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基因对胶质瘤增殖与侵袭力的影响

脑胶质瘤为神经外科的常见病、多发病 ,约占颅内肿瘤的 45 % ,其主要治疗手段包括手术切除、化学治疗与放射治疗 ,其生存时间短、预后差 ,平均生存时间不足 1年 ,这与其具有高增殖性及高侵袭性有关。本实验应用腺病毒作为载体介导抑癌基因PTEN体外转染人脑胶质瘤细胞U87,检测其细胞增殖力与体外侵袭力的改变 ,进而探讨PTEN作为胶质瘤基因治疗靶点的可能性。分别用的Ad PTEN(实验病毒 )、Ad LacZ(对照病毒 )感染人胶质瘤细胞U87(病毒滴度为 5× 1 0 8pfu/ml,MOI为 5 0 ) ,同时用无病毒感染的U87作空白对照 ,首先用RT PCR检测PTE…     

野生型PTEN基因对胶质细胞瘤侵袭力的影响

目的观察转入野生型PTEN基因的胶质瘤细胞体外侵袭力改变,探索PTEN基因对胶质瘤细胞影响方式.方法构建野生型PTEN基因的pcDNA3.1 Hygro(-)真核质粒载体,并用于转染PTEN基因失活的U251细胞系,采用重组基底膜侵袭模型,观察转染前后细胞侵袭力的改变.结果转染PTEN基因可以明显抑制U251细胞的侵袭力.结论 PTEN基因可通过抑制胶质瘤细胞的侵袭力达到抑制肿瘤生长.     

IL-24对B16细胞生长抑制作用的体内外实验研究

目的:构建白细胞介素24(Interleukin24,IL24)真核表达质粒,研究其体内外表达对肿瘤细胞的生长抑制作用。方法:采用重组DNA技术构建IL24真核表达质粒pEGFPIL24。用脂质体法将重组质粒及空载体体外转染B16细胞,再经激光扫描共聚焦显微镜(LSM)观察其表达,用MTT法检测B16细胞的体外增殖能力,用流式细胞仪检测细胞周期。小鼠实体瘤模型研究基因转染对肿瘤的体内生长抑制作用。结果:pEGFPIL24转染B16细胞后,LSM可观察到IL24在细胞中的表达。转染IL24基因的B16细胞体外增殖能力明显受到抑制,细胞被阻滞在G2/M期。与对照组相比,IL24基因治疗组肿瘤在体内的生长受到明显抑制,P<0.05。结论:IL24基因转染的肿瘤细胞体外生长受抑。于荷瘤小鼠的瘤内注射pEGFPIL24可抑制肿瘤生长,提示IL24具有明显的体内外抗肿瘤作用。     

PTEN基因转染对白血病细胞VEGF调控作用的影响

目的:探讨在白血病细胞中与张力蛋白同源的10号染色体缺失的磷酸酶基因(phosphatase and tensin hemology deleted on chromosome ten gene,PTEN)对血管内皮生长因子(vascular endothelial growth factor,VEGF)及其受体1(VEGF receptor 1,VEGFR1)调控作用的影响.方法:将携带有野生型PTEN及绿色荧光蛋白(green fluorescent protein,GFP)基因的腺病毒(Ad-PTEN-GFP)及空载体腺病毒(Ad-GFP)转染人慢性粒细胞白血病急变细胞株K562,实时荧光定量PCR法检测不同转染组细胞中PTEN、VEGF和VEGF1 mRNA表达水平,Western印迹法检测PTEN、VEGF、Akt和磷酸化Akt蛋白的表达水平,并采用Transwell小室侵袭实验检测不同转染组细胞的侵袭性.通过MTT实验及FCM法检测PTEN基因对脐静脉内皮细胞株ECV304增殖和凋亡的影响.通过鸡胚尿囊膜(chick chorioallantoic membrane,CAM)体内血管生长实验检测PTEN基因对鸡胚血管生成的影响.结果:与空载体腺病毒(Ad-GFP)相比,Ad-PTEN-GFP 转染人白血病细胞K562后,VEGF及其受体的表达被明显抑制,并呈剂量依赖性负相关;同时,Ad-PTEN-GFP 转染后K562细胞侵袭能力明显减弱.PTEN基因转染能够抑制血管内皮细胞ECV304增殖,并促进其细胞凋亡,细胞周期阻滞在S期.PTEN基因转染可明显抑制CAM血管生长.结论:肿瘤抑制基因PTEN能够抑制内皮细胞增殖和白血病细胞侵袭,其作用机制可能是负调控白血病细胞VEGF表达以及抑制肿瘤血管新生.     

PTEN在人脑胶质瘤细胞中的表达及其对细胞增殖的影响

背景与目的:PTEN基因的缺失及突变与多种肿瘤有关,而脑胶质瘤是与PTEN基因变异关系最为密切的恶性肿瘤之一.本研究旨在观察PTEN基因对人脑胶质瘤细胞生物学特性的影响,为PTEN用于胶质瘤基因治疗提供科学的实验依据.方法:(1)应用RT-PCR方法扩增人脑胶质瘤细胞U251、SHG-44中PTEN基因,序列测定分析.(2)采用阳离子聚合物转染试剂,将携带野生型PTEN基因的重组真核表达载体质粒转染至胶质瘤细胞,G418筛选出稳定转染的细胞并扩增培养.通过细胞形态学、细胞生长曲线观察PTEN基因表达对细胞形态和增殖的影响,应用Western blot、免疫细胞化学法检测相关蛋白的表达.结果:(1)胶质瘤细胞U251和SHG-44的PTEN mRNA分别存在着突变型、缺失型两种表达方式.(2)稳定转染的U251和SHG-44细胞株的生长曲线显示细胞增殖明显受抑制,第7天细胞计数分别为未转染对照组细胞数的39.1%、27.8%;Westem blot显示稳定转染细胞株有外源性PTEN蛋白的表达;细胞免疫化学法显示胶质纤维酸性蛋白(glial fibrillary acidic protein,GFAP)表达量增加,从而表现出对细胞形态影响的差异,稳定转染的U251细胞出现向星形细胞分化的特征,SHG-44细胞形态转染前后无明显改变.结论:恢复野生型PTEN基因表达对胶质瘤细胞存在差异性的诱导分化作用.     

抗CD3或B7.1与CD28共刺激PBLs膜表面分子表达与肝癌细胞凋亡的相关性

为探讨腺病毒载体介导的外源性Rb基因导入对膀胱癌细胞生长的抑制作用,为膀胱癌的基因治疗提供实验依据,构建了Rb基因的复制缺陷型重组腺病毒载体.体外转染人膀胱癌细胞株EJ.应用免疫组化法、免疫印迹及聚合酶链反应技术检测外源性Rb基因腺病毒载体的转染效率及Rb基因表达效果;用流式细胞仪分析细胞周期;以细胞计数及同位素掺入技术观察外源性Rb基因对EJ细胞生长的抑制效果.结果显示腺病毒载体可有效地将外源基因导入EJ细胞.Rb基因重组腺病毒载体转染细胞后,胞内DNA合成减少,细胞生长受到抑制.流式细胞仪分析显示68%的EJ细胞生长停滞在GO/GI期.结果提示,外源性Rb基因重组腺病毒载体转染膀胱癌细胞可有效抑制细胞生长     

反义microRNA-221与反义microRNA-222抑制人脑胶质瘤细胞的体外与体内生长

目的 探讨敲低microRNA(miR)-221和miR-222表达以抑制人脑胶质瘤U251细胞生长的作用及其机制.方法 脂质体介导转染反义寡聚核苷酸(AS-miR-221和AS-miR-222)于人脑胶质瘤细胞U251.采用Northern blot鉴定转染后U251细胞的miR-221和miR-222表达水平;四甲基偶氮唑蓝(MTT)法评价AS-miR-221和AS-miR-222抑制U251细胞生长的作用;Transwell实验检测细胞侵袭能力;流式细胞术检测细胞周期的分布和凋亡;Western blot检测转染后U251细胞相关蛋白表达的变化,并用AS-miR-221和AS-miR-222治疗裸鼠皮下移植瘤,观察其在活体内对肿瘤生长的抑制作用.结果 Northern blot检测结果 显示,AS-miR-221和AS-miR-222共转染后,肿瘤细胞miR-221和miR-222表达明显下降,细胞生长速度降低,细胞穿过率为14.5%,细胞周期出现G0/G1期阻滞,凋亡率(13.7%)增高,并可见connexin43、p27、PUMA、caspase-3、PTEN、TIMP3和Bax等相关蛋白表达增高,而bcl-2表达降低,p53无明显变化.经AS-miR-221和AS-miR-222治疗后,裸鼠皮下移植瘤生长明显受抑.结论 AS-miR-221和AS-miR-222共转染可抑制U251细胞的增殖与侵袭,miR-221和miR-222可以作为人脑胶质瘤基因治疗的侯选靶点.     

腺病毒介导野生型p53基因对人白血病细胞系HL-60、K_(562)生长的抑制作用

目的探索肿瘤抑制基因ｐ５３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ｐ５３ｃＤＮＡ表达。转染后Ａｄ／ｐ５３病毒对ＨＬ－６０细胞和Ｋ５６２细胞的生长有抑制作用。随着Ａｄ／ｐ５３病毒浓度加大,ＨＬ－６０和Ｋ５６２细胞的ＯＤ值越低即生长抑制程度越大。经Ａｄ／ｐ５３病毒转染的ＨＬ－６０和Ｋ５６２细胞均有明显的细胞凋亡峰出现,对照细胞则无。细胞周期分析无明显异常发现。结论重组腺病毒载体介导的野生型ｐ５３基因在体外对人白血病细胞系ＨＬ－６０和Ｋ５６２的生长有抑制作用,能导致细胞凋亡的发生。     

腺病毒介导的PTEN对A549肺癌细胞体内外生长的影响

目的：研究携带第10号染色体缺失的磷酸脂酶和张力蛋白同源基因（phosphatase and tensin homologue-deleted chromosome ten gene，PTEN）的腺病毒表达载体对A549肺癌细胞体内外生长的抑制作用。方法：从人外周血淋巴细胞中通过RT—PCR扩增出PTEN基因片段，将其克隆到pAdTrack—CMV转移载体上，构建了PTEN重组腺病毒载体。体外用MTT法检测Ad—PTEN对A549肺癌细胞生长的抑制作用，以流式细胞术检测肿瘤细胞的周期和凋亡率。建立荷瘤裸鼠模型，体内检测Ad—PTEN对A549肺癌细胞移植瘤生长的影响，以免疫组化法检测移植瘤中微血管密度。结果：克隆的P陋Ⅳ基因测序结果与基因Bank数据库完全相符。Ad—PTEN体外感染A549肺癌细胞48h后其凋亡率为10．5％，明显高于对照细胞；4d后细胞生长与对照组细胞相比抑制了57％。肺癌细胞移植瘤治疗结束时，Ad—PTEN组瘤重为（0．58±0．29）g，对照组瘤重为（1．42±0．24）g，其生长抑制达59％（P〈0．05）；同时移植瘤中微血管密度降低约49％（P〈0．05）。结论：成功构建的Ad—PTEN腺病毒载体能在体内外抑制A549肺癌细胞及其移植瘤的生长。     

Combination gene therapy with PTEN and EGFR siRNA suppresses U251 malignant glioma cell growth in vitro and in vivo

The over-expression/amplification of the epidermal growth factor receptor (EGFR) gene and mutation/deletion of tumor suppressor PTEN gene are main genetic changes identified in glioblastomas. These two genetic changes play a critical role in the formation of many malignant tumors and have been shown to be the important therapeutic targets. In this study, we used an expression plasmid that expresses small hairpin RNA-targeting sequences of human EGFR and wild-type PTEN cDNA to examine the growth inhibitive effects in U251 glioma cells. It was found that down-regulation of EGFR expression and up-regulation of PTEN expression resulted in the suppression of cell proliferation, arrest of cell cycle, reduction in cell invasion and promotion of cell apoptosis in vitro. In addition, the growth of the subcutaneous U251 glioma in the nude mice treated with expression plasmid was significantly inhibited. Our results demonstrated that the expression plasmid could exert proliferation and invasion inhibition effects on U251 cells in vitro and in vivo. It suggested that combinatory gene therapy targeting EGFR and PTEN would be a new strategy in gene therapy of glioblastoma.     

Suppression of invasion in human U87 glioma cells by adenovirus-mediated co-transfer of TIMP-2 and PTEN gene

TIMPs and PTEN are known to be inhibitors of the invasive activities of malignant glioma. But there has been no literature reported concerning the effect of combined gene transfer of these two genes on invasiveness of glioma. This study was designed to evaluate the effect of adenovirus-mediated in vitro gene transfer of tissue inhibitor of metalloproteinases-2 (TIMP-2) and phosphatase and tensin homology deleted on chromosome ten (PTEN) on invasion of human U87 glioma cells. The mRNA and protein expressions of TIMP-2 and PTEN in U87 cells infected with AdTIMP-2 and AdPTEN were determined by RT-PCR and Western blot, respectively. The relative activity of MMP-2 and MMP-9 were measured by Gelatin zymogram and invasion of U87 in vitro were detected using Boyden chamber. The number of invasion cell of U87, U87 infected with Ad-gal, AdPTEN, AdTIMP-2 and AdPTEN/TIMP-2 was 55.63+/-13.27, 48.27+/-14.75, 35.27+/-10.94, 27.37+/-12.81, and 19.17+/-5.45, respectively. In vitro invasiveness of glioma cells was significantly inhibited by infection with AdTIMP-2 and/or AdPTEN, which was not consistent with the change of MMPs activity. And in the combinated group, the inhibition effect was more remarkable than in single group. Our studies suggest that adenovirus-mediated combined TIMP-2 and PTEN gene therapy is possibly useful for anti-invasion therapy of malignant glioma.     

外源型P14ARF基因对人脑胶质瘤细胞生长的影响

目的：研究Ｐ１４ＡＲＦ基因对人脑胶质瘤细胞株（Ｕ－２５１）生长的影响。方法：用脂质体介导携带Ｐ１４ＡＰＦ的真核表达载体转染Ｕ－２５１细胞,观察其对该细胞株的生长、增殖周期等生物学行为的影响。结果：导入Ｐ１４ＡＰＦ胶质瘤细胞Ｕ－２５１的生长有明显的抑制作用。     

人PTEN基因表达载体的构建及其在U251细胞中的表达

背景与目的:克隆人野生型和突变型PTEN的cDNA并构建其表达载体,探讨该表达质粒在人星形胶质瘤细胞U251中的表达情况.材料与方法:分别从新鲜胎盘组织和结肠癌组织中提取总RNA,采用RT-PCR的方法扩增人野生型和突变型PTEN基因全长cDNA,分别克隆入pMD 18-T载体上,经PCR、酶切鉴定均为阳性的克隆,进行核苷酸序列分析.再分别将两段基因定向克隆入pcDNA3.1( )载体中构建表达载体,转染入U251细胞.结果:成功克隆了人野生型及突变型PTEN cDNA并成功构建其表达载体,发现突变型PTEN蛋白对细胞生长无明显抑制作用,而野生型PTEN蛋白对细胞生长有明显抑制作用.结论:PTEN可能能抑制肿瘤细胞生长,为后进一步开展PTEN对肿瘤相关功能的研究奠定基础.     

EGB761对U87胶质瘤细胞的促凋亡作用

目的:研究银杏叶提取物EGB761对U87胶质瘤细胞的促凋亡作用。方法:EGB761干预U87胶质瘤细胞后,CCK-8观察50、100和200mg/L不同浓度EGB761作用下U87细胞的增殖状况,流式细胞仪分析用AnnexinV-FITC和PI双染检测不同浓度EGB761对U87胶质瘤细胞凋亡的影响。结果:EGB761可抑制U87胶质瘤细胞的增殖,呈浓度和时间依赖性反应;EGB761 100mg/L、200mg/L干预U87胶质瘤细胞后,流式细胞仪检测显示细胞凋亡率明显升高（P<0.05）。结论:EGB761可诱导U87胶质瘤细胞凋亡。     

PTEN gene transfer in human malignant glioma: sensitization to irradiation and CD95L-induced apoptosis.

The tumor suppressor gene PTEN (MMAC1, TEP1) encodes a dual-specificity phosphatase and is considered a progression-associated target of genetic alterations in human gliomas. Recently, it has been reported that the introduction of wild type PTEN into glioma cells containing endogenous mutant PTEN alleles (U87MG, LN-308), but not in those which retain wild-type PTEN (LN-18, LN-229), causes growth suppression and inhibits cellular migration, spreading and focal adhesion. Here, we show that PTEN gene transfer has no effect on the chemosensitivity of the four cell lines. Further, a correlational analysis of the endogenous PTEN status of 12 human glioma cell lines with their sensitivity to seven different cancer chemotherapy drugs reveals no link between PTEN and chemosensitivity. In contrast, ectopic expression of wild type PTEN, but not the PTEN(G129R) mutant, in PTEN-mutant gliomas markedly sensitizes these cells to irradiation and to CD95-ligand (CD95L)-induced apoptosis. PTEN-mediated facilitation of CD95L-induced apoptosis is associated with enhanced CD95L-evoked caspase 3 activity. Protein kinase B (PKB/Akt), previously shown to inhibit CD95L-induced apoptosis in nonglial COS7 cells, is inactivated by dephosphorylation. Interestingly, both PTEN-mutant U87MG and PTEN-wild-type LN-229 cells contain phosphorylated PKB constitutively. Wild-type PTEN gene transfer promotes dephosphorylation of PKB specifically in U87MG cells but not in LN-229 cells. Sensitization of U87MG cells to CD95L-apoptosis by wild-type PTEN is blocked by insulin-like growth factor-1 (IGF-1). The protection by IGF-1 is inhibited by the phosphoinositide 3-OH (PI 3) kinase inhibitor, wortmannin. Although PKB is a down-stream target of PI 3 kinase, the protection by IGF-1 was not associated with the reconstitution of PKB phosphorylation. Thus, PTEN may sensitize human malignant glioma cells to CD95L-induced apoptosis in a PI 3 kinase-dependent manner that may not require PKB phosphorylation.     

Adenoviral p16/CDKN2 gene transfer to malignant glioma: role of p16 in growth,invasion, and senescence

In gliomas, a high frequency of homozygous p16 gene deletions have been demonstrated, which are believed to be linked with malignant progression. The aim of this study was to assess the role of p16 in growth, invasion, and senescence. The human glioma cell lines U87 MG and U373 MG were transduced with Ad-p16, and cell viability was assessed by trypan blue staining. To examine the mechanism of cell growth inhibition, cell cycle analyses and annexin assays were performed. The invasive potential of Ad-p16 transduced cells was evaluated using a Matrigel invasion assay, and trimolecular complex (MMP-2/MT1-MMP/TIMP-2) synthesis was proven by zymography and Western blotting. To establish the link between p16 and cell senescence, we stained for Senescence-Associated beta-galactosidase activity. A cell proliferation assay demonstrated that Ad-p16 treatment significantly inhibits cell growth. Moreover, this cell growth inhibition was induced by cell cycle arrest, not by apoptosis. In vitro treatment of malignant glioma cells with Ad-p16 significantly decreased their invasive potential by Matrigel invasion assay. However, we were unable to demonstrate any differences in the constitutive productions and secretions of MMP-2, MT1-MMP, and TIMP-2, among the mock-treated, Ad-lacZ-transduced, and Ad-p16-transduced cells. p16 expression caused an enlargement of all cells, and these were morphologically similar to senescent cells. Staining for Senescence-Associated beta-galactosidase activity showed that the enlarged cells stained positively. Taken together these data strongly suggest that the anti-cancer effect of p16 is modulated by p16-mediated cell cycle arrest and by the induction of senescence.     

Radiosensitizing potential of the selective cyclooygenase-2 (COX-2) inhibitor meloxicam on human glioma cells

The COX-2 protein is frequently overexpressed in human malignant gliomas. This expression has been associated with their aggressive growth characteristics and poor prognosis for patients. Targeting the COX-2 pathway might improve glioma therapy. In this study, the effects of the selective COX-2 inhibitor meloxicam alone and in combination with irradiation were investigated on human glioma cells in vitro. A panel of three glioma cell lines (D384, U87 and U251) was used in the experiments from which U87 cells expressed constitutive COX-2. The response to meloxicam and irradiation (dose-range of 0–6 Gy) was determined by the clonogenic assay, cell proliferation was evaluated by growth analysis and cell cycle distribution by FACS. 24–72 h exposure to 250–750 μM meloxicam resulted in a time and dose dependent growth inhibition with an almost complete inhibition after 24 h for all cell lines. Exposure to 750 μM meloxicam for 24 h increased the fraction of cells in the radiosensitive G₂/M cell cycle phase in D384 (18–27%) and U251 (17–41%) cells. 750 μM meloxicam resulted in radiosensitization of D384 (DMF:2.19) and U87 (DMF:1.25) cells, but not U251 cells (DMF:1.08). The selective COX-2 inhibitor meloxicam exerted COX-2 independent growth inhibition and radiosensitization of human glioma cells.