Ultraviolet irradiation transforms C3H10T1/2 cells to a unique, suppressible phenotype

Transformation of C3H10T1/2 cells by exposure to ultraviolet (UV) irradiation followed by tetradecanoyl phorbol acetate (TPA) has been used as a model of two-stage carcinogenesis. However, cells cloned from UV-TPA-induced foci (UV-TDTx cells) had a unique phenotype. Cloned UV-TDTx cells appeared transformed in pure culture but were unable to form foci when cocultured with C3H10T1/2 cells. However, in the presence of TPA, UV-TDTx cells form foci in mixed culture with C3H10T1/2 cells. This phenotype was the only one observed for UV-TPA transformants. These data suggest that communal suppression of cell division is a discrete phenomenon that must be overcome as one step in the multistage process of transformation, and this protocol permits the routine isolation of transformed cells responsive to density-dependent growth suppression.     

Immunologic inhibition of ultraviolet radiation-induced tumor suppressor cell activity

Long-term exposure of C3H mice to ultraviolet radiation resulted in the formation of suppressor T cells that recognize ultraviolet radiation-induced regressor skin cancers as a class before the appearance of overt tumors. Administration of monoclonal antibodies to the product of the I-Jk subregion of the major histocompatibility complex or low doses of cyclophosphamide in vivo inhibited the development or activity of these cells. This activity of the monoclonal antibody was eliminated by adsorption on B10.BR (I-Jk) but not B10.D2 (I-Jd) splenocytes. These findings provide evidence that elements expressing the I-J determinant are important in regulating the host response prior to the overt development of ultraviolet radiation-induced skin cancers and suggest novel therapeutic approaches to malignancies or other diseases involving suppressor T cells in their pathogenesis.     

Phagocytes as carcinogens: malignant transformation produced by human neutrophils

In a study of the relation between chronic inflammation and carcinogenesis, C3H mouse fibroblasts of the 10T 1/2 clone 8 line (10T 1/2 cells) were exposed to human neutrophils stimulated to synthesize reactive oxygen intermediates or to a cell-free enzymatic system generating superoxide (xanthine oxidase plus hypoxanthine). After exposure, the 10T 1/2 cells were either placed in tissue culture or immediately injected into athymic nude mice. Both malignant and benign tumors developed in the mice injected with treated cells, but not in those injected with control cells; in one instance cells grown from one of the benign tumors subsequently developed a malignant phenotype. Malignant transformation was also observed in treated cells in the experiments in vitro.     

Epidermal-growth-factor-dependent transformation by a human EGF receptor proto-oncogene

The epidermal growth factor (EGF) receptor gene EGFR has been placed in a retrovirus vector to examine the growth properties of cells that experimentally overproduce a full-length EGF receptor. NIH 3T3 cells transfected with the viral DNA or infected with the corresponding rescued retrovirus developed a fully transformed phenotype in vitro that required both functional EGFR expression and the presence of EGF in the growth medium. Cells expressing 4 x 10(5) EGF receptors formed tumors in nude mice, while control cells did not. Therefore, the EGFR retrovirus, which had a titer on NIH 3T3 cells that was greater than 10(7) focus-forming units per milliliter, can efficiently transfer and express this gene, and increased numbers of EGF receptors can contribute to the transformed phenotype.     

Trxs过量表达对UV-B胁迫的大麦幼苗叶片抗氧化酶活性的影响

以过量表达硫氧还蛋白s基因(Trxs)的转基因大麦(LSY-11-1-1,T)及其非转基因对照(LSY,CK)为材料,采用水培法研究了UV-B胁迫条件下转基因大麦幼苗(UVB-T)与对照(UVB-CK)叶片中Fv/Fm值、H2O2与MDA含量以及抗氧化酶活性的变化.结果表明,UV-B处理引起大麦幼苗叶片Fv/Fm值降低,H2O2和MDA含量升高,过氧化物歧化酶(SOD)、过氧化物酶(POD)、谷胱甘肽过氧化物酶(GSH-PX)与过氧化氢酶(CAT)等抗氧化酶活性出现不同程度的增加;UVB-T的Fv/Fm值极显著高于UVB-CK,而H2O2和MDA含量均极显著低于UVB-CK,同时,其上述抗氧化酶活性普遍高于UVB-CK,而且UVB-T的CAT活性变化趋势与UVB-CK明显不同,其CAT出现2次活性高峰,而UVB-CK仅出现1次.     

Role of the guanosine triphosphatase Rac2 in T helper 1 cell differentiation

T helper 1 (TH1) cells mediate cellular immunity, whereas TH2 cells potentiate antiparasite and humoral immunity. We used a complementary DNA subtraction method, representational display analysis, to show that the small guanosine triphosphatase Rac2 is expressed selectively in murine TH1 cells. Rac induces the interferon-gamma (IFN-gamma) promoter through cooperative activation of the nuclear factor kappa B and p38 mitogen-activated protein kinase pathways. Tetracycline-regulated transgenic mice expressing constitutively active Rac2 in T cells exhibited enhanced IFN-gamma production. Dominant-negative Rac inhibited IFN-gamma production in murine T cells. Moreover, T cells from Rac2-/- mice showed decreased IFN-gamma production under TH1 conditions in vitro. Thus, Rac2 activates TH1-specific signaling and IFN-gamma gene expression.     

New method for detecting cellular transforming genes

Tumor induction in athymic nude mice can be used to detect dominant transforming genes in cellular DNA. Mouse NIH 3T3 cells freshly transfected with either cloned Moloney sarcoma proviral DNA or cellular DNA's derived from virally transformed cells induced tumors when injected into athymic nu/nu mice. Tumors were also induced by cells transfected with DNA from two tumor-derived and one chemically transformed human cell lines. The mouse tumors induced by human cell line DNA's contained human DNA sequences, and DNA derived from these tumors was capable of inducing both tumors and foci on subsequent transfection. Tumor induction in nude mice represents a useful new method for the detection and selection of cells transformed by cellular oncogenes.     

环形泰勒虫感染细胞抑制性消减文库的构建及ESTs分析

【目的】环形泰勒虫（Theileria annulata）可以感染牛B淋巴细胞、树突状细胞和巨噬细胞,并可使被感染细胞发生转化,体外培养时可获得类似肿瘤样细胞的无限增殖能力。本研究通过构建环形泰勒虫转化和非转化宿主细胞抑制性消减文库,筛选环形泰勒虫裂殖体基因及转化细胞与宿主正常细胞之间的差异表达基因。【方法】以环形泰勒虫裂殖体转化细胞和非感染牛外周血单个核细胞（即非转化宿主细胞）为试验材料,提取总RNA和mRNA,反转录合成cDNA;然后分别作为tester和driver,进行抑制性消减杂交（SSH）,构建环形泰勒虫转化和非转化宿主细胞抑制性消减文库;对随机挑取的阳性克隆进行测序及生物信息学分析,并利用实时荧光定量PCR对文库部分差异基因进行验证。【结果】通过对文库454个阳性克隆进行测序,共获得了有效表达序列标签（ESTs）364条,大小主要分布在350-1 200 bp之间;将这些ESTs序列在GenBank中进行BLASTn网上序列比对,其匹配于192条基因序列;其中包括环形泰勒虫序列60条(11条为虫体蛋白酶基因序列、10条为膜蛋白基因序列、7条为假设蛋白基因序列、5条为凋亡相关虫体蛋白基因序列、5条为核糖体蛋白基因序列、4条为细胞周期蛋白基因序列、4条为虫体抗原基因序列和虫体其它基因序列14条)、牛基因序列131条（其中19条为肿瘤相关蛋白基因序列）和1条未知序列;其中已有功能注释的ESTs 285条,再将已知功能的ESTs通过WEGO软件从生物学过程（biological process）、细胞组成（cellular component）和分子功能（molecular function）3个部分进行GO聚类分析,结果表明,宿主细胞差异基因主要集中在细胞、细胞进程、结合、催化、代谢进程、生物调节等功能方面。另外,通过对部分环形泰勒虫裂殖体转化宿主细胞差异基因进行荧光定量PCR分析,结果表明,肿瘤相关基因HCLS1和SENP5在转化宿主细胞中转录水平分别是非感染牛PBMCs(非转化宿主细胞)的2.06和1.32倍。【结论】利用SSH技术,成功构建了环形泰勒虫感染宿主细胞的抑制性消减文库,筛选获得了转化宿主细胞和环形泰勒虫裂殖体的表达基因,并通过荧光定量PCR分析获得了2个可能参与宿主细胞转化的基因,为进步探索环形泰勒虫与宿主细胞相互作用的分子机制奠定了基础。     

伊氏锥虫细胞工程苗的研究

试验采用体外培养技术,通过接受γ-射线和紫外线辐照的影响,定向选育突变虫株。首次对伊氏锥虫的细胞工程苗进行了研究。试验结果表明受γ-射线或紫外线辐照的虫体接种小白鼠后,潜伏期和病程与对照组相比差异极显著(P<0.01)。辐照虫体体外培养后接种小白鼠,潜隐期和病程均与对照组相似。经紫外线处理后辐照的伊氏锥虫转入用小鼠腹腔巨噬细胞作支持细胞的培养基内培养,培养7～10d使突变表型表达。然后加入嘌呤类似物继续培养2周,筛选出基因突变株,建立细胞系。小白鼠试验测得该细胞系LD_(50)为10~(-3.5)·mL~(-1),比强毒株毒力减少60倍,其免疫保护率达90%。     

Ectopic expression of the serotonin 1c receptor and the triggering of malignant transformation

Neurotransmitter receptors are usually restricted to neuronal cells, but the signaling pathways activated by these receptors are widely distributed in both neural and non-neural cells. The functional consequences of activating a brain-specific neurotransmitter receptor, the serotonin 5HT1c receptor, in the unnatural environment of a fibroblast were examined. Introduction of functional 5HT1c receptors into NIH 3T3 cells results, at high frequency, in the generation of transformed foci. Moreover, the generation and maintenance of transformed foci requires continued activation of the serotonin receptor. In addition, the injection of cells derived from transformed foci into nude mice results in the generation of tumors. The serotonin 5HT1c receptor therefore functions as a protooncogene when expressed in NIH 3T3 fibroblasts.     

氢化可的松对小鼠T淋巴细胞增殖的抑制作用

[目的]探讨氢化可的松对小鼠T淋巴细胞增殖功能的影响。[方法]用淋巴细胞分离液无菌制备小鼠脾淋巴细胞,于RPMI1640的完全培养基中加入ConA后进行转化。用流式细胞仪检测T细胞活化分子CD25和CD69及其共表达率。将转化的淋巴细胞分成不加氢化可的松的对照组和加入不同浓度的氢化可的松组,在各时间点取样计数,绘制其生长曲线。[结果]经ConA刺激3 d的T细胞上CD25和CD69分子及其共表达率最高,分别达73.5%、58%和32%。生长曲线显示,各组细胞在加氢化可的松后第2 d达到对数生长期,10-5、10-6、10-7mol/L浓度的氢化可的松对活化小鼠T淋巴细胞增殖都有抑制作用,但各浓度组间无显著差异（P〉0.05）,而与对照组相比有显著差异（P〈0.05）。[结论]在一定的浓度范围内氢化可的松对小鼠T淋巴细胞的增殖具有抑制作用,这为细胞移植免疫耐受的诱导提供了试验依据。     

Suppressor T lymphocytes control the development of primary skin cancers in ultraviolet-irradiated mice

Exposure of mice to ultraviolet radiation results in the development of suppressor T lymphocytes in lymphoid organs, followed by the appearance of primary skin cancers. The presence or absence of these suppressor lymphocytes determines whether or not primary cancers will develop in the ultraviolet-irradiated skin. This demonstrates the importance of immunological regulatory pathways in carcinogenesis and provides an example of immunological surveillance.     

环境UV-B辐射增强对叶用杜仲林叶片光合作用与活性物质含量的影响

[目的]探讨环境紫外(UV-B)辐射对叶用杜仲叶片光合作用和活性物质含量及膜脂过氧化的影响,为叶用杜仲林的生态培育提供理论依据。[方法]在不同紫外(UV-B)辐射强度处理[CK:+0;T1:+1.40 kJ/(m~2·d);T2:+2.81 kJ/(m~2·d)]下,以5年生杜仲为研究对象,采用单因素方差分析与相关分析方法研究净光合速率、光合色素、次生代谢产物及自由基含量差异及相关性。[结果]UV-B辐射强度增加显著降低杜仲叶片光合参数净光合速率(Pn)、气孔导度(Gs)、胞间CO_2浓度(Ci)、蒸腾速率(Tr)和叶绿素a/b,显著提高了叶绿素a、叶绿素b、类胡萝卜素和总叶绿素含量,T1处理的叶绿素含量最高[(1.15±0.049)mg/g],T2处理的类胡萝卜含量最高[(0.036 0±0.001 6)mg/g];UV-B辐射强度增加显著降低杜仲叶片部分次生代谢产物桃叶珊瑚苷、京尼平苷酸、京尼平苷和绿原酸含量,且最大变化值之间分别相差20.74、61.23、16.16和13.63倍,同时也显著提高了部分次生代谢产物京尼平、黄酮、总酚和单宁含量,且T1处理的京尼平、黄酮、总酚和单宁含量分别是CK处理的1.21、1.11、1.34和1.37倍,总酚与单宁含量以T2处理最高,分别为(1.290 0±0.023 0)和(17.30±1.44)μg/g;UV-B辐射增强显著增加了MDA、H_2O_2和O_2·~-含量,且T1处理的MDA含量与O_2·~-含量较CK处理组分别高39.46%、70.48%;T2处理的H_2O_2含量较CK处理组高43.51%。[结论]UV-B辐射显著影响杜仲叶片光合参数及绿原酸、桃叶珊瑚苷、京尼平苷和京尼平苷酸等主要活性物质含量,因此可以通过调节环境UV-B辐射强度以提高叶用杜仲林的经济效益。     

猪链球菌2型灭活疫苗对小鼠的免疫效果评价

在前期筛选出2株毒力强、抗原性好、遗传稳定的猪链球菌2型(SS2)疫苗菌株(HF2、HF3株)并分别制成灭活疫苗(采用ISA 201 VG矿物油佐剂)的基础上,进一步与SS2商品化灭活疫苗(HA9801株,铝胶佐剂)同步免疫小鼠,利用间接ELISA、流式细胞术、免疫攻毒试验、细菌定植试验和病理组织学观察等方法测定小鼠血清中IgG抗体效价,细胞因子含量(IL-4、IL-10、IFN-γ、TNF-β、MCP-1),外周血中CD4⁺/CD3⁺、CD8⁺/CD3⁺ T细胞亚群含量,攻毒保护率,组织荷菌数和病理组织变化等指标。结果显示,二免7 d后,HF2、HF3灭活疫苗组和HA9801商品化灭活疫苗组的血清IgG抗体效价分别为1:25 600、1:12 800、1:25 600;HF2灭活疫苗组的IL-4、IL-10含量显著高于HF3灭活疫苗组和HA9801商品化灭活疫苗组,IFN-γ、TNF-β含量显著高于HF3灭活疫苗组,但MCP-1含量显著低于HF3灭活疫苗组;HF2和HF3灭活疫苗组的CD4⁺/CD3⁺ T细胞比率均显著低于HA9801商品化灭活疫苗组,但CD8⁺/CD3⁺ T细胞比率差异不显著;HF2、HF3灭活疫苗和HA9801商品化灭活疫苗对小鼠的攻毒保护率均为100%;HF2灭活疫苗组小鼠的肺、脾脏组织荷菌数显著低于HF3灭活疫苗组;HF2灭活疫苗组小鼠的肺、肾、脾脏病理变化较HF3灭活疫苗组和HA9801商品化灭活疫苗组轻微。综合结果表明,由SS2(HF2株)制备的ISA 201 VG佐剂灭活疫苗免疫小鼠后不仅可产生较强的免疫应答,还可完全抵抗强毒株的攻击。     

The ras oncogenes increase the intrinsic resistance of NIH 3T3 cells to ionizing radiation

Identification of genes that function to protect cells from radiation damage is an essential step in understanding the molecular mechanisms by which mammalian cells cope with ionizing radiation. The intrinsic radiation resistance (D0) of NIH 3T3 cells was markedly and significantly increased by transformation with ras oncogenes activated by missense mutations. This radiobiologic activity appeared to be a specific consequence of the ras mutations rather than of transformation, since revertant cells that contained functional ras genes (but were no longer phenotypically transformed) retained their increased D0's.     

肠出血性大肠杆菌O157∶H7 toxB基因缺失株的构建

为了明确肠出血性大肠杆菌(Enterohemorrhagic Escherichia coli)O157∶H7大质粒pO157编码的蛋白质ToxB与O157∶H7在肠道黏附、定殖的关系,构建含有toxB基因同源臂的重组自杀性质粒pMEG375-BN-Kan-BC,转化SM10宿主菌获得重组SM10(pMEG375-BN-Kan-BC)。将SM10(pMEG375-BN-Kan-BC)与O157∶H7进行混合培养,通过同源重组,获得杂交toxB基因缺失株。用体外接种的HEp-2细胞和体内感染链霉素处理的小鼠,明确缺失株黏附定殖能力。经PCR和Western blot鉴定,toxB基因被卡那霉素(Kan+)选择标记基因表达盒所代替。O157∶H7(△toxB)生长特性与亲本株相比无明显差异。O157∶H7(△toxB)与亲本株相比,对HEp-2细胞的黏附能力降低了2倍。口服感染小鼠后,O157∶H7(△toxB)第3 d粪便排菌量仅为3.1×105 CFU,持续排菌11 d;而亲本株排菌量高达4.03×107 CFU,持续排菌时间超过14 d。     

不同剂量的紫外照射结合热处理对龙眼保鲜效果的研究

以龙眼品种‘石硖’为试材,研究不同剂量的紫外照射结合热处理对龙眼果实采后的保鲜效果。在10 min55℃热处理的基础上,以紫外杀菌灯为辐射源,对果实进行辐射剂量分别为24、30、42 J/m2紫外照射。置于常温25℃下贮藏。发现55℃热处理10 min结合30 J/m2紫外照射处理能延缓果实失重、可溶性固形物和可溶性蛋白含量的降低,减缓维生素C的消耗,抑制果实丙二醛(MDA)含量和多酚氧化酶(PPO)活性的积累,且在贮藏期内该处理果实的超氧化物歧化酶(SOD)活性均明显较高。结果表明,55℃热处理10 min结合30 J/m2紫外照射处理可以有效延缓龙眼果实的采后衰老进程,保持果实品质。     

Sporozoite vaccine induces genetically restricted T cell elimination of malaria from hepatocytes

The target of the CD8+ T cell-dependent immunity that protects mice immunized with irradiation-attenuated malaria sporozoites has not been established. Immune BALB/c mice were shown to develop malaria-specific, CD8+ T cell-dependent inflammatory infiltrates in their livers after challenge with Plasmodium berghei sporozoites. Spleen cells from immune BALB/c and C57BL/6 mice eliminated hepatocytes infected with the liver stage of P. berghei in vitro. The activity against infected hepatocytes is not inhibited by antibodies to interferon-gamma and is not present in culture supernatants. It is genetically restricted, an indication that malaria antigens on the hepatocyte surface are recognized by immune T effector cells. Subunit vaccine development will require identification of the antigens recognized by these T cells and a method of immunization that induces such immunity.     

Contact-dependent demyelination by Mycobacterium leprae in the absence of immune cells

Demyelination results in severe disability in many neurodegenerative diseases and nervous system infections, and it is typically mediated by inflammatory responses. Mycobacterium leprae, the causative organism of leprosy, induced rapid demyelination by a contact-dependent mechanism in the absence of immune cells in an in vitro nerve tissue culture model and in Rag1-knockout (Rag1-/-) mice, which lack mature B and T lymphocytes. Myelinated Schwann cells were resistant to M. leprae invasion but undergo demyelination upon bacterial attachment, whereas nonmyelinated Schwann cells harbor intracellular M. leprae in large numbers. During M. leprae-induced demyelination, Schwann cells proliferate significantly both in vitro and in vivo and generate a more nonmyelinated phenotype, thereby securing the intracellular niche for M. leprae.     

UV-B辐射对葡萄叶片光合色素和类黄酮含量的影响

[目的]研究UV-B辐射对葡萄叶片光合色素和类黄酮含量的影响,以期为研究UV-B辐射增强对葡萄光合作用的影响提供依据。[方法]试验在室外条件下,以酿酒葡萄＂赤霞珠＂（Cabernet Sauvignon）为材料,通过增加不同强度的UV-B辐射,测量葡萄叶片光合色素和类黄酮含量的影响。[结果]低辐射（T1）处理下,叶绿素a、叶绿素b、总叶绿素、类胡萝卜素含量平均值分别比对照增加了5%、2%、4%、3%;高辐射（T2）处理下,叶绿素a、叶绿素b、总叶绿素、类胡萝卜素含量分别比对照增加了11%、9%、10%、7%,其中,叶绿素a差异达显著水平。类黄酮的含量也随着UV-B辐射天数延长而显著增加,T1、T2平均值分别比对照增加了13%、9%。[结论]葡萄对UV-B辐射增强具有较强的适应性,一定范围内适当增加UV-B辐射可能不会对葡萄叶片的光合作用产生不利的影响。