腺病毒介导PTEN基因抗脑胶质瘤作用的实验研究

目的：探讨10号染色体上缺失的磷酸酶和张力蛋白同源物(phosphatase and tensin homology deleted on chromosome ten，PTEN)对人脑胶质瘤细胞的抗增殖作用，包括细胞周期改变、抗血管生成的作用。方法：用含PTEN基因的重组腺病毒载体，体外转染人脑胶质瘤细胞U87，用RTPCR、Western blot检测基因及蛋白的表达。通过细胞生长试验、流式细胞仪分析技术检测转染细胞的增殖、细胞周期的改变，用MTT法检测PTEN对人脐静脉内皮细胞生长的影响，体内实验检测其致瘤能力。结果：RT-PCR、Western blot检测转染AdPTEN病毒后的U87细胞PTEN表达由阴性转阳性，转染后对U87细胞的体外生长有明显抑制作用，使细胞出现G0～G1期阻滞，并抑制其体内肿瘤生长。含PTEN的上清对内皮细胞生长有明显抑制作用。体内实验表明，AdPTEN治疗组可明显抑制肿瘤的生长。结论：重组腺病毒载体介导的PTEN基因在体内外对人脑胶质瘤细胞L187的生长有抑制作用，并抑制肿瘤血管生成，具有显著的抗胶质瘤作用。     

腺病毒介导PTEN基因抗脑胶质瘤作用的实验研究

目的 :探讨 10号染色体上缺失的磷酸酶和张力蛋白同源物 (phosphataseandtensinhomologydeletedonchromo someten ,PTEN )对人脑胶质瘤细胞的抗增殖作用 ,包括细胞周期改变、抗血管生成的作用。方法 :用含PTEN基因的重组腺病毒载体 ,体外转染人脑胶质瘤细胞U87,用RT PCR、Westernblot检测基因及蛋白的表达。通过细胞生长试验、流式细胞仪分析技术检测转染细胞的增殖、细胞周期的改变 ,用MTT法检测PTEN对人脐静脉内皮细胞生长的影响 ,体内实验检测其致瘤能力。结果 :RT PCR、Westernblot检测转染AdPTEN病毒后的U87细胞PTEN表达由阴性转阳性 ,转染后对U87细胞的体外生长有明显抑制作用 ,使细胞出现G0 ～G1期阻滞 ,并抑制其体内肿瘤生长。含PTEN的上清对内皮细胞生长有明显抑制作用。体内实验表明 ,AdPTEN治疗组可明显抑制肿瘤的生长。结论 :重组腺病毒载体介导的PTEN基因在体内外对人脑胶质瘤细胞U87的生长有抑制作用 ,并抑制肿瘤血管生成 ,具有显著的抗胶质瘤作用     

RNasin对脑胶质瘤细胞生长的抑制作用

核糖核酸酶抑制因子（ＲｉｂｏｎｕｃｌｅａｓｅＩｎｈｉｂｉｔｏｒ，ＲＮａｓｉｎ）是体内的一种糖蛋白，对多种实体性肿瘤生长有明显的抑制作用。本文采用从人胎盘组织中提取制备的ＲＮａｓｉｎ，体内、外观察其对人ＳＨＧ４４及大鼠Ｃ６胶质瘤细胞生长的抑制作用。１材...     

雷公藤单体体外抑制胶质细胞的实验研究

目的：研究雷公藤单体对胶质瘤细胞的体外抑制作用。方法：通过MTT法测定3种雷公藤单体（甲素，红素和Wilforol A)对胶质瘤细胞株SHG44、C6、U251的体外抑制作用；应用免疫组化法观察雷公藤甲素与雷公藤红素后SHG44胶质瘤细胞bax、bcl-2蛋白表达的变化。结果：雷公藤二萜类单体雷公藤甲素对胶质瘤细胞有极明显的抑制作用；雷公藤三萜类单体中红素的抑制作用次之，两者均使SHG44细胞bax表达增加，bcl-2表达下降。结论：雷公藤甲素与雷公藤红素对胶质瘤细胞有明显的抑制作用，其作用与促进bax表达、抑制bcl-2表达，导致细胞凋亡有关。     

血管抑素抑制人脑胶质瘤生长及病理学研究

目的：观察人血管抑素（Angiostatin, AS）对小鼠脑胶质瘤皮下移植瘤生长的抑制作用。方法：用MTT法观察AS对血管内皮细胞系ECV304和人脑胶质瘤细胞系TJ905增殖的影响；建立荷G422脑胶质瘤小鼠皮下移植模型，皮下注射AS，计算瘤体比和抑瘤率。结果： (1)AS对ECV304的抑制作用随着剂量的增加而增强，AS对TJ905无影响；(2)动物实验显示，AS剂量为10 mg·kg-1·d-1和50 mg·kg-1·d-1时抑瘤率分别为21.8%和84.3%；(3)免疫组化Ⅷ因子染色表明实验组与对照组之间在血管发育及数量方面均有差别。结论： AS在体外对胶质瘤细胞无抑制作用，在体内能通过抑制血管内皮细胞增殖而抑制胶质瘤的生长。     

血管生成抑制素对C6脑胶质瘤的抑瘤效应

目的探讨不同剂量的血管生成抑制素（Angiostatin）在胶质瘤治疗中的抑制血管生成作用。方法应用不同剂量（100ng、1000ng）的血管生成抑制素分别作用于体外培养的大鼠C6脑胶质瘤细胞系和C6/SD大鼠脑胶质瘤模型,分别计算细胞存活率和抑瘤生成率,SABC免疫组织化学技术检测体内胶质瘤中的微血管密度。结果不同剂量的血管生成抑制素均对体外培养的胶质瘤细胞的生长不产生明显抑制作用;对体内生长的胶质瘤则有显著抑制作用,而且较高剂量的Angiostatin的抑制作用更为明显,抑瘤率最高达65.3％（P<0.01）。经不同剂量的血管生成抑制素处理的脑胶质瘤中微血管密度较对照组降低（P<0.01）。结论血管生成抑制素能抑制C6脑胶质瘤的血管生成,且较高剂量实验组的作用更为明显,此研究对血管生成抑制素抑制C6胶质瘤的生长机制研究奠定了一定基础。     

IFN-α和TGF-β1对卵巢癌细胞SKOV3端粒酶活性的影响

目的：观察IFN－α和TGF－β1对卵巢癌细胞系SKOV3端粒酶活性和凋亡的影响，探讨它们在肿瘤治疗中的作用机制。方法：用IFN－α和KTGF－β1分别处理SKOV3细胞，然后采用端粒酶PCR ELISA方法检测不同处理时间细胞的端粒酶活性表达，同时利用流式细胞仪观察处理前后SKOV3细胞的凋亡情况。用裸鼠体内成瘤实验及免疫组化，检测转染TGF－β1基因对SKOV3细胞生长及端粒酶活性的影响。结果：IFN－α对SKOV3细胞生长有抑制作用，可明显下调SKOV3细胞端粒酶活性；而TGF－β1作用则相反，在体内外对SKOV3细胞生长均有促进作用，可明显上调SKOV3细胞端粒酶活性；IFN－α和TGF－β1均不诱导SKOV3细胞发生凋亡。结论：抑制端粒酶活性是IFN－α抗卵巢癌治疗的一个重要机制；TGF－β1在体内外对SKOV3细胞生长均有促进作用。     

TRAIL真核表达治疗肝细胞癌作用的研究

目的：构建鼠源TRAIL（mTRAIL）真核表达质粒。研究其体内、外表达对肝癌细胞的诱导凋亡作用，抑制肝癌生长作用及与重组人FN多肽真核表达质粒pCH510协同抑制肝癌生长作用。方法：采用RTPCR及重组DNA技术构建mTRAIL真核表达质粒；体内、外进行基因转染；用流式细胞仪检测肝癌细胞凋亡率，用TdTmediated dUTP nick end labeling (TUNEL)法及免疫组织化学技术检测肝癌细胞凋亡；小鼠实体瘤块模型研究基因转染抑制肝癌生长作用。结果：用RTPCR方法自小鼠脾细胞RNA扩增出TRAIL基因的全长cDNA，并克隆至真核表达载体pcDNA3.1中获重组质粒pX1；以pX1转染BHK细胞株后攻击肝癌细胞株H22细胞，可检测到肝癌细胞凋亡；肌肉内注射转染质粒DNA pX1，通过诱导肿瘤细胞凋亡抑制肝癌生长；pX1与FN多肽真核表达质粒pCH510有协同抑制肝癌生长作用。结论：质粒pX1可在细胞及小鼠体内表达；体内、外表达可诱导肝癌细胞凋亡并可通过该机制抑制肝癌生长；与FN多肽真核表达质粒pCH510有协同抑制肝癌生长作用。     

人突变p27基因对大肠癌细胞SW480生长的影响

目的：观察人突变p27基因（p27mt）对人大肠癌细胞生长的影响，探讨p27mt基因在大肠癌基因治疗中的作用机制。方法：以携带突变p27基因复制缺陷型腺病毒（Ad—p27mt）为载体，转染大肠癌细胞SW480；用Western blot方法检测p27mt蛋白的表达；细胞计数法检测p27mt对SW480细胞生长的抑制作用；用流式细胞仪检测细胞周期：用DNA片段分析法检测细胞凋亡。结果：通过免疫印迹分析Ad—p27mt转染SW480细胞后，p27在细胞中出现了蛋白高表达。PI染色流式细胞仪检测77．96％的细胞阻滞于G0/G1期，而Ad—LacZ组及空白对照组分别为27．57％和25．29％；生长曲线显示Ad—p27mt对细胞生长就有明显的抑制作用；DNA片段分析示p27mt基因可诱导细胞的凋亡。结论：p27mt对细胞周期有明显的阻滞作用，主要阻滞于G0／G1期；p27mt基因对细胞的生长抑制机制与诱导细胞凋亡和细胞周期的阻滞有关。     

p53基因对胶质瘤细胞系U251生长抑制作用的研究

背景与目的：肿瘤抑制基因p53是调节多种与细胞周期、凋亡、DNA修复等有关基因表达的转录因子。p53基因在大约30％的胶质瘤中发生突变，在胶质瘤的发生和发展中起重要作用。本文主要探讨野生型p53基因过表达对脑胶质瘤细胞系U251细胞生长抑制的机制。方法：通过p53腺病毒表达载体pAdCMV-p53及空载体pAdCMV-lacZ分别感染U251细胞系，RT-PCR及Westem blot方法检测转染效率；并通过MTT检测生长抑制率、流式细胞仪检测细胞周期及TUNEL检测分析细胞凋亡等指标观察p53基因对U251细胞生长的影响。结果：MOI为100时，野生型p53基因的过表达可引起U251细胞G0、G1期阻滞、诱导U251细胞凋亡以及引起U251细胞生长抑制。结论：p53基因可以通过细胞周期G0、G1期阻滞及诱导细胞凋亡抑制胶质瘤细胞系U251的生长。     

IL-24基因对大鼠胶质瘤细胞生长状况的影响

目的　探讨IL 2 4基因对C6大鼠胶质瘤细胞生长状况的影响。方法　应用逆转录病毒载体 ,将IL 2 4基因导入C6细胞 ,经G4 18筛选后获得表达IL 2 4分子的阳性细胞克隆C6 /IL 2 4 ;用RT PCR方法检测目的基因表达 ;四甲基偶氮唑蓝 (MTT)法检测细胞体外增殖状况 ,流式细胞技术检测细胞的增殖活性 ,并制作荷瘤动物模型 ,观察C6 /IL 2 4和C6细胞的体内致瘤性。结果　RT PCR检测表明 ,外源IL 2 4基因于mRNA水平在C6 /IL 2 4细胞已获得稳定表达。C6 /IL 2 4细胞系的体外增殖性较亲代C6细胞明显下降 ,流式细胞术检测其细胞增殖指数 (PI)为 (2 9.71± 0 .89) %。 9只接种C6 /IL 2 4细胞的实验组大鼠中 ,6只颅内成瘤 ,肿瘤体积为 (14 .0 8± 9.81)mm3 ,明显小于接种C6细胞大鼠的肿瘤体积 (P <0 .0 5 )。结论　外源性IL 2 4基因可部分抑制胶质瘤细胞异常增殖的肿瘤特性。     

TAM对C6细胞生长与侵袭能力的影响

目的:通过观察TAM对体外培养C6细胞生长状况、迁移能力与MMP-2蛋白表达的影响,为TAM用于胶质瘤的治疗提供理论依据。方法:观察不同剂量TAM对大鼠C6胶质瘤细胞生长曲线、细胞倍增时间及细胞形态的影响,研究TAM对C6细胞的生长抑制作用;细胞划痕法检测不同剂量TAM对C6细胞迁移能力的影响;FITC标记的可激活穿膜肽检测不同剂量TAM对C6细胞MMP-2蛋白表达的影响。结果:TAM显著抑制C6胶质瘤细胞的生长,且存在剂量、时间依赖关系;TAM显著抑制C6细胞迁移,存在剂量依赖关系;TAM对C6细胞活性MMP-2蛋白的表达未见明显影响。结论:虽然TAM不能抑制C6细胞MMP-2蛋白的表达,但可显著抑制其生长与迁移,因此有望用于胶质瘤的化学治疗。     

Sodium phenylacetate (NaPa) improves the TAM effect on glioblastoma experimental tumors by inducing cell growth arrest and apoptosis

BACKGROUND: Multiform glioblastomas represent the most aggressive brain tumors. Here, the cooperative effects of sodium phenylacetate (NaPa) and/or tamoxifen (TAM) on CNS1 and 9L glioblastoma cell lines in vitro and in an experimental animal tumor model were investigated. MATERIALS AND METHODS: The drug effects on cell cycle and apoptosis were investigated by flow cytometry. CNS1 cells were implanted subcutaneously in nude mice to form tumors which were then treated with NaPa, TAM or NaPa/TAM. RESULTS: A significant inhibitory effect of NaPa on the two glioma cell lines (LD50 of 10 mM) was observed. 10(-5) M of TAM inhibited approximately 35% of 9L cell growth, and 90% of CNS1 cell growth. When a combination of both drugs included 10(-9) M of TAM, inhibition of about 50% of 9L cell growth and 75% of CNS1 cell growth occurred. The NaPa/TAM combined treatment increased the number of G0/G1 arrested cells and apoptotic cells as compared to treatments with NaPa or TAM alone. Inhibition of CNS1 tumor growth were observed after a two week treatment with NaPa (32 mg/kg/day) or TAM (6 mg/kg/day). CONCLUSION: These results showed a synergistic effect between these two drugs on tumor cell proliferation, caused by cell cycle arrest in the G0/G1 phase and by induction of apoptosis.     

单用TAM及联合应用5-FU对结肠癌细胞株生长抑制作用及增殖凋亡的影响

目的探讨他莫昔芬(tamoxifen,TAM)单独或联合5-FU化疗对结肠癌细胞株生长抑制作用及凋亡的影响.方法应用MTT法,比较单纯应用TAM和TAM与5-FU联合对SW480、HT29细胞株生长抑制作用,通过药物量-效关系曲线,观察TAM有无化疗增敏作用,同时检测PCNA和AI,探讨TAM联合5-FU对增殖、凋亡的影响.结果单独应用TAM对SW480、HT29细胞株无生长抑制作用;TAM联合5-FU在一定浓度时可抑制SW480和HT29细胞的生长,提高对5-FU的敏感性,起到化疗增敏作用.通过对PCNA和AI 2个指标的检测,随着浓度的增加和时间的延长,TAM联合5-FU抑制细胞的增殖和促进细胞的凋亡均呈上升趋势.结论一定浓度的TAM能增加人结肠癌细胞株SW480、HT29对化疗药物5-FU的敏感性,抑制细胞增殖,促进细胞凋亡.     

Combined antitumor activity of 7-Hydroxystaurosporine (UCN-01) and Tamoxifen against human breast carcinoma<Emphasis Type="Italic">in Vitro</Emphasis> and<Emphasis Type="Italic">in Vivo</Emphasis>

BACKGROUND: 7-Hydroxystaurosporine (UCN-01) was originally isolated as a protein kinase C inhibitor and has shown antitumor activity against several human cancer cell lines. UCN-01 inhibits cell cycle progression from the G1 to the S phase and is associated with inhibition of cyclin-dependent kinase (CDK) activity and induction of intrinsic CDK inhibitor p21, leading to dephosphorylation of retinoblastoma (Rb) protein. Tamoxifen (TAM) traps cancer cells in the G1 phase, suggesting that the mechanism of action of TAM is similar to that of UCN-01. The present study was conducted to assess the antitumor activity of UCN-01 combined with TAM against human breast carcinoma cells in vitro and in vivo. MATERIALS AND METHODS: MCF-7 cells were treated with UCN-01, TAM, or UCN-01 combined with TAM at various concentrations in vitro. The antitumor effect was evaluated as the inhibition rate (I.R.%) by MTT assay. Two human breast carcinoma xenografts in nude mice, MCF-7 and Br-10, were treated with UCN-01, TAM or both agents together. The expression of p21 and the phosphorylation status of Rb protein in MCF-7 cells were detected by Western blotting. RESULTS: UCN-01 or TAM alone inhibited the proliferation of MCF-7 cells in a concentration-dependent manner. Combined treatment with UCN-01 followed by TAM inhibited the growth of MCF-7 cells synergistically and no significant differences in cytotoxicity were observed between the different sequences of UCN-01/TAM and TAM/UCN-01. Combination treatment with UCN-01 and TAM against MCF-7 and Br-10 in vivo exhibited superior antitumor effects compared with either agent treatment alone. Although 0.1 microg UCN-01 per ml (I.R.: 48.1%) or 2 microM TAM (I.R.: 31%) induced p21 expression, phosphorylation of Rb protein was not inhibited. However, combination treatment with UCN-01 and TAM at the same concentrations resulted in an I.R. of 67% and dephosphorylation of Rb protein. CONCLUSION: The present study suggests that combining UCN-01 and TAM could result in augmented cytotoxicity because of their similar mechanism of action. This combination may have potential clinical applications for breast cancer treatment, by reducing the toxicity of UCN-01.     

Effect of tamoxifen on DNA synthesis and proliferation of human malignant glioma lines in vitro

Previous studies in our laboratory have shown that proliferation of human malignant gliomas in vitro depends in part upon the activation of protein kinase C (PKC) and, conversely, can be blocked by inhibitors of PKC. Here, we examined the effect of tamoxifen, a known PKC inhibitor, on DNA synthesis and proliferation of an established human glioma line (U138) and two low passage cultures of explanted human glioblastomas. Tamoxifen produced a profound, dose-dependent inhibition of both [3H] thymidine incorporation and cell proliferation, with a 50% effective dose of 20 ng/ml under serum-free conditions and 50 to 200 ng/ml in the presence of 10% serum. These tumors were estrogen receptor negative and showed no mitogenic response to estradiol. Furthermore, concentrations of estradiol as high as 10 micrograms/ml had no effect on the tamoxifen-induced inhibition. This suggests that the mechanism of growth inhibition by tamoxifen in these gliomas did not involve an estrogen receptor-mediated process but may instead result from its inhibition of PKC. In view of the profound effect of tamoxifen on cultured gliomas at concentrations that can safely be achieved therapeutically, further in vitro and in vivo studies of this agent are warranted.     

VEGF抗体治疗胶质瘤的实验研究

目的 以血管内皮生长因子（ｖａｓｃｕｌａｒ ｅｎｄｏｔｈｅｌｉａｌ ｇｒｏｗｔｈｆａｃｔｏｒ,ＶＥＧＦ）抗体进行抗血管生成治疗胶原瘤的研究。方法 应用ＶＥＧＦ抗体分别作用于Ｃ６胶原瘤细胞系的体外培养细胞和动物模型,计算细胞存活率及抑溜率,ＡＢＣ免疫组化技术检测体内生长胶质瘤中微血管密度。结果 ＶＥＧＦ抗体不抑制体外培养细胞生长,细胞存活率为１００％。对体内生长的胶质瘤则有显著抑制作用且呈剂量依赖关     

Effects of Genistein and Synergistic Action in Combination with Tamoxifen on the HepG2 Human Hepatocellular Carcinoma Cell Line

Introduction: The flavonoids comprise a diverse group of polyphenolic compounds with antioxidant activity that is present in edible plants like soybeans and soy products. In vivo studies have concentrated on the effects of flavonoids on cancer and genistein (GE), a soy-derived isoflavone, has been reported to reduce prostate, colon, hepatic and breast adenocarcinoma risk. Tamoxifen (TAM) is an important drug for cancer treatment worldwide, which can induce apoptosis in various cancers, including examples in the liver, breast and ovaries. The aim of the present study was to evaluate the effects of GE and TAM, alone and in combination, on proliferation and apoptosis in the human hepatocellular carcinoma (HCC) HepG2 cell line. Materials and Methods: HepG 2 cells were treated with GE, TAM and GE/TAM and then MTT and flow cytometry assays were conducted to determine effects on viability and apoptosis, respectively. Results: GE and TAM inhibited cell proliferation and induced apoptosis in the HepG 2 cell lines. Discussion: Our findings clearly indicated that GE and TAM may exert inhibitory and apoptotic effects in liver cancer cells. Conclusion: GE and TAM can significantly inhibit growth of HCC cells and play a significant role in apoptosis.     

鸦胆子油乳注射液对C6胶质瘤细胞作用的实验研究

背景与目的:鸦胆子油乳注射液在临床上用于许多肿瘤的治疗,然而其确切的分子机制目前尚不完全清楚。本研究探讨鸦胆子油乳注射液对C6胶质瘤细胞增殖作用的影响。方法:体外培养大鼠C6胶质瘤细胞,用MTT比色法检测鸦胆子油乳注射液对C6胶质瘤细胞的抑制作用。用免疫组化检测鸦胆子油乳注射液对C6胶质瘤细胞caspase-3表达的影响。结果:MTT比色法显示鸦胆子油乳注射液对C6胶质瘤细胞的抑制率呈剂量依赖性。免疫组化显示随鸦胆子油乳注射液浓度增加,caspase-3表达明显增多。结论:体外鸦胆子油乳注射液能增加C6胶质瘤细胞凋亡基因caspase-3表达,抑制细胞增殖。