哺乳动物细胞对丝裂霉素诱导的DNA链间交联的修复

目的:探讨哺乳动物细胞能否修复DNA链间交联(interstrand crosslink,ICL).方法:构建带有丝裂霉素(mitomycin C,MMC)交联的质粒pCMV-Luc/ICL,将该交联质粒转染无修复酶缺陷和有修复酶缺陷的哺乳动物细胞系,观察细胞对MMC链间交联的修复能力.结果:(1)无酶缺陷的哺乳动物细胞对DNA的修复能力很强,即使缺乏同源序列,也能有效地去除DNA链间交联;(2)修复过程需要许多酶类参与,其中与核苷酸切除修复(nucleotide excision repair,NER)相关的酶类起着关键性作用,提示NER参与MMC链间交联的修复过程;(3)序列分析表明,非同源性重组修复为易错修复(error-prone repair).结论:哺乳动物细胞能有效地修复MMC诱导的DNA链间交联,核苷酸切除修复是重要的修复途径.     

冬凌草甲素对哺乳动物细胞DNA损伤作用的影响

目的　探讨冬凌草甲素对哺乳动物细胞DNA损伤的抑制作用。方法　用小鼠骨髓嗜多染红细胞(PCE)微核试验和大白鼠肝原代细胞非程序DNA合成(unscheduledDNAsynthesis ,UDS)实验检测冬凌草甲素对哺乳动物细胞DNA损伤的影响。结果　冬凌草甲素可明显地抑制环磷酰胺(CP)诱导的小鼠骨髓PCE微核发生率(P <0 .0 1)及盐酸氧芥(NH2 ·HCl)诱导的小鼠肝原代细胞UDS(P<0 .0 1)。结论　冬凌草甲素具有明显的抑制哺乳动物细胞DNA损伤的作用。     

载体介导的哺乳动物细胞RNA干扰技术与基因治疗

RNA干扰(RNA interference,RNAi)是生物在进化过程中,抵御病毒感染及由重复序列和突变引起基因组不稳定性的保护机制.RNAi是由双链RNA引发的靶基因mRNA降解而导致基因转录后沉默的现象,研究者利用这一现象对要研究的基因进行抑制,从而形成了RNAi技术.RNAi具有特异高效的特点,因此该技术已在研究基因功能和进行基因治疗中得到广泛应用.RNAi的实现途径有多种,每种方式都各有优缺点.     

Ubiquitous Presence in Mammalian Cells of Enzymatic Activity Specifically Cleaving 8-Hydroxyguanine-containing DNA

Here we report the finding of enzymatic activity that specifically cleaves DNA containing 8-hydroxyguanine (oh⁸Gua) residues in various mammalian cells. To detect this activity, we used a synthetic double-stranded DNA containing a single oh⁸Gua at a defined position as the substrate, and analyzed the products of enzymatic digestion by polyacrylamide gel electrophoresis. Two cleavage sites near the oh⁸Gua residue were detected with partially purified fractions from cow brain and rat liver, and also with preparations from all mammalian tissues examined. These results suggest that enzymatic activity for the removal of oh⁸Gua from DNA is widely distributed in mammalian cells.     

抗人CD3人/鼠嵌合抗体基因的构建

为了降低鼠源抗人CD3单抗的免疫原性．增加其在人体内的生物活性及治疗作用，使该抗体能更广泛更有效地长期多次用于人体治疗肿瘤、器官移植排斥反应及自身免疫性疾病。本文采用PCR技术从分泌抗CD3单抗的杂交瘤细胞HIT3a的mRNA中分离克隆了抗体的轻重链可变区基因的cDNA。以此轻重链可变区cDNA为特异探针从HIT3a基因文库中分离带有调控序列的功能性轻重链可变区基因，并将其插入到含有人k轻链及人71重链恒定区基因的哺乳动物表达载体中成功地构建了抗人CD3人／鼠轻重链嵌合抗体基因，为研制人抗CD3入／鼠嵌合抗体完成了关键性的第一步。     

Cancer Stem Cells and Response to Therapy

The cancer stem cell (CSC) model states that cancers are organized in cellular hierarchies, which explains the functional heterogeneity often seen in tumors. Like normal tissue stem cells, CSCs are capable of self-renewal,either by symmetric or asymmetric cell division, and have the exclusive ability to reproduce malignant tumors indefinitely. Current systemic cancer therapies frequently fail to eliminate advanced tumors, which may be dueto their inability to effectively target CSC populations. It has been shown that embryonic pathways such as Wnt, Hedgehog, and Notch control self-renewal and cell fate decisions of stem cells and progenitor cells. These are evolutionary conserved pathways, involved in CSC maintenance. Targeting these pathways may be effective in eradicating CSCs and preventing chemotherapy or radiotherapy resistance.     

A Variant Schmidt-Ruppin Strain of Rous Sarcoma Virus with Increased Affinity for Mammalian Cells

SR-RSV-D(H), a variant virus with extremely high tropism for mammalian cells, was isolated by passage of the Schmidt-Ruppin strain of Rous sarcoma virus of subgroup D (SR-RSV-D) through hamster cells. This variant virus has acquired an altered envelope glycoprotein, encoded by the env gene, that has high affinity for receptors on the surface of mammalian cells. The variant virus transforms rat cells at about 100 times the efficiency of the parental virus, SR-RSV-D(S), as assayed by focus formation. Addition of amphotericin B (Fungizone) to the medium at a concentration of 0.2 μg/ml completely inhibited rat cell transformation by SR-RSV-D(H), possibly by blocking virus penetration into the cells, whereas the drug showed no inhibitory effect on transformation of chick embryo flbroblast (CEF) cells by the variant virus or on transformation of rat cells by the parental virus. The efficiency of transformation of rat cells by the variant virus was much less than its efficiency of transformation of CEF cells. Analysis of infection of rat cells suggested that the virus can infect rat cells as efficiently as CEF cells but that rat cells were not transformed by the virus as fully as CEF cells because of inefficiency of some post-penetrational step involved in viral gene expression. The finding that El AY cells, rat cells expressing adenovirus El A gene, were transformed by SR-RSV-D(H) as efficiently as CEF cells supports this conclusion and suggests that expression of the E1A gene in rat cells may overcome the defect in the transforming step(s) in rat cells.     

树突状细胞体外诱导抗肿瘤免疫

目的以树突状细胞（DC）在体外诱导抗肝癌免疫。方法自肝癌患者外周血中分离DC;以粒／巨噬细胞集落刺激因子（GM－CSF）及白介素-4（IL－4）联合刺激DC;以人肝癌细胞系HepG2细胞的肿瘤相关抗原（TAA）激活DC;DC诱导自体T淋巴细胞增殖、分化为细胞毒性T细胞（CTL）;检测CTL及其上清液对HepG2细胞、BEL－7402细胞、LOVO细胞及HOS－8603细胞的细胞毒作用。结果经人肝癌细胞系HePG2细胞的TAA激活并经GM－CSF及IL－4联合刺激后,肝癌患者外周血DC能够诱导自体T淋巴细胞增殖分化为CTL,该CTL及其上清液对HePG2细胞均有高效杀伤作用（杀伤率分别为92％±10％和41％±8％）,对BEL－7402细胞亦有较强的杀伤力（杀伤率分别为56％±10％和31％±9％）,对SGC－7901细胞、LOVO细胞及HOS－8603细胞则无明显的细胞毒作用。结论肝癌患者外周血DC体外能够诱导高效而特异抗肝癌免疫。提示DC可能在肿瘤治疗及预防其复发和转移中发挥重要作用。     

Leukemia Propagating Cells Rebuild an Evolving Niche in Response to Therapy

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Radiobiologic Response of Cho-Ki Cells Treated with Vitamin A

Treatment of CHO-KI cells with vitamin A altered their response to subsequent gamma irradiation. In general longterm preincubation with low doses of the vitamin caused a relative increase in the number of cells surviving a given radiation dose. The effect resulted in an increase in the D₀ of the survival curve. Long or short term exposure to high concentrations of the vitamin caused a decrease in the number of surviving cells leading to a decrease in the extrapolation number of the survival curve. Recovery of cells from radiation damage, assessed using the split dose technique, was also impaired by vitamin A pretreatment. A mechanism involving repair of potentially lethal damage may explain the protective effect of vitamin A since this was highly dependent on the cell density of cultures at the time of irradiation. However, in view of the data showing that the vitamin A concentrations necessary to alter the radiation survival curve shoulder caused a significant release of sialic acid into the medium, a mechanism involving membrane stability may account for both the reduction in repair/recovery capacity of the treated cells and the radioprotective effect.     

Overexpression of Proliferating Cell Nuclear Antigen in Mammalian Cells Negates Growth Arrest by Serum Starvation and Cell Contact

Proliferating cell nuclear antigen (PCNA) functions as a processivity factor for DNA polymerase δ, and is expressed at high levels in growing normal and tumor cells. To clarify the relationship between cell proliferation and PCNA expression, we generated NIH-3T3 cells that overexpress PCNA and analyzed the phenotype of these cells. The resulting 3T3-PCNA cells, which overexpressed PCNA, were found to proliferate beyond the saturation density of the parental NIH-3T3 cells. Although NIH-3T3 cell proliferation is arrested under serum starvation conditions, 3T3-PCNA cell proliferation is not arrested by serum starvation. The expression levels of cdk2, cdk4 and cdk6 were the same in 3T3-PCNA and NIH-3T3 cells. The activity of cdk4 was identical for both cell types. However, the activity of cdk2 was higher in serum-starved 3T3-PCNA cells than in NIH-3T3 cells, although the expression of cyclin E decreased in both types of cells, suggesting that increases in cdk2 activity are related to negation of growth arrest in 3T3-PCNA cells. These results indicate that increases in PCNA expression lead to the disruption of growth control and may lead to malignant transformation.