目的 研究凋亡抑制基因XIAP与抑癌基因p53在非小细胞肺癌中的表达及两者的相关性.方法 转染特异短发夹样RNA(XIAP shRNA)序列的表达载体至非小细胞肺癌细胞A549中,应用半定量PT PCR方法测定转染前后细胞中XIAP与p53的表达情况并进行对比.结果 PT-PCR结果显示XIAP在阳性转染组的表达较阴性转染组、未转染组细胞表达明显降低；p53在阳性转染组表达升高,加入顺铂后,阳性转染组XIAP mRNA表达较阴性转染组、未转染组细胞降低,p53在阳性转染加顺铂组的表达明显提高.结论 非小细胞肺癌中XIAP与p53呈负相关,高表达的XIAP抑制野生型p53的表达,反之XIAP受到抑制后野生型p53表达增高,促进非小细胞肺癌细胞凋亡.

Objective To study the expression of antiapoptosis gene XIAP and tumor suppressor gene p53 in non-small cell lung cancer as well as their respective dependencies.Methods The specific short hairpin RNA (XIAP shRNA) expression vector sequence was transfected to A549 cell.The expressions of XIAP and p53 in transfected cells was determined by semi quantitative RT-PCR.Results PT-PCR results showed that the expression of XIAP in positive transfection group was significantly decreased than that in negative transfection group and untransfected group.The expression of p53 was elevated in the positive transfection group.After the addition of cisplatin,XIAP mRNA expression in positive transfection group was lower than that in negative transfection group and untransfected group,and p53 mRNA expression increased in positive transfection plus cisplatin group.Conclusions XIAP and p53 are negatively correlated.High expression of XIAP inhibits expression of wild type p53,whereas wild-type p53 expressio

目的设计合成有效的靶向Toll样受体4(TLR4)基因的小干扰RNA(siRNA)表达载体,并筛选出TLR4基因稳定沉默的血管平滑肌细胞(SMC)。方法依据RNA干扰(RNAi)序列设计原则,以TLR4基因为靶基因,合成3对小发夹RNA(shRNA)寡核苷酸链,经退火、与线性化pSilence 2.1-U6 neo质粒连接,酶切及测序进行鉴定。脂质体法转染血管SMC,新霉素(G418)加压筛选并收集稳定表达质粒的SMC。RT-PCR及Western blot法检测收集的SMC中TLR4 mRNA和蛋白表达,以确定siRNA对TLR4的抑制效率。结果测序鉴定插入的发夹样序列正确,成功构建了TLR4基因siRNA表达载体;并获得了稳定沉默TLR4的血管SMC。RT-PCR及Western blot证实RNA干扰TLR4基因后,SMC中TLR4 mRNA和蛋白表达均明显减低,其中pSilence2.1-siTLR4-1的抑制作用最强。结论成功构建了靶向TLR4基因的siRNA表达载体,并有效抑制血管SMC中TLR4表达。

Objective To construct vectors expressing small interfering RNA targeting the Toll like receptor-4 (TLR4) gene and obtain TLR4 knock downed vascular smooth muscle cells (SMC). Methods Three small hairpin RNA (shRNA) targeting the TLR4 gene were designed, synthesized and cloned into the pSilence 2.1-U6 neo vector. Positive clones were verified with double enzyme digestion and sequencing. Then the recombinants were transfected to SMC by the cationic lipid method respectively.SMC were stably transfected with an expression plasmid and screened by G418. TLR4 mRNA and protein expression were detected by RT-PCR and Western blot methods. Results The pSilence2.1-siTLR4 ex-pression vectors were successfully constructed and a TLR4 knock-downed SMC cell line was established. RT-PCR and Western blot analysis confirmed that the expression of TLR4 was significantly down-regulated in the infected SMC cell line, and pSilence2.1-siTLR4-1was the most efficacious recombinant vector.Conclusion Recombinant

背景：端粒酶反转录酶对端粒酶活化起重要作用。 目的：利用pLentilox3.7.U6载体构建靶向大鼠脊髓星形胶质细胞端粒酶反转录酶基因的短发夹RNA干扰分子表达载体并鉴定其作用。 方法：选择端粒酶反转录酶基因上两段序列体外合成RNA干扰分子的正义链和反义链模板序列，经退火成互补双链，与线性化pLentilox3.7.U6载体连接、转化大肠杆菌和序列测定，应用蛋白质印迹和免疫荧光技术，在体外培养的大鼠脊髓星形胶质细胞模型上验证构建的干扰载体抑制端粒酶反转录酶基因表达的效果。 结果与结论：蛋白质印迹和免疫荧光检测结果表明，重组质粒干扰组中星形胶质细胞端粒酶反转录酶均呈低表达。结果证实，实验成功构建了针对大鼠脊髓星形胶质细胞端粒酶反转录酶基因的短发夹RNA质粒表达载体，此载体能有效抑制体外培养的大鼠脊髓星形胶质细胞端粒酶反转录酶的表达。

BACKGROUND:Telomerase reverse transcriptase (TERT) plays an important role in telomerase activation. OBJECTIVE:To construct the targeting short hairpin RNA plasmid vector expressing TERT gene from astrocytes by using pLentilox3.7.U6. METHODS:By using two sequences from TERT gene, we synthetized sense and antisense strand template sequences of RNA interference molecular in vitro, and then obtained the complementary strands through annealing procedure. We connected the strands with pLentilox3.7.U6 that was sequenced and transfected into the Escherichia coli. In the end, we tested its effect of reducing the TERT gene expressing by using cultured astrocytes from rat spinal cord in vitro through western blot and immunofluorescence technique. RESULTS AND CONCLUSION:Western blot and immunofluorescence assay showed that, compared with the control group, the interference groups had a lower TERT expression in astrocytes. The targeting short hairpin RNA plasmid vector expressing TERT gene is usef

为了构建可在人喉癌细胞中稳定表达IFI16基因短发夹RNA(shRNA)的表达载体,设计合成的IFI16基因shRNA片段,连接到经BamHI和EcoRI双酶切的pGreenPuroTMshRNA表达载体中,连接产物转化大肠杆菌后挑取几个抗性菌落,用PCR技术初步进行鉴定,经PCR初步鉴定为重组质粒的一个重组子DNA用测序进一步鉴定,测序结果显示成功构建了IFI16基因shRNA的表达载体pGreenPuro-IFI16shRNA.

To construct the vector that can stably express IFI16 shRNA in human laryngocarcinoma cells ,the well designed and correctly synthesized IFI16 shRNA fragment was used to ligate into the pGreenPuro TM shRNA expressing vector that predigested with BamH I and EcoR I .Subsequently ,the recombinant vector was used to transform the E .coli and then the PCR was performed to identify the resistant colonies .Besides ,the identified recombinant DNA was subjected to sequence for the further identification .Finally ,the IFI16 shRNA expressing vector named pGreenPuro-IFI16 shRNA was correctly constructed as circumstantiated by the sequencing result .

探讨BubR1基因在紫杉醇治疗肿瘤中的作用。方法:构建针对BUBR1基因的特异性短发夹RNA载体,导入人肝癌细胞株HepG-2,转染48 h后,运用实时荧光定量PCR、Western印迹法评价RNA干扰效果;BubR1表达下调的肝癌细胞及对照组细胞用不同浓度(1、3、10、30、100、300、1000和3000 nmol/L)的紫杉醇处理,MTT法检测细胞增殖情况。结果:shRNA-BubR1可有效抑制BubR1的mRNA、蛋白水平以及内源性表达。MTT结果显示,BubR1表达下调的细胞在不同浓度紫杉醇作用下的增殖抑制率与对照组相比明显增加(P0.05),并呈现剂量依赖性。结论:下调BubR1的表达能增强肝癌细胞对紫杉醇的敏感性。

Objective To investigate the role of BubR1 cin the therapeutic effects of paclitaxel ontumor. Methods P1asmid vector containing BubR1 specific shRNA was constructed and eleetroporated into humancervix cancer HepG-2 cells. RFQ-PCR, Western blotting method were used to evaluate the silencing em ciency of RNA interference after 48 h.HepG-2 cells with down-regulated BubR1 and control cells were treated with paclitaxe1 at different concentrations (1,3,10,30,l0o,30o,1 000 and 3 000 nmol/L).The proliferation of cells were measured by MTI1 assay.Results BubR1 specific shRNA effectively inhibited the mRNA and protein expression of BubR1 in HepG-2 cells and endogenous expression.The resultsof MTT indicated that the growth inhibition rate was obvi-ously increased in those cells with down-regulated BubR1 compared with control cells (P<0.05) and the growth inhibition effect was in a dose-dependent manner. Conclusion Down-regulation of BubR1geneexpression can enhance the sensitivity of cervical canc