促肝细胞生长素诱导人肝癌细胞（BEL－7402）凋亡

本文从细胞学、ＤＮＡ凝胶电泳、流式细胞术三方面研究促肝细胞生长素（ｐＨＧＦ）在体外对肝癌细胞增殖活性的影响。结果表明：ｐＨＧＦ对肝癌ＢＥＬ－７４０２细胞增殖有抑制作用，并存在剂量和时间相关性。其４８ｈ的半数抑制浓度（ＩＤ５０）为０．３７ｍｇ／ｍｌ±０．０４ｍｇ／ｍｌ。而３７℃灭活的ｐＨＧＦ对ＢＥＬ－７４０２细胞增殖在１５ｈ无抑制作用，在２４和４８ｈ抑制作用很弱（ＩＤ５０＞１．５ｍｇ／ｍｌ）。ＤＮＡ凝胶电泳结果表明，ｐＨＧＦ可诱导ＢＥＬ７４０２细胞产生细胞凋亡（Ａｐｏｐｔｏｓｉｓ）。流式细胞术（ＦＣＭ）结果显示：ｐＨＧＦ抑制ＢＥＬ－７４０２细胞增殖过程是先使细胞停留在Ｇ０／Ｇ１期，继而诱导细胞产生凋亡。后两项结果均显示ｐＨＧＦ对人肝癌细胞凋亡的诱导里时间和剂量相关性。     

胎肝提取物对培养中4株肝癌细胞增殖和分化的影响

目的：观察４月胎肝提取物对ＱＧＹ－７７０３、ＢＥＬ－７４０２、ＳＭＭＣ－７７２１人肝癌细胞和ＨｅｐＡ小鼠肝瘤细胞增殖和分化的影响。方法：从４月胎肝中制备提取物,采用［３Ｈ］－ＴｄＲ掺入法和ＭＴＴ比色法。结果：１００～５００ｍｇ／Ｌ的胎肝提取物能明显抑制ＱＧＹ－７７０３、ＢＥＬ－７４０２、ＳＭＭＣ－７７２１、ＨｅｐＡ等肝癌细胞的ＤＮＡ合成和脱氢酶活力。对ＤＮＡ合成的半数抑制浓度（ＩＣ５０）分别为３４６．９３、１５１．１８、１８２．３７、２３１．０３ｍｇ／Ｌ;对脱氢酶活力的抑制弱于对ＤＮＡ合成的抑制,ＩＣ５０分别为１３１８．１、１５９９．２、７９３．８、８１３．１ｍｇ／Ｌ。结论：胎肝提取物能够抑制上述４种细胞增殖和促进分化。     

蟾蜍灵诱导人胃癌细胞凋亡的实验研究

用ＭＴＴ法、形态学观察法、ＤＮＡ电泳和流式细胞仪观察蟾蜍灵（Ｂｕｆａｌｉｎ）诱导的胃癌细胞凋亡。结果表明,０．０１ｕｍｏｌ／Ｌ蟾蜍灵作用ＭＧｃ－８０３细胞４８ｈ,即可显著抑制细胞生长,ＩＣ５０值约为０．１ｕｍｏｌ／Ｌ。琼脂糖凝胶电泳获得梯形图谱,汉式细胞仪ＤＮＡ直方图上出现典型的亚二倍体峰,凋亡主要发生在Ｇ１／Ｇ０期,细胞凋亡率随药物浓度和作用时间的增加而增高,以上结果提示,ｂｕｆａｌｉｎ对胃癌细     

血管内皮生长因子的免疫学测定法

目的：检测肿瘤细胞条件培养液中ＶＥＧＦ的表达量。方法：在表达及分离纯化ＶＥＧＦ的基础上,成功地制备了ＶＥＧＦ抗体,以夹心法测定ＶＥＧＦ,ＶＥＧＦ测定范围为０．０２～２００．００ｎｇ／ｍｌ,灵敏度可达０．０２ｎｇ／ｍｌ,批内及批间变异均小于１０％,回收率平均为１０２．４％。其中人胃癌细胞ＭＧＣ８０３、ＢＧＣ８２３、乳癌ＮＭＣＦ－７及肝癌ＢＥＬ－７４０２分泌上清中ＶＥＧＦ的含量分别为６．５０、０．４５     

单细胞电泳用于检测人精子DNA链断裂

目的 引进精子单细胞凝胶电泳（ｓｉｎｇｌｅ－ｇｅｌ ｅｌｅｃｔｒｏｐｈｅｒｏｓｉｓ,ＳＣＧＥ）实验方法,用以检测人类精子ＤＮＡ链断裂。方法 载有精子和凝胶的玻片首先在ｐＨ１０的弱碱性裂解液中作用１ｈ,精子核分别用１０ｍｍｏｌ／Ｌ二硫苏糖醇处理１ｈ,１０μｇ／ｍｌ ＲＮＡ酶处理４ｈ,２００μｇ／ｍｇ蛋白酶Ｋ处理１５ｈ。精子核用１５μｇ／ｍｌ的ＥＢ染色５ｍｉｎ。计数彗星细胞百分率,并用本试验室建立的精     

恒定磁场协同阿霉素诱导肝癌细胞凋亡的研究

目的：探讨恒定磁场联合阿霉素对诱导肝癌细胞凋亡的影响。方法：采用流式细胞仪碘化丙啶染色法和活细胞荧光染色法（Ｈｏｅｃｈｓｔ染色法），观察不同处理因素对细胞凋亡率的影响。结果：两种实验方法均显示０５ｍｇ／Ｌ的阿霉素与对照组比较没有显著差异（Ｐ＞００５），而单独用恒定磁场或１０ｍｇ／Ｌ的阿霉素与对照组比较有显著性差异（Ｐ＜００５）。应用恒定磁场后发现，０５ｍｇ／Ｌ和１０ｍｇ／Ｌ浓度的阿霉素诱导ＨｅｐＧ－２细胞凋亡的结果与对照组相比，均有显著差异（Ｐ＜００５）。结论：恒定磁场具有增强阿霉素诱导肝癌细胞凋亡的作用     

去甲斑蝥素诱导人肝癌BEL-7402细胞凋亡的研究

目的研究新型抗癌药物去甲斑蝥素抑制人肝癌（ＢＥＬ－７４０２）细胞生长的机制。方法从细胞的形态学观察和ＤＮＡ琼脂糖凝胶电泳等方面考察去甲斑蝥素作用人肝癌细胞的特点,并进一步用免疫细胞化学及Ｗｅｓｔｅｒｎｂｌｏｔｉｎｇ等分析手段研究癌基因蛋白Ｂｃｌ－２在此过程中的表达情况。结果１０ｍｇ／Ｌ去甲斑蝥素作用人肝癌细胞２４ｈ,部分细胞出现固缩、细胞质膜突起、核染色质异常凝集等,并伴有膜包被的凋亡小体的形成。ＤＮＡ琼脂糖凝胶电泳显示,处理组部分细胞的ＤＮＡ已降解成以约２００ｂｐ为基数的ＤＮＡ片断,呈现典型的“梯状”带纹。免疫细胞化学及Ｗｅｓｔｅｒｎｂｌｏｔｉｎｇ分析显示该过程伴随癌基因蛋白Ｂｃｌ－２表达的减弱。结论新型抗癌药物去甲斑蝥素可以诱导人肝癌细胞发生凋亡,该凋亡过程与癌基因蛋白Ｂｃｌ－２表达下调相关     

杂色曲霉素诱导体外培养人外周血淋巴细胞凋亡的研究

目的：研究我国肿瘤高发区饮食污染霉菌毒素－杂色曲霉素（ＳＴ）对人免疫机能的可能影响。方法：流式细胞术（ＦＣＭ）、形态学观察及ＤＮＡ电泳。结果：ＳＴ可诱导体外培养的人外周血淋巴细胞发生凋亡,并且在一定时间（２￣４８ｈ）及剂量（０．１２５￣２．０ｍｇ／Ｌ）范围内呈正相关关系。光镜及透射电镜观察均发现了凋亡细胞典型的形态学变化,如核染色质凝集边集,凋亡小体形成等;ＤＮＡ琼脂糖凝胶电泳也出现特征性的ＤＮＡ     

去甲斑蝥素诱导人肝癌BEL—7402细胞凋亡的研究

目的 研究新型抗癌药物去甲斑螯素抑制人肝癌（ＢＥＬ－７４０２）细胞生长的机制。方法 从细胞的形态学观察和ＤＮＡ琼脂糖凝胶电泳等方面考察去甲斑螯素作用人肝癌细胞的特点，并进一步用免疫细胞化学及Ｗｅｓｔｅｒｎ ｂｌｏｔｔｉｎｇ等分析手段研究癌基因蛋白Ｂｃｌ－２在此过程中的表达情况。结果 １０ｍｇ／Ｌ去甲斑螯素作用人肝癌细胞２４ｈ，部分细胞出现固缩、细胞质膜突起、核染色质异常凝集等，并伴有膜包被的凋亡小     

人粒细胞集落刺激因子在家蚕细胞及幼虫中的高效表达

含信号肽序列的ｈＧ－ＣＳＦｃＤＮＡ基因克隆于Ｍ１３ｍｐ１８中，测序表明其基因序列与高活性ｈＧ－ＣＳＦｃＤＮＡ一致。将ｈＧ－ＣＳＦ基因插入家蚕核型多角体病毒（ＢｍＮＰＶ）转移载体ｐＢＥ２８４，经与野生型病毒ＤＮＡ共转染家蚕细胞后，通过体内同源重组的方式构建了重组病毒ＢｍＮＰＶ－Ｇ－ＣＳＦ，Ｓｏｕｔｈｅｒｎ印迹杂交表明重组病毒ＤＮＡ中含Ｇ－ＣＳＦ基因片段。重组病毒感染单层ＢｍＮ细胞后第四天表达量达１． ２×１０６ＣＦＵ／ｍｌ培养液，Ｗｅｓｔｅｒｎ－ｂｌｏｔ分析可见分子量为１９×１０３左右一条带。家蚕幼虫感染重组病毒后第四天表达量达１．４×１０７ＣＦＵ／ｍｌ血淋巴（ｈｅｍｏｌｙｍｐｈ）。     

Humoral inhibitors of the immune response in uremia. I. Effect of serum and of the supernatant of spleen cultures from uremic rats on the mixed lymphocyte reaction.

The effects of the serum of rats with experimentally induced chronic renal failure and of the supernatant of cultured spleen cells obtained from these animals were tested in the mixed lymphocyte reaction (MLR). Serum of uremic rats with blood urea nitrogen levels greater than 35 mg. per 100 ml. and supernatant of spleen cultures from animals with blood urea nitrogen levels greater than 60 mg. per 100 ml. were found to be inhibitory to the MLR. The inhibitory activity of uremic serum was directly correlated with blood urea nitrogen levels. The inhibitory activities both of uremic serum and of the supernatant of cultured uremic spleen cells were further studied and compared. Dialysis in vitro does not remove the suppressing effect of serum or of supernatant on the MLR. Irreversible inhibition of the MLR is observed after 48 hours' exposure to the serum or to the supernatant; this inhibition occurs whether the MLR culture is exposed during the first 48 hours or last 48 hours of incubation. In some uremic rats the inhibitory activity of the serum disappears or is weakened after splenectomy, despite a continued rise in blood urea nitrogen levels.     

甲钴胺体外诱导大鼠骨髓间充质干细胞向神经元样细胞分化

背景：目前骨髓间充质干细胞移植到脊髓损伤动物体内后对脊髓损伤的恢复效果非常有限。甲钴胺是治疗神经系统疾病及损伤的常见药物,其对骨髓间充质干细胞的影响尚不清楚。 目的：探讨甲钴胺体外定向诱导骨髓间充质干细胞向神经元样细胞分化的可行性,观察分化后的细胞增殖和生长情况。 方法：取大鼠胫腓骨骨髓,采用密度梯度离心贴壁细胞培养法分离、培养骨髓间充质干细胞,取第四五代骨髓间充质干细胞,分别以25,50,100 mg/L的甲钴胺进行诱导分化24,48和72 h。倒置相差显微镜下连续观察细胞形态学变化,MTT法检测细胞活性,RT-PCR和Western blot法检测特异性标志物Nestin和NSE的表达。 结果与结论：甲钴胺诱导骨髓间充质干细胞后大部分细胞变成神经元样细胞。与对照组比较,甲钴胺诱导后细胞活性无明显变化。不同剂量甲钴胺诱导48 h后,Nestin和NSE在mRNA和蛋白水平表达均上调,其中100 mg/L组表达上调最明显;100 mg/L 甲钴胺诱导24,48和72 h后,Nestin和NSE在mRNA和蛋白水平表达均上调,其中72 h表达上调最明显。说明甲钴胺可定向诱导骨髓间充质干细胞向神经元样细胞分化,100 mg/L为甲钴胺的较佳诱导浓度。     

Con A-induced suppressor cells in man. I. Induction and characterization of suppressor cells

Con A-induced suppressor cells were studied in autologous co-cultures of normal human lymphocytes. Lymphocytes pretreated with 5 µg/ml, 20 µg/ml, or 50 µg/ml of Con A were cultured for 24, 48 and 72 hours. Subsequently, the whole mononuclear (MN) cell subpopulation or T-enriched, B-enriched, and monocyte-enriched fractions were added to freshly obtained lymphocytes, stimulated with 5 µg/ml of Con A and cultured for a further 96 hours. MN cells pretreated with 5 µg/ml of Con A did not significantly affect the DNA synthesis in co-cultures with unfractionated MN cells, while cells pretreated with 20 µg/ml or 50µg/ml for 48 and 72, but not for 24 hours, possessed the suppressive activity. The suppressor cells were found in T-, but not B-enriched subpopulations. Moreover, suppression was induced by T-enriched fraction from MN cells pretreated with 5 µg/ml of Con A, while unfractionated cells failed to exhibit inhibitory activity. The degree of T-cell-induced suppression was dose-dependent. Irradiation with 3000 R diminished the suppressive activity of cells derived from 48-hour cultures and abrogated the activity of cells from 72-hour cultures.The present data indirectly prove that Con A-induced suppressor cells are radiosensitive T lymphocytes. The observation that induction of suppressor cells by Con A is dose- and time-dependent provides the further insight into regulatory immunological mechanism in humans.     

羟基喜树碱抑制体外培养兔晶体上皮细胞的生长

为研究羟基喜树碱（HCPT）对体外培养兔晶体上皮细胞（RLECs)生长的作用,取2－3代的RLECs在96孔板中培养24h后,用不同浓度的HCPT分别作用24及72h,用甲基噻唑四唑（Methyl thiazolyal tetrazolium,MTT)比色测定法观察其对RLECs增殖的影响,结果显示HCPT能抑制RLECs的增殖,24h的半数抑制量（ID50）为96．041mg/L,72h的ID50为1．34mg/L;且HCPT还可抑制RLECs的贴壁,提示HCPT可能在用于降低后发性白内障的发生方面有一定作用。     

鲨鱼软骨制剂对人不同细胞生长抑制作用及其机制的探讨

目的　研究鲨鱼软骨制剂 (SCP)对人胃癌细胞 (MGC80 3)、红白血病细胞 (K5 6 2 )、人结肠高分化腺癌细胞 (THC 890 8)、人乳腺癌细胞 (MCF 7)和人脐静脉血管内皮细胞 (VEC340 )的生长抑制作用 ,探讨其作用机制。方法　采用盐酸胍抽提 ,丙酮分级沉淀 ,烘干 ,微孔滤膜过滤等步骤从鲨鱼软骨粉中提取鲨鱼软骨制剂 (SCP) ;MTT法检测不同浓度的SCP对体外培养的MGC80 3细胞系、K5 6 2细胞系、THC 890 8细胞系、MCF 7细胞系和VEC340细胞系的生长抑制作用 ;Hoechst33342 /PI荧光双染法检测细胞凋亡。结果　SCP明显抑制MGC80 3、K5 6 2、THC 890 8、MCF 7和VEC340五种细胞系的生长 ,其IC50 值分别为 0 5mg/ml、0 7mg/ml、1mg/ml、1 5mg/ml和 2mg/ml。凋亡细胞比例在用药后 4 8h分别达 79 9%、4 4 6 %、78 9%、6 3 3%和 4 4 8%。结论　SCP能直接抑制不同肿瘤细胞生长 ,又能抑制血管内皮细胞的生长 ,其作用机制与细胞凋亡有关。     

In vivo cytokinesis blocked micronucleus assay with carbendazim in rat fibroblasts and comparison with in vitro assays

A successful in vivo application of the cytokinesis blocked micronucleus assay for the detection of aneuploidy induced by carbendazim (CARB) was carried out in the granuloma pouch assay. This was performed in two ways: (i) in vivo exposure of the skin fibroblasts to cytochalasin B (cytB) and CARB, by simultaneous injection of both substances into the pouch; (ii) in vivo exposure to CARB followed by in vitro culturing of the fibroblasts in the presence of cytB. Only the first assay was successful. Injection of cytB (with or without the test compound) into the pouch resulted in the induction of binucleate cells in vivo, up to a maximum of 5% at 1 mg cytB/pouch. After injection of CARB (0-50 or 0-10 mg/pouch) and cytB (1 mg) into the pouch, aneuploidy was determined in the isolated binucleate fibroblasts by fluorescence in situ hybridization with a general centromeric probe and combinations of chromosome-specific probes (19p + 19q, 4q + Yq). With all probes, the induction of chromosome loss and/or non-disjunction by CARB was very pronounced; at 10 mg CARB/pouch the total malsegregation frequency of chromosomes 4, 19 and Y was approximately 300/1000 binucleate cells. In an in vitro cytokinesis block assay with CARB (0-2.5 microg/ml) in primary skin fibroblasts the induced aneuploidy frequencies were as high as observed in the in vivo assay. The use of two probes for chromosome 19, which enabled the scoring of chromosome breaks in addition to aneuploidy, revealed no significant induction of chromosome breaks by CARB. The frequency of polyploid mononucleate and binucleate cells was decreased after CARB treatment, in both the in vivo and in vitro assays. However, in an additional in vitro assay without cytB a major induction of polyploidy from 2.5 microg/ml CARB and above was observed, showing that cytB may interfere with polyploidy induction.     

Antitumor activity and underlying mechanisms of ganopoly, the refined polysaccharides extracted from Ganoderma lucidum, in mice

Ganopoly is an aqueous polysaccharide fraction extracted from G. lucidum by patented biochemical technique and has been marketed as an over-the-counter product for chronic diseases including cancer and hepatopathy in many Asian countries. This study was undertaken to explore the anti-tumour effect and the underlying mechanisms of Ganopoly in mice and human tumor cell lines. The maximum tolerated dose (MTD) of Ganopoly in mice was estimated to be 100 mg/kg from a pilot study. Treatment of mice with oral Ganopoly for 10 days significantly reduced the tumour weight of sarcoma-180 in a dose-dependent manner, with inhibition rates of 32.3, 48.2 and 84.9% and growth delays of 1.5, 3.5, and 13.1 days at 20, 50, and 100 mg/kg, respectively. Incubation of Ganopoly at 0.05-1.0 mg/ml for 48 hours showed little or negligible cytotoxicity against human tumor CaSki, SiHa, Hep3B, HepG2, HCT116 HT29, and MCF7 cells in vitro. In contrast, 10 mg/ml of Ganopoly caused significant cytotoxicity in all tumour cells tested except MCF7, with marked apoptotic effect observed in CaSki, HepG2, and HCT116 cells, as indicated by nuclear staining and DNA fragmentation. In addition, Ganopoly enhanced concanavalin A-stimulated proliferation of murine splenocytes by 35.3% at 10 mg/ml, and stimulated the production of nitric oxide in thioglycollate-primed murine peritoneal macrophages in a concentration-dependent manner over 0.05-10 mg/ml. Addition of Ganopoly at 1 mg/ ml to murine peritoneal macrophages also potentiated lipopolysaccharide-induced nitric oxide production by 64.2%. Treatment of healthy mice or mice bearing sarsoma-180 with oral Ganopoly over 20-100 mg/kg for 7 day significantly increased the expression of both TNF-alpha and IFN-gamma (at both mRNA and protein levels) in splenocytes in a dose-dependent manner. Moreover, treatment of Ganopoly over 20-100 mg/kg significantly increased cytotoxic T lymphocyte cytotoxicity and NK activity in mice. The overall findings indicated that Ganopoly had antitumor activity with a broad spectrum of immuno-modulating activities and may represent a novel promising immunotherapeutic agent in cancer treatment.     

In vitro effect of the extract and the 1,7-dihydroxy-2,3-dimethoxy xanthone from Polygala cyparissias on the contractions induced by inflammatory mediators and ovalbumin in normal and actively sensitised trachea from guinea pig

OBJECTIVE: This study describes the in vitro action of the hydroalcoholic extract and the 1,7-dihydroxy-2,3-dimethoxy xanthone isolated from P. cyparissias on agonist and ovalbumin induced contractions in trachea, from normal and actively sensitised guinea pigs. RESULTS: The hydroalcoholic extract of P. cyparissias (0.125 to 1 mg/ml), incubated with the guinea-pig trachea for 20 min, had no effect on the resting tone of the preparations, but caused a concentration-dependent, reversible and non competitive inhibition of contractions induced by acetylcholine, histamine, compound 48/80, bradykinin, substance P, prostaglandin E2 and the stable analogue of thromboxane A2 mimetic U 46619. The calculated mean IC50 values for the hydroalcoholic extract were: 0.37, 0.51, 0.06, 0.32, 0.48, 0.3 and 0.17 mg/ml, respectively. Also, the extract of P. cyparissias (0.125 to 0.5 mg/ml) antagonised, in a graded manner (IC50 of 0.46 mg/ml) ovalbumin-induced contractions in guinea-pig trachea obtained from animals which had been actively sensitised to this antigen. Pre-incubation of the preparations with the purifed xanthone isolated from P. cyparssias (2.5 to 80 microg/ml; 10.0 to 310.0 microM) caused significant and concentration-dependent, reversible and noncompetitive inhibition of the contractile responses elicited by acetylcholine, histamine, bradykinin, substance P, U 46619 and prostaglandin E2. The calculated mean IC50 values for these effects were: 132.0, 73.0, 9.2, 32.0, 110.6 and 66.0 microM, respectively. At very high concentrations (I55.0-620.0 microM) the xanthone also antagonised contraction induced by KCl in guinea-pig trachea (IC50 of 190.0 microM). CONCLUSIONS: Taken together these and our previous in vivo results are consistent with the view that the active principles present in P. cyparissias, including the 1,7-dihydroxy-2,3-dimethoxy xanthone, antagonise, in a non competitive but, reversible manner the contractions induced by chemical inflammatory mediators in the guinea pig trachea in vitro. Thus, these results might explain at least in part, the medicinal use of this plant in the management of inflammation, asthma and allergy.     

抗癌生物活性肽对人乳腺癌nm231细胞的增殖抑制作用

 目的 探讨抗癌生物活性肽（ACBP）对人乳腺癌 nm231细胞的作用及其机制。 方法 nm231 细胞经不同浓度（0.05、0.10、0.20、 0.25 µg/ml）ACBP 作用 72 h，以噻唑蓝（MTT）法测定细胞增殖活性。以 0.25 µg/ml 的 ACBP 分别作用于 nm231 细胞 4、6、24、48、72 h，行光镜和透射电镜（作用 48 h 细胞）观察。应用 DNA 凝胶电泳和磷脂结合蛋白 V（Annexin V，AV）/碘化丙啶（PI）双标记染色流式细胞术等方法，观察 0.25 µg/ml 的 ACBP 作用不同时间对nm231 细胞凋亡发生及作用 48 h 对细胞周期的影响。以上各项检测均设以 RPMI 1640 培养液取代 ACBP 的对照组。 结果 浓度为 0.1 µg/ml 的 ACBP 即对 nm231 细胞的增殖有明显抑制作用，抑制率为 10.3%，抑制作用具有浓度依赖性，ACBP 浓度为 0.25 µg/ml 时抑制率达 35.1%。光镜观察显示，经 0.25 µg/ml 的 ACBP 作用 24 h，细胞出现凋亡特征；作用 48 h，光镜及电镜下均可见大量的典型凋亡细胞。DNA 凝胶电泳显示，经 0.25 µg/ml 的ACBP 作用 24 h 的细胞出现 DNA 断裂；作用 48 h 的细胞出现典型的梯形电泳图谱。流式细胞术分析显示，ACBP 作用后AV（+）/ PI（－）细胞和 AV（+）/ PI（+）细胞比例均随培养时间延长而增大；作用 48 h 的 nm231细胞 G0/1 期细胞比例高于对照组（P < 0.01），而细胞增殖指数低于对照组（P < 0.01）。 结论 ACBP 可抑制 nm231 细胞的增殖，其机制与抑制nm231 细胞的 DNA 合成和诱导细胞凋亡相关。