetic analysis of high animal and plant was discussed.A good results were obtained when we adopted this method in the teaching.     

医疗机构合理用药评价模型的构建研究

目的 针对医疗机构的合理用药水平进行评价研究。方法 根据医疗机构合理用药的具体要求,构建医疗机构合理用药评价指标体系,采用基于模糊群决策的方法和多指标评价分析法构建医疗机构合理用药评价模型。结果 构建了基于模糊群决策的医疗机构合理用药评价模型,并通过实例分析证明了评价模型的可行性。结论 建立的基于模糊群决策的医疗机构合理用药评价模型能够对医疗机构的合理用药水平进行科学评价,为提高医疗机构合理用药水平奠定基础。     

The mechanism of hatching of eggs of Haemonchus contortus

Fluid collected from hatching eggs of Haemonchus contortus contained a lipase which hydrolysed 2-naphthyl laurate (about 0·7 μmol naphthol freed /h/10⁶ eggs). The fluid also hydrolysed l-leucinamide (about 2·3 μmol leucine freed/h/10⁶ eggs). The fluid when added to normal or heated eggs caused ‘hatching’. ‘Hatching’ also occurred in exsheathing fluid from infective juveniles and in a preparation of pancreatic lipase containing leucine aminopeptidase. A purified mammalian leucine aminopeptidase in combination with several different lipases did not attack egg shells.The ‘spontaneous’ hatching of eggs of H. contortus was strongly inhibited by 1,10-phenanthroline, 10^?3M, and this inhibition was reversed by Zn²⁺. However, the inhibition of ‘hatching’ of eggs in externally applied hatching fluid, or the hydrolysis of leucinamide in hatching fluid was generally less marked.