DAPT suppresses the proliferation of human glioma cell line SHG-44

Objective:To explore the suppressing effect ofγ-secretase inhibitor DAPT on proliferation of human glioma cell line SHG-44 in vitro and its mechanism.Methods:The SHG-44 cell was treated by DAPT with different concentration.The proliferation of cells was detected by MTT assay;cell cycle and TSC of CD133^+were determined by flow cytometry analysis technique;the key factor in Notch signaling pathway(Notch-1,Delta-1,Hes-1)was measured by reverse transcrip tase-polymerase chain reaction and western blotting.Results:DAPT inhibited the growth and proliferation of SHG-44 cells significantly(P<0.05).And the inhibiting effect on SHG-44 cells produced by DAPT showed a dose-dependent manner.DAPT increased the rate of cells in G_0/G_1 phase of SHG-44 cells,while it decreased the rate of cells in S phase.TSC of CD133^+was significantly reduced after DAPT treated SHC-44 cells.The expression of protein and mRNA of Notch-1,Delta-1 and Hes-1 were gradually downregulated with the increase of DAPT doses.Conclusions:DAPT can downregulate these key factor in Notch signaling pathway,reduce the TSC of CD133+and inhibit the proliferation of SHC-44 cells.

Effects of all-trans retinoic acid on metabolic gene expression in the glioma cell line SHG-44

BACKGROUND： Genetic abnormalities and changes in gene expression have been shown in various grades of glioma. However, the relationship between gene expression patterns and pathways related to malignant transformation of glioma remains poorly understood. OBJECTIVE： To screen differentially expressed genes between normal and all-trans retinoic acid-treated glioma cell line SHG-44 cells with a complementary DNA （cDNA） microarray. DESIGN, TIME AND SETTING： The genomics, in vitro study was performed at the Laboratory of Neurobiology, Third Military Medical University of Chinese PLA, China from January to October 2007. MATERIALS： The glioma cell line SHG-44 was provided by the Third Military Medical University of Chinese PLA. AII-trans retinoic acid was purchased from Sigma, USA. cDNA microarray was purchased from City University of Hong Kong. METHODS： The glioma cell line SHG-44 was treated with 10 μmol/L all-trans retinoic acid for 3 days Differentiation-related genes were determined using cDNA microarray. MAIN OUTCOME MEASURES： Gene expression patterns were compared between normal and all-trans retinoic acid-treated SHG-44 cells. Differentially expressed genes were randomly selected and determined by Northern blot analysis. RESULTS： Northern blot analysis revealed downregulated RPL 13 gene expression and upregulated SOD2 gene expression, which was identical to cDNA microarray results. Five differentially expressed genes （TPI1, BPGM, ALDOA, LDHA, and RRM1） were shown to be involved in cell metabolism, in six metabolic pathways. Four differentially expressed genes （TPI1, BPGM, ALDOA, and LDHA） were associated with carbohydrate metabolism, such as fructose metabolism, pyruvic acid metabolism, pentose phosphate pathway, glycolysis, and gluconeogenesis. One differentially expressed gene （RRM1） was correlated with purine and pyrimidine metabolism. CONCLUSION： Five metabolic genes （TPI1, BPGM, ALDOA, LDHA, and RRM1）, which participate in cell carbohydrate and nucleotide metabolism, were shown to closely correlate with glioma development.

Inhibition of all-trans retinoic acid on MDM2 gene expression in astrocytoma cell line SHG-44

Objective To investigate the impact of all-trans retinoic acid （ATRA） on MDM2 gene expression in astrocytoma cell line SHG-44, and to provide basic data for further research on the progression mechanism and gene therapy of human astrocytoma. Methods The differential expressions of MDM2 gene and protein in SHG-44 cells were detected by cDNA microarray and Western blot, respectively, before and after treatment of ATRA. The expressions of MDM2 protein in WHO grade Ⅱ and grade Ⅳ astrocytomas were determined by immunohistochemical streptavidin-peroxidase method. Some differentially expressed genes were selected randomly for Northern blot analysis. Results The intensity ratio of ATRA-treated to untreated SHG-44 cell was 0.37 in the cDNA microarray, suggesting that the expression of MDM2 gene was down-regulated in SHG-44 cells after treatment with ATRA. Some genes differentially expressed in the microarray were confirmed by Northern blot. Western blot demonstrated that the optical density ratios of MDM2 to β-actin in ATRA-treated and untreated SHG-44 were 14.02±0.35 and 21.40±0.58 （t = 24.728, P = 0.000）, respectively, suggesting that the expression of MDM2 protein was inhibited in ATRA-treated SHG-44 cells. Moreover, the percentages of MDM2-positive protein were 24.00% （6/25） and 56.52% （13/23） （x^2 = 5.298, P = 0.021） in WHO grade Ⅱ and grade Ⅳ astrocytomas, respectively, suggesting that the expression of MDM2 protein may increase along with the elevation of astrocytoma malignancy. Conclusion ATRA can inhibit MDM2 gene expression in SHG-44 cells, and MDM2 is related to astrocytoma progression.

立体定向大鼠脑内SHG-44人脑胶质瘤模型的建立

目的 :探讨建立大鼠 SHG- 4 4脑胶质瘤模型的方法。方法 :选用雄性 Wistar大鼠 ,接种前连续 3d用地塞米松 1 mg/ 1 0 0 g体重灌胃 ;在立体定向条件下选取大鼠脑右侧尾状核区为靶点 ,接种 2× 1 0 5个处于对数生长期的 SHG- 4 4细胞 ,接种后观察大鼠生长状态 ,分别于第 1、 2周进行核磁共振 (MRI)检查。在实验第 2周时解剖标本 ,行组织病理和 GFAP免疫组化检查。结果 :接种 1周后核磁共振检查 ,脑内形成实体瘤 ;HE染色组织病理证实是胶质瘤 ,GFAP免疫组化阳性 ;成瘤率约 6 0 %。结论 :用预先免疫抑制的方法 。

人脑胶质瘤细胞SHG-44照射后COX-2表达与放射敏感性的关系

目的:探讨人脑胶质瘤细胞SHG-44照射存活后代细胞对放射敏感性的变化,以及环氧合酶-2(COX-2)的表达情况,为临床使用COX-2抑制剂提供理论依据。方法:采用克隆形成率研究SHG-44及SHG-44照射存活后代细胞的放射敏感性;并分别用RT-PCR方法及免疫组化的方法检测COX-2基因mRNA和蛋白的表达情况。结果:与SHG-44细胞相比,SHG-44照射后代细胞对放射的敏感性下降,COX-2 mRNA及蛋白表达均升高。COX-2表达水平与SHG-44照射后代细胞放射敏感性呈负相关。结论:辐射诱导了SHG-44细胞COX-2表达的升高,使SHG-44照射后代细胞对放射敏感性下降,COX-2表达的升高可能是导致其辐射耐受的原因之一。

吴茱萸碱对胶质瘤SHG-44细胞凋亡的促进作用及其机制

目的研究吴茱萸碱对人胶质瘤SHG-44细胞凋亡的促进用及其机制。方法将体外培养的胶质瘤SHG-44细胞分为对照组、吴茱萸碱组(根据吴茱萸碱浓度不同分为3个亚组),CCK-8法观察细胞活性,流式细胞仪测定细胞凋亡率,Hoechst 33258核染色法观察细胞核凋亡,透射电镜观察细胞形态学的变化,Western blot法检测胶质瘤SHG-44细胞内Cleaved Caspase-3及Cleaved Caspase-9的蛋白表达。结果吴茱萸碱对胶质瘤SHG-44细胞增殖有抑制作用。流式细胞仪及Hoechst 33258核染色法的检测结果表明,随着吴茱萸碱作用浓度的递增,胶质瘤SHG-44细胞凋亡率呈剂量依赖性递增;吴茱萸碱作用后可使Cleaved Caspase-3及Cleaved Caspase-9蛋白的表达升高。结论吴茱萸碱通过改变Cleaved Caspase-3及Cleaved Caspase-9蛋白的表达,抑制胶质瘤SHG-44细胞增殖并促进其凋亡。

九节龙皂苷诱导胶质瘤SHG-44细胞凋亡及其机制研究

目的研究九节龙皂苷对胶质瘤SHG-44细胞潜在的治疗作用及其机制。方法用四甲基偶氨唑蓝(MTT)法检测不同剂量九节龙皂苷于不同时间(6、12、24、72h)对人胶质瘤SHG-44细胞活性的影响和细胞流式术检测SGH-44细胞调亡情况;Western-blot检测caspase-3和Bcl-2在SHG-44细胞中的表达情况。结果流式细胞仪检测显示,随着九节龙皂苷浓度的增大和时间延长,SHG-44细胞的凋亡率明显上升,Western-blot结果提示九节龙皂苷下调了凋亡抑制蛋白Bcl-2的表达并激活了凋亡蛋白caspase-3,九节龙皂苷明显抑制SHG-44细胞的生长与增殖。结论九节龙皂苷引起胶质瘤细胞大量凋亡,具有显著的抗肿瘤作用,通过调控caspase-3和Bcl-2诱导胶质瘤SHG-44细胞凋亡。

芳香化酶在胶质母细胞瘤细胞系SHG-44细胞中的表达及调控

目的 研究芳香化酶细胞色素P45 0 (AROM)及雌激素受体 (ER α)在胶质母细胞瘤细胞系SHG 4 4细胞中的基因表达。 方法 细胞培养、免疫细胞化学染色、原位杂交染色及RT PCR技术。 结果 在SHG 4 4细胞中分别检测到AROM及ER α的表达 ,进一步发现SHG 4 4细胞中AROM的表达是由多个组织特异性启动子驱动基因的转录。 结论 可能为中枢神经系统肿瘤发生的激素调节提供新的资料。

miRNA-34a对胶质瘤SHG-44细胞增殖和凋亡的影响

目的:研究微小RNA-34a(microRNA-34a,miR-34a)在人脑胶质瘤组织中的表达以及其对胶质瘤SHG-44细胞增殖和凋亡的影响。方法:20例胶质瘤组织标本均取自于青岛医学院附属医院神经外科(2007年01月至2010年12月),作为对照的正常脑组织取自5位重症脑外伤需行减压手术的患者。Real-time PCR检测胶质瘤组织中miR-34a的表达。体外转染miR-34a mimics至SHG-44细胞中,MTT实验、流式细胞术检测SHG-44细胞的增殖、细胞周期及凋亡。结果:miR-34a在人脑胶质瘤组织中的表达量明显低于正常脑组织,其在Ⅲ、Ⅳ期胶质瘤组织中表达量明显低于Ⅰ、Ⅱ期胶质瘤组织。miR-34amimics体外转染组与空白组相比,其细胞增殖抑制率明显提高[(37.24±5.72)%vs(4.19±0.63)%,P<0.01];miR-34amimics转染组SHG-44细胞G1期比例明显高于空白对照组[(61.78±2.01)%vs(50.91±1.19)%,P<0.05];且miR-34a转染组细胞凋亡率与空白组细胞相比显著升高[(15.28±3.65)%,vs(2.07±0.84)%,P<0.01]。结论:miR-34a在人脑胶质瘤组织中低表达,miR-34a可抑制SHG-44细胞的增殖、诱导细胞周期阻滞和细胞凋亡。

他莫昔芬对SHG-44人胶质瘤细胞增殖的抑制作用研究

目的:探讨他莫昔芬对人胶质瘤细胞SHG-44生长的作用及其机制。方法:SHG-44细胞PKC和雌激素受体(ER)的表达用免疫组化,细胞活性分析用四唑盐比色试验,细胞增殖和凋亡通过流氏细胞仪检测,氯通通电流的记录应用全细胞膜片钳技术。结果:细胞PKC表达阳性,ER表达阴性,加入他莫昔芬后,SHG-44细胞变老、脱落,细胞总数减少,G2/M期细胞增多,凋亡细胞比例增加,氯离子通道电流受到抑制。结论:他莫昔芬对人胶质瘤细胞SHG-44有明显的抑制作用,其机制可能是通过对PKC及氯通道的抑制。

顺铂对鼠胶质瘤SHG-44细胞的增殖抑制和辐射增敏作用

目的:观察顺铂对鼠胶质瘤SHG-44细胞的抑制及放射增敏作用,并探讨其作用机制。方法:不同浓度紫杉醇(0、0.625μg/ml、1.25μg/ml、2.5μg/ml和5μg/ml)作用于SHG-44细胞,MTT法检测顺铂对SHG-44细胞的增殖抑制作用;克隆形成法检测细胞辐射敏感性变化,流式细胞术检测细胞凋亡百分率变化。结果:顺铂有明显的抑制SHG-44细胞增殖和提高其放射敏感性的作用,顺铂可提高3 Gy、6 Gy照射24 h后SHG-44细胞的凋亡率。结论:顺铂对人胶质瘤SH-44细胞具有杀伤及放射增敏作用。其可能机制是加强射线对肿瘤细胞的诱导凋亡效应,增加其杀伤肿瘤作用。

针对MAGE-1基因RNAi在恶性胶质瘤SHG-44细胞中的作用研究

目的构建抑制黑色素瘤抗原-1(MAGE-1)的siRNA表达载体,鉴定其在人恶性胶质瘤细胞系SHG-44细胞中对MAGE-1基因表达的干涉作用。方法化学合成2对编码短发夹RNA序列的靶向MAGE-1基因寡核苷酸链,克隆到经BglⅡ、HindⅢ双酶切的pSUPER载体上,重组构建RNA干涉(RNAi)质粒载体。利用逆转录多聚酶链反应(RT-PCR)、流式细胞术和荧光显微镜,检测经稳定转染后SHG-44细胞中MAGE-1的表达,以了解siRNA的干扰效果。结果重组构建的pSUPER-MAGE-1载体经双酶切电泳及插入基因片段序列分析,表明寡核苷酸链成功插入到预计位点,并且序列与预期完全一致。稳定转染后G418筛选出的SHG-44多克隆细胞MAGE-1的表达经RT-PCR、流式细胞术和荧光显微镜检测,2对siRNA均有较明显的干涉作用。结论成功构建了针对MAGE-1基因的siRNA表达载体,抑制SHG-44细胞中的MAGE-1分子的表达。

他莫昔芬对SHG-44人胶质瘤细胞钠通道的抑制作用

目的:研究他莫昔芬对SHG-44胶质瘤细胞钠通道电流的作用。方法:采用全细胞膜片钳方法记录SHG-44细胞的钠通道电流,并观察施用不同浓度的他莫昔芬后电流的变化。结果:该钠通道电流特性为内向电流、快速激活失活,他莫昔芬能够明显阻断该电流,该阻断具有剂量依赖性及电压依赖性。在0 mV时,8μmol/L他莫昔芬对钾电流抑制率为69%。半数抑制浓度(IC50)为5.54μmol/L。结论:他莫昔芬可明显阻断SHG-44胶质瘤细胞上的钠通道,这可能是他莫昔芬抑制胶质瘤细胞增殖的机制之一。

他莫昔芬抑制胶质瘤细胞系SHG-44增殖及钠通道电流

目的研究他莫昔芬对SHG-44胶质瘤细胞的增殖及其细胞膜上钠通道电流的作用。方法用四唑盐比色试验分析细胞活性,通过流式细胞仪检测细胞增殖和凋亡。以全细胞膜片钳法记录SHG-44细胞的钠通道电流。结果加入他莫昔芬后,SHG-44细胞变老、脱落,细胞总数减少。他莫昔芬组G2/M期细胞较对照组增多,凋亡细胞比例增加。钠通道电流特性为内向电流、快速激活失活。他莫昔芬可剂量依赖性及电压依赖性阻断该电流。在0mV时,8μmol/L他莫昔芬对钠通道电流抑制率为69%。半数抑制浓度(IC50)为5.54μmol/L。结论他莫昔芬可明显阻断SHG-44胶质瘤细胞上的钠通道,这可能是其抑制胶质瘤细胞增殖的机制之一。

他莫昔芬对SHG-44胶质瘤细胞氯通道的抑制作用

目的研究氯通道阻断剂他莫昔芬(tamoxifen)对SHG-44胶质瘤细胞电压依赖性氯通道电流的作用。方法采用全细胞膜片钳方法记录SHG-44细胞的电压依赖性氯通道电流,并观察施用不同浓度的他莫昔芬后电流的变化。结果该电流特性为外向整流、不失活,他莫昔芬能够明显阻断该电流,该阻断具有剂量依赖性及电压依赖性。在+100 mV时,μM及5μM他莫昔芬对氯电流抑制率分别为48%及89%。结论他莫昔芬可明显阻断SHC-44胶质瘤细胞上的电压依赖性氯通道。

参麦注射液诱导人脑胶质瘤SHG-44细胞凋亡的影响

目的观察参麦注射液体内外对人脑胶质瘤SHG-44细胞生长的抑制及诱导凋亡的作用。方法常规培养细胞,分别加入10,20,40,80,160,320μg·m L-1参麦注射液,分别于培养48,72,96 h后采用噻唑蓝(MTT)法检测参麦注射液对SHG-44细胞增殖的抑制作用;Annexin V-FITC/PI法检测对细胞凋亡的影响;建立SHG-44细胞裸鼠异种移植瘤模型,随机分为模型对照组、顺铂组及参麦注射液(20,40,80 mg·kg-1)3个剂量组,每日腹腔注射给药,观察荷瘤裸鼠的一般活动状况以及进食量;12 d后处死裸鼠,剥瘤称定质量并计算抑瘤率;取组织瘤块,免疫组织化学法检测基因蛋白Caspase-9、Caspase-12、Fas、Survivin含量。结果参麦注射液6个剂量组对SHG-44细胞的增殖均具有明显抑制作用,该抑制率具有时间和剂量依赖性;3个剂量的参麦注射液均能明显诱导SHG-44细胞凋亡;参麦注射液腹腔注射给药可明显抑制荷瘤裸鼠移植瘤的生长;促进Caspase-9、Caspase-12、Fas表达,抑制Survivin的表达。结论参麦注射液体内外均可抑制人脑胶质瘤SHG-44细胞的生长,并诱导其凋亡,其诱导凋亡的机制可能与上调Caspase-9、Caspase-12、Fas水平,下调Survivin有关。

Growth and radiosensitivity of irradiated human glioma cell progeny

BACKGROUND： Progenitors of the immortalized human glioma cell line, SHG-44, are significantly less sensitive to irradiation. Two hypotheses regarding the mechanism of this effect exist： several studies have suggested that there is a subgroup with different radiosensitivities in identical cell group, and the progenitors of irradiate is a adaptive response subgroup, so its radiosensitivity is descend. A second hypothesis suggests that irradiated glioma progeny have a stronger ability to repair DNA damage. This would suggest that when progeny are continuously irradiated, resistance to irradiation-induced DNA increases, and radiosensitivity decreases. OBJECTIVE： To investigate radiosensitivity and growth features after irradiation to progeny of the human glioma cell line SHG-44. DESIGN, TIME AND SETTING： A randomized, controlled experiment, which was performed at the Department of Radiology Laboratory, the First Hospital Affiliated to Soochow University, between September 2004 and January 2006. MATERIALS： The glioma cell line SHG-44 was provided by the Institute of Neuroscience, First Affiliated Hospital of Suzhou University. Propidium iodide reagent was provided by Coulter Corporation. A linear accelerator, KD-2 type, was provided by Siemens, Germany. The flow cytometer EPICS-XL was provided by Coulter Corporation. METHODS： Brain glioma SHG-44 cells were divided into four groups： SHG-44, SHG-44-2, SHG-44-6, and SHG-44-10 . The SHG-44-2, SHG-44-6, and SHG-44-10 cells were vertically irradiated with varying doses of 2, 6 and 10 Gy by a linear accelerator （6 MVX）. The cells were passaged for 15 generations and cultured in RPMI-1640 culture media. MAIN OUTCOME MEASURES： Community re-double time, mean lethal dose （D0）, extrapolation number （N）, fraction surviving fraction irradiated by 2 Gy dose （SF2）, quasi-threshold dose （Dq）, and cell cycle. RESULTS： The Population doubling time （PDT） of SHG-44-2, SHG-44-6, and SHG-44-10 cell groups was not significant （P = 0.052）. Compared to these three groups, the PDT of the SHG-44 cell group was significantly difference （F = 7.878, P 〈 0.002）. SHG-44 cell clone ratewas 26.5%, and SHG-44-10 cell group was 15.5%. The SHG-44-10 cell group also exhibited radiosensitivity, but was less than the radiosensitivity of the SHG-44 cell group. Compared to the SHG-44 cell group, the ratio of the G2/M phase was decreased in the SHG-44-10 cell group, and the radio of S phase was increased. The SHG-44 and SHG-44-10 cell groups were irradiated with 8 Gy. After 12 hours, the G2/M ratio was compared to pre-irradiation times, indicating a significantly higher ratio in the pre-irradiated groups （P 〈 0.01）. The cells between S HG-44 and SHG-44-10 groups were harvested 12 hours after irradiation： G2 phase of SHG-44-10 cells was arrested and the G2/M ratio was increased, which was intensified with increasing irradiation doses. CONCLUSION： In the present study, the proliferation delay and decreased radiosensitivity were confirmed in progeny of irradiated human glioma cells, and radiosensitivity was dose-dependent.

The effects of acetaminophen combined with radiation on the radiosensitivity of irradiated human glioma cell progeny

Objective： To study the effects of acetaminophen （ACE） combined with radiation on the progeny of the human glioma cell line SHG-44, and to investigate if ACE may be an useful therapeutic radiosensitivity agent in the treatment of recurrent human glioma. Methods： A randomized, controlled experiment, was performed at the Department of Radiology Laboratory, the First Hospital Affiliated to Soochow University, between September 2004 and January 2006. Brain glioma SHG-44 cells were divided into three groups： SHG-44, SHG-44-10, and SHG-44-10 ＋ ACE cells groups. The SHG-44-10 cells group was irradiated with dose of 10 Gy by a linear accelerator （6 MVX）. It was passaged for 15 generations and cultured in RPMI-1640 culture media. Then SHG-44-10 ＋ ACE cells group was treated with ACE. Measures： Community re-double time, mean lethal dose （DO）, extrapolation number （N）, fraction surviving fraction irradiated by 2 Gy dose （SF2）, quasi-threshold dose （Dq）, and cell cycle. Results： The SF2 of the SHG-44, SHG-44-10, and SHG-44-10 ＋ ACE cells groups were 70.8%, 80.6% and 45.2%, respectively, with significance （P = 0.040）. The SHG-44-10 and SHG-44-10 ＋ ACE cells groups were irradiated with 8 Gy. After 12 hours, the G2/M ratio of the SHG-44-10 and SHG-44-10 ＋ ACE cells groups were indicating significantly higher ratio compared to pre-irradiated groups （P 〈 0.01）. After 24 hours, the G2/M ratio of the SHG-44-10 cells group decreased rapidly, while the ratio of the SHG-44-10 ＋ ACE cells group still maintained in high level. Conclusion： In the present study, Subtoxic dose of ACE increased the radiosensitivity of the progeny of irradiated human glioma cell. ACE may be an useful radiosensitivity agent in the treatment of recrudescent human malignant glioma.

^(60)Coγ射线照射联合三氧化二砷对人脑胶质瘤SHG44细胞DNA损伤的研究

目的研究60Coγ射线照射联合As2O3对SHG44细胞的生长抑制作用。方法以SHG44细胞为实验对象,3H-TdR掺入法研究As2O3和照射对SHG44细胞DNA合成率的影响;用单细胞凝胶电泳法,以DNA彗星尾长及尾部DNA百分含量来比较As2O3、照射分别单独作用及它们联合作用对细胞DNA的损伤。结果联合组SHG44细胞存活率低于As2O3(P<0.01);3组均诱导DNA损伤,联合作用组对细胞DNA的损伤程度明显高于照射及As2O3单独作用组。结论电离辐射和As2O3联合应用对SHG44细胞的杀灭作用均强于电离辐射和As2O3单独作用。

^(125)I对体外培养的胶质瘤细胞C-myc、p53基因表达及细胞增殖的影响

目的探讨125I对体外培养的胶质瘤细胞SHG-44C-myc、p53基因表达及细胞增殖的影响。方法利用免疫组化技术检测经125粒子放射处理3d后,SHG-44细胞的C-myc蛋白表达与p53蛋白表达水平。结果经1粒、3粒125粒子放射处理3d后,SHG-44细胞C-myc蛋白表达明显减弱,而p53蛋白表达显著增强。二者呈负相关。结论125I可以通过影响C-myc基因与p53基因的表达抑制人脑恶性胶质瘤细胞株SHG-44增殖,从而抑制胶质瘤的生长。