端粒酶反义cDNA对乳腺癌细胞恶性表型的影响

 为了进一步探讨端粒酶在肿瘤发生中的作用 ,实验应用反义核酸技术研究端粒酶反义cDNA对乳腺癌细胞MCF 7恶性表型的影响 采用的方法包括 :基因重组、脂质体共转染法获得端粒酶反义重组病毒 ,病毒感染MCF 7后 ,检测细胞的生长曲线、细胞周期及集落形成能力 结果表明 ,与对照组细胞相比 ,反义病毒感染后的MCF 7细胞恶性表型明显降低 提示端粒酶RNA的反义cDNA的导入 ,可以显著抑制乳腺癌细胞恶性表型     

端粒酶反义cDNA对乳腺癌细胞损伤修复能力的影响

 利用反义核酸技术将端粒酶RNA的全长cDNA反向导入乳腺癌MCF 7细胞基因组中 .通过单细胞凝胶电泳实验发现 ,其DNA受H2 O2 损伤后的修复能力下降 .但端粒酶活性抑制为何引起其DNA损伤修复能力下降的原因尚待进一步研究 .     

端粒酶RNA反义基因对肝癌细胞的影响

用RT-PCR的方法钓取端粒酶RNA基因的cDNA,并将其反向插入到逆转录病毒载体pLNCX上,构建hTR基因的反义表达质粒。将质粒经脂质体介导转染人肝癌SMMG-7721细胞中表达。结果表明hTR反义基因的表达有效地封闭或抑制肝癌细胞的端粒酶活性,抑制细胞的生长和增殖,延长细胞的倍增时间并促进细胞凋亡。     

下调c—erbB—2对细胞DNA修复和凋亡的影响

将有义和反义c-erbB-2逆转录病毒体分别经脂质体包裹后转染入人胚肺二倍体成纤维细胞（2BS）。Southern印迹杂交表明,外源c-erbB-2 cDNA在转染细胞中已成功整合入基因组中。Northern印迹杂交显示,有义转染细胞的erbB-2表达上升57％,反义转染细胞erbB-2表达下降48％。与对照和空载体转染细胞相比,反义转染细胞的DNA损伤修理能力显著下降,凋亡可诱导性降低。这和我们观察到的反义转染细胞提早出现衰老表型相一致。     

ω-6脂肪酸脱氢酶基因在乳腺癌细胞内的表达和作用

为探讨ω- 6脂肪酸脱氢酶基因fat -1在人类乳腺癌细胞MCF- 7中表达和对其生长的作用,将fat -1基因插入到腺病毒载体中,构建腺病毒重组载体(Ad·GFP·fat1) .通过包装细胞系(2 93)产生重组腺病毒,感染MCF 7细胞.用核糖核酸酶保护性分析技术,检测fat -1基因在MCF- 7细胞内的表达,细胞增殖试剂盒(MTT)和凋亡染色试剂盒染色分析fat 1基因对MCF- 7细胞增殖和凋亡的影响,用酶联免疫分析花生酸类(eicosanoids)前列腺素E2 (prostaglandinE2 )的含量.结果显示,腺病毒介导的fat- 1基因能在MCF- 7细胞内有效异源表达,抑制MCF -7细胞的增殖且导致凋亡,前列腺素的含量也明显地减少.结果说明,fat- 1基因在乳腺癌的基因治疗中具有良好利用价值.     

雌激素受体α短发夹RNA慢病毒载体的构建及其对乳腺癌干细胞的影响

目的:构建特异抑制雌激素受体α(ERα)基因的短发夹RNA(sh RNA)慢病毒表达载体,检测其对ERα的干扰效果,并探讨其对MCF7细胞中乳腺癌干细胞含量的影响。方法:以人的ERα核酸序列为靶标,根据sh RNA设计原则设计并化学合成特异的sh RNA序列,经退火处理后与p SIH-H1质粒连接,转化大肠杆菌DH5α后挑取单克隆并测序;将该克隆进行病毒包装并感染乳腺癌MCF7细胞,用嘌呤霉素进行筛选,最终得到稳定克隆;收取细胞,用Western印迹和q RT-PCR检测ERα在m RNA及蛋白水平上的变化,流式细胞术检测敲低ERα能否影响MCF7乳腺癌干细胞的含量。结果:经测序分析表明目标sh RNA序列成功插入载体,q RT-PCR检测稳定克隆,发现该sh RNA能有效抑制目的基因的m RNA转录水平,同时Western印迹检测发现内源性ERα量明显下降;流式细胞术检测发现ERα敲低的MCF7稳定克隆中雌激素诱导的乳腺癌干细胞含量上升的现象被明显抑制。结论:设计并构建的抑制ERα的sh RNA能够有效抑制ERα的表达,并下调MCF7细胞中乳腺癌干细胞的含量,为研究ERα在乳腺癌干细胞中的功能及机制奠定了实验基础。     

人同源盒基因NKX3.1对前列腺癌细胞的诱导凋亡作用

构建人同源盒基因NKX3.1 cDNA真核表达载体,研究其在前列腺癌细胞PC-3、LNCaP 中的表达及对细胞的促凋亡作用.以人前列腺癌细胞LNCaP细胞中的总RNA为模板,RT-PCR扩增NKX3.1基因全长编码片段,将NKX3.1 cDNA重组到真核表达载体pcDNA3.1（+）中; 将pcDNA3.1-NKX3.1表达载体瞬时转染前列腺癌细胞PC-3和LNCaP 细胞,用RT-PCR和Western印迹检测NKX3.1 cDNA在转录水平和蛋白水平的表达;绘制细胞生长曲线,观察NKX3.1对前列腺癌细胞增殖的抑制作用;用DNA/ladder和流式细胞术检测NKX3.1对前列腺癌细胞凋亡的影响,进一步用RT PCR检测凋亡相关基因caspase3、caspase8、caspase9、Apaf1、survivin和Bcl2表达的变化.人同源盒基因NKX3.1 cDNA真核表达载体pcDNA3.1-NKX3.1经酶切及测序鉴定正确. pcDNA3.1-NKX3.1转染PC-3和LNCaP细胞后,经RT-PCR和Western印迹证明能有效表达NKX3.1.生长曲线显示,前列腺癌细胞转染NKX3.1 cDNA后细胞增殖受到抑制;前列腺癌细胞转染NKX3.1 cDNA 48 h后,DNA电泳呈现具有凋亡特征的DNA ladder;流式细胞术检测出现明显凋亡峰;RT-PCR检测凋亡相关基因.结果显示,caspase3、caspase8、caspase9基因表达明显增加,Bcl2基因表达明显减少.本研究成功构建了真核表达载体pcDNA3.1 NKX3.1, 转染PC3和LNCaP细胞后能有效表达,并对细胞具有诱导凋亡作用     

c-myc表达对顺铂诱导人胶质瘤细胞系BT_(325)凋亡的影响

研究 c- m yc在化疗药物顺铂诱导肿瘤细胞凋亡中的作用。在原有已构建好 c- myc反义表达载体 pc DNA3- MYC并成功转染 BT32 5 细胞的基础上 ,用顺铂诱导细胞凋亡。采用 TUNEL技术并从光镜、电镜、荧光显微镜及激光共聚焦显微镜水平观察 BT32 5 细胞凋亡的形态学特征 ;流式细胞仪检测实验组 (即 pc DNA3- MYC转染的细胞 )及对照组 (包括未转染或仅转染空载体 pc DNA 3的细胞 )凋亡率的异同。结果显示 :与转染空载体 pc DNA3及未转染组的细胞相比 ,在转染 pc DNA 3-MYC的 BT32 5 细胞中 ,顺铂诱导的细胞凋亡作用明显受阻 ,结果表明 :肿瘤细胞内异常表达的 c- myc在顺铂诱导肿瘤细胞凋亡的过程中有重要作用。     

反义hTR抑制人胰腺癌P3细胞系端粒酶活性和增殖的研究

设计针对端粒酶RNA模板序列并经硫代磷酸修饰的正义、反义和随机序列寡核苷酸,以正常人成纤维细胞作对照,观察其对人胰腺癌细胞端粒酶活性和细胞生长增殖的影响作用以及对正常细胞是否有毒副作用。结果表明：针对hTR模板区的反义寡核苷酸能降低胰腺癌P3细胞端粒酶活性,抑制细胞生长,促进细胞周期改变并诱导细胞发生凋亡,而且对正常细胞没有明显的毒副作用。因此我们认为利用反义技术封闭hTR基因很可能成为治疗肿瘤的安全、有效手段之一。 Abstract:This paper is to investigate PS-ODN's (antisense-PS-ODN of hTR,sense-PS-ODN of hTR and random sequence) effects on telomerase activity and proliferation of P3 pancreatic cancer cells,and to find a novel method for gene therapy of pancreatic cancer.The results indicate that the anti-hTR complementary to the template region of hTR is sufficient to inhibit P3 cell telomerase activity and cell proliferation in vitro,and as a result,they can lead to a profound induction of programmed cell death.Telomerase represents an interesting and promising anticancer drug target and antitelomerase technology may have potential significance in tumor therapy.     

山杏叶乙酸乙酯提取物调控乳腺癌MCF7细胞增殖和凋亡的机制研究

为探究山杏叶乙酸乙酯提取物对乳腺癌MCF7细胞株增殖和凋亡的影响,本研究采用CCK8法检测山杏叶提取物对MCF7细胞增殖的抑制作用,流式细胞仪检测细胞凋亡率,倒置荧光显微镜和流式细胞仪分别检测胞内活性氧(ROS)的水平变化,RT-PCR检测细胞周期及凋亡相关基因的表达情况,试剂盒检测caspase-3的活性。实验表明,山杏叶提取物可降低MCF7细胞存活率,促进细胞凋亡,增加胞内ROS水平。同时上调和Bax,下调Bcl-2,增强caspase-3活性,并降低CDK4、CyclinE和CyclinD1的表达。综上说明山杏叶提取物可通过调控周期蛋白的表达来抑制MCF7细胞的增殖,并通过caspase途径和提升ROS水平来诱导MCF7细胞的凋亡。     

端粒酶RNA的反义cDNA对乳腺癌细胞端区长度的影响

应用反义核酸技术,将端粒酶ＲＮＡ的ｃＤＮＡ反向插入整合型腺病毒载体ｐＡｄＥ１ＣＭＶＩＴＲＥＸｎｅｏ,与ｐＢ－ＨＧ１１Ｘ共转染２９３细胞反获得反义重组病毒。将此反义重组病毒感染乳腺癌细胞ＭＣＦ－７后,使细胞端区长度缩短。     

Effect of sodium butyrate on human breast cancer cell lines

We have investigated the effects exerted by sodium butyrate (NaBu) on the growth and cell cycle perturbations of four human breast cancer cell lines (MCF7, T47D, MDA-MB231 and BT20) with different steroid receptor profiles. Moreover, since one of the supposed mechanisms of action for NaBu activity involves the induction of apoptosis, we have studied the effects of NaBu on DNA fragmentation by agarose gel electrophoresis and flow cytometry. In all investigated cell lines, NaBu exerted a time- and dose-dependent inhibition of growth and caused a maximum inhibitory effect (85% to 90%) at the concentration of 2.5 m m . The inhibition was already evident after 3 days of treatment. The antiproliferative effect of NaBu was associated with a persistent block of cells in the G₂M phase. The block was associated with apoptosis only in oestrogen-receptor positive cell lines. The inhibiting effect of NaBu in hormone-dependent and independent cell lines and its ability to induce apoptosis through a cell cycle perturbation in hormone-dependent cell lines may have important implications in the treatment of human tumours including breast cancer.     

Inhibition of sodium butyrate-induced apoptosis in recombinant Chinese hamster ovary cells by constitutively expressing antisense RNA of caspase-3

Sodium butyrate (NaBu) can enhance the expression of genes controlled by some of the mammalian promoters, but it can also inhibit cell growth and induce cellular apoptosis. Thus, the beneficial effect of using a higher concentration of NaBu on a foreign protein expression is compromised by its cytotoxic effect on cell growth. To overcome this cytotoxic effect of NaBu, the expression vector of antisense RNA of caspase-3 was constructed and transfected to recombinant Chinese hamster ovary (rCHO) cells producing a humanized antibody. Using this antisense RNA strategy, rCHO cells (B3) producing a low level of caspase-3 proenzyme were established. When batch cultures of both B3 cells and control cells transfected with antisense RNA-deficient plasmid were performed in the absence of NaBu, both cells showed similar profiles of cell growth and antibody production. Compared with control cell culture, under the condition of 5 mM NaBu addition at the exponential growth phase, expression of antisense RNA of caspase-3 significantly suppressed the NaBu-induced apoptosis of B3 cells and extended culture longevity by >2 days if the culture was terminated at cell viability of 50%. However, compared with control cell culture, the final antibody concentration of B3 cell culture was not increased in the presence of NaBu, which may be due to the loss of cellular metabolic capability resulted from the depolarization of mitochondrial membrane. Taken together, this study suggests that, although expression of antisense RNA of caspase-3 does not improve antibody productivity of rCHO cells, it can suppress NaBu-induced apoptotic cell death of rCHO cells and thereby may reduce problems associated with cellular disintegration.     

外源p21~(WAF1)转染对人成纤维细胞凋亡的影响

构建了有义和反义ｐ２１ＷＡＦ１ 逆转录病毒表达载体, 分别经脂质体包裹后转染人胚肺二倍体成纤维细胞（２ＢＳ）。Ｓｏｕｔｈｅｒｎ 印迹杂交证实转染细胞中外源ｐ２１ ＷＡＦ１ｃＤＮＡ 已整合入基因组中。与空载体转染细胞相比, 有义转染细胞的ｐ２１ＷＡＦ１ ｍ ＲＮＡ 表达上升; 细胞增殖速度明显减慢; 对丁酸钠诱导凋亡的敏感性降低, 表现在细胞存活率升高, 核ＤＮＡ 梯状断裂片段出现的时间滞后, 断裂片段浓度下降, 流式细胞计检测的凋亡峰面积缩小。而反义转染细胞的ｐ２１ＷＡＦ１ ｍ ＲＮＡ表达下降; 细胞增殖速度较快; 对丁酸钠诱导凋亡的敏感性上升, 有关表现与有义转染细胞相反。说明２ＢＳ细胞内ｐ２１ＷＡＦ１ 的表达量与其被丁酸钠诱导凋亡的能力呈负相关。     

H18杂交瘤抗凋亡能力的改造

利用PCR从pGEMTbcl-X_L质粒中获得bcl-X_L基因,构建真核表达载体pEF-bcl-X^{L,脂质体法转染杂交瘤细胞,G418筛选稳定表达株,Western blotting检测目的蛋白表达,流式细胞仪检测Bcl-X_L提高杂交瘤抗正丁酸钠诱导凋亡的功能。 将构建的编码鼠bcl-X_L基因的真核表达载体pEF-bcl-X_L,转染H18细胞后,获得稳定的表达株细胞;稳定表达Bcl-X_L的细胞具有抗正丁酸钠诱导凋亡的功能。鼠bcl-X_L基因在杂交瘤细胞中稳定表达,提高了杂交瘤抗凋亡的能力,对高密度大规模培养杂交瘤细胞具有重要意义。}     

Overexpression of bcl-2 inhibits sodium butyrate-induced apoptosis in Chinese hamster ovary cells resulting in enhanced humanized antibody production

Sodium butyrate (NaBu) can enhance the expression of genes from some of the mammalian promoters including cytomegalovirus (CMV) and simian virus 40 (SV40), but it can also inhibit cell growth and induce cellular apoptosis. Thus, the beneficial effect of using a higher concentration of NaBu on a foreign protein expression is compromised by its cytotoxic effect on cell growth. To overcome this cytotoxic effect of NaBu, a survival protein, human Bcl-2, was overexpressed in recombinant Chinese hamster ovary (CHO) cells (SH2-0.32), producing a humanized antibody directed against the S surface antigen of hepatitis B virus. When batch cultures of both control cells transfected with bcl-2-deficient plasmid (SH2-0.32-Deltabcl-2) and cells transfected with bcl-2 expression plasmid (14C6-bcl-2) were performed in the absence of NaBu, both cells showed similar profiles of cell viability and antibody production. Compared with the SH2-0.32-Deltabcl-2 culture, under the condition of NaBu addition at the exponential growth phase, overexpression of the bcl-2 gene considerably suppressed the NaBu-induced apoptosis of 14C6-bcl-2 by inhibiting caspase 3 activity and extending culture longevity by >2 days. As a result, the final antibody concentration of 14C6-bcl-2 culture was twofold higher than that of SH2-0.32-Deltabcl-2 culture in the presence of NaBu and threefold higher than that of SH2-0.32-Deltabcl-2 and 14C6-bcl-2 cultures in the absence of NaBu.     

大鼠bcl-x_L cDNA的克隆和高滴度重组bcl-x_L腺相关病毒的制备

 以RT PCR法从大鼠脑组织中克隆bcl XL 基因 ,将其定向插入带rep cap基因和neu基因的三功能腺相关病毒 (AAV)载体 ,转染HeLa细胞并以G4 18筛选 ,然后用腺病毒感染筛选后的细胞克隆 ,包装成重组腺相关病毒 .用带rep cap基因的C12细胞筛选和滴定产生重组腺相关病毒的细胞克隆 ,粗制细胞裂解物中病毒滴度最高只有 3× 10 5IU ml.从产病毒量较高的克隆大量制备重组病毒 ,经肝素柱高压液相亲和层析法纯化、浓缩病毒后获得了高达 4× 10 11IU ml的重组病毒 .为研究脑缺血动物模型中BCL XL 的抗脑细胞凋亡作用打下了基础     

Helper-free stocks of recombinant adeno-associated viruses: normal integration does not require viral gene expression.

A method is described for the production of recombinant adeno-associated virus (AAV) stocks that contain no detectable wild-type helper AAV. The recombinant viruses contained only the terminal 191 nucleotides of the AAV chromosome bracketing a nonviral marker gene. trans-Acting AAV functions were provided by a helper DNA in which the terminal 191 nucleotides of the AAV chromosome were substituted with adenovirus terminal sequences. Although the helper DNA did not appear to replicate, it expressed AAV functions at a substantially higher level than did DNA molecules that contained neither AAV nor adenovirus termini. Since the recombinant viruses with AAV termini contained no sequence homology to the helper DNA, no wild-type AAV was generated by homologous recombination within infected cells. Since the terminal region of the AAV chromosome is required for replication and encapsidation, only recombinant DNAs were amplified and packaged into AAV virions. When human cells were infected at a high multiplicity with a recombinant virus carrying a drug resistance marker gene, approximately 70% of the infected cells gave rise to colonies stably expressing the marker. The recombinant virus gene was then used to generate drug-resistant human cell lines subsequent to infection. These cells contained stably integrated copies of the recombinant viral DNA which could be excised, replicated, and encapsidated by infection with wild-type AAV plus adenovirus. Thus, AAV gene expression is not required for normal integration of an infecting DNA containing AAV termini.