介绍一种简易细胞培养液

根据中等卫校的特点,开展人体外周血淋巴细胞培养和染色体制备实验很有必要,通常该实验多采用RPMI1640和小牛血清做培养液。因以上材料价格贵,且不易购买,故开设该实验有困难。我们经数十次的反复对比试验,证明可用单纯猪血清做细胞培养液,现介绍如下: (一)准备将所需玻璃器皿洗净、烤干经高压灭菌备用。配制1％植物血球凝集素(PHA)、0.85％无菌生理盐水、双抗液(每毫升含青霉素、链霉素各5000单位)各适量。     

板蓝根提取物对小鼠免疫功能的影响

目的 研究板蓝根提取物对小鼠免疫功能的影响.方法 称重法检测小鼠免疫器官指数,血球分析仪分析小鼠外周血免疫细胞数量,比浊法检测小鼠血清中溶菌酶含量,溶血空斑形成实验检测B淋巴细胞抗体形成.结果 板蓝根提取物能明显增加小鼠免疫器官指数,提高小鼠外周血中免疫细胞数量.与对照组相比,外周血中淋巴细胞和白细胞数量分别增加(8.4±1.42)％和(10.52±1.63)％,血清中溶菌酶含量增加(10.82±1.08)％,溶血空斑形成数量增加(13.9±1.3)个/107脾细胞.结论 板蓝根提取物可提高小鼠的体液免疫和细胞免疫等功能.     

甲2巨球蛋白制剂对正常人外周血淋巴细胞周期的影响

采用外周血淋巴细胞姐妹染色单体差别法(SCE)检测技术,研究甲_2巨球蛋白制剂α_2M对人外周血淋巴细胞周期的影响,在人外周血淋巴细胞培养中加入α_2M制剂浓度在≤0.5mg/ml培养液时,第一次细胞周期(M_1)、第二次细胞周期(M_2)、第三次细胞周期(M_3)、细胞增殖速度指数(PRI)改变不大。在加入的α_2M制剂浓度(?)1.0 mg/ml培养液时,M_1百分数增加(r=0.977,P<0.001)、M_2百分数也随α_2M浓度递增而增高(r=0.956,P<0.001)而M_3百分数则随α_2M浓度递增而减少(r=-0.979,P<0.001)、细胞增殖速度指数则随α_2M浓度递增而减少(r=-0.983,P<0.01),表明在人外周血淋巴细胞离体培养时,当每毫升培养液中α_2M浓度等于或大于1mg时,细胞分裂周期延缓。讨论了它的意义和可能作用的环节。     

EPSTEIN-BARR病毒血清抗体阳性健康人外周血体外培养“自发性”转化的研究

对35名Epstein-Barr病毒血清抗体阳性健康人的外周血淋巴细胞进行了体外培养,观察B淋巴细胞感染了EB病毒后所导致“自发性”转化的情况。由于在培养基中加入了免疫抑制剂环胞菌素A,使“自发性”转化的发生率由26.7％提高到74.3％,说明了在血清抗体阳性健康人的外周血液中,EB病毒感染的B淋巴细胞的数量远比既往文献报道的高。     

禽类染色体制备方法的比较研究

以引进品种尼克红鸡为研究对象,对制备禽类染色体的外周血淋巴细胞培养法、羽髓法和改进的骨髓法进行了比较研究。结果表明：(1)外周血淋巴细胞培养法和羽髓细胞培养法制备染色体均未获得成功,因其操作复杂,技术条件要求高,周期长,成本高,可能不适宜于禽类的染色体制备。(2)直接羽髓法和改进的直接骨髓法以及骨髓短期孵育法却获得了成功,由于操作简便。周期短,成本低,适宜于禽类的染色体制备。     

刺五加多糖对断奶仔猪外周血淋巴细胞信号传导的影响

为了初步探讨刺五加多糖(ASPS)调节仔猪免疫功能的作用机制,试验研究了ASPS对体外培养的仔猪外周血淋巴细胞信号传导的影响。试验取28日龄断奶仔猪的外周血制备淋巴细胞悬液,分离其中的T、B淋巴细胞,分别与培养体系终浓度为0、40、80、160、320μg/mL ASPS共同孵育,MTT法测定T、B淋巴细胞转化率;在培养体系中加入TLR4抗体初步探讨ASPS影响淋巴细胞的信号传导途径。结果表明,ASPS能显著促进体外培养的仔猪外周血T淋巴细胞转化率以及淋巴细胞IL-2的分泌量(P0.05),而对B淋巴细胞转化率和IL-4水平无显著影响(P0.05)。当培养体系中无TLR4抗体时,ASPS对淋巴细胞分泌TNF-α、NO、iNOS和NF-κB因子水平有显著影响(P0.05),而当培养体系中加入TLR4抗体后,ASPS对上述指标无显著影响(P0.05)。上述结果提示,T细胞可能是ASPS直接作用的靶细胞之一,ASPS调节淋巴细胞免疫功能可能是通过TLR4/NF-κB信号通路起作用。     

柯萨奇B组病毒感染与胰岛素依赖型糖尿病关系的研究

探讨柯萨奇B组病毒(CBV)感染与胰岛素依赖型糖尿病之间的关系。用PT-PCR方法检测38例IDDM患者外周血中CBV-RNA,同时用ELISA法测定血清中CBV特异性IgM。结果显示,病例组38例患者外周血CBV-RNA阳性率为42.10％,血清中CBV-IgM阳性率为36.84％。对照组的阳性率分别为7.5％和5％。两组比较有显著性差别(P<0.05)。说明胰岛素依赖型糖尿病与柯萨奇B组病毒感染有关。     

穿山甲(Manis Pentadactyla aurita)的核型分析

穿山甲(Manis pentadactyla aurita)的核型尚未见报道过,我们在1982年观察了1只雄性穿山甲核型,现报道如下。 一、材料和方法 进行核型分析的穿山甲获自贵州,空运抵京饲养了数日,抽取其静脉血做淋巴细胞培养。培养液的配制是RPMI-1640和小牛血清按4:1的比例混合,加入适量的谷氨酰胺溶液。PHA选用Difco PHA M型。培养温度为36℃,培养时间为72个小时。与此同时,我们还取其骨髓细胞作体外短期培养,培养温度为室温,培养时间为2小时。无论是淋巴细胞培养还是骨髓细胞短期培养均采用空气干燥法制片。     

中华大蟾蜍(Bufo bufo gagarizans)血淋巴细胞的体外培养及其转化

本文报道了中华大蟾蜍血淋巴细胞的培养及其在植物血球凝集素(PHA)的刺激下所引起的转化,并初步查明了转化的淋巴细胞的S期、G_2期所占时间。用氚标记放射自显影手段测定了细胞的转化率。用姐妹染色单体区分着色的方法决定细胞的分裂次数。实验结果表明,在新鲜血液中有0.1—0.2％的白细胞具有合成DNA的能力。培养3天后,淋巴细胞转化率为14.8％,第5天达48％。培养的第6天有6—10％为第2次分裂。在秋水仙素处理3小时的情况下,有丝分裂指数为7％,延长处理时间至10—15小时,则平均达10％。最高有丝分裂指数为29％。转化淋巴细胞的S期为16小时,G_2期为3小时。     

金丝猴的染色体组型

本研究通过外周血淋巴细胞培养的方法,对二只金丝猴的染色体组型和染色体带型进行了分析。现已确证,金丝猴的2倍体细胞的染色体数目为2n=44。雄性为XY,雌性为XX。在染色体组型分析中,测量和计算了每一染色体的相对长度,臂比和着丝点指数。染色体和染色单体的“自发”畸变率分别为0.67％和2％。从姬姆萨(Giemsa)氏带型分析表明,每一对同源染色体都有自己的特殊带型,因此所有的染色体都能予以识别,并能准确无误的配对。     

Growth of cultured blood cells in the presence of the cryoprotector polyethylene oxide 400

The effect of polyethylene oxide 400 (PEO-400) cryoprotector on the mitotic activity of human peripheral blood leukocytes stimulated with PHA was studied in a culture. The cytologic peculiarities of the blood cell growth on addition of PEO-400 prior to cultivation are manifested in the preservation of the transformation capacity and mitosis despite a significant reduction of the mitotic activity in comparison with control cultures.     

The frequency of 6-thioguanine-resistant human peripheral blood lymphocytes as determined by flow cytometry and by clonal propagation

Human peripheral blood lymphocytes were selected for 6-thioguanine resistance in short-term cultures. Resistant cells, defined as cells capable of incorporating tritiated thymidine under the selective conditions, were flow-cytometrically differentiated with respect to their DNA content. This was carried out by sorting at two stages of the cell cycle, before and after mid-S-stage, yielding frequencies of resistant cells in the range of 10(-4) and 10(-5), respectively. Observed frequencies for cells from the whole cell cycle spectrum and for cells cultured according to the long-term protocol, the clonal assay, were in the range of 10(-4) and 10(-5), respectively. Our interpretation of these results is that the over-representation of tritiated thymidine-labelled cells occurring before mid-S-stage after short-term culture reflects less resistant cells or phenocopies which are probably eliminated during long-term culture with the clonal assay, hence leading to a decreased frequency of 6-thioguanine-resistant cells. We conclude, therefore, that short-term culture in combination with flow cytometric sorting after mid-S-stage in the cell cycle can be used as an alternative to the clonal assay for the determination of fully 6-thioguanine-resistant human peripheral lymphocytes.     

单核细胞促进人外周血淋巴细胞离体增殖的自由基机理

在人外周血淋巴细胞培养体系中加入不同数量的单核细胞共同培养,以~3H-TdR掺入法和细胞色素C还原法分别测定淋巴细胞增殖能力和培养介质中O_2~-浓度;加入NADPH氧化酶抑制剂DPI实验性地下调O_2~-浓度,观察淋巴细胞增殖能力的变化。结果显示加入单核细胞可增加培养介质中O_2~-浓度,进而促进淋巴细胞增殖,提示O_2~-浓度的升高是淋巴细胞和单核细胞共培养体系中淋巴细胞增殖的重要因素。     

Changes in gravity inhibit lymphocyte locomotion through type I collagen

Summary Immunity relies on the circulation of lymphocytes through many different tissues including blood vessels, lymphatic channels, and lymphoid organs. The ability of lymphocytes to traverse the interstitium in both nonlymphoid and lymphoid tissues can be determined in vitro by assaying their capacity to locomote through Type I collagen. In an attempt to characterize potential causes of microgravity-induced immunosuppression, we investigated the effects of simulated microgravity on human lymphocyte function in vitro using a specialized rotating-wall vessel culture system developed at the Johnson Space Center. This very low shear culture system randomizes gravitational vectors and provides an in vitro approximation of microgravity. In the randomized gravity of the rotating-wall vessel culture system, peripheral blood lymphocytes did not locomote through Type I collagen, whereas static cultures supported normal movement. Although cells remained viable during the entire culture period, peripheral blood lymphocytes transferred to unit gravity (static culture) after 6 h in the rotating-wall vessel culture system were slow to recover and locomote into collagen matrix. After 72 h in the rotating-wall vessel culture system and an additional 72 h in static culture, peripheral blood lymphocytes did not recover their ability to locomote. Loss of locomotory activity in rotating-wall vessel cultures appears to be related to changes in the activation state of the lymphocytes and the expression of adhesion molecules. Culture in the rotating-wall vessel system blunted the ability of peripheral blood lymphocytes to respond to polyclonal activation with phytohemagglutinin. Locomotory response remained intact when peripheral blood lymphocytes were activated by anti-CD3 antibody and interleukin-2 prior to introduction into the rotating-wall vessel culture system. Thus, in addition to the systemic stress factors that may affect immunity, isolated lymphocytes respond to gravitational changes by ceasing locomotion through model interstitium. These in vitro investigations suggest that microgravity induces non-stress-related changes in cell function that may be critical to immunity. Preliminary analysis of locomotion in true microgravity revealed a substantial inhibition of cellular movement in Type I collagen. Thus, the rotating-wall vessel culture system provides a model for analyzing the microgravity-induced inhibition of lymphocyte locomotion and the investigation of the mechanisms related to lymphocyte movement.     

Immune mechanism of rats on Nippostrongylus brasiliensis in vitro. II. The influence of lymphocytes and peritoneal cells

Nippostrongylus were collected from the intestines of rats 6 days p.i. and kept under sterile conditions in cultures. Serum, lymphocytes and peritoneal cells of immune or non-infected animals were added in various combinations to the culture media. The culture media were changed 2-3 times in an experimental period of 10 days, resp. serum and cells were added. The lymphocytes were isolated from the peripheral blood or from the mesenterial lymph nodes whereas the mononuclear cells were obtained from the peritoneal cavity. Serum and lymphocytes from the peripheral blood both from immune and non-infected rats, had no increased lethal effect on Nippostrongylus. The highest lethality rate of adults (65-68%) was achieved in cultures with peritoneal cells and lymphocytes from the lymph nodes of sensitized rats. Serum of infected or non-infected animals had no influence on adult Nippostrongylus in cultures with these cell combinations. In the controls without any cell-supplements the survival rate of the parasites was up to 88%.     

The quantity of associated acrocentric chromosomes in human lymphocytes passing through 1st, 2d and 3d mitoses in culture

The reproductive ability of lymphocytes of peripheral blood with the usage of 5-bromine-deoxyuridine has been studied in 8 healthy children at the age of 5-6 years. Single second mitoses occurred in 48 hour cultures (6.5%), in 72 hour cultures the frequency of the first, second and third mitoses was equal, in 96 hour cultures the third mitoses dominated. Consequent divisions of lymphocytes were accompanied by a decrease in associative acrocentric chromosome, in average by 25%, within one mitotic cycle, while in mitoses of a given ordinal number the frequency of associations did not depend on the duration of cultivation. The fixation of the culture at the 48th hour of cultivation makes it possible to take into account the frequency of associations of acrocentric chromosomes without calculation of the ordinal number of mitosis because of an significant amount of second mitoses at this time, and of a sufficient value of the mitotic index (4.6 +/- 0.5%) necessary for cytogenetic analysis.     

Detection of HLA-DRw (Ia-like) antigens on human T lymphocytes grown in tissue culture.

Human T lymphocytes that proliferate in the presence of conditioned medium from PHA-stimulated allogeneic peripheral blood cells were shown to express IPA antigens after the 8th culture day. Ia antigens as detected by xenogeneic antisera were present on 80 to 90% of the cultured cells which were also strongly reactive with xenogeneic antisera defining a human T cell antigen and formed E rosettes. Control cultures with PHA or no conditioned medium expressed T cell but not Ia antigens. These cultured T cells also express the same HLA-DRw determinants as the B cells of the donor they were derived from. Absorption of xenogeneic Ia, and HLA-DRw alloantisera with cultured T cells completely removed the reactivity of these sera for enriched peripheral blood B lymphocytes from normal donors.     

Characterization of feline whole-blood cultures and determination of the frequency of radiation-induced dicentrics in human and feline lymphocytes.

The Ham's F-10 and PHA culture system was applied to whole feline blood and cell-cycle characteristics such as DNA synthesis and mitotic indices were studied. The results are comparable to those obtained from human whole-blood cultures. The yields of dicentrics were also determined in lymphocytes from X-irradiated human and feline blood. The ratio between the experimental yields of dicentrics in human as compared to feline lymphocytes was 1:0.27.     

Transformation of human embryonic cells by the Rous sarcoma virus and the polyomavirus depending on the mitotic cycle phase]

The whole cycle of skin-muscle embryonic human tissue culture is 18 hours, with phases S, G1, G2 and M being 7, 6, 4 and 1 hour, respectively. The mitotic index of this culture is 28%. The maximum sensitivity of these synchronized cell cultures to transforming activity of the Rous and Sindai viruses was observed in phase S. The infection of synchronized primary embryonic human fibroblasts in phase S with the polyoma virus together with the Sindai virus has resulted in single cases of transformation. Similar results were obtained with non-synchronized human cultures.     

Chromosome damage by dothistromin in human peripheral blood lymphocyte cultures: a comparison with aflatoxin B1

The clastogenic potential of the pine tree fungal toxin dothistromin was studied by metaphase chromosome analysis of stimulated human peripheral blood lymphocytes exposed in vitro. The frequency of gaps, breaks, deletions and exchanges was scored in a series of cultures from 3 different donors. 50 cells were analysed for each dose level on coded slides. Testing was performed with and without added metabolic activation (as S9 mix) and aflatoxin B1 was used as a positive control in all experiments. Dothistromin caused a dose-dependent increase in the frequency of gaps and deletions which was not dependent on added metabolic activation. Even at high doses of dothistromin only a very small number of complex exchange-type aberrations were seen. This is in contrast to aflatoxin B1 where such aberrations were seen at low dose levels and especially in cultures to which S9 mix was added. High doses of dothistromin caused culture toxicity manifesting as haemolysis of the donor red blood cells and reduction of mitotic index. Culture toxicity occurred without a marked increase in aberration frequency. This toxicity may be masking any major potential for clastogenicity by dothistromin.