Survivin基因启动子调控的肿瘤特异性表达载体的构建

目的：扩增survivin基因启动子,构建survivin启动子调控的真核表达载体,验证其在肿瘤细胞的特异性表达。方法：以Hep-2细胞基因组DNA为模板,PCR扩增survivin启动子;利用核酸内切酶双酶切sur-vivin启动子基因片段和pShuttle质粒,构建载体pSurp,酶切和测序鉴定;分别双酶切pSurp载体和pEGFP-C1载体,构建survivin启动子调控的真核表达载体pSurp-EGFP,脂质体转染Hep-2细胞及血管内皮细胞ECV304,荧光显微镜下观察EGFP的表达,Western blot方法验证该基因的蛋白表达。结果：成功扩增survivin基因启动子并构建survivin基因启动子调控的pSurp-EGFP真核表达载体。脂质体法转染Hep-2细胞和血管内皮细胞ECV304,在荧光显微镜下见Hep-2细胞发出较强的绿色荧光,而ECV304细胞没有绿色荧光;Western blot的结果也确证了EGFP蛋白仅在Hep-2细胞表达。结论：Survivin启动子的成功克隆与其真核表达载体的构建,为肿瘤的特异性基因治疗奠定了实验基础。     

Survivin启动子与肿瘤

利用特异性启动子介导的基因治疗是肿瘤靶向性治疗的一种有效途径.Survivin启动子既有肿瘤细胞的靶向性,又有增殖性血管内皮细胞的靶向性,且能有效介导目的基因表达,展示了其在肿瘤靶向性治疗研究中的良好前景.     

CMVE-PEG3启动子在前列腺癌DU145细胞中的活性鉴定

[目的]比较CMVE-PEG3p、PEG-3启动子在DU145细胞中的启动活性,为前列腺癌靶向性基因治疗提供依据.[方法]用PCR法扩增CMV增强子、PEG-3启动子;在真核表达质粒pShuttle-EGFP基础上分别构建2种启动子调控的、以绿色荧光蛋白为目的基因的真核表达质粒pShuttle-CMVE-PEG3p-EGFP、pShuttle-PEG3p-EGFP.将重组质粒用脂质体分别转染前列腺癌DU145细胞和人正常前列腺上皮细胞RWPE-1,72h后用Imagepro-Plus6.0分析2种启动子在相同时间内启动绿色荧光蛋白的表达水平.[结果]重组质粒在DU145细胞中观察到了绿色荧光,在RWPE-1细胞中无绿色荧光.pShuttle-CMVE-PEG3p-EGFP质粒在DU145细胞中表达强于pShuttle-PEG3p-EGFP.2种质粒在DU145细胞中的荧光强度(IOD)分别为246.22、130.93.[结论]所克隆的CMVE-PEG3p启动子和PEG-3启动子在前列腺癌DU145细胞中均表现出肿瘤特异性,其中CMVE-PEG3p启动子具有更强的启动效应,有望开发成为前列腺癌靶向性基因治疗的工具.     

survivin启动子的克隆及在HeLa细胞中的特异生物活性

目的: 构建带有survivin启动子的pGL3Basic真核表达载体，探讨survivin启动子在HeLa细胞中的特异表达活性。 方法: 采用PCR技术扩增survivin启动子，插入pGL3Basic载体，构建携带survivin启动子的pGL3Basic真核表达载体（pGL3Basic/ Surp）。纯化pGL3Basic/ Surp质粒，用脂质体法转染HeLa细胞和正常血管内皮细胞ECV304，48 h后收集转染细胞与荧光素酶底物反应，检测荧光素酶活性。 结果: 成功克隆1 kb survivin基因启动子，并构建了携带有survivin基因启动子的pGL3Basic真核表达载体，转染后的Hela细胞荧光素酶活性为2074.2±78.5，而ECV304荧光素酶活性为9.7±1.1。 结论： 成功克隆的survivin启动子在HeLa细胞中表现出较高的肿瘤特异性活性，为进一步开发肿瘤的靶向基因治疗奠定了基础     

Survivin基因启动子调控的肿瘤特异性表达载体的构建

目的:扩增survivin基因启动子,构建survivin启动子调控的真核表达载体,验证其在肿瘤细胞的特异性表达。方法:以Hep-2细胞基因组DNA为模板,PCR扩增survivin启动子;利用核酸内切酶双酶切sur-vivin启动子基因片段和pShuttle质粒,构建载体pSurp,酶切和测序鉴定;分别双酶切pSurp载体和pEGFP-C1载体,构建survivin启动子调控的真核表达载体pSurp-EGFP,脂质体转染Hep-2细胞及血管内皮细胞ECV304,荧光显微镜下观察EGFP的表达,Western blot方法验证该基因的蛋白表达。结果:成功扩增survivin基因启动子并构建survivin基因启动子调控的pSurp-EGFP真核表达载体。脂质体法转染Hep-2细胞和血管内皮细胞ECV304,在荧光显微镜下见Hep-2细胞发出较强的绿色荧光,而ECV304细胞没有绿色荧光;Western blot的结果也确证了EGFP蛋白仅在Hep-2细胞表达。结论:Survivin启动子的成功克隆与其真核表达载体的构建,为肿瘤的特异性基因治疗奠定了实验基础。     

Survivin基因及其在肿瘤研究中的意义

Survivin基因是一种新的凋亡抑制基因，在肿瘤形成过程中与细胞凋亡的抑制和细胞增生有关，早期出现于肿瘤组织细胞中，并成为某些肿瘤预后不良的标志。在体内和体外研究中，针对Survivin的反义核体酸及其突变体可以诱导细胞凋亡，降低肿瘤生产的能力，增强肿瘤细胞对化疗药物的敏感性，因此该基因有可能成为肿瘤诊断和治疗的新的靶位。     

PEG-3启动子在前列腺癌细胞DU145中的活性鉴定

目的探讨PEG-3启动子在前列腺癌细胞DU145中的启动活性。方法用PCR法扩增PEG-3启动子;在真核表达质粒pShuttle-CMVp-EGFP基础上构建PEG-3启动子调控的、转录绿色荧光蛋白基因的真核表达质粒pShuttle-PEG3p-EGFP。将2种质粒(pShuttle-PEG3p-EGFP和pShuttle-CMVp-EGFP)用脂质体分别转染前列腺癌细胞DU145和人正常前列腺上皮细胞RWPE-1,72 h后在荧光显微镜下用Im-agepro-Plus 6.0分析2种启动子启动绿色荧光蛋白基因的转录活性。结果重组质粒在前列腺癌细胞DU145中观察到了绿色荧光,在人正常前列腺上皮细胞RWPE-1细胞中无绿色荧光。pShuttle-PEG3p-EGFP和pShuttle-CMVp-EGFP 2种质粒在前列腺癌细胞DU145中的荧光强度分别为403.50、130.93。结论所克隆的PEG-3启动子表现出较强的肿瘤特异性,在前列腺癌细胞DU145中有一定的启动活性,可望开发成为肿瘤靶向性基因治疗工具。     

G蛋白调节子16对胶质瘤C6细胞周期的调节

目的 探讨G蛋白调节子16（RGS16）对胶质瘤C6细胞周期的影响。方法 利用脂质体介导法将RGS16基因导入C6细胞中，在倒置显微镜下观察细胞形态变化和贴壁生长情况；免疫细胞化学法检测转染前后RGS16蛋白的表达情况；流式细胞仪检测转染pCMV5-RGS16和pCMV5质粒后每隔12h后的细胞周期变化。结果 转染pCMV5-RGS16质粒24h后30．0％细胞贴壁性降低，突起收缩，细胞变圆，72h之后细胞又恢复正常；RGS16蛋白的表达呈时相性，36h时表达率最高（阳性率为13．0％），72h表达终止；C6细胞的各期细胞比例变化与RGS16蛋白表达对应，在36h时G1期比例从转染前的70．5％降低到60．2％，S期比例从20．9％增加到34．9％；在48h时G1期增加到76．2％，S期减少到11．4％；72h各期恢复到正常比例。对照组细胞转染前后形态变化不明显，RGS16蛋白表达阴性，细胞周期变化不明显。结论 RGS16能促进C6细胞周期的运行。     

组织特异性启动子及其在肿瘤基因治疗中的应用

随着基因治疗研究的深入，愈来愈多的组织特异性启动子已被用于调控目的基因在靶器官中的表达，尤其是携带治疗性基因可以实现对肿瘤组织的特异性杀伤作用，组织特异性启动子的应用已成为基因治疗中的一个重要手段，现综述各系统启动子的分类及其应用。     

Nuclear survivin expression predicts poorer prognosis in glioblastoma

Survivin is a member of the inhibitor of apoptosis family, and is expressed in various malignant tumors. Survivin overexpression has been reported to be a poorer prognostic factor in various malignancies. However, the prognostic value of survivin expression in patients with glioblastoma is still controversial. Therefore, in this study the role of survivin as a predictor for survival was investigated in patients with glioblastoma. Tissue specimens were obtained from 66 patients with glioblastoma treated with radiotherapy. Survivin expression was detected by an immunohistochemical method. Nuclear and cytoplasm survivin scores were defined by using the cell positivity and staining intensity. The scores were defined as follows, 0 (no staining), 1 (less than 50% of cell positivity and any staining), 2 (more than 50% of cell positivity and weak to moderate intensity) and 3 (more than 50% of cell positivity and strong intensity). The correlation between survivin scores and the overall survival rate was evaluated. Nuclear and cytoplasm survivin staining were noted in 47 and 58 patients, respectively. The number of patients with nuclear survivin score of 0, 1, 2 and 3, were 19 (28.8%), 26 (39.4%), 9 (13.6%) and 12 (18.2%), respectively. The 3-year overall survival rate of the nuclear survivin score 3 was 0%, significantly lower than the 11.6% of the nuclear survivin score ≤2 (P = 0.0003). Cytoplasm survivin score did not correlate with the prognosis. Nuclear survivin expression may be a useful biomarker for predicting prognosis in patients with glioblastoma.     

siRNA抑制survivin基因周期的影响表达对HeLa细胞

目的设计靶向survivin基因的干涉片段,构建真核表达载体pSUPER／survivin并转染宫颈癌HeLa细胞,探讨siRNA抑制survivin表达对联合1射线宫颈癌HeLa细胞周期的影响。方法化学合成干涉引物,退火获得survivin基因的干涉片段,连入pSUPER载体并测序。将测序正确的pSUPER／survivin载体转染HeLa细胞株,RT—PCR法和流式细胞术检测HeLa细胞中survivin基因的表达,流式细胞术检测转染联合＇射线照射后各组细胞的细胞周期数据。结果本实验成功的构建了pSUPER／survivin载体。转染pSUPER／survivin的HeLa细胞其survivin基因表达水平明显低于未转染的HeLa细胞及转染空载体pSUPER的HeLa细胞。1射线照射后,转染pSUPER／survivin的HeLa细胞发生了S期细胞比例下降,及G2／M期阻滞。结论成功的构建了pSUPER／survivin载体,siRNA干涉survivin可以作为提高放射敏感性的潜在分子靶点,通过减少S期细胞比例,促进肿瘤细胞凋亡,通过增加G2／M期阻滞增加放射线的杀伤。     

存活素siRNA表达质粒的构建及其对MCF-7细胞细胞周期和增殖的调控

背景与目的：存活素特异地在肿瘤组织中过表达,许多报道表明抑制它的功能对肿瘤治疗有利。RNA干扰被证明是一项能有效而特异地抑制基因表达的新技术。本研究的目的在于构建存活素基因的小分子干扰RNA(siRNA)表达质粒,并检测该质粒在乳腺癌细胞中的生物学功能。方法：应用含有u6启动子的mu6pro载体构建存活素siRNA质粒,RT-PCR和Western blot法检测该质粒转染MCF-7细胞后存活素表达的变化,流式细胞仪和MTT法分别检测其对细胞周期和对细胞增殖的影响。结果：存活素siRNA质粒明显下调MCF-7细胞中存活素的表达,阻断细胞周期在G1期,显著抑制细胞增殖,促进细胞凋亡。结论：通过RNA干扰,存活素的表达在mRNA和蛋白质水平上被下调。     

Radiation-induced mitotic cell death and glioblastoma radioresistance: A new regulating pathway controlled by integrin-linked kinase,hypoxia-inducible factor 1alpha and survivin in U87 cells

We have previously shown that integrin-linked kinase (ILK) regulates U87 glioblastoma cell radioresistance by modulating the main radiation-induced cell death mechanism in solid tumours, the mitotic cell death. To decipher the biological pathways involved in these mechanisms, we constructed a U87 glioblastoma cell model expressing an inducible shRNA directed against ILK (U87shILK). We then demonstrated that silencing ILK enhanced radiation-induced centrosome overduplication, leading to radiation-induced mitotic cell death. In this model, ionising radiations induce hypoxia-inducible factor 1alpha (HIF-1α) stabilisation which is inhibited by silencing ILK. Moreover, silencing HIF-1α in U87 cells reduced the surviving fraction after 2 Gy irradiation by increasing cell sensitivity to radiation-induced mitotic cell death and centrosome amplification. Because it is known that HIF-1α controls survivin expression, we then looked at the ILK silencing effect on survivin expression. We show that survivin expression is decreased in U87shILK cells. Furthermore, treating U87 cells with the specific survivin suppressor YM155 significantly increased the percentage of giant multinucleated cells, centrosomal overduplication and thus U87 cell radiosensitivity.In consequence, we decipher here a new pathway of glioma radioresistance via the regulation of radiation-induced centrosome duplication and therefore mitotic cell death by ILK, HIF-1α and survivin. This work identifies new targets in glioblastoma with the intention of radiosensitising these highly radioresistant tumours.     

Survivin在羟基喜树碱诱导胃癌细胞株BGC-823凋亡过程中的变化及其意义

背景与目的抗肿瘤药物主要通过诱导细胞凋亡发挥作用,化疗耐药性产生可能与凋亡相关基因表达异常有关。本文探讨凋亡抑制基因survivin在胃癌化疗耐药中的作用。方法21.96mmol/L羟基喜树碱处理胃癌细胞株BGC-823,FCM及TUNEL检测细胞凋亡,RT-PCR和免疫组化检测survivin表达。结果与对照组相比,羟基喜树碱能诱导胃癌细胞株BGC-823凋亡。[凋亡率FCM(12.68±0.17)%vs(3.35±0.13)%(P<0.01),凋亡指数(AI)TUNEL法(16.00±1.23)%vs(2.78±0.84)%(P<0.01)],同时诱导BGC-823细胞Survivin表达上调[mRNA(1.489±0.041)vs(0.756±0.037)(P<0.05),蛋白(0.928±0.046)vs(0.303±0.032)(P<0.01)]。结论Survivin基因可能和胃癌细胞获得性化疗耐药有关。     

PTEN基因转染对卵巢癌A2780细胞周期和增殖能力影响的研究

目的:研究外源性PTEN基因稳定转染对人卵巢癌A2780细胞周期和增殖能力的影响。方法:构建PTEN基因的野生型真核表达质粒pEGFP-C1-WT-PTEN和突变型表达质粒pEG-FP-C1-C124A-PTEN,以脂质体介导法转染体外培养的人卵巢癌细胞株A2780,同时转染空载体pEGFP-C1质粒,以未转染细胞为对照(转染成功后分别命名为WT-PTEN/A2780组、C124A-PTEN/A2780组、pEGFP-C1/A2-780组和A2780组。应用RT-PCR、Western blot方法分析目的基因及其蛋白表达,并采用MTT法和流式细胞术检测细胞增殖和细胞周期。结果:与对照细胞相比,WT-PTEN/A2780组和C124A-PTEN/A2780组PTEN mRNA及PTEN蛋白出现明显的高表达,与对照组细胞相比差异有统计学意义(t=5.300,P=0.034;t=11.963,P=0.007;t=7.869,P=0.016;t=22.421,P=0.002);WT-PTEN/A2780组细胞生长速度明显慢于未转染A2780细胞,然而C124A-PTEN/A2780组和pEGFP-C1/A2780组细胞生长速度无明显变化。流式细胞术显示WT-PTEN/A2780细胞G1期细胞数增加到(71.18±4.34)%,S期细胞数变为(17.48±1.96)%,与对照组相比差异有统计学意义(t=19.508,P=0.003;t=25.354,P=0.002),提示从G1期到S期发生抑制。结论:野生型PTEN可依赖其磷酸酶活性使A2780细胞在G1/S期发生细胞周期阻滞,并抑制A2780细胞增殖。     

Isolation of cancer stem-like cells from a side population of a human glioblastoma cell line,SK-MG-1

Accumulating evidence suggests that in several types of brain tumors, including glioma, only a phenotypic subset of tumor cells called brain cancer stem cells (BCSCs) may be capable of initiating tumor growth. Recently, the isolation of side population (SP) cells using Hoechst dye has become a useful method for obtaining cancer stem cells in various tumors. In this study, we isolated cancer stem-like cells from human glioma cell lines using the SP technique. Flow cytometry analysis revealed that SK-MG-1, a human glioblastoma cell line, contained the largest number of SP cells among the five glioma cell lines that were analyzed. The SP cells had a self-renewal ability and were capable of forming spheres in a neurosphere culture medium containing EGF and FGF2. Spheres derived from the SP cells differentiated into three different lineage cells: neurons, astrocytes and oligodendrocytes. RT-PCR analysis revealed that the SP cells expressed a neural stem cell marker, Nestin. The SP cells generated tumors in the brains of NOD/SCID mice at 8 weeks after implantation, whereas the non-SP cells did not generate any tumors in the brain. These results indicate that SP cells isolated from SK-MG-1 possess the properties of cancer stem cells, including their self-renewal ability, multi-lineage differentiation, and tumorigenicity. Therefore, the SP cells from SK-MG-1 may be useful for analyzing BCSCs because of the ease with which they can be handled and their yield.     

鼠乳腺癌细胞SX-9放射敏感性与细胞周期研究

通过对小鼠乳腺癌放射敏感细胞SX 9照射前、后细胞周期分布及其 p5 3基因状态等研究 ,初步探讨其放射敏感性与照射后细胞周期阻滞的关系。方法　利用克隆形成法比较SX 9与SR 1细胞照射后细胞存活份数及流式细胞术研究其细胞周期分布的差异 ;RT PCR克隆并鉴定p5 3基因 ;DNA电泳、流式细胞术和苔盼蓝染色研究细胞死亡方式。 结果　SX 9细胞对γ射线高度敏感 ,照射后G1 期阻滞消失 ,但仍存在G2 期阻滞 ;其 p5 3基因第 5外显子存在突变 ,DNA电泳检测不到DNAladder;苔盼蓝染色检测到的细胞死亡比例与对照组无显著差异。结论　γ射线照射放射敏感细胞SX 9细胞周期改变与其亲本细胞SR 1明显不同。SX 9亲本细胞SR 1细胞存在G1 ,G2 期阻滞。DNA电泳及苔盼蓝染色检测结果与SX 9细胞相同。结果表明SX 9细胞对放射敏感不是由于照射后细胞凋亡所致 ,而是由于其失去增殖能力引起增殖死亡的结果。     

Paradoxical expression of anti-apoptotic and MRP genes on cancer stem-like cell isolated from TJ905 glioblastoma multiforme cell line

Existence of cancer stem cell is regarded as a main reason for metastasis and/or recurrences. In this study, the cancer stem-like cell derived from TJ905 glioblastoma multiforme cell line was isolated successfully. However, it was observed that generating rate of the cancer stem-like cells was lower than that of TJ905 cells, that expression of the anti-apoptotic and multidrug resistance-associated protein (MRP) genes were paradoxical to the literatures, which showed the uncertainty of cancer stem cells, and that some stem cell was not the solo factor to maintain tumor growth and resist apoptosis and anti-tumor drugs.