浙江蝮蛇蛇毒成分的分离及其对K562细胞的增殖抑制作用

[目的]分离纯化浙江蝮蛇蛇毒主要组分并分析其对抑制白血病细胞株K562的细胞增殖作用。[方法]采用DEAE-Cellulose、CM-Sephadex C-50和Sephadex G-75柱层析,以离子交换和凝胶过滤法从蝮蛇蛇毒中分离纯化主要组分;SDS-PAGE检测其纯度和估计其分子量;MTT及FCM方法检测蛇毒主要组分对白血病细胞的增殖抑制。[结果]从浙江蝮蛇蛇毒中分离纯化得到一分子量约为30kD组分,该组分对白血病细胞具有较强的细胞增殖抑制作用,且呈剂量依赖性关系,5.0、10.0mmol/L浓度时对K562细胞增殖抑制率分别达26.32%和32.28%,并使细胞阻滞于G2/M期。[结论]浙江蝮蛇蛇毒可抑制白血病细胞的增殖,其有较好的应用前景。     

8-氯腺苷诱导多种肿瘤细胞凋亡

目的研究８－氯腺苷（８ＣＡ）对肿瘤细胞生长和凋亡的作用。方法以不同浓度的８ＣＡ处理在体外培养的肿瘤细胞株,使用ＭＴＴ法测定细胞生长曲线,以光学显微镜、流式细胞仪和ＤＮＡ片段化分析细胞凋亡。结果１０μｍｏｌ／Ｌ８ＣＡ处理４８小时,可显著抑制肿瘤细胞的增殖。ＭＴＴ曲线测定发现,所试细胞对８ＣＡ的敏感程度依次为：ＨＬ－６０白血病细胞系最敏感,ＭＧｃ－８０３胃癌细胞系次之,ＮＩＨ３Ｔ３正常纤维母细胞最不敏感。进一步研究发现,８ＣＡ抑制ＨＬ－６０和ＭＧｃ－８０３肿瘤细胞生长的机制是诱导肿瘤细胞凋亡（ａｐｏｐｔｏｓｉｓ）,肿瘤细胞具有典型的细胞凋亡形态特征,基因组ＤＮＡ发生断裂,并在琼脂糖凝胶中呈现梯形图谱,流式细胞仪分析可检出凋亡峰。结论８ＣＡ可作为细胞凋亡诱导剂用于肿瘤治疗。     

LGI1基因对胶质瘤细胞作用的研究

目的:研究富亮氨酸胶质瘤失活基因1(LGI1)与胶质瘤细胞生长及胶质瘤细胞凋亡的关系。方法:构建pcDNA3.1/LGI1质粒,采用脂质体介导的方法转染LGI1缺失的胶质瘤细胞系。RT-PCR法和免疫组化法检测转染细胞的LGI1基因表达,MTT法检测转染细胞的增殖活性,AnnevinV-FITC法检测转染细胞的凋亡。结果:pcDNA3.1/LGI1转染胶质瘤细胞系A172后,A172细胞LGI1mRNA和蛋白表达阳性;细胞增殖活性受到抑制,MTT吸收值在转染后24和48小时下降,与正常对照组相比有统计学意义(P<0.05);转染后24小时早期凋亡细胞较多,48小时后早期凋亡减少。结论:LGI1基因可抑制肿瘤细胞的增殖,诱导细胞凋亡。     

人端粒酶逆转录酶(hTERT)特异性siRNA对乳腺癌细胞hTERT水平及细胞生长影响的研究

背景与目的:在肿瘤细胞中端粒酶高水平的表达是肿瘤细胞永生化的重要因素。本研究利用人端粒酶逆转录酶(hTERT)的小干扰RNA(siRNA)特异性地抑制乳腺癌细胞的hTERT,通过研究该效应对细胞生长、凋亡和永生化相关基因的影响,来进一步阐述端粒酶在肿瘤细胞永生化可能的作用。方法:通过实时定量RT-PCR(Real-TimeRT-PCR)测定各乳腺癌细胞系中内源性hTERT的表达水平及测定RNA干扰对与细胞永生化相关基因的调节;通过脂质体转染法将hTERTsiRNA导入MCF7细胞;通过细胞生长曲线和流式细胞仪(FACS)评价该特异性的siRNA所诱导的hTERT水平下调对细胞生长和凋亡的影响。结果:所检测的乳腺癌细胞系中,hTERT均相对高表达。hTERTsiRNA可抑制MCF7中hTERT的水平,并在抑制后的第4天开始表现出显著的细胞生长抑制,并出现凋亡,以及多个细胞永生化相关的基因,如RAC1、PCYT2、FDFT1和ATP5G2,表达水平均下调50%以上。结论:hTERTsiRNA可特异性地抑制hTERTmRNA水平,从而抑制细胞生长,导致细胞凋亡,并下调多个细胞永生化基因。     

腺病毒介导野生型p53基因对人白血病细胞系HL—60,K562生长的抑制作用

目的 探索肿瘤抑制基因Ｐ５２对人白血病细胞系的生长抑制作用和促凋亡作用。方法 选用含野生型ｐ５３基因的重组腺病毒载体，体外感染人白血病细胞系ＨＬ－６０，Ｋ５６２。通过细胞生长曲线，ＤＮＡ检测及流式细胞仪分析检测转染细胞的增殖受抑制和细胞凋亡的发生。结果 ＰＣＲ检测转染后ＨＬ－６０和Ｋ５６２细胞ｐ５３ ｃＤＮＡ表达。     

白花蛇舌草对U14宫颈癌抗肿瘤作用的实验研究

目的 探讨白花蛇舌草(HD)对U14宫颈癌细胞移植瘤的抑制作用及可能的分子生物学机制.方法 建立裸鼠U14宫颈癌模型,用不同剂量白花蛇舌草进行治疗,并以顺铂治疗组为阳性对照,通过HE染色观察肿瘤组织病理变化,流式细胞仪检测肿瘤细胞凋亡情况,采用PCR-TRAR-ELISA方法定量检测细胞端粒酶活性水平.结果 HD对U14宫颈癌细胞移植瘤生长有明显抑制作用,并可诱导U14细胞凋亡,经中、高剂量的HD治疗后U14细胞端粒酶活性明显下降,与阴性对照组比较差异显著(P＜0.01),与阳性对照组比较无明显差异(p＞0.05).结论 一定剂量HD具有诱导U14宫颈癌细胞凋亡和抑制肿瘤细胞端粒酶活性的作用,这可能是其抑制肿瘤生长的重要分子生物学机制.     

鲨鱼软骨制剂对三种细胞系增殖抑制作用的比较

目的比较鲨鱼软骨制剂(SCP)对人结肠高分化腺癌细胞(THC-8908)、人红白血病细胞(K562)和人乳腺癌细胞(MCF-7)的生长抑制作用,探讨其抗癌作用机制.方法采用MTT法检测不同浓度SCP对体外培养的THC-8908细胞、K562细胞和MCF-7细胞的细胞毒作用.Hoechst33342/PI荧光双染色方法检测细胞凋亡.结果SCP明显抑制THC-8908、K562和MCF-7三种细胞系的生长,其IC50值分别为1mg/m1、0.7mg/ml和1.5mg/ml.凋亡细胞比例在用药后48h分别达78.85%、44.55%和63.32%.结论SCP能直接抑制肿瘤细胞生长.     

泰素帝诱导人肺腺癌细胞凋亡的实验研究

目的 探讨泰素帝诱导人肺腺癌细胞凋亡的作用机制。方法 应用形态学方法、流式细胞术（ＦＡＣＳ）、ＤＮＡ凝胶电泳、ＡｎｎｅｘｉｎＶ标记等方法检测细胞凋亡的发生,应用ＲＴ－ＰＣＲ检测凋亡相关基因Ｆａｓ、ｂｃｌ－２、ｃ－ｍｙｃ表达的变化。结果 泰素帝对人肺腺癌细胞系Ａ５４９细胞的生长有明显的抑制作用,使细胞分裂阻滞于Ｇ２Ｍ期,并诱导肿瘤细胞发生凋亡。凋亡细胞表现为细胞固缩,核染色质聚集或断裂,ＤＮＡ凝胶电     

雄黄抑制卵巢癌细胞株COC1增殖和诱导凋亡的体外研究

目的:研究雄黄对卵巢癌细胞株COC1的抑制增殖及诱导凋亡作用。方法:用不同浓度的雄黄溶液作用于人卵巢癌细胞株COC1,于不同时间点收集细胞,MTT法检测肿瘤细胞生长抑制率;流式细胞仪检测细胞凋亡率及分析细胞周期的变化;原位末端标记法(TUNEL)观察细胞凋亡形态学特征。结果:不同浓度雄黄溶液对COC1细胞有不同程度的生长抑制作用。其生长抑制率具有浓度和时间依赖性。并有明显周期特异性生长抑制作用。结论:雄黄溶液对于COC1细胞具有诱导凋亡、抑制增殖的作用,诱导肿瘤细胞发生G1期阻滞是雄黄抑制卵巢癌细胞生长作用的可能机制之一。     

单纯加温与加温联用顺铂对人卵巢癌细胞增殖及凋亡的影响

目的探讨单纯加温与联用顺铂(DDP)对卵巢癌细胞增殖及凋亡的影响.方法采用MTT法检测加温及加温联合DDP对卵巢癌DDP敏感细胞系skov3和DDP耐药细胞系SK-OV-3细胞增殖的抑制效应,分析加温与DDP在抑制卵巢癌细胞增殖中的相互作用;采用TUNEL和流式细胞术分析加温及加温联合DDP对卵巢癌细胞周期和凋亡的影响,探讨热化疗的作用机制.结果 42℃以上加温可显著抑制卵巢癌DDP敏感和耐药细胞系细胞增殖(P<0.001),且与加热温度和时间之间存在剂量效应关系.加热与DDP在抑制卵巢癌细胞增殖上表现为相加或协同作用,在skov3细胞系以相加作用(0.85≤q≤1.15)为主,而在SK-OV-3细胞系则主要表现为协同作用(q>1.15).加温可诱导卵巢癌细胞凋亡,并降低S期细胞比率和增加G0/G1期细胞比率.加温与DDP在诱导skov3细胞凋亡上表现为相加作用(0.85≤q≤1.15).41～42℃、30～90分钟加温与2.5 μg/ml DDP可协同诱导SK-OV-3细胞凋亡(q>1.15).结论热诱导肿瘤细胞凋亡并将细胞阻滞于G0/G1期是热疗杀伤卵巢癌细胞的重要机制.加温与DDP在诱导卵巢癌细胞凋亡和抑制细胞增殖等作用上具有相加或协同效应.     

Detection of circulating hepatoma D23 antigen and immune complexes in tumour bearer serum

Serum from rats bearing progressively growing aminoazo dye-induced rat hepatomata has been fractionated by Sephadex G150 gel filtration chromatography and isolated fractions have been examined by indirect membrane immunofluorescence techniques to detect tumour specific antigen and antibody. Hepatoma D23-specific antigenic activity was associated with material (of approximate molecular weight <150,000) isolated in the included volume of the gel at pH 7·3. The fraction excluded from the gel (of approximate molecular weight >150,000) was adjusted to pH 3·0 and further separated by Sephadex G150 gel filtration chromatography at pH 3·0 into gel included and excluded fractions. Hepatoma D23 specific antibody, demonstrable by membrane immunofluorescence staining of hepatoma D23 cells, was found to be eluted in the excluded volume and specific antigenic activity was retarded into the included volume of the gel. These results indicate that hepatoma D23 bearer serum contains free circulating tumour specific antigen in excess, together with specific immune complexes. The presence of these factors in tumour bearer serum is discussed in terms of “blocking” phenomena whereby serum factors may protect tumour cells from sensitized lymphocyte cytotoxic attack.     

Studies on a naturally occurring human antibody active against mouse Landschutz ascites tumor cells. II. Identification of the antibody as IgM and its partial purification

1. The cytotoxic antibody in normal human serum, active against mouse tumor cells, was identified as a 19S macroglobulin by means of Sephadex gel filtration, DEAE-cellulose chromatography and density-gradient centrifugation procedures. 2. A purified IgM fraction was isolated from normal human serum. The cytotoxic activity of this fraction, as tested against mouse tumor cells, was enriched 20–80 times over that of the unfractionated serum. The purified IgM showed mainly one precipitin line on immunoelectrophoretic analysis. In the ultracentrifuge the 19S peak comprised about 85% of the total protein.     

Human thymus-leukemia-associated antigen: isolation and partial characterization.

Human thymus-leukemia-associated antigen (HThy-L), a saline-soluble antigen, was previously detected by immunodiffusion (but not on the cell surface) in significant quantity in extracts of normal thymocytes, cells from cultured T-cell lines, and erythrocyte-rosette-positive leukemia blasts. Two species of HThy-L were identified and isolated from normal human thymus tissue after extraction in tris buffer, ammonium sulfate fractionation, acid precipitation of inactive fractions, DEAE-cellulose (DE-52) chromatography, Sephadex G-100 gel filtration, and carboxymethyl-cellulose (CM-52) chromatography; On Sephadex G-100, both HThy-L species had a similar molecular weight (40,000--50,000), but they eluted in different positions on DE-52 and CM-52. Analysis by sodium dodecyl sulfate polyacrylamide gel electrophoresis showed that each of the 2 HThy-L species contained 2 components with molecular weights of approximately 43,000 and 23,000. Further purification of HThy-L on Sephadex G-50 showed that the 43,000-dalton component possessed HThy-L activity.     

Egg lectin of Rana japonica and its receptor glycoprotein of Ehrlich tumor cells

Egg lectin of Rana japonica, which specifically agglutinates transformed cells but does not agglutinate nontransformed cells and erythrocytes, has been isolated by gel filtration and successive ion-exchange chromatographies on diethylaminoethyl cellulose and carboxymethylcellulose columns and has been characterized as a homogeneous carbohydrate-free protein with a relative molecular weight of 13,500. The lectin, at a concentration of 1 microgram/0.1 ml, causes obvious cytoagglutination of various transformed and tumor cell. The receptor of the Erlich ascites tumor cells which inhibits the lectin-induced agglutination of the Ehrlich ascites tumor cells has been isolated and characterized. The receptor was solubilized from Ehrlich ascites carcinoma cells by treating a tumor cell suspension with insolubilized trypsin, and the solubilized receptor was isolated by gel filtration through Sephadex G-100, followed by ion-exchange chromatography on diethylaminoethyl cellulose. The receptor was identified as a homogeneous glycoprotein having about 25% carbohydrate. The receptor, at a concentration of 4 microgram/0.1 ml, completely inhibited the cytoagglutination of the Ehrlich carcinoma cells caused by three agglutination doses (about 3 microgram/0.1 ml) of the R. japonica lectin.     

Differential sensitivity to Ad5 E1B-21kD and Bcl-2 proteins of apoptin-induced versus p53-induced apoptosis

Apoptin, a small protein derived from chicken anemia virus (CAV),induces apoptosis in human tumor cell lines regardless of whetherthese express p53 or not. We examined whether the small adenovirus5 E1B protein of 21 kDa (E1B-21KD, also called E1B-19kD) andBcl-2 could inhibit apoptin-induced apoptosis in human tumorcell lines and compared this with p53-induced apoptosis. E1B-21kD,but not Bcl-2, was found to inhibit apoptin-induced apoptosisin the osteosarcoma cell lines U2OS and Saos-2. However, neitherexpression of E1B-21kD nor of Bcl-2 resulted in inhibition ofapoptin-induced apoptosis in Hep3B hepatoma cells and kidneyrhabdoid tumor G401 cells. Both Bcl-2 and Ad5 E1B-21kD wereable to inhibit p53-induced apoptosis in the human tumor celllines Saos-2 and Hep3B. In Saos-2 and U2OS, but not in Hep3Band G401, expression of E1B-21kD leads to retention of apoptinin the cytoplasm, in that way preventing its nuclear function.These results indicate that proteins inhibiting the p53-inducedapoptotic pathway do not block apoptin-induced apoptosis ordo so only in a cell type-specific manner. The apoptin-inducedapoptotic pathway is distinct from that induced by p53 and,therefore, apoptin is a potential antitumor agent.     

A tumour-associated antigen from the pleural effusion of patients with squamous-cell carcinoma of lung

A fraction showing tumour-associated antigenic properties has been isolated from pleural effusions of patients with squamous-cell carcinoma of the lung. Purification of the material was accomplished by ion-exchange and affinity chromatography, and by immunoabsorbents. The antigenic activity was monitored by its inhibitory capacity in a specific complement-dependent cytotoxic system. The final fraction has a mol. wt. of approximately 1.7 X 10(5), as judged by gel filtration on Sephadex G200, and the main component appears to be a glycoprotein with N-acetyl-D-glucosamine groups. The most purified antigen preparation exhibited a highly selective capacity to inhibit in the cytotoxic assay and to bind, when labelled with 125I, to 2 specific antisera. The active fractions isolated from pleural effusions fully crossreacted with fractions prepared from squamous-cell carcinoma extracts. CEA and bacterial antigens were not detected in the material, and the presence of alpha-fetoprotein, HLA and blood-group antigens may be ruled out on account of their respective molecular weights.     

An orally active Amazonian plant extract (BIRM) inhibits prostate cancer growth and metastasis

PURPOSE: Poor efficacy of conventional chemotherapeutic drugs against metastatic hormone-refractory prostate cancer (CaP) drives patients to try "alternative medicine". The antitumor activity of one such agent, "BIRM" (biological immune response modulator; "Simple Ecuadorian Oral Solution: an extract of an Amazonian plant"), was characterized in vitro and in vivo using established CaP cell lines and a tumor model. METHODS: The cytotoxicity of BIRM in four human and one rat CaP cell line was evaluated using cell proliferation-inhibition and clonogenic survival assays. BIRM-induced apoptosis, alterations in cell cycle phase progression and inhibition of the extracellular matrix-degrading enzyme hyaluronidase were also investigated in these cells. The in vivo efficacy of BIRM was evaluated in rats with subcutaneous tumor implants of Dunning EGFP-MAT LyLu cells. The active species in BIRM were characterized by gel filtration chromatography. RESULTS: BIRM inhibited cell proliferation and clonogenic growth of the CaP cells (IC(50) about 8.0 microl/ml). It increased cell accumulation in the G(0)/G(1) phase by 33.8% and decreased the proportion of cells in S phase by 54.6%. Apoptotic cell death in BIRM-treated cells was associated with activation of cell death-associated caspases. BIRM inhibited the activity of hyaluronidase, a hyaluronic acid-degrading enzyme, at 1 microl/ml. Treatment of MAT LyLu tumor-bearing rats with BIRM by oral gavage resulted in a significant decrease in tumor incidence (50%), tumor growth rate (18.6+/-1.3 days for 1 cc tumor growth in control rats and 25.7+/-2.6 days in BIRM-treated rats), and only one out of six BIRM-treated rats versus four out of six in the control group developed lung metastasis. Three active ingredients in BIRM with a relative molecular mass (Mr) of >or=3500 were identified by ultracentrifugation and gel filtration chromatography and were found to be resistant to proteinase and heat (100 degrees C). CONCLUSION: The plant extract BIRM contains antitumor compounds of Mr >or=3500 with potent antiproliferative activity in vitro and in vivo against prostate cancer cells.     

Preparation of colony stimulating factors from human placental conditioned medium

Human placental conditioned medium is able to stimulate human bone marrow cells to form neutrophilic granulocyte-monocyte colonies and eosinophil colonies in agar cultures. Methods are described for preparing highly active preparations (capable of supramaximal stimulation of colony formation) by partial purification of the conditioned medium using calcium phosphate absorption and gel filtration chromatography. Partially purified calcium phosphate gel eluates appear to increase in specific activity after concentration by ultrafiltration. This phenomenon was not observed with the active fractions after further purification on Sephadex G-100. Fractionation of the conditioned medium using gel filtration and hydrophobic chromatography (Cibacron Blue-Sepharose) showed that distinct and partially separable factors were responsible for stimulating granulocyte-macrophage and eosinophil colony formation. From the gel filtration data the apparent molecular weight of the eosinophil colony stimulating factor was higher than the molecular weight of the granulocyte-macrophage colony stimulating factor. Cibacron Blue-Sepharose chromatography led to the separation of one molecular species of granulocyte-macrophage colony stimulating factor from eosinophil colony stimulating factor so that a specific stimulus for human neutrophilic granulocyte and/or monocyte-macrophage progenitors is now available.     

Identification and purification of a human lung tumor-associated antigen from a primary lung tumor

A human lung tumor-associated protein has been purified from an extract of a human small cell carcinoma of the lung and shown by Ouchterlony double diffusion analysis to be antigenically identical to a component which was previously demonstrated in 84 of 98 lung tumor extracts of all histological types but absent from extracts of normal adult and fetal lung, other normal tissues, and tumors of other organs. These studies utilized xenoantisera raised against a pool of lung tumor extracts which were exhaustively adsorbed with normal serum and tissue extracts. A radial immunodiffusion assay developed for the antigen permitted its quantitation throughout the course of isolation. Purification was accomplished by ion-exchange chromatography, gel filtration, and affinity immunoadsorption. By ion-exchange chromatography, the proteins appeared to be quite heterogeneous, with immunological reactivity detected in three different peaks. However, all the active components were immunologically identical. Gel filtration of the major antigenic component from diethylaminoethyl cellulose similarly demonstrated a further fractionation into several active, immunologically identical forms. These results suggest a charge-size isomeric relationship among the various forms, all of which possess a common and identical antigenic site. The major component was isolated throughout the purification scheme. The final product represented 9% of the input activity, produced a single, although broad, protein-staining region on 7% polyacrylamide gels which was coincident with antigenic activity, and exhibited immunological identity with the antigen in the crude extract as well as with that in an extract from another lung tumor.     

Isolation and identification of a tumour reducing component from mistletoe extract (Iscador)

Using a combination of gel filtration and paper chromatography, a tumour reducing component from mistletoe extract (Iscador) was isolated and identified to be a peptide of approximate molecular weight 5000. The isolated peptide reduced the solid tumour induced by Dalton's lymphoma ascites tumour cells (DLA cells) in mice. The isolated component was very cytotoxic to the DLA cells but was not cytotoxic to normal lymphocytes, indicating a cell dependent specificity.