太白银莲花皂苷B对胶质瘤细胞生长抑制的研究

目的:太白银莲花皂苷B(Anemone taipaiensis saponin B)是第一次从太白银莲花中经过系统化学分析和分离鉴定的皂苷之一,所以它的生物学效应目前仍然不清楚。在本研究中,我们首次体外研究太白银莲花皂苷B对胶质瘤细胞系的生物学效应,观察它对胶质瘤细胞增殖的的抑制作用。方法:采用四甲基偶氮唑蓝(MTT)法测定太白银莲花皂苷B对胶质瘤细胞生长曲线的影响,Hoechst 33342细胞核染色后荧光显微镜观察,采用光学显微镜观察细胞的形态学变化。结果:MTT实验结果显示太白银莲花皂苷B对胶质瘤细胞U87MG和U251MG有强烈的生长抑制作用,且具有剂量依赖性,应用SPSS18.0统计软件得出太白银莲花皂苷B对U87MG细胞72 h的抑制浓度为IC25=5.2μmol/L,IC50=6.7μmol/L and IC75=8.7μmol/L,U251细胞的抑制浓度为IC25=6.2μmol/L,IC50=7.9μmol/L and IC75=10.5μmol/L。Hoechst 33342细胞核染色荧光显微镜观察以及光学显微镜下细胞形态观察显示出典型的凋亡细胞形态学特征,经过皂苷B处理后,细胞皱缩成圆球形,细胞核碎裂或者致密浓染,向核膜边缘聚集,染色质浓缩为半月状、车轮状或者马蹄状,凋亡小体出现。这些特征在24 h时更明显。结论:体外实验初步显示,太白银莲花皂苷B对U87MG和U251MG细胞具有明显的增殖抑制作用,并具有促凋亡作用。     

紫草素对人脑胶质瘤U251 细胞增殖和凋亡的影响

目的:观察紫草素对体外培养的人脑胶质瘤U251细胞的增殖和凋亡的影响。方法:应用MTT法和Annexin V-FITC/PI双染流式细胞术分别检测2.5、5、10μM/L的紫草素对U251细胞的体外抑杀作用以及凋亡诱导作用,进一步应用Western blot方法检测紫草素对凋亡相关蛋白Bcl-2及Bax表达水平的影响。结果:紫草素对人U251胶质瘤细胞具有明显的增殖抑制作用,且呈一定的剂量依赖性和时间依赖性。紫草素可明显上调U251细胞Bax的表达,下调Bcl-2的表达,与对照组相比存在显著性差异(P0.05)。结论:紫草素对人胶质瘤U251细胞具有明显的抑制增殖和促进凋亡作用。     

姜黄素诱导人脑胶质瘤细胞U251分化的实验研究

目的探讨姜黄素对体外培养的人胶质瘤细胞U251的诱导分化作用及其机制。方法将人脑胶质瘤U251细胞分为对照组和药物组,对照组以含10%小牛血清的DMEM培养基常规培养,药物组用16μmol/L姜黄素（本实验先前的研究结果显示,姜黄素作用于U251细胞48h的半数抑制浓度为16μmol/L）处理。倒置显微镜观察细胞形态学变化;流式细胞仪检测细胞周期变化;免疫细胞化学法检测波形蛋白（vimentin）表达变化;免疫印迹（Westernblot）法检测细胞外调节蛋白激酶（ERK）、磷酸化细胞外调节蛋白激酶（pERK）和胶质纤维酸性蛋白（GFAP）表达变化。结果姜黄素作用96h后,与对照组相比,U251细胞突起明显增多变细长。姜黄素培养48h,S期细胞由28.53%明显降低至20.35%（P=0.015）;vimentin强阳性细胞百分率由90%明显降低至50%（P=0.009）;GFAP蛋白与内参β-actin表达量的比值由0.22明显升高至0.50（P=0.01）。Westernblot法显示,姜黄素分别作用于U251细胞48和96h,ERK蛋白水平无明显改变,pERK/ERK分别为0.35和0.22,较对照组明显减少（P=0.03和0.007）。结论姜黄素对体外培养的胶质瘤U251细胞有显著的诱导分化作用,其机制可能与抑制异常激活的ERK信号通路有关。     

莪术醇对人脑胶质瘤U251细胞增殖及凋亡作用的影响

目的:探讨莪术醇对人脑胶质瘤U251细胞增殖及凋亡作用的影响。方法:采用MTT法和流式细胞仪检测法检测不同浓度下莪术醇(12.5、25、50、100μg/ml)对U251细胞增殖和凋亡作用的影响。结果:MTT结果显示,不同浓度的莪术醇(12.5、25、50、100μg/ml)增殖率明显低于对照组(P0.05);流式细胞仪检测显示,不同浓度莪术醇(12.5、25、50、100μg/ml)的凋亡率较对照组明显升高(P0.05)。结论:莪术醇具有抑制U251细胞增殖和促进U251细胞凋亡的作用。     

白藜芦醇对人胶质瘤细胞迁移能力的影响

目的:探讨白藜芦醇对人胶质瘤U251细胞迁移能力的影响。方法:常规培养人胶质瘤U251细胞,取对数生长期细胞进行实验。采用细胞划痕实验检测经终浓度为50和100μg/ml的白藜芦醇刺激后的人胶质瘤U251细胞的迁移距离。结果:细胞划痕实验结果显示,对照组、50μg/ml白藜芦醇组和100μg/ml白藜芦醇组刺激的U251细胞迁移率分别为(25.7±2)%、(19.8±7)%和(8.3±2)%,均有显著性差异(p0.05)。结论:白藜芦醇具有抑制人胶质瘤U251细胞迁移的作用。     

人参皂苷Rh2对大鼠C6胶质瘤细胞Siah-1、Synaptophysin、MMP9及VEGF表达的影响

摘要 目的：探讨人参皂苷Rh2对大鼠C6胶质瘤细胞Siah-1、突触素（Synaptophysin）、基质金属蛋白酶-9（MMP-9）血管内皮生长因子（VEGF）表达的影响。方法：将大鼠C6胶质瘤细胞分为对照组、人参皂苷Rh2低剂量组（16 μg/mL）、人参皂苷Rh2中剂量组（32 μg/mL）、人参皂苷Rh2高剂量组（48 μg/mL），CCK-8法和平板克隆实验检测细胞增殖；流式细胞术检测细胞凋亡；Transwell检测细胞侵袭；实时荧光定量-聚合酶链式反应（qRT-PCR）检测C6胶质瘤细胞中VEGF、Siah-1、Synaptophysin、MMP-9 mRNA表达；蛋白质印迹法（Western blot）检测C6胶质瘤细胞中VEGF、Siah-1、Synaptophysin、MMP-9蛋白表达。结果：与对照组比较，人参皂苷Rh2低剂量组、人参皂苷Rh2中剂量组、人参皂苷Rh2高剂量组大鼠C6胶质瘤细胞OD₄₅₀值（24 h、48 h）、克隆形成率、细胞侵袭数、VEGF、Synaptophysin、MMP-9 mRNA及蛋白表达降低，细胞凋亡率、Siah-1 mRNA及蛋白表达升高，且呈剂量依赖性（P<0.05）。结论：人参皂苷Rh2可能通过上调Siah-1，下调VEGF、Synaptophysin、MMP-9表达来抑制大鼠C6胶质瘤细胞增殖与侵袭，促进细胞凋亡。     

端粒酶抑制剂对不同非整倍体状态的人结肠癌HCT116细胞增殖及凋亡的影响

该文研究端粒酶在人结肠癌HCT116高非整倍体变异组及低非整倍体变异组细胞中的表达差异及其端粒酶抑制剂3′-叠氮-3′-脱氧胸苷(3′-Azido-3′-deoxythymidine,AZT)对2组细胞增殖及凋亡的影响。取HCT116细胞加入盐酸强力霉素16 h后撤药,称为高非整倍体变异组;另取HCT116细胞不作任何处理,设为对照组,称为低非整倍体变异组;在撤药后第11 d,采用100、250μmol/L的AZT处理2组细胞72 h,并分别设立空白对照组(未加AZT的高非整倍体变异组及低非整倍体变异组)。采用染色体滴定法进行染色体计数。采用Western blot检测MAD2L1、PUMA、BAX、P21、γ-H2AX蛋白质水平。采用荧光定量PCR检测h TERT、PUMA、BAX、NOXA、P21基因表达,端粒酶活性试剂盒检测端粒酶活性,CCK-8法检测细胞生存率。实验结果表明,采用盐酸强力霉素诱导HCT116细胞的非整倍体率可达到77.33%;高非整倍体变异组细胞h TERT基因的表达及端粒酶活性明显高于低非整倍体变异组;加入AZT后,高非整倍体变异组P21、γ-H2AX蛋白质水平上升程度较整倍体明显,低非整倍体变异组PUMA、BAX蛋白质水平上升程度较高非整倍体变异组明显。该研究表明,盐酸强力霉素可以诱导非整倍体形成,高非整倍体变异组的端粒酶活性及h TERT基因表达高于低非整倍体变异组,AZT可以对高非整倍体变异组和低非整倍体变异组细胞产生增殖抑制作用、DNA损伤作用、细胞周期阻滞作用、诱导凋亡作用。     

丹酚酸B通过抑制线粒体功能增加胶质瘤细胞的放疗敏感性

目的:研究药用植物丹参的根茎提取成分丹酚酸B对人胶质瘤U251细胞的放疗增敏作用,并探讨其可能的分子机制。方法:使用1μM浓度的丹酚酸B处理胶质瘤U251细胞,并用等量PBS建立对照组,使用射线照射建立放射治疗模型。MTT法检测细胞活力;流式细胞术检测细胞凋亡;荧光染色检测活性氧ROS含量及线粒体肿胀程度。结果:丹酚酸B能够显著降低射线照射后U251细胞活力,并增加其凋亡(P0.05)。丹酚酸B能够显著增加射线照射后U251细胞活性氧ROS的产生,并增加线粒体的肿胀程度(P0.05)。结论:丹酚酸B能够通过诱导内源性凋亡增加胶质瘤细胞的放疗敏感性,这种作用可能是通过抑制线粒体功能而实现的。     

滇重楼茎叶总皂苷抗肝癌HepG2细胞活性

探究滇重楼茎叶总皂苷对肝癌HepG2细胞增殖的抑制、细胞周期阻滞及诱导细胞凋亡作用。从滇重楼地上茎叶提取总皂苷,配制成浓度为10μg/m L、20μg/m L、40μg/m L、80μg/m L和160μg/m L的总皂苷提取物处理HepG2细胞。总皂苷提取物对细胞增殖的抑制作用采用MTT法检测;对细胞周期阻滞作用采用流式细胞术检测;诱导细胞凋亡的作用采用细胞核荧光染色、流式细胞术和caspase-3活性试剂盒检测。结果表明,滇重楼茎叶总皂苷提取物能显著抑制细胞增殖,且具有时间、剂量依赖效应,能阻滞细胞周期于S期,并能诱导细胞凋亡。但其诱导凋亡作用仅高剂量组(≥80μg/m L)效果显著,低剂量组(80μg/m L)不显著。     

EPA诱导人肝癌细胞SMMC-7721凋亡及端粒酶活性调控机制研究

目的:探究二十碳五烯酸(Eicosa Pentaenoic Acid.EPA)对SMMC-7721人肝癌细胞的凋亡、端粒逆转录酶h TERT的调控作用及端粒酶表达活性的影响。方法:体外培养SMMC-7721人肝癌细胞,用不同浓度的EPA(0μM、25μM、50μM、100μM、200μM)作用于SMMC-7721肝癌细胞(24 h、48 h、72 h)后,显微镜下观察其形态学变化;应用MTT法检测SMMC-7721肝癌细胞细胞增殖变化情况;Western-blot法检测h TERT、Bax、Bcl-2蛋白表达水平变化;Real Time-PCR检测h TERTm RNA的表达变化;ELISA法检测SMMC-7721肝癌细胞端粒酶活性的表达水平。结果:EPA可诱导肝癌细胞SMMC-7721发生细胞凋亡,具有明显的时间计量依赖关系。在此过程中Bcl-2蛋白表达的降低和Bax蛋白表达上调,同时端粒酶逆转录酶h TERT蛋白及其m RNA的表达水平和端粒酶活性均明显降低。结论:抑制端粒酶逆转录酶基因(h TERTm RNA)表达而抑制端粒酶的活性、诱导癌细胞凋亡,可能是EPA的抗癌作用机制之一。     

Honokiol crosses BBB and BCSFB, and inhibits brain tumor growth in rat 9L intracerebral gliosarcoma model and human U251 xenograft glioma model

Background

Gliosarcoma is one of the most common malignant brain tumors, and anti-angiogenesis is a promising approach for the treatment of gliosarcoma. However, chemotherapy is obstructed by the physical obstacle formed by the blood-brain barrier (BBB) and blood-cerebrospinal fluid barrier (BCSFB). Honokiol has been known to possess potent activities in the central nervous system diseases, and anti-angiogenic and anti-tumor properties. Here, we hypothesized that honokiol could cross the BBB and BCSFB for the treatment of gliosarcoma.

Methodologies

We first evaluated the abilities of honokiol to cross the BBB and BCSFB by measuring the penetration of honokiol into brain and blood-cerebrospinal fluid, and compared the honokiol amount taken up by brain with that by other tissues. Then we investigated the effect of honokiol on the growth inhibition of rat 9L gliosarcoma cells and human U251 glioma cells in vitro. Finally we established rat 9L intracerebral gliosarcoma model in Fisher 344 rats and human U251 xenograft glioma model in nude mice to investigate the anti-tumor activity.

Principal Findings

We showed for the first time that honokiol could effectively cross BBB and BCSFB. The ratios of brain/plasma concentration were respectively 1.29, 2.54, 2.56 and 2.72 at 5, 30, 60 and 120 min. And about 10% of honokiol in plasma crossed BCSFB into cerebrospinal fluid (CSF). In vitro, honokiol produced dose-dependent inhibition of the growth of rat 9L gliosarcoma cells and human U251 glioma cells with IC₅₀ of 15.61 µg/mL and 16.38 µg/mL, respectively. In vivo, treatment with 20 mg/kg body weight of honokiol (honokiol was given twice per week for 3 weeks by intravenous injection) resulted in significant reduction of tumor volume (112.70±10.16 mm³) compared with vehicle group (238.63±19.69 mm³, P = 0.000), with 52.77% inhibiting rate in rat 9L intracerebral gliosarcoma model, and (1450.83±348.36 mm³) compared with vehicle group (2914.17±780.52 mm³, P = 0.002), with 50.21% inhibiting rate in human U251 xenograft glioma model. Honokiol also significantly improved the survival over vehicle group in the two models (P<0.05).

Conclusions/Significance

This study provided the first evidence that honokiol could effectively cross BBB and BCSFB and inhibit brain tumor growth in rat 9L intracerebral gliosarcoma model and human U251 xenograft glioma model. It suggested a significant strategy for offering a potential new therapy for the treatment of gliosarcoma.     

Activity of the Human Telomerase Catalytic Subunit (hTERT) Gene Promoter Could Be Increased by the SV40 Enhancer

Telomerase is a ribonucleoprotein complex of which the function is to add telomeric repeats to chromosomal ends. Telomerase consists of two essential components, the telomerase RNA template (hTR) and the catalytic subunit (hTERT). hTERT is expressed only in cells and tissues positive for telomerase activity, i.e., tumor and fetal cells. The aim of this study is to test the increased telomerase promoter activity for cancer gene therapy in adenovirus vector. We cloned the hTERT promoter in place of the SV40 promoter in the pGL3-contol vector to be increased by the SV40 enhancer sequences, resulting in strong expression of luc+ only in telomerase positive cancer cells. Then we transfected the constructed plasmid into a normal human cell line and several cancer cell lines. Through these experiments, we identified the selective and increased expression of the luciferase gene controlled by the hTERT promoter and the SV40 enhancer in the telomerase positive cancer cell lines. To investigate the possibility of utilizing the hTERT promoter and the SV40 enhancer in targeted cancer gene therapy, we constructed an adenovirus vector expressing HSV-TK controlled by the hTERT promoter and the SV40 enhancer for the induction of specific telomerase positive cancer cell death. NSCLC cells infected by Ad-hT-TK-enh were more significantly suppressed and induced apoptosis than those infected by Ad-hT-TK. Telomerase is activated in 80～90% of cancers, so adenovirus with increasing telomerase promoter activity might be used for targeted cancer gene therapy using suicide genes. These results show that the hTERT promoter and the SV40 enhancer might be used for targeted cancer gene therapy.     

Adenovirus-mediated suicide SCLC gene therapy using the increased activity of the hTERT promoter by the MMRE and SV40 enhancer

Telomerase is a ribonucleoprotein complex of which the function is to add telomeric repeats to chromosomal ends. Telomerase consists of two essential components, the telomerase RNA template (hTR) and the catalytic subunit (hTERT). hTERT is expressed only in cells and tissues positive for telomerase activity, i.e., tumor or stem cells. The aim of this study was to use increased telomerase promoter activity in small-cell lung cancer (SCLC) gene therapy. The hTERT promoter and Myc-Max response elements (MMRE) in pGL3-Control vector containing SV40 enhancer resulted in strong expression of the luciferase gene only in telomerase positive and myc overexpressing SCLC cell line but not in normal human cell line. To investigate the possibility of the utilization of the MMRE, hTERT promoter, and SV40 enhancer in targeted SCLC gene therapy, adenovirus vector expressing HSV-TK controlled by the MMRE, hTERT promoter, and SV40 enhancer for the induction of telomerase positive and myc-overexpressing cancer specific cell death was constructed. SCLC cells infected with Ad-MMRE-hT-TK-enh were significantly suppressed and induced apoptosis more than those of Ad-hT-TK or Ad-hT-TK-enh infected cells. Telomerase and c-myc are activated in 60 approximately 80% of SCLC, so the increased activity of telomerase promoter can be used for targeted SCLC gene therapy. These results show that the MMRE, hTERT promoter, and SV40 enhancer can be used in SCLC targeted cancer gene therapy.     

Activity of the human telomerase catalytic subunit (hTERT) gene promoter could be increased by the SV40 enhancer

Telomerase is a ribonucleoprotein complex of which the function is to add telomeric repeats to chromosomal ends. Telomerase consists of two essential components, the telomerase RNA template (hTR) and the catalytic subunit (hTERT). hTERT is expressed only in cells and tissues positive for telomerase activity, i.e., tumor and fetal cells. The aim of this study is to test the increased telomerase promoter activity for cancer gene therapy in adenovirus vector. We cloned the hTERT promoter in place of the SV40 promoter in the pGL3-contol vector to be increased by the SV40 enhancer sequences, resulting in strong expression of luc+ only in telomerase positive cancer cells. Then we transfected the constructed plasmid into a normal human cell line and several cancer cell lines. Through these experiments, we identified the selective and increased expression of the luciferase gene controlled by the hTERT promoter and the SV40 enhancer in the telomerase positive cancer cell lines. To investigate the possibility of utilizing the hTERT promoter and the SV40 enhancer in targeted cancer gene therapy, we constructed an adenovirus vector expressing HSV-TK controlled by the hTERT promoter and the SV40 enhancer for the induction of specific telomerase positive cancer cell death. NSCLC cells infected by Ad-hT-TK-enh were more significantly suppressed and induced apoptosis than those infected by Ad-hT-TK. Telomerase is activated in 80 approximately 90% of cancers, so adenovirus with increasing telomerase promoter activity might be used for targeted cancer gene therapy using suicide genes. These results show that the hTERT promoter and the SV40 enhancer might be used for targeted cancer gene therapy.     

藏红花素诱导人胶质瘤U251细胞内质网应激性凋亡的研究

目的：研究藏红花素对人胶质瘤U251细胞的促凋亡作用和可能的机制。方法：不同浓度藏红花素处理U251细胞后,MTT法检测细胞活力,TUNEL染色观察细胞凋亡情况。结果：①藏红花素显著抑制U251细胞的增殖,并诱导其发生凋亡。②藏红花素增加了U251细胞胞浆内钙离子的含量,并上调了内质网分子伴侣GRP78的表达。③藏红花素处理后的U251细胞内质网相关凋亡分子CHOP,Caspase-4,JNK活性明显增高。结论：藏红花素通过诱导内质网应激性凋亡抑制人胶质瘤U251细胞的增殖。