小鼠腹腔细胞作为人杂交瘤细胞饲养细胞的实验研究

在人杂变瘤细胞克隆化过程中，常规采用人外周血淋巴细胞(PBL)作为饲养细胞．但因PBL制备比较繁琐，来源有限，给实际工作带来不便。我们试用小鼠腹腔细胞作饲养细胞，并与PBL对比，证明两对人杂变瘤细胞的克隆化率及用性率没有显性差别。现将结果报告如下。     

细胞舍生

细胞舍生山西医学院（０３０００１）何泽涌，杨美林（接上期）以糖皮质激素，或抗ＣＤ３—ＴＣＲ抗体，或与调控Ｃａ２＋通道相关受体的配体作为信号，对胸腺细胞膜作用后，都使细胞质Ｃａ２＋浓度持续增高，细胞核内的核酸内切酶随之活化，细胞舍生。因此认为细胞质Ｃａ...     

细胞舍生

细胞舍生山西医学院（０３０００１）何泽涌，杨美林（一）细胞舍生是细胞的一种死亡方式。这是在体内正常的微环境中，通过细胞自身内部的某些生化反应，如某种蛋白质的合成，或某种酶的活化等等而致的细胞死亡。细胞舍生是有选择性的，即在体内同一微环境内，有的细胞舍...     

高浓度大鼠肝实质细胞与非实质细胞共培养的细胞功能研究

目的:高浓度肝实质细胞与非实质细胞原代共培养,研究其功能活性。方法:应用原位胶原酶灌流法分离大鼠肝细胞,获得有活性的肝细胞,并应用高浓度实质和非实质肝细胞共培养的方法原代培养（共培养组）,并以微囊肝细胞培养为对照组（微囊组）进行了比较。结果:两组均维持白蛋白分泌、尿素合成功能7天;共培养组的白蛋白分泌与微囊组一样为下降趋势,共培养组从（0.870±0.102）降至（0.492±0.040）g·L-1·10-6cells·24h-1,微囊组从（1.147±0.099）降至（0.375±0.012）g·L-1·10-6cells·24h-1;共培养组的肝细胞第4天后维持在一个较稳定的水平,而微囊组肝细胞前3天明显高于共培养组,而后两天显著低于共培养组（P<0.05）。共培养组的尿素合成功能,由（4.50±0.56）降至（4.37±0.19）μmol·L-1·10-6cells·90min-1,微囊组由（5.42±0.81）降至（3.60±0.33）μmol·L-1·10-6cells·90min-1,前3天微囊组高于共培养组,后2天共培养组明显高于微囊组（P<0.05）。共培养2～7天肝细胞的对氨基苯甲酸（PABA）浓度稳定在 7.2～9.9mg/L·10-6cells·24h-1。结论:高浓度肝实质细胞与非实质细胞共培养,可使肝细胞的特异性功能维持7d,而且较微囊肝细胞更适合应用于中空纤维舱型生物人工肝。     

T细胞淋巴瘤细胞凋亡的基因调控

凋亡（Apoptosis）是细胞坏死之外的一种细胞死亡方式，即所谓细胞程序化死亡（Programmedcelldeath），是指正常细胞的“生理性”死亡过程。它是一个有序的过程，是以细胞内源性内切酶活性增加为代表的一系列生化代谢的变化[1]。细胞先是接收、识别某些特殊的生理或病理性刺激信号，然后启动细胞特有的基因或基因群，通过mRNA的转录，合成一组有致死效应的蛋白质，从而导致细胞的解体死亡[1]。这是一种细胞的“自杀”行为，与细胞的增殖和分化一样，对维持组织的自身稳定具有重要的积极作用。一、调亡的基因调控细胞的调亡受到多种因素的…     

静脉血管平滑肌细胞中的多倍体细胞分析

目的探讨流式细胞术分析静脉血管平滑肌细胞多倍体细胞的方法和意义。方法选取普通级健康成年日本大耳白兔2只,雄性,8月龄,大约3 kg;普通级SD大鼠2只,雄性,8周龄,大约200 g。人大隐静脉为2例心脏冠状动脉旁路移植术中修补血管所得。在麻醉日本大耳白兔和大鼠后开腹腔获取下腔静脉,采用多种酶组合的消化液消化兔、大鼠以及人的静脉血管得到适合流式细胞检测的静脉血管平滑肌单细胞悬液,用碘化丙啶标记细胞,利用细胞流式仪分析兔、大鼠以及人的静脉血管平滑肌单细胞悬液各5 000个细胞,检测细胞DNA含量,DNA含量翻倍的细胞为多倍体细胞。并利用荧光原位杂交技术（FISH）验证阳性对照HEK293细胞系中的多倍体的存在。结果流式结果显示分析的5 000个细胞中,阳性对照HEK293细胞的多倍体细胞为1 355个（27.1%）,从2例患者中取得的大隐静脉多倍体细胞为310个（6.2%）和250个（5.0%）;2只大鼠的下腔静脉平滑肌细胞中多倍体细胞为360个（7.2%）和450个（9%）;2只兔的下腔静脉平滑肌细胞中多倍体细胞分别为270个（5.4%）和305个（6.1%）。结论成年的静脉血管包括人大隐静脉的平滑肌细胞中普遍存在一定比例的多倍体细胞。     

细胞缝隙连接对乳腺癌细胞紫杉醇敏感性的影响

目的：探讨细胞缝隙连接对乳腺癌细胞紫杉醇敏感性的影响。方法：以乳腺癌T47D细胞为研究对象,采用CCK8法检测细胞毒性。结果：紫杉醇（TAX）的细胞毒性有细胞密度依赖性,与低密度相比,细胞在高密度时的毒性显著增加。这说明,细胞在高密度有缝隙连接（Gap Junction,GJ）形成的情况下,TAX的细胞毒性显著增加。此外,细胞在高密度的情况下,分别采用维甲酸（RA）和甘草次酸（18-α-GA）增强或抑制GJ功能,结果发现,RA组的TAX细胞毒性显著高于正常组,相反,给予18-α-GA抑制GJ功能后,TAX的细胞毒性显著降低。结论：增强GJ功能可显著提高乳腺癌细胞对TAX的敏感性。GJ可能是提高TAX对乳腺癌疗效的新靶点。     

Bis(2-chloroethyl)sulfide (BCES) disturbs the progression of rat keratinocytes through the cell cycle

Epidermal basal keratinocytes are the primary target in BCES-induced cutaneous injury. DNA synthesis is inhibited by exposure to BCES which could relate to the mustard's cytotoxic effect. The effects of BCES on the cell cycle in keratinocytes synchronized by aphidicolin were investigated. Primary keratinocytes synchronized at the Gl/S boundary entered the S, G2, M, and G1 phases at successive times after release from the block. When cells were exposed to 1, 10, or 50 μM BCES in different phases of the cell cycle, cells in the S phase were more sensitive to BCES than cells in the other phases. Keratinocytes exposed to 1 μM BCES at the Gl/S boundary exhibited a prolongation of the S phase and a block in the G2 phase. When these cells were exposed to 10 or 50 μM BCES, they did not enter the S phase for up to 12 h and the incorporation of thymidine into DNA was inhibited. These results suggest that the blocks in the G2 and G1 phases relate to the cytotoxic effect of BCES on the germinative population of epidermal keratinocytes.     

Modulation of anthracycline resistance by reserpine in P388 leukemia cells

The activity of reserpine and a possible mechanism by which it reverses the resistance to both doxorubicin and pirarubicin in doxorubicin-resistant P388 leukemia (P388/DOX) cells were examined in vitro. During 48 hr drug-exposure, the sensitivity of doxorubicin and pirarubicin were potentiated markedly when reserpine was present at the concentration of 1 microgram/ml, which is not toxic to P388 leukemia (P388/S) cells. However, reserpine had little effect on the cytotoxicity of doxorubicin and pirarubicin in the sensitive parent cell. Reserpine at 0.5-20 micrograms/ml increased intracellular accumulation of doxorubicin and pirarubicin in the drug-resistant cells. The potentiating action of reserpine was stronger when the cells were preincubated with reserpine within 30 min. Efflux of doxorubicin and pirarubicin was greater in drug-resistant cells compared to sensitive cells. This enhanced efflux of drug resulted in a decrease in the intracellular accumulation of doxorubicin in the drug-resistant cells. When the resistant cells were exposed to 2 micrograms/ml of reserpine, this enhanced efflux was blocked. A similar effect of reserpine on doxorubicin was seen with the efflux pattern of pirarubicin. From the measurements of drug uptake and efflux, it seems that like other multiple drug resistance modifiers, reserpine modulates anthracycline resistance by increasing intracellular accumulation of drug.     

The cytokinetic and cytotoxic effects of ICRF-159 and ICRF-187 in vitro and ICRF-187 in human bone marrow in vivo

The cytotoxic and cytokinetic effects of ICRF-159 and its d-enantiomer ICRF-187 have been examined in vitro. The effects of both agents were identical. Cytotoxicity is dependent on both the drug concentration and the duration of drug exposure. Drug exposure for twice the cell cycle time is necessary for maximum effect. Cytotoxicity is also dependent upon the rate of cell proliferation. A rapidly growing cell population is more sensitive to brief drug exposure than a slowly growing population. The cytokinetic effects were studied using flow cytometry, determination of [3H]-thymidine incorporation and mitotic index. ICRF-159/187 appears to act only during the G2 phase of the cell cycle. There is no detectable delay in cell passage through the G1/S boundary or in transit through S phase. Inhibition of DNA synthesis occurs only after the G2 block prevents subsequent entry of cells in S phase. A fraction of the cells, depending upon drug concentration, undergo further DNA synthesis without cell division, resulting in a tetrapoid cell population. The cytokinetic effects were determined in the bone marrow of patients receiving ICRF-187. All dose-rates produced G2/M accumulation in the marrow with depletion of S phase cells. One patient was given a single injection of 1.0 gm/M2 . G2/M accumulation was observed 24 h after treatment, with recovery to a pretreatment DNA cycle distribution 24 h later. These studies suggest that a continuous drug infusion, or intermittent infusions timed to allow the normal cell population to recover, may produce superior clinical activity with this agent. A Phase I study of such an intermittent schedule is indicated.     

Inhibitory effect of substituted dextrans on MCF7 human breast cancer cell growth in vitro.

Substituted dextrans can reproduce some of the properties of heparin and can thus be used to alter cellular growth. We studied the effect of heparin (H108), dextran (D), carboxymethylbenzylamide dextran (CMDB) and carboxymethylbenzylamide sulfonate dextran (CMDBS) on the growth of human mammary cells of the MCF7 tumor line. The cells were cultured in minimum Eagle's medium containing 2% fetal calf serum without biopolymer, or with increasing concentrations of H108, D, CMDB or CMDBS. Growth curves were accurately based on cell counting using a Coulter counter. Cell distribution in the various phases of the cycle was analyzed by flow cytometry. Dose-dependent growth inhibitory effects (400-4000 micrograms/ml) were observed. The effect on MCF7 tumor cells was most apparent with CMDBS. The percentage of cells in the S phase decreased with preferential blocking in the G0/G1 phase. Pre-clinical studies can be anticipated as there is an absence of in vivo toxicity.     

The effects of cell cycle position on the cytotoxicity and mutagenicity of benzo(a)pyrene in cultured Chinese hamster cells

The induction of cytotoxicity and mutation to 6-thioguanine resistance (6TGr) by S9-activated benzo(a)pyrene (B(a)P) was studied in asynchronized and synchronized Chinese hamster V79 cells. After treatment of asynchronized populations with B(a)P (0.25-2 micrograms/ml) in the presence of S9 for 3 h, the number of 6TGr cells increased. The increase was concentration-dependent up to 2 micrograms/ml, and was accompanied by a concomitant concentration-dependent decrease in cell survival. Synchronized cells were treated with B(a)P for 2 h at 2-h intervals after release from the G1/S block by hydroxyurea (HU). The cytotoxicity of 2 micrograms/ml of B(a)P was maximal at 0 h after HU release, i.e., G1/S phase, and also at 2 h after HU release, i.e., early S phase. Thereafter, it decreased with the progression of the cell cycle. Similarly, treatment with B(a)P at 0 h and 2 h after HU release resulted in the maximum incidence of 6TGr mutants, after which the incidence showed a decrease from 4-10 h after HU release. These results indicate that the cells in G1/S and early S phase are highly susceptible to cytotoxic and mutagenic damage induced by B(a)P and suggest the presence of a specific hot spot in the cell cycle for mutagenesis by the carcinogen B(a)P in cultured hamster cells.     

Sensitivity of different phases of the cell cycle to selected hepatotoxins in cultured liver-derived (BL9L) cells

1. Many toxins are active against dividing cells and cytofluorometric analysis of synchronized dividing liver-derived (BL9L) cells has been employed to study the relative sensitivity of the G1(G0), S and G2/M phases of the cell cycle to selected hepatotoxins. 2. The cytotoxic metal beryllium, which inhibits cell division, caused a specific block at the G1 phase of the cell cycle. 3. Dehydroretronecine, an antimitotic metabolite of the hepatotoxic plant pyrrolizidine alkaloids, retarded progression of cells through the cell cycle with a consistent accumulation at the late S to G2 phase. 4. Exposure of cells to aflatoxin B1-8,9-epoxide, the putative carcinogenic metabolite of the hepatocarcinogen aflatoxin B1, particularly during the early period of S phase, produced morphologically transformed cells.     

Inhibitory effect of tumor cell proliferation and induction of G2/M cell cycle arrest by panaxytriol

Panaxytriol, a polyacetylenic compound, isolated from red ginseng (Panax ginseng C.A. Meyer), was studied to determine its effects on the growth and cell cycle of tumor cell lines. The compound showed both significant cytotoxicity and inhibition of DNA syntheses in various tumor cells tested. For P388D1, a mouse lymphoma cell line, IC50 values for cytotoxicity and inhibition of DNA synthesis were 3.1 and 0.7 microg/ml, respectively. The cytotoxic effect of panaxytriol was both time- and dose-dependent. It also induced the cell cycle arrest of P388D1 at the G2/M phase, which was measured through flow cytometry. Particularly, the proportion of cells in the G2/M phase of the cell cycle increased from 9 % to 26 and 48 %, respectively, after 24 and 36 h exposure to panaxytriol at 5 microg/ml. There were corresponding decreases in the proportion of cells at the G0/G1 phase. The S phase also decreased during the 36-h treatment.     

Differential effects of NAD, nicotinamide and related compounds upon growth and nucleoside incorporation in human cells

Two human melanoma cell lines, MM96 and MM127, were found to be highly sensitive to the toxicity of adenosine (D50 100-150 micrograms/ml) compared with other melanoma lines. HeLa cells and a lymphoblastoid line (D50 greater than 500 micrograms/ml). The MM127 line was also sensitive to NAD (D50 41 micrograms/ml) compared with the other lines (D50 greater than 400 micrograms/ml), and accumulated three-fold more NAD-derived isotopic label. Nicotinamide exhibited little toxicity in any cell type (D50 greater than 400 micrograms/ml); 25-100 micrograms/ml nicotinamide greatly increased the plating efficiency of melanoma cells and fibroblasts when low levels of foetal calf serum were used. The toxicity of DNA-damaging agents (alkylating agents and u.v.) in melanoma cells was not reduced in the presence of NAD, adenosine or nicotinamide. Studies of the effects of the latter compounds upon the incorporation of deoxynucleosides showed that: (a) melanoma cells have lower purine pools than fibroblasts; (b) [3H]deoxyguanosine incorporation was inhibited more than [3H]deoxyadenosine incorporation; (c) incorporation of [3H]deoxyadenosine and [3H]deoxyguanosine into RNA was inhibited by adenosine, thus providing a method for determination of guanine-specific DNA repair; and (d) NAD enhanced thymidine incorporation in intact melanoma cells but not in fibroblasts, in a pattern similar to the release from template restriction previously reported for permeabilised tumour cells.     

雷公藤甲素对宫颈癌Hele细胞增殖的抑制作用

目的研究雷公藤甲素对宫颈癌Hele细胞增殖的抑制作用。方法采用MTT方法检测雷公藤甲素对体外培养的宫颈癌Hele细胞增殖的影响,运用流式细胞术检测细胞周期,RT-PcR分析细胞周期蛋白B1（cyclinBl）mRNA的表达;Western-blot检测cyclinBl、P34cdc2。和磷酸化P34cdc2（phosphorcdc2）蛋白的表达。结果雷公藤甲素浓度依赖性（0．5～4．0／lg．mL-1）抑制体外培养的宫颈癌Hele细胞,干扰Hele细胞周期。细胞周期阻滞于s期和G：／M期,s期和G2／M期的百分率上升;同时G0／G1细胞百分率降低;RT-PCR结果显示,雷公藤甲素（0．5～4．0／ag．mL-1）显著抑制cyclinB1mRNA的表达;Western-blot结果显示,雷公藤甲素（0．5～4．0gg．mL-1）作用24h后cyclinB1蛋白表达开始不同程度降低,P34cdc2。变化较小,雷公藤甲素作用12h后磷酸化P34cdc2。蛋白水平明显改变。结论雷公藤甲素可以显著抑制宫颈癌Hele细胞体外增殖,其机制与细胞周期阻滞于s期和影响细胞周期因子cyclinBl的表达和改变P34cdc2。磷酸化有关。     

Effects of methylacetylenic putrescine, an ornithine decarboxylase inhibitor and potential novel anticancer agent, on human and mouse cancer cell lines

Sensitivity of several human and mouse cancer cell lines to methylacetylenic putrescine (MAP) was evaluated using clonogenic, sulforhodamine B and cell counting assays. The effects of MAP on cell morphology, cell cycle phase distribution and changes in polyamine metabolism of xenografted MCF-7 and MDA-MB-231 human mammary tumor cells were also investigated. On the basis of IC50 values, BHT-101 human thyroid carcinoma cells were the most sensitive (9 micrograms/ml), followed by P388 mouse lymphoma (32 micrograms/ml), MCF-7 (48 micrograms/ml) and MDA-MB-231 (110 micrograms/ml) human breast carcinoma cell lines. MAP treatment led to accumulation of P388 cells in G1 phase. At higher doses, the cytoplasm of the cells became vacuolated followed by apoptosis. The foamy cytoplasm may suggest a rare type of cell death (Clarke III type) called non-apoptotic programmed cell death. MAP treatment resulted in a total inhibition of ornithine decarboxylase (ODC) activity with a concomitant decrease of intracellular polyamine (mostly putrescine and spermidine) content in the breast cancer cells, whilst the spermine concentration was shown to increase. MAP proved at least 10 times more potent than the formerly studied DL-alpha-difluoromethylornithine making it an attractive candidate for clinical testing.