滋养细胞肿瘤PP_（11）、PP_（19）、PP_（25）和PP_（26）的免疫

通过免疫组织化学方法研究了４种胎盘蛋白（ＰＰ１１、ＰＰ１９ＰＰ２５和ＰＰ２６）在滋养细胞肿瘤中的表达情况。发现ＰＰ１１在恶性葡萄胎中的表达明显低于葡萄胎（Ｐ＜０．０５），ＰＰ２５和ＰＰ２６在绒癌中的表达低于葡萄胎（Ｐ＜０．０５），ＰＰ１９在各种滋养细胞肿瘤中皆有强表达，提示联合检测４种胎盘蛋白有助于滋养细胞肿瘤间的鉴别诊断。ＰＰ１９还是中间型滋养细胞肿瘤的优良标志物。     

体视学应用（生物医学）培训班（第一轮通知）

各有关单位及专家学者： 医学研究的主要方向之一就是对各种生物结构进行形态学研究,尤其是定量形态学研究。目前国内广泛应用的传统定量方法的主要缺陷是缺乏随机抽样,运用二维定量技术及只获得密度信息。现代体视学方法是一系列基于二维切片或投影图像的全面观察而获得所观察结构三维定量信息的精确手段。     

GENOTOXIC EFFECT OF CIGARETTE SMOKE CONDENSATE (CSC) ON HUMAN DIPLOID CELL 2BS STRAIN

A series of bioassays such as sister chromatid exchange frequencies ( SCE.), chromosomal aberration ( CA ), micronuclel rate (MN) and cell-cycle delay have been used to detecting the genotoxic effect of cigarette smoke condensate (CSC) on human diploid cell 2BS strain. The results suggested that a higher SCE, ( 17. 0/ cell) was observed In 2BS cells treated with CSC at 100 μg/ml, as compared with 6. 9/cell of the background (P<0. 001). CA rate was significantly increased from 4% to 36% In cells treated with 10 μg/ml CSC (P< 0.001). MN rate varied from 9 -26‰ In cells treated with CSC compared to that of control (6‰). Meanwhile, the cell-cycle of cells was markedly delayed by CSC. The survival rate of 2BS cells declined to 59. 6% for treatment with CSC at 200 μg/ ml. There was a dose-effect response In SCE., CA, MN rate. We proposed that active oxygen might responsible for genotoxiclty of CSC on cells.     

Detection of direct mutagenicity of cigarette smoke condensate in mammalian cells

Mutagenicity of cigarette smoke condensate (CSC) and the acidic, basic and neutral fractions of CSC was examined in the AL hybrid cell, a Chinese hamster ovary cell containing one human chromosome 11. Since the human chromosome 11 is not necessary for survival of the AL cells, mutations involving large deletions and chromosomal loss by non-dysjunction are non-lethal events that are detectable by loss of human cell surface antigens (a1, a2 and a3) encoded by genes on chromosome 11p (a1 and a3) and 11q (a2) through an antibody-complement lysis assay. Exposure of AL cells to CSC without exogenous metabolic activation caused a dose-dependent cytotoxicity and mutagenicity. Mutagenicity also increased with time of incubation up to 3 h with a maximum of 300 a1- mutants/10(5) survivors (250% above background; P less than 0.0005) after incubation with 100 micrograms/ml CSC. Cytotoxicity and mutagenicity of CSC were inversely proportional to cell density. Fifty percent lethal doses for the acidic, basic and neutral fractions of CSC after 3 h of incubation were 30, 100 and 240 micrograms/ml respectively, and the acidic fraction at a concentration of 25 micrograms/ml induced 350 a1- mutants/10(5) survivors (230% above background; P less than 0.0005); the basic and neutral fractions were less mutagenic. These results indicate that CSC and fractions of CSC can directly produce a spectrum of mutations, through both deletional and non-dysjunctional mechanisms of a kind known to lead to inactivation of tumor suppressor genes.     

Effect of cigarette smoke condensate on unscheduled DNA synthesis (UDS) in human lung fibroblast cells

The effect of cigarette smoke condensate (CSC) on UDS in human lung fibroblast cells (2BS) was studied. The results showed that CSC induced a marked increase in UDS of 2BS cells in a dose-dependent manner. 3H/14C ratio was 1.77 +/- 0.34 at a dose of 125 micrograms/ml of CSC as compared with 1.33 +/- 0.04 of the control. Catalase, at doses of 40, 60 units/ml, could significantly inhibit UDS induced by CSC (75 micrograms/ml), but after inactivation catalase had no effect. It is suggested that the increase in UDS is due to free radicals generated from CSC.     

Inducing effect of hematoporphyrin derivative (HpD) on cell sister chromatid exchanges (SCE) in vitro

The mutagenic effect of HpD on cell SCE and the reactions of cell SCE to different sources of light combined with HpD were studied using V79 cells. There were 6 doses of HpD: 1 microgram/ml, 3 micrograms/ml, 5 micrograms/ml, 10 micrograms/ml, 50 micrograms/ml and 100 micrograms/ml. The dose of 5 micrograms/ml is equal to the maximum dose of HpD used in the clinic (HpD per milliliter of patient's blood). Our experiments demonstrated that when the cells were cultured in the dark and HpD was added to the medium no more than 5 micrograms/ml, the SCE frequencies were not increased. The cells were irradiated with different sources of light without HpD, both the fluorescence and ultraviolet light could promote SCE but the light of daylight lamp and red light did not increase it. But when HpD was added into culture medium at the dose of less than 5 micrograms/ml, every light could increase the cell SCE intensively except the daylight lamp light. The red light was more notable than the others by relation analysis.     

Effect of beta-carotene on sister chromatid exchanges induced by MNNG and aflatoxin B1 in V79 cells

In order to elucidate the mechanism of tumor prevention of beta-carotene, its effect on sister chromatid exchanges (SCE) induced by MNNG in cultured V79 cells, under condition free of the enzyme system to convert beta-carotene into vitamin A, was studied. It was found that SCE level was significantly increased by high doses beta-carotene (10(-5)-10(-4) M) and the enhancement of SCE was restored to its original level by addition of alpha-tocopherol (final concentration 2 micrograms/ml). This may be due to the latter inhibiting the oxidation of beta-carotene and reducing the amount of oxidated carotene, which is toxic for cultured cells. Combination of beta-carotene and alpha-tocopherol at low doses inhibited SCE induced by MNNG (P less than 0.05) but no protective activity was observed when used separately. It was also found that beta-carotene (2 x 10(-7) M) and retinol (16 micrograms/ml) inhibited SCE induced by aflatoxin B1, which is activated by S-9 mixture. The present data clearly show that the antitumor activity of beta-carotene may be attributed to both itself and its degraded compound vitamin A, and may take part in the initiation of carcinogenesis. Combination of beta-carotene and other cancer preventive drugs is more effective and safer than it used individually.     

免疫磁珠分选与顺铂富集喉癌Hep-2细胞系中肿瘤干细胞的比较

目的比较免疫磁珠分选（MACS）及化疗药物顺铂（DDP）筛选富集喉癌Hep-2细胞系肿瘤干细胞的效果。方法以CD133MACS、DDP作用两种方法来富集喉癌Hep-2细胞系中的肿瘤干细胞,并以流式细胞术（FCM）检测处理后CD133^＋细胞的百分率。同时观察细胞形态学的改变,判断两种方法分选后细胞对后续实验的影响。结果经FCM检测,MACS分选喉癌Hep-2,CD133^＋细胞得率为64．33％,不同浓度DDP作用于喉癌Hep-2细胞48h,FCM检测CD133^＋细胞有不同的得率,其中质量浓度为4μg／ml时,所得CD133^＋≥细胞百分率最高,为50．7％。MACS与DDP各组比较差异均有统计学意义（P〈0．01）;两种方法处理后MACS组细胞的存活状态要好于DDP组。结论MACS分选纯度较高,对细胞损伤小,适合后续培养;但分选前耗时长,每次分选仅能用于一种Marker。DDP筛选简单易行,符合临床肿瘤干细胞（CSC）抵抗化疗的模式。但阳性细胞得率与细胞毒性成正比。MACS和DDP筛选肿瘤干细胞各有其优势及适用范围,实验中可根据不同目的来确定所用筛选方法。     

Sister chromatid exchange induction by diaziquone in human and mouse lymphocytes following both in vivo and in vitro exposures

Diaziquone (AZQ) (NSC 182986), a lipid-soluble benzoquinone derivative currently being tested as an experimental chemotherapeutic agent, was used to treat mouse and human peripheral blood lymphocytes (PBLs) to determine its genotoxic potential by examination of sister chromatid exchange (SCE) induction. In vitro exposure to AZQ caused a linear increase in SCE in both mouse and human PBLs, with mouse PBLs being about twice as sensitive as the human cells. The lowest in vitro concentration found to induce a significant effect on SCE frequency was 0.3 micrograms/ml in mice and 1.0 micrograms/ml in human PBLs. Mice exposed by either i.p. or i.v. injection showed similar dose-related linear increases in SCE frequencies in their PBLs. After i.v. administration of AZQ, splenocytes from treated mice showed approximately the same SCE frequency as found in the PBLs. In general, AZQ caused a slowing of cell cycling in vivo while giving inconsistent responses in vitro. AZQ did cause a dose-related decrease in the number of recoverable mononuclear lymphocytes in mice treated in vivo. Contrary to the in vitro studies, comparison of SCE responses in mice with those previously observed in brain tumor patients undergoing chemotherapy with AZQ (Kligerman et al., Cancer Res., 47: 631-635, 1987) revealed AZQ was a much more potent SCE inducer in humans than in mice.     

Biochemical and morphological effects of cigarette smoke condensate and its fractions on normal human bronchial epithelial cells in vitro

We investigated the effect of cigarette smoke condensate (CSC), two basic fractions (BIa and BIb) of CSC, the ethanol-extracted weakly acidic fraction (WAe), and the methanol-extracted neutral fraction (Nmeoh) on the clonal growth rate, plasminogen activator (PA) activity, cross-linked envelope (CLE) formation, and ornithine decarboxylase activity, epidermal growth factor (EGF) binding, thiol levels, and DNA single strand breaks in cultured human bronchial cells. Neither CSC nor any of the fractions were mitogenic over the range 0.01-100 micrograms/ml. All were growth inhibitory at higher concentrations. The 40% growth inhibitory concentrations for CSC, BIa, BIb, WAe, and Nmeoh were 10, 10, 10, 3, and 1 micrograms/ml, respectively. Effects on CLE formation, morphology, PA, and ornithine decarboxylase activities, EGF binding, and thiol levels were evaluated using 40% growth inhibitory concentrations. We found that CSC and all fractions caused an increased formation of CLEs, from a baseline of 0.5% in the untreated cells to a maximum increase of 25% induced by Nmeoh. A squamous morphological change was observed within 1 h after exposure to Nmeoh, WAe, and CSC. The BIa and BIb fractions had little effect. Only Nmeoh increased PA significantly, from 2.5 +/- 0.4 to 5.1 +/- 0.3 units/mg cellular protein. CSC and the WAe and Nmeoh (Nmeoh greater than WAe greater than CSC) fractions caused a decrease in EGF binding, in each case reaching a maximum effect after a 10-12-h incubation. This effect on EGF binding was further characterized in the case of Nmeoh. In untreated normal human bronchial epithelial cells, by Scatchard analysis the kd was 2.0 nM and there were 1.2 X 10(5) receptors/cell. In cells incubated in medium containing Nmeoh (3 micrograms/ml) the kd was 3.2 nM and there were 1.1 X 10(5) receptors/cell. Thus, inhibition of EGF binding by Nmeoh was due primarily to a decrease in the affinity. At the 40% growth inhibitory concentrations neither CSC nor any of the fractions significantly affected intracellular thiol levels. While a 3-h incubation in medium containing CSC caused significant DNA single strand breaks only at a concentration of 100 micrograms/ml, Nmeoh caused a marked effect at 5 micrograms/ml. Neither CSC nor any of the fractions had an effect on ornithine decarboxylase activity. Due to the effects of the Nmeoh fraction on growth, morphology, EGF binding, PA activity, and formation of single strand breaks we consider it to be the most likely portion of CSC to contain compounds with actions similar to those of the phorbol ester, indole alkaloid, and polyacetate tumor promoters.     

Disparate cytogenetic responses of peripheral blood and spleen lymphocytes to ethenoadenine nucleotides in vitro; maximal expression in splenic lymphocytes under conditions of enhanced membrane permeabilization

Exogenously supplied 1,N6-ethenoadenosine triphosphate (epsilon-ATP) and 1,N6-ethenodeoxyadenosine triphosphate (epsilon-dATP) are potent inducers of sister chromatid exchanges (SCEs) in murine spleen lymphocytes but not in peripheral blood lymphocytes cultured in vitro. Data suggest that spleen lymphocyte membranes are inherently more permeable than blood lymphocytes to transient uptake of epsilon-ATP and epsilon-dATP. The effect of media pH and divalent cations on SCE frequency and chromosomal aberrations in spleen cells pulse-treated with epsilon-ATP were studied. The most dramatic responses were observed at pH 8.0 in Ca2+/Mg2(+)-free Hank's balanced salt solution (HBSS). Under the latter conditions, SCE and chromosomal aberration responses (mean +/- SD) of lymphocytes from replicate mice were 69.4 +/- 13.1 SCE/cell and 49 +/- 8.5% of cells with aberrations respectively. Chromosomal aberrations included multiple complex breakage and rearrangements. In HBSS containing Ca2+ (0.575 mM) and Mg2+ (0.4 mM) in concentrations equivalent to those in RPMI 1640, maximum SCE and aberration responses of 31.8 and 28% were observed in cells treated at pH 6.0. Similarly, maximum SCE frequencies (46 +/- 1.6 SCE/cell) and percentage of cells with aberrations (8 +/- 1.4%) were present in spleen cells treated at pH 6.0 in RPMI media. SCEs and aberrations decreased with increasing pH in either media containing divalent cations. In Ca2+/Mg2(+)-free HBSS, the highest mitotic index and fastest cell cycling were seen at pH 6.0. Mitotic indices dropped dramatically at pH 7.4 but recovered considerably at pH 8.0, in spite of a high frequency of cells containing aberrant chromosomes. The most dramatic cytotoxicity occurred at pH 6.0 in HBSS containing Ca2+ and Mg2+. Decreased cytotoxicity was apparent at higher pH and in RPMI medium. Conditions for optimal growth of control cells were obtained following pulse-treatment in Ca2+/Mg2(+)-free HBSS medium at high pH (8.0). Because of the dramatic cytogenetic toxicity of exogenously supplied epsilon-ATP, and the ubiquitous occurrence and biological importance of intracellular ATP, the latter should be considered a potential target for adduct formation by electrophilic metabolites of carcinogenic agents.     

Effects of tumor promoters and cocarcinogens on growth and differentiation of cultured human esophageal epithelial cells

The acute effects of 12-O-tetradecanoylphorbol-13-acetate [(TPA) CAS: 56937-68-9], T-2 toxin (CAS: 21259-20-1), capsaicin (CAS: 404-86-4), cigarette smoke condensate (CSC), and ethanol (CAS: 3807-77-0) were examined in secondary cultured human esophageal epithelial cells in serum-free LHC-8 medium. Effects were evaluated by morphology and measurement of clonal growth rate (population doublings per day), cross-linked envelope (CLE) formation, and the enzymatic activities of ornithine decarboxylase (ODC) and plasminogen activator (PA). All compounds tested were inhibitory to clonal growth; concentrations causing 50% growth inhibition were estimated as 10 nM TPA, 6 nM T-2 toxin, 40 microM capsaicin, 8 micrograms CSC/ml, 540 mM ethanol, and 0.8 microgram CSC/ml with 220 mM ethanol. None of the compounds tested induced CLE formation, although calcium ionophore (A23187) could induce CLE in at least 60% of the cells. TPA (10 and 100 nM) decreased the ODC activity of cells, and capsaicin (100 microM) induced ODC by 220%. TPA (1-100 nM) and capsaicin (100 microM) also induced PA activity. Slight increases in ODC activity by CSC (10 micrograms/ml), CSC (1 microgram/ml) with ethanol, and T-2 toxin (1 nM) were observed, but PA activity was not affected by these compounds. The results indicated that the response of human esophageal epithelial cells to TPA is both similar to and different from that reported for human epidermal and bronchial cells in vitro. Enhancement of PA activity and decrease in ODC by TPA are found in all three human epithelial cell types. However, these changes are not associated in esophageal cells with increased CLE formation as reported in studies with the use of bronchial and epidermal epithelial cells. The results from these acute studies provide the basis for designing in vitro carcinogenesis investigations using these agents.