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1.
目的 构建表达STK11(serine/threonine kinase 11,STK11)基因和报告基因EGFP融合蛋
白的重组质粒,并转染肺癌A549和H460细胞株,检测其对肺癌细胞迁移能力的影响。方法 构建重
组质粒pEGFP-STK11,双酶切和测序进行鉴定。重组质粒转染A549和H460细胞株,荧光显微镜观察
EGFP蛋白表达情况,Western blot检测STK11蛋白表达情况,划痕实验检测其对肺癌细胞迁移能力的
影响。结果 酶切和测序结果表明pEGFP-STK11重组质粒构建成功;在转染A549和H460细胞后24h观
察到绿色荧光最强;Western blot结果显示STK11蛋白在转染后24 h条带最深;划痕实验显示,转染组
细胞迁移距离小于0.9%氯化钠溶液对照组(P<0.05)。结论 成功构建STK11基因重组质粒,并能转
染A549和H460细胞,转染后对肺癌细胞迁移能力起一定抑制作用。 相似文献
2.
人微管不稳定蛋白基因真核表达载体的构建与表达及对食管癌细胞的作用 总被引:1,自引:0,他引:1
目的 构建人微管不稳定蛋白基因(stathmin)真核表达载体,研究其对食管癌细胞EC9706的作用.方法 采用逆转录聚合酶链反应(RT-PCR)扩增stathmin cDNA,克隆至pMDl8-T载体并酶切鉴定后,亚克隆至pEGFP-C2真核表达载体.将测序鉴定正确的重组载体和空载体经脂质体转染EC9706细胞,G418筛选获得稳定转染的细胞株.通过荧光显微镜观察转染细胞荧光蛋白的表达,采用Western blot检测转染细胞中增强型荧光蛋白(EGFP)和stathmin融合蛋白的表达.选取稳定转染的细胞株,采用细胞计数法绘制细胞生长曲线,采用流式细胞仪分析细胞的增殖状态,平板克隆形成实验、裸鼠移植瘤实验分析转染细胞成瘤性.结果 RT-PCR扩增获得450 bp的cDNA编码序列;克隆至pMD18-T载体,经酶切鉴定后获反向插入质粒;亚克隆至pEGFP-C2载体后,经酶切与测序鉴定,重组载体pEGFP-stathmin构建成功.重组载体和空载体转染EC9706细胞,G418筛选获得稳定转染的细胞株.通过荧光显微镜观察到,转染细胞中呈现绿色荧光;经Western blot证实,重组载体表达46 000的融合蛋白.与空载体转染细胞相比,转染重组载体的EC9706细胞形态变大,增殖速度减慢,细胞分裂阻滞于G2/M期,细胞克隆形成数减少,裸鼠体内成瘤性降低(P<0.05).结论 构建的真核表达载体pEGFP-stathmin在食管癌EC9706细胞中能够稳定表达,并抑制肿瘤细胞的增殖和成瘤性. 相似文献
3.
survivin基因RNAi逆转录病毒载体设计与构建方法实验研究 总被引:4,自引:0,他引:4
目的 设计和构建survivin基因的表达si RNA逆转录病毒重组载体,探讨胶质瘤分子病因及用于基因治疗的可行性。方法 利用在线软件si Direct设计干扰survivin基因靶序列,合成回文DNA序列退火后克隆至线性化p SUPER质粒载体,重组质粒载体双酶切电泳鉴定和测序分析,再转染phoenix细胞产生病毒转染低分化SHG4 4 - 9胶质瘤细胞株。利用NIH3T3细胞测定病毒滴度。Western blot测定转染后SHG4 4 - 9细胞survivin表达量。结果 p SU PER表达si RNA重组质粒载体经双酶切电泳鉴定和DNA测序分析,证实插入6 0 bp序列与原序列一致,位置正确。测定p SUPER.retro- S1、p SU PER.retro- S2病毒滴度值分别为5 .5×10 5CFU/ m l和5 .75×10 5CFU/ ml,干扰效率分别为70 .5 %和接近10 0 .0 %。结论 survivin基因的表达si RNA逆转录病毒重组载体的构建成功,不但为研究胶质瘤分子病因和基因治疗提供了有用工具,而且也为研究高表达survivin的其它肿瘤构建了新的平台。 相似文献
4.
目的:构建腺病毒纤维蛋白基因的真核表达质粒,并检测其在真核细胞中的表达,为腺病毒靶向性载体构建创造条件。方法:采用限制性内切酶技术,酶切腺病毒骨架质粒pAdEasy-1,经多次亚克隆,最后完成克隆纤维蛋白基因并构建其真核表达质粒,用脂质体法将构建好的真核表达质粒瞬时转染COS-7细胞,于转染后72h,用Western blot法检测所表达的蛋白。结果:克隆成功纤维蛋白基因,并构建纤维蛋白基因真核表达质粒pcDNA/Fiber,经限制性内切酶酶切鉴定及测序证实了其正确性,Western blot结果显示,新表达蛋白在变性条件下大小为62kD,而在非变性条件下为186kD。结论:纤维蛋白基因真核表达质粒能在真核细胞中表达,产物具有三聚体结构。可用于腺病毒靶向性载体的构建。 相似文献
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目的:建立稳定的基因工程细胞系.方法:将含有人肿瘤坏死因子α(hTNFα)全长cDNA插入表达载体pSNAV2.0,产生重组质粒pSNAV2.0-TNFα,利用阳离子脂质体介导将其转染到人HEK-293细胞中,G418筛选阳性克隆hTNF/293,采用Western blot检测转染细胞上清中hTNF蛋白的表达.结果:通过Sal I和EcoR I双酶切、PCR及测序鉴定证明:在质粒pSNAV2.0中插入片段正确.采用RT-PCR和Western blot法检测表明HEK-293细胞培养上清中有hTNFct蛋白表达,分子量为17KDa.结论:成功构建重组质粒pSNAV2.0-TNFα,真核表达载体构建正确,转染HEK-293细胞后可有效分泌hTNFα蛋白,并能分泌到细胞外. 相似文献
6.
目的:构建miR-513a-5p慢病毒过表达载体,转染人骨肉瘤细胞株,观察miR-513a-5p对人骨肉瘤细胞放疗敏感性的影响。方法:PCR法扩增人miR-513a-5p基因,克隆入pLentis-CMV-GFP-MCS-PGK-PURO载体获得重组质粒pLentis-miR513a,双酶切鉴定并测序后将正确的重组质粒和对照质粒转染293FT细胞制备慢病毒,分别转染骨肉瘤HOS和U2OS细胞,qRT-PCR法及荧光显微镜鉴定转染结果。克隆形成实验、MTT法检测miR-513a-5p高表达HOS和U2OS细胞在X射线照射下细胞存活情况。结果:双酶切及测序结果确定成功构建miR-513a-5p慢病毒载体pLentis-miR513a。qRT-PCR结果提示,转染骨肉瘤细胞株后miR-513a-5p表达显著升高。克隆形成实验结果显示miR-513a-5p高表达后骨肉瘤细胞在X射线照射下细胞增殖减慢。MTT结果提示miR-513a-5p高表达骨肉瘤细胞经X射线照射后细胞存活减少。结论:成功构建了miR-513a-5p慢病毒载体,建立了高效稳定表达miR-513a-5p的骨肉瘤细胞株,高表达miR-513a-5p能显著增加X射线照射后骨肉瘤细胞的放疗敏感性。 相似文献
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目的:构建真核表达载体pEGFP—BLCAP并转染骨肉瘤细胞SOSP—M。方法:从培养的人骨肉瘤细胞系SOSP—M细胞中提取总RNA,经RT—PCR获得BLCAP基因的cDNA,测序正确后插入真核表达载体pEGFP—C2中。构建的重组质粒经脂质体介导转染SOSP—M细胞,经观察荧光蛋白表达和Western blot鉴定目的蛋白在转染后的SOSP—M细胞中的表达情况。结果:RT—PCR成功地扩增出一条约280bp的片段,经限制性内切酶分析和DNA序列测定证实目的基因cDNA已插入重组质粒;荧光显微镜下观察到转染后的SOSP—M细胞发出较强绿色荧光,Western blot证明BLCAP能以融合蛋白的形式在SOSP—M细胞中获得表达。结论:构建了真核表达载体pEGFP—BLCAP并成功表达目的蛋白。 相似文献
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目的:构建白血病相关基因LRP16真核表达质粒,并检测其在人子宫内膜癌HEC-1-B细胞中的表达。方法:从人子宫内膜癌HEC-1-B细胞中提取总RNA,应用PCR技术,扩增获得LRP16基因编码序列片段,克隆入真核表达载体EX-Y2069-M29,对重组质粒进行酶切和测序鉴定后,以脂质体介导法转染至HEC-1-B细胞,采用Western blot法检测LRP16蛋白的表达。结果:酶切和测序结果证明LRP16基因真核表达质粒EX-Y2069-M29的DNA序列完全正确,将其转染HEC-1-B细胞后,LRP16蛋白表达明显增加。结论:LRP16基因重组真核表达质粒EX-Y2069-M29构建成功,并能在HEC-1-B细胞中表达,为进一步研究LRP16基因奠定了基础。 相似文献
9.
目的:构建VEGF-C基因RNA干扰(RNAi)的真核细胞表达载体。方法:以VEGF-C为靶基因,以pGenSil-l质粒为载体,设计构建重组体,根据GenBank数据库提供的VEGF-C基因核苷酸序列,按照Tuschl设计原则,选择设计两条带发夹结构的核苷酸序列,克隆到空载体pGenSil-l中,转化DH5α菌株,提取质粒,进行限制性内切酶酶切鉴定和测序分析,将重组的pGenSil-VEGF-C质粒转染LOVO细胞48小时,检测其对LOVO细胞VEGF-C蛋白与mRNA表达的影响。结果:经酶切鉴定筛选出的重组体测序结果与目的序列完全一致,重组载体显著降低Lovo细胞VEGF-C的蛋白和mRNA表达,重组载体构建成功。结论:利用RNAi技术可成功构建抑制VEGF-C表达的小干扰RNA重组体。 相似文献
10.
目的:构建慢病毒干扰LSD1表达的载体并鉴定。方法:设计并合成3对针对LSD1基因的shRNA链,退火形成双链,与酶切的质粒连接,转化DH5a菌株,提取质粒,进行酶切鉴定和测序分析。脂质体法转染人胃癌MKN-28细胞,通过荧光倒置显微镜判定转染效率,并通过real time-PCR法检测细胞中LSD1基因的表达水平。结果:经酶切鉴定筛选出的重组质粒测序结果与目的序列相同,重组质粒构建成功;转染胃癌MKN-28细胞后,LSD1基因在mRNA水平的表达降低。结论:成功构建了靶向LSD1基因的shRNA慢病毒载体,并筛选出1种对LSD1基因有显著抑制作用的shRNA。 相似文献
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目的 以真核表达质粒pmU6为基础,针对bi 1基因构建在细胞内表达短发夹状 RNA (shRNA) 的质粒载体,并观察它们对CNE 2Z、CNE 1、HO8910PM、HO8910细胞株生长增殖的影响。方法 人工合成bi 1寡核苷酸链,退火、定向克隆入pmU6载体中产生重组质粒,将重组质粒转染到细胞中,MTT比色法观察转染试剂及质粒载体对细胞生长增殖的影响。结果 与未处理组相比,bi 1shRNA对CNE 2Z、CNE 1、HO8910PM细胞株的生长增殖有明显的抑制作用,而对HO8910细胞株的生长增殖无明显抑制作用。结论 重组质粒能在细胞内表达shRNA,产生RNA干扰(RNA interference, RNAi)效应并特异性抑制靶细胞的生长增殖,为质粒介导的RNAi技术应用于鼻咽癌和卵巢癌的基因治疗提供一定的理论依据。 相似文献
13.
Shanzhou Duan Ying Tsai Peter Keng Yongbing Chen Soo Ok Lee Yuhchyau Chen 《Oncotarget》2015,6(29):27651-27660
Cisplatin-based chemotherapy is currently the most effective treatment regimen for non-small cell lung cancer (NSCLC), but eventually tumor resistance develops which limits its success. The potential implication of IL-6 signaling in the cisplatin resistance of NSCLC was explored by testing whether NSCLC cells with different levels of intracellular IL-6 show different responses to the cytotoxic treatment of cisplatin. When the cisplatin cytotoxicity of the IL-6 knocked down human NSCLC cells (A549IL-6si and H157IL-6si) were compared with their corresponding scramble control cells (A549sc and H157sc), higher cisplatin cytotoxicity was found in IL-6 si cells than sc cells. Subcutaneous xenograft mouse models were developed using a pair of A549sc and A549IL-6si cells. When the tumor grew to about 400 mm2, mice were treated with cisplatin and tumor regression was monitored. Higher tumor regression was detected in the A549IL-6si xenografts compared to A549sc xenografts following cisplatin treatment. Immunostaining study results from tumor tissues also supported this finding. Expression of anti-apoptotic proteins Bcl-2 and Mcl-1 and DNA repair associated molecules ATM, CHK1, TP73, p53, and ERCC1 were significantly up regulated in cisplatin-treated A549sc and H157sc cells, but no increase was detected in A549IL-6si and H157IL-6si cells. Further inhibitor studies revealed that up regulation of these molecules by IL-6 may be through activation of IL-6 downstream signaling pathways like Akt, MAPK, Stat3, and Erk. These results provide potential for combining cisplatin and inhibitors of IL-6 signaling or its downstream signaling pathway as a future therapeutic approach in preventing development of cisplatin resistant NSCLC tumors. 相似文献
14.
Differences in DNA alkylation products formed in sensitive and resistant human glioma cells treated with N-(2-chloroethyl)-N-nitrosourea 总被引:4,自引:0,他引:4
The distribution of alkylated deoxynucleosides and bases has been determined in the DNA of a sensitive and a resistant human glioma-derived cell line exposed to therapeutic levels of [3H]N-(2-chloroethyl)-N-nitrosourea in vitro. The resistant cell line is 5-fold less sensitive to the cytotoxic effects of N-(2-chloroethyl)-N-nitrosourea and 8-fold less sensitive to sister chromatid exchange than the sensitive cell line. In comparison with the sensitive cells, DNA from the resistant cells contains much less of the cross-link, 1-(3-deoxycytidyl),2-(1-deoxyguanosinyl)ethane. DNA from the resistant cells also contains significantly fewer minor base modifications. The decrease in 1-(3-deoxycytidyl),2-(1-deoxyguanosinyl)ethane cross-link formation is probably explained by the higher level of O6-alkyltransferase in the resistant cell line. The lower levels of other DNA modifications could be explained by the presence of higher levels of other DNA repair activities. 相似文献
15.
DNA breakage in human lung carcinoma cells and nuclei that are naturally sensitive or resistant to etoposide and teniposide 总被引:3,自引:0,他引:3
Evidence suggests that the anticancer agents etoposide (VP16-213) and teniposide (VM26) produce DNA breaks and cytotoxicity by interaction with type II topoisomerase. Therefore, levels of type II topoisomerase may influence sensitivity to VP16-213 and VM26. We have characterized four lung carcinoma-derived cell lines for natural sensitivity or resistance to VP16-213 and VM26. Included in this study were two small cell lung carcinoma lines (SW900 and SW1271), an adenocarcinoma line (A549), and a large cell carcinoma (H157). SW1271 was the most sensitive line with a median inhibitory concentration for cell proliferation of 0.5 microM for VM26 and 2.7 microM for VP16-213, and SW900 was the most resistant with median inhibitory concentration values of 2.0 and 16 microM, respectively. A549 and H157 cells were intermediate in sensitivity to these drugs. Alkaline elution techniques were used to study in vivo formation and repair of single and double strand DNA breaks. Single strand DNA breaks were observed in SW1271 cells exposed to as little as 10 nM VM26 or 100 nM VP16-213 for 1 h, whereas SW900 cells required exposure to 10-fold higher concentrations of VM26 or VP16-213 to produce similar results. Single strand DNA breaks predominated only in SW1271 and A549 cells and then, only at low drug concentrations, whereas the ratios between single and double strand DNA breaks decreased at higher drug concentrations. Plots of cytotoxicity versus single and double strand DNA breakage revealed that cytotoxicity produced by both drugs was more closely related to double strand DNA break formation in all four cell lines. DNA breaks appeared rapidly upon addition of drug, reaching plateaus in DNA breaks within 30 min, and repair of both single and double strand DNA breaks occurred rapidly with time to repair one-half of the DNA breaks of 20 to 60 min in all four cell lines upon removal of drug, arguing against repair as a mechanism for drug resistance. DNA breakage was also observed in nuclei isolated from SW900 and SW1271 cells in similar magnitude to that observed in the respective cells. Results indicate that DNA breakage plateaus may reflect a steady-state equilibrium established between the drug and its nuclear target, possibly type II topoisomerase, and suggest that natural resistance to VP16-213 and VM26 may be due to different enzyme levels in sensitive and naturally resistant cells. 相似文献
16.
目的:构建可被单纯疱疹病毒感染的新型细胞系B16-ova-HVEM,体外验证目的基因的表达并初步探究其成瘤后,应用Ⅱ型溶瘤单纯疱疹病毒(oHSV2)的治疗效果。方法构建表达单纯疱疹病毒受体(HVEM)的质粒载体,脂质体转染B16-ova细胞,嘌呤霉素筛选表达HVEM的阳性克隆。体外验证后进行体内检测,B16-ova-HVEM荷瘤C57BL/6小鼠,成瘤后分为两组,每组5只小鼠,给予oHSV2治疗为oHSV2治疗组,荷瘤未治疗的小鼠为对照组。治疗后测量肿瘤大小,观察小鼠生存期;流式细胞术检测两组小鼠外周血中CD4+、CD8+T细胞和髓系来源的抑制性细胞(MDSC)比例变化。结果质粒DNA测序和酶切鉴定结果均显示成功构建含HVEM的质粒;荧光显微镜下可观察到筛选后细胞发绿色荧光;流式细胞术检测筛选后细胞GFP阳性率为98.3%;RT-PCR验证结果显示筛选后细胞含有目的基因HVEM;oHSV2在感染复数(MOI)为0.1、0.5和1的情况下均能明显感染筛选出的细胞;动物实验显示,oHSV2体内治疗后抑瘤效果显著(P﹤0.001);流式检测结果显示oHSV2治疗后小鼠外周血中CD4+、CD8+T细胞比例明显高于对照组(P﹤0.01),而MDSC比例明显低于对照组(P﹤0.01)。结论成功构建B16-ova-HVEM细胞系,其既可被OT-1小鼠T细胞特异性识别又可被单纯疱疹病毒感染,为肿瘤特异性免疫治疗和溶瘤病毒联合治疗的研究奠定基础。 相似文献
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Cell cycle genes as targets of retinoid induced ovarian tumor cell growth suppression. 总被引:4,自引:0,他引:4
D Zhang S Vuocolo V Masciullo T Sava A Giordano D R Soprano K J Soprano 《Oncogene》2001,20(55):7935-7944
We have examined the effect of all-trans-retinoic acid (RA) on cell cycle gene expression in RA sensitive CA-OV3 and RA resistant SK-OV3 ovarian carcinoma cell lines. Gene expression was analysed by multiprobe RNAse protection, Western blotting and in vitro kinase assays. No differences were observed between RA sensitive and RA resistant ovarian carcinoma cells in the levels of expression of many cell cycle genes including cyclin A, B and E, cdk 2,4 and 6, E2F-1, E2F-2, E2F-3, E2F-4, E2F-5, DP-1 and DP-2. However, RA sensitive CA-OV3 cells expressed higher levels of p53, p27, p21, and p16 compared to RA resistant SK-OV3 cells. In addition, RA treatment of CA-OV3 cells resulted in a significant decrease in hyperphosphorylated RB and RB-2/p130 and corresponding significant increases in the levels of hypophosphorylated and/or partially phosphorylated RB-2/p130 protein and hypophosphorylated RB. Also, RA treatment increased expression of the cdk inhibitor p27 and decreased activity of cdk 2, cdk 4 and cdk 6. Finally, amounts of p27-cyclin E and RB-2/p130-E2F4 complexes were found to increase in CA-OV3 cells growth arrested by RA. These results suggest that the pocket protein pathways are critical targets for retinoid suppression of ovarian carcinoma cell growth. 相似文献
20.
Ueno K Hirata H Majid S Chen Y Zaman MS Tabatabai ZL Hinoda Y Dahiya R 《Molecular carcinogenesis》2011,50(6):449-457
The Wnt signaling pathway is activated in most cancers while Wnt antagonist genes are inactivated. However, the functional significance and mechanisms of inactivation of Wnt antagonist Dkk-3 gene in renal cell carcinoma (RCC) has not been reported. In this study, we examined potential epigenetic mechanisms regulating Dkk-3 expression in RCC cells and whether Dkk-3 expression affects cell growth and apoptosis. The expression of Dkk-3 is regulated by histone modification rather than CpG island DNA methylation in renal cancer cells. Renal cancer cell proliferation was significantly inhibited and apoptosis was promoted in Dkk-3 transfected renal cancer cells. Dkk-3 did not inhibit the Wnt/beta-catenin signaling pathway but induced apoptosis via the noncanonical JNK pathway in renal cancer cells. Expression of p21, MDM-2, and Puma genes were increased after transfecting RCC cell lines with a Dkk-3 expression plasmid. Overexpression of Dkk-3 induced G(0)/G(1) arrest together with an increase in p21 expression. Growth of stable Dkk-3 transfected cells in nude mice was decreased compared to controls. Our data show for the first time that mRNA expression of Dkk-3 is regulated by histone modification and that Dkk-3 inhibits renal cancer growth through modulation of cell cycle and apoptotic pathways. 相似文献