鸡红细胞融合最适条件的探讨

目的：探讨快速、有效的细胞融合条件.方法：用鸡红细胞为材料,聚乙二醇（Mw=4000）为诱导剂,诱导鸡红细胞融合.结果：鸡红细胞融合的最适温度为39℃,最适时间为15min.结论：在该条件下,同时用Giemsa染液对融合细胞染色,实验观察效果明显.     

聚乙二醇诱导鸡红细胞融合条件的优化

目的:探讨聚乙二醇诱导鸡红细胞融合最适条件.方法:以鸡红细胞为材料,聚乙二醇为诱导剂,从细胞密度、融合温度、聚乙二醇浓度及反应时间进行单因素实验,并设计3因素3水平的正交实验计算细胞融合率.结果:鸡红细胞融合的最佳条件是:细胞密度为2×106个/ml,聚乙二醇浓度为35%,反应温度为35℃,反应时间40min.结论:在该条件下,细胞融合率最高达33.06%.     

蟾蜍红细胞融合条件的正交设计优化

目的：用聚乙二醇法探讨蟾蜍红细胞融合的最佳条件。方法：采用3因素3水平的正交实验法,以蟾蜍红细胞为材料,从聚乙二醇浓度、反应时间、反应温度三方面计算细胞融合率,探讨和比较蟾蜍红细胞融合的最适条件及影响因素。结果：蟾蜍红细胞融合的最佳条件是：选用50%的聚乙二醇,反应温度为37℃,反应时间为15min。     

鸡胚与胎鼠的成纤维细胞条件培养液促进成肌细胞增殖和融合的比较

用培养过鸡胚(来亨鸡)或胎鼠(ICR小鼠)肌组织的成纤维细胞的条件培养液,定量地研究它们对胎鼠或鸡胚的成肌细胞的增殖和融合的影响。所得结果如下:(1) 胎鼠的成纤维细胞条件培养液促进胎鼠或鸡胚成肌细胞增殖,分别为对照组的2.65倍,(P<0.001)或2.35倍,(P<0.01);(2) 鸡胚的成纤维细胞条件培养液促进鸡胚或胎鼠的成肌细胞增殖,分别为对照组的2.66倍,(P<0.01)或2.17倍,(P<0.01);(3) 胎鼠的成纤维细胞条件培养液增加胎鼠或鸡胚的成肌细胞的融合率,分别为对照组的1.9倍或2.6倍;鸡胚的成纤维细胞条件培养液只增加鸡胚成肌细胞的融合率,为对照组的2.1倍,但对胎鼠成肌细胞的融合无明显的影响。 实验结果提示:成纤维细胞条件培养液促进成肌细胞的增殖,两种动物间无明显的差异,但在融合上却有一定的种属特异性。     

荷斯坦种公牛血清替代补体溶血活性研究

本文将国外脊椎动物血清补体溶血活性标准测定方法,运用到荷斯坦种公牛研究中,首次建立了测定荷斯坦种公牛血清补体溶血ACH50的方法。种公牛血清经相应靶红细胞吸附后,可溶解悬浮在EGTAMgGVB缓冲液中的正常的兔血红细胞、人A,B,AB,O型红细胞,小鼠、大鼠、鸡红细胞,但对绵羊、山羊、猪红细胞溶血活性较低;对奶牛红细胞无溶血活性。且发现种公牛血清的溶血活性和靶红细胞的动物种类在系统发育上和种公牛的亲缘关系远近没有直接联系。种公牛血清在EGTAMgGVB缓冲液中对兔血红细胞发生溶血的最适条件是:温度是37℃,最适pH是7.3-7.4,最适Mg2 的浓度是4mmol/L,最适孵育时间为90min。溶血活性是二价离子依赖、热敏感(溶血活性热灭活温度是56℃)。种公牛血清对兔血红细胞的溶血活性在受到酵母聚糖、甲胺、肼、EDTA、鸡抗酵母聚糖牛血清结合物抗血清处理时,溶血活性可全部或部分消失,溶血活性抑制程度与补体抑制剂浓度相关。我们运用建立的标准溶血方法并以兔血红细胞作为指示细胞检测不同年龄的53头种公牛血清补体替代途径的溶血活性,溶血值在13.2-44.3u/ml之间,还发现不同年龄组公牛之间溶血活性有随年龄增加而逐步增大趋势,但差异不显著(P>0.05),在4-5岁公牛群中达到最大值。对种公牛血清补体系统溶血水平进行系统研究,一方面可以填补国内在此领域研究空白,另一方面也利于种公牛疾病监测、控制,此外也为兽医临床诊断试剂的研制提供新的技术手段。     

火鸡新城疫病毒的分离与鉴定

1988年北京某火鸡场暴发新城疫,从病鸡脾、脑组织分离到新城疫病毒(NDV)强毒株。现简要报告如下。 将病火鸡的脾和脑研磨,用生理盐水1:5稀释,接种9日龄非免疫鸡胚3只,收集24小时后死亡的鸡胚尿囊液,即为待测病毒。低温冰箱保存备用,红细胞为非免疫鸡红细胞,生理盐水洗4次,配成0.5％浓度,4℃冰箱保存。     

鸡菌的液体培养及其多糖物质研究

采用不同的液体培养基,探索了鸡堆放(土从)菌(Termitomyces albuminosus)人工培养的最适条件,多糖形成的动态变化,并对鸡(土从)多糖进行了分离、纯化,及其免疫活性测定。结果表明:鸡(土从)菌从碳源玉米淀粉,氮源花生蛋白,生长因子麸皮浸汁,pH4.7,25℃为最适生长条件;Sephadex-G100分离多糖出现两个峰;鸡(土从)多糖作为刺激原对人淋巴细胞转化有促进作用。     

一种来源于脆弱类杆菌的α-半乳糖苷酶的理化性质及其酶解B型红细胞的工艺研究

利用α-半乳糖苷酶去除红细胞表面的B抗原是制备通用O型红细胞的有效方法.本文在克隆表达纯化脆弱类杆菌来源的α-半乳糖苷酶的基础上对其理化性质进行了研究,该酶的分子量为64908Da,等电点在7.12～7.30之间,最适温度为41℃,最适pH为5.6～6.0,其理化性质适合用于B型红细胞的血型改造;为了确定高效、快速、温和的酶解条件,本文对酶解B型红细胞的工艺进行了优化.通过研究缓冲液对酶与红细胞结合的影响,确定了最佳酶解缓冲液为250mmol/L甘氨酸和3mmol/LNaCl,pH6.8;酶解的最适红细胞压积为40%,酶解温度为26℃,酶解时间为1h.利用优化的酶解工艺获得的B-ECORBCs形态及结构功能指标均正常,流式细胞结果证明其B抗原和H抗原标记率与O型红细胞相当,说明制备B-ECORBCs的工艺已成熟.这种工艺具有酶用量少、酶解条件温和、制备过程简单和时间短等优势,具有很好的临床应用前景.     

酿酒酵母与球形红假单胞菌原生质体跨界融合研究

测定了球形红假单胞菌原核细胞与酿酒酵母真核细胞的原生质体形成、再生及融合最佳组合条件。以溶菌酶ＥＤＴＡ反应系统处理球形红假单胞菌Ｐ９４７９,最适脱壁条件为：溶菌酶浓度０.５ｍｇ／ｍｌ,ＥＤＴＡ浓度为０.２％,蔗糖浓度２０％,酶作用时间为４０ｍｉｎ;此条件下原生质体形率为７８.９％,再生率为１１.２％。用蜗牛酶巯基乙醇反应系统处理酿酒酵母Ｙ９４０７,最适脱壁条件为：蜗牛酶浓度１.０％,巯基乙醇浓度０.１％,蔗糖浓度２０％,酶作用时间为３０ｍｉｎ;此条件下酵母原生质体形成率为９９.８％,再生率为９.７％。用聚乙二醇诱导Ｐ９４７９与Ｙ９４０７的原生质体发生融合,当聚乙二醇的浓度为３０％,Ｃａ２＋浓度为５０ｍｍｏｌ／Ｌ,ｐＨ为６.５,时间为１０ｍｉｎ时,有最高的融合率为７.６×１０－６。     

鸡Zong菌的液体培养及其多糖物质研究

采用不同的液体培养基,探索了鸡Ｚｏｎｇ菌人工培养的最适条件,多糖形成的动态变化,并对鸡Ｚｏｎｇ多糖进行了分离、纯化,及其免疫活性测定。结果表明：鸡Ｚｏｎｇ菌从碳源玉米淀粉,氮源花生蛋白,生长因子麸皮浸汁,ｐＨ４．７,２５℃为最适生长条件;Ｓｅｐｈａｄｅｘ－Ｇ１００分离多糖出现两个峰;鸡Ｚｏｎｇ多糖作为刺激原对人淋巴细胞转化有促进作用。     

葡萄糖浓度对细胞融合效果的影响

目的:细胞融合是细胞生物学领域近30年来得到迅速发展的一项新兴技术手段,因其操作简便、人工可控等优点在研究核质互作、肿瘤发生、疫苗研发和培育新型生物品种等方面均有广泛应用。其中,利用聚乙二醇(PEG)进行化学融合是细胞融合中最为常用且简便的技术手段。PEG化学融合效果受到多种因素影响,如PEG浓度、Ca2+、Mg2+、pH值等,然而对于糖类物质在细胞融合中的影响未见报道。本文旨在为了更全面了解PEG法诱导的化学细胞融合,通过优化融合条件以提高化学细胞融合效率。方法:选取鸡血血细胞为材料,通过改变原Hanks缓冲液中葡萄糖浓度,观察比较各组细胞融合率,探究葡萄糖浓度在化学细胞融合中的影响,并通过对比结果获得了对于鸡血血细胞应采用的最适葡萄糖浓度区间。结果:对于鸡血血细胞融合实验,葡萄糖浓度在10-14 mmol/L范围内细胞融合效率较原Hanks液配方高2倍左右。结论:葡萄糖对细胞融合效果具有一定的影响,可以通过调节葡萄糖浓度提高细胞融合率,从而为PEG化学细胞融合提供一种更为优化的方案。     

四种鱼类外周血红细胞细胞周期及DNA含量

通过流式细胞仪测量了兴国红鲤（Ｃｙｐｒｉｎｕｓ ｃａｒｐｉｏ Ｖａｒ． Ｓｉｎｇｕｏｎｅｎｓｉｓ）、奥利亚罗非鱼（Ｓａｒｏｔｈｅｒｏｄｏｎ ａｕｒｅａ）、 鳙（Ａｒｉｓｔｉｃｈｔｈｙｓ ｎｏｂｉｌｉｓ Ｒ．）和团头鲂（Ｍｅｇａｌｏｂｒａｍａ ａｍｂｌｙｃｅｐｈａｌａ Ｙｉｎ．）红细胞的ＤＮＡ含量,四种鱼与鸡红细胞ＤＮＡ含量的比值分别为１·６５、０．９６、０．９１、１·１５,以鸡红细胞ＤＮＡ含量２．３ｐｇ／Ｎ计算,四种鱼二倍体体细胞的 ＤＮＡ含量分别为３．８０ ｐｇ／Ｎ, ２． ２２ｐｇ／Ｎ、２． ０８ｐｇ／Ｎ、 ２． ６６ｐｇ／Ｎ。另外,四种鱼外周血红细胞分别有２６％、 ２６％、 ２３％、 ２４％的细胞处于 ＤＮＡ合成期（Ｓ）,ＤＮＡ合成后期（Ｇ２）和分裂期（Ｍ）,这表明这四种鱼类的外周血红细胞并非失去分裂能力的特化细胞群,它们表现了较强的细胞周期现象。这有可能是这几种硬骨鱼类外周血仍具有造血功能的缘故。     

Myoblast-mediated fusion-injection: a new technique for introduction of macromolecules specifically into living skeletal muscle cells

A new technique for the introduction of macromolecules specifically into living skeletal muscle cells has been developed by a modification of the red blood cell ghost-mediated fusion-injection technique [M. Furusawa (1980) Int. Rev. Cytol. 62, 29-67]. Fluorescein-labeled bovine serum albumin (FITC-BSA) was introduced into chicken skeletal muscle myoblasts by the human red blood cell-mediated fusion-injection method in the presence of polyethylene glycol. Myoblasts loaded with FITC-BSA were then purified by a fluorescence cell sorter and cocultured with myotubes. Specific cell fusion between myoblasts and myotubes occurred under normal culture conditions and BSA was successfully introduced into living myotubes. This technique may provide a new method not only for the study of a given macromolecule's function in living muscle cells but also for therapeutic purposes such as muscle-specific drug delivery.     

应用双光子显微镜和流式细胞仪定性及定量确定小鼠巨噬细胞的吞噬功能

建立了流式细胞仪和双光子激光共聚焦荧光显微镜进行定性和定量检测小鼠巨噬细胞吞噬鸡红细胞的方法,并同传统光学显微镜细胞化学染色观察方法相比较,探讨其检测巨噬细胞吞噬效应的优越性。常规方法获取小鼠腹腔和脾脏巨噬细胞,制备巨噬细胞悬液。常规制备鸡红细胞,计数并调整活细胞数,用5-二醋酸羧基荧光素琥珀酸单胞菌酯(5-carboxyfluorescein diacetate succinimidyl ester,CFSE)染色,与巨噬细胞共温育一定时间后,小鼠巨噬细胞特异性荧光抗体F4/80标记巨噬细胞。应用流式细胞仪检测巨噬细胞中CFSE阳性百分率来表示巨噬细胞吞噬率;应用双光子显微镜观察被吞噬的CFSE阳性鸡红细胞动态分布情况。同时,采用传统光学显微镜吉姆萨染色观察巨噬细胞吞噬百分率。结果显示,流式细胞仪结合双光子显微镜检测巨噬细胞吞噬率与传统的显微镜计数法比较,两者有明显的正相关性。双光子显微镜和流式细胞仪可以定性与定量检测巨噬细胞吞噬功能,该方法具有灵敏、快捷、重复性好以及准确率高的特点,是进行免疫学研究的可行方法。     

Cytotoxic immune cells with specificity for defined soluble antigens. I. Assay with antigen-coated target cells

An in vitro cytotoxic system is described, in which immune cells specific for a given soluble antigen exert a specific cytotoxic effect on target cells to which this antigen has been covalently linked. The nature of the target cell is important in this system. When antigen-coated P 815-X2 mastocytoma cells and antigen-coated chicken red blood cells were incubated for several hours in culture medium at 37 °C, the presence of membrane-bound antigen could still be demonstrated on the latter, but not on the former target cells. This might be the reason why antigen-specific target cell destruction by specific immune cells was observed only with antigen-coated chicken red blood cells as target cells. The specificity of the cytotoxic effect was controlled in each experiment in a criss-cross way by using two non cross-reacting antigens both as immunogens and for coating the target cells. Specific cytotoxicity was demonstrable with both guinea pig and mouse immune cells and with different kinds of antigens: foreign proteins, hapten-heterologous protein conjugates and hapten-autologous protein conjugates.     

Utilization of a differential hemoglobin elution procedure as a rapid assay for identification of embryonic and adult chicken red blood cells.

1. A hemoglobin elution-staining procedure has been developed for distinguishing embryonic chick red blood cells from adult chicken red blood cells. 2. Adult hemoglobin is eluted from red blood cells with 1.9 M potassium phosphate buffer, pH 7.2; whereas, embryonic hemoglobin is retained within the cells and gives positive staining with erythrosin B. 3. The hemoglobin elution-staining pattern during development can be correlated with two embryonic hemoglobins as detected by polyacrylamide gel electrophoresis. 4. The series of red blood cells staining with erythrosin B correspond to the primary erythrocyte series suggesting that hemoglobin expression during development is correlated with different cell populations.     

Ontogenic evolution of chicken red cell Fc receptor

The ontogenic evolution of chicken red cell Fc receptor was studied in red cells from different age chicken embryos, baby chicken, and adult chicken. The Fc receptor binding capacity for ligands, the number of Fc receptors by red cell, and the association constant between receptor and ligand were analyzed. The Fc receptor is expressed in the red cell surface of 6-day chicken embryo and its binding capacity for ligand—minimal at this moment—is increased in the 8-day chicken embryo red cells. The 12-day chicken embryo erythrocytes binding capacity is similar to the adult chicken red cells. The number of Fc receptors by red cell increase with the age of chicken embryo. After 9 days this number is not modified and it is the same as in adult chicken. Variations of Ko and binding capacity for ligands show a similar evolution in embryogenic development. From these data we suggest that although on Day 9 the number of receptors per cell is the same as in adult chicken, the receptors are not completely exposed at this time and as a consequence, their binding capacity for ligands is lower than in adult chicken erythrocytes.     

Expression of an onco-developmental antegen among avian species.

Rabbit antisera directed against an onco-developmental antigen on chicken red blood cells have been serologically dissected through specific adsorptions. It is now possible to detect 13 antigenic determinants with the fractionated antisera. The onco-developmental antigen referred to as chicken fetal-leukemic antigen (CFA) is fetal-specific in the white Leghorn chicken, being present on the embryonic but not adult peripheral red blood cells of non-being present on the embryonic but not adult peripheral red blood cells of non-leukemic birds. However, one or more of the onco-developmental antigenic determinants have been detected on adult peripheral red blood cells of non-Gallus avian species, as well as on red blood cells from two adult chicken varieties. For phylogenetic purposes, red blood cells from avian species were characterized for their combinations of CFA determinants. Comparisons among species revealed specific patterns of antigenic expression within phylogenetic groups. Several CFA determinants were restricted in their occurrence to species within a single family, and one determinant was found in all cases where CFA was expressed. The distribution of CFA determinants was used to determine immunological distances among four Galliform species. These distances agreed with the immunological relationships established using different serological markers.