靶向SMPD1基因的RNA干扰对人成纤维细胞凋亡的保护作用

 【目的】以人包皮成纤维细胞（HFF）为模型,通过系统比较针对不同靶点的小干扰RNA（siRNA）对酸性鞘磷脂酶1（SMPD1）基因的沉默效果,筛选获得最有效的小干扰RNA序列,同时观察沉默SMPD1对细胞凋亡的保护作用。【方法】设计合成三对靶向SMPD1基因的小干扰RNA作为实验组,同时设立阴性对照组和脂质体组（lipofectamine 2000）,瞬时转染原代培养的HFF细胞,采用荧光定量RT-PCR法及Western blot测定SMPD1表达抑制情况,并用化疗药物丝裂霉素诱导细胞凋亡,MTT法检测siRNA转染对细胞的保护作用。【结果】小干扰RNA1,2,3对SMPD1基因mRNA的抑制效率分别为96.3%,39%,63.2%,以siRNA1最高,最佳浓度为50nmol/L,最佳作用时间为48h。SMPD1蛋白在48h出现沉默效果,96h开始恢复。50nmol/L的siRNA1可以有效抑制化疗药物丝裂霉素引起的细胞凋亡,细胞存活率为73.8%,与对照组存活率65.4%比较差异具有统计学意义。【结论】三对siRNA均具有一定的靶基因沉默效果,其中siRNA1效果最佳,沉默效率达到96.3%;靶向SMPD1基因的siRNA能有效抑制人成纤维细胞的凋亡。     

靶向SMPD1基因的RNA干扰对人成纤维细胞凋亡的保护作用

目的】以人包皮成纤维细胞（HFF）为模型,通过系统比较针对不同靶点的小干扰RNA（siRNA）对酸性鞘磷脂酶1（SMPD1）基因的沉默效果,筛选获得最有效的小干扰RNA序列,同时观察沉默SMPD1对细胞凋亡的保护作用。【方法】设计合成三对靶向SMPD1基因的小干扰RNA作为实验组,同时设立阴性对照组和脂质体组（lipofectamine 2000）,瞬时转染原代培养的HFF细胞,采用荧光定量RT-PCR法及Western blot测定SMPD1表达抑制情况,并用化疗药物丝裂霉素诱导细胞凋亡,MTT法检测siRNA转染对细胞的保护作用。【结果】小干扰RNA1,2,3对SMPD1基因mRNA的抑制效率分别为96.3%,39%,63.2%,以siRNA1最高,最佳浓度为50nmol/L,最佳作用时间为48h。SMPD1蛋白在48h出现沉默效果,96h开始恢复。50nmol/L的siRNA1可以有效抑制化疗药物丝裂霉素引起的细胞凋亡,细胞存活率为73.8%,与对照组存活率65.4%比较差异具有统计学意义。【结论】三对siRNA均具有一定的靶基因沉默效果,其中siRNA1效果最佳,沉默效率达到96.3%;靶向SMPD1基因的siRNA能有效抑制人成纤维细胞的凋亡。     

Enhanced Chemotherapy Sensitivity of Human Colon Cancer Cells to 5-Fluorouracil by siRNA Recombinant Expression Vector Targeting Survivin Gene

Objective To investigate the effects of small interfering RNA （siRNA） recombinant expression vector targeting survivin gene on chemotherapy sensitivity of human colon cancer cells to 5-fluorouracil. Methods siRNA recombinant expression vector targeting survivin gene was constructed and transfected into human colon cancer cell lines LOVO. After 48 hours of transfection, cells were harvested for analysis of survivin mRNA and protein expressions using RT-PCR and Western blot. In addition, after human colon cancer cell lines were treated with Survivin siRNA and/or 5-fluorouracil, MTT assay and flow cytometry were used to analyze cell proliferation and apoptosis. Results Restriction endonuclease analysis confirmed that siRNA recombinant expression vector targeting survivin gene was successfully constructed. Inhibitory ratios of survivin mRNA and protein expressions by Survivin siRNA were 36.33% and 44.65%, respectively. Survivin siRNA combined with 5-fluorouracil significantly increased the cell proliferation inhibitory ratio and apoptosis ratio compared with 5-fluorouracil treatin~ alone （P〈0.05）. Conclusion The siRNA recombinant expression vector targeting survivin gene can inhibit the expression of survivin gene, and enhance chemotherapy sensitivity of human colon cancer cells to 5- fluorouracil.     

Effects of Livin gene RNA interference on apoptosis of cervical cancer Hela cells and enhanced sensitivity to cisplatin

The recombinant plasmids pGenesil-1-BIRC71 and pGenesil-1-BIRC72 were transfected into Hela cells and cisplatin was added with different concentrations in order to study the inhibitory effects of Livin gene, increase the apoptosis induced by cisplatin, and detect the expression ofBcl-2, Bax, caspase-3, and survivin genes. The pGenesil-1-BIRC71 and pGenesil-1-BIRC72 were transfected into Hela cells, and the expression levels of Livin, Bcl-2, Bax, caspase-3, and survivin genes were detected by using fluorescence quantitative real-time PCR. Then cisplatin at different concentrations （3.0, 6.0 and 9.9 μg/mL） was added into the transfected Hela cells, and 24, and 48 h later, the apoptosis rate was measured by flow cytometry. After transfection of pGenesil-l-BIRC71 and pGenesil-1-BIRC72 into Hela cells, the expression level of Livin gene was obviously reduced, and the apoptosis rate was significantly increased in transfection group as compared with control group （P〈0.05）. Cisplatin could increase the apoptosis rate in a dose- and time-dependent manner. After cisplatin was added, the expression levels of Bcl-2 mRNA were reduced, and those of Bax, caspase-3, and survivin mRNA were increased in transfection group as compared with those in control group （P〈0.05）. It was concluded that shRNA expression vector targeting Livin gene could inhibit the expression of Livin gene in Hela cells and enhance the apoptosis induced by cisplatin, which was related to the decreased expression of Bcl-2 and activation of Bax and caspase-3. Survivin might play an important role as an antagonist in the process of apoptosis induction.     

靶向性血小板衍生生长因子siRNA表达质粒的构建及鉴定

目的 构建靶向性血小板衍生生长因子(PDGF)B链的小分子干扰RNA(siRNA)表达质粒,为进一步研究PDGF基因功能奠定基础.方法 根据PDGF-B链序列设计合成含靶向PDGF基因siRNA转录模板的茎环结构,与两端分别有Bam HI、HindIII酶切位点的pSilencer3.1-Hlhygro质粒连接,转化大肠杆菌,扩增、纯化得到所需质粒,通过琼脂糖凝胶电泳及基因测序鉴定其分子量及插入片段的序列.结果 PCR扩增片段与预期结果相符,双酶切证实PDGF siRNA表达载体克隆构建成功,插入片段测序结果与合成的siRNA结果一致.结论 成功构建了靶向性PDGF基因的siRNAs表达质粒. Abstract： Objective To construct the small interfering RNA (siRNA) expressing plasmids targeting platelet-derived growth factor(PDGF)and study the function of PDGF with RNA interference technology. Methods Two complementary 63-nt oligonucleotides targeting PDGF were synthesized with 5 single-stranded over-hangs according to the PDGF-B gene sequence in the Genebank and the kit manual,which were ligated with the linearized pSilencer3.1-Hlhygro.The plasmids were transformed into DH5α bacteria to amplify and then purified.The purified plasmids were identified by gel electrophoresis and sequencing.Results PDGF siRNA expression vectors were successfully constructed and identified by double endonuclease digestion.Sequence analysis of inserted fragment revealed the same sequence as synthesized siRNA oligonucleotides.Conclusions siRNA expression plasmids targeting PDGF have been successfully constructed.     

3种不同的survivin siRNA对EJ28细胞增殖和凋亡的影响

目的 观察靶向survivin的不同siRNA对EJ28细胞增殖和凋亡的影响.方法 通过化学合成方法合成3对siRNA和1对荧光标记的阴性对照siRNA.转染荧光标记的siRNA后,在荧光显微镜下观察转染效率.实验分为siRNA164、siRNA167、siRNA389、阴性对照组、脂质体对照组和细胞对照组.转染后24、48 h和72 h,MTT法检测siRNA对EJ28细胞增殖的影响,流式细胞术检测凋亡率.结果 靶向survivin的序列特异性siRNA均能抑制EJ28细胞的增殖,其中以siRNA164抑制细胞的增殖能力最强(P＜0.05),转染48 h后的抑制率最高[(41.32±2.54)%];转染48 h后,siRNA164组凋亡率最高[(13.20±0.25)%].结论 靶向survivin的RNAi技术在膀胱癌的基因治疗中可能具有一定的价值.     

Inhibition of survivin expression and mechanisms of reversing drug-resistance of human lung adenocarcinoma cells by siRNA

Background Survivin, a member of the inhibitor of apoptosis protein （IAP） family, overexpresses in tumor cells and not expresses in terminally differentiated adult tissues. This study aimed to investigate the effects of survivin-specific siRNA on cell proliferation, apoptosis and chemosensitivity to cisplatin in vitro and in vivo and explore the mechanisms about decreasing expression of survivin in reversing cancer cells resistance to chemotherapeutic drug.Methods Survivin-specific siRNA was transfected into A549/DDP cells. The expression of survivin and lung resistance-related protein （LRP） mRNA levels were determined by RT-PCR, chemosensitivity of A549/DDP （cisplatin）cells to cisplatin was determined by MTT assay, and apoptosis and cell cycle were determined by flow cytometry （FCM）.The protein expression levels of survivin, LRP, cyclin-D1, caspase-3 and bcl-2 were determined by Western blotting analyses. The effect of survivin siRNA inhibition on tumor growth was studied in athymic nude mice in vivo.Results Survivin-specific siRNA efficiently down-regulated survivin expression. The cell cycle was arrested at G2/M phase, and apoptosis was obviously found. Inhibition of survivin expression could make the IC50 and drug-resistant index of cisplatin decrease, and enhance the cancer cells sensitivity to cisplatin. After transfection by survivin-specific siRNA, expression of LRP and cyclin-D1 were downregulated, caspase-3 expression was upregulated, bcl-2 expression had no obvious change. The animal experiment confirmed knockdown of survivin could inhibit the tumor growth.Conclusions Survivin-specific siRNA can efficiently suppress the expression of survivin, increase apoptosis, inhibit cells proliferation and enhance the chemosensitivity to cisplatin in vitro and in vivo. Suppression of survivin expression helping to reverse drug-resistance may have relationship with downregulation of LRP and upregulation of caspase-3.Anti-tumor strategies based on the inhibition of survivin may be useful in targeting lung adenocarcinomas.     

Effect of down-regulation of TRPC6 on the puromycin aminonucleoside-induced apoptosis of mouse podocytes

Eukaryotic expression vectors carrying the small hairpin RNA （shRNA） for TRPC6 mRNA were constructed, and the effects of knocking-down TRPC6 on puromycin aminonucleoside （PAN）-induced apoptosis of mouse podocytes were observed. Two eukaryotic expression vectors containing small hairpin structure targeting TRPC6 named pGCsi-TRPC6A and pGCsi-TRPC6B were designed and synthesized. The plasmids were transfected into conditionally immortalized murine podocyte cell line by liposome. The changes in the TRPC6 mRNA and protein expression were observed by RT-PCR and Western blot after 48 h. Cultured podocytes were divided into four groups： control group, PAN treatment group, PAN treatment＋shRNA transfection group, and PAN treatment＋negative control group. The expression of Bax and Bcl-2 mRNA and proteins was detected by RT-PCR and Western-blot respectively. The apoptotic rate of podocytes was measured by flow cytometry. The results showed that the expression of TRPC6 mRNA and protein was decreased in the podocytes when transfected with pGCsi-TRPC6A, and pGCsi-TRPC6B. The expression of Bax was increased, and that of Bcl-2 was decreased at protein and mRNA levels in the podocytes after treated with PAN for 48 h. These changes was attenuated by knocking-down TRPC6. Knocking-down TRPC6 could effectively decrease the PAN-induced apoptosis of podocytes. It was concluded that TRPC6 may play an important role in the PAN-induced apoptosis of podocytes. Knocking-down TRPC6 gene could effectively prevent the podocytes from apoptosis induced by PAN.     

Saturated Fatty Acid Induces Insulin Resistance Partially Through Nucleotide-binding Oligomerization Domain 1 Signaling Pathway in Adipocytes

Objective To investigate the potential role of nucleotide-binding oligomerization domain 1 （NOD1）, a component of the innate immune system, in mediating lipid-induced insulin resistance in adipocytes. Methods Adipocytes from Toll-like receptor 4 deficiency mice were used for stimulation experiments. The effect of oleate/palmitate mixture on nuclear factor-κB （NF-κB） activation was analyzed by reporter plasmid assay. The release of proinflammatory chemokine/cytokines production was determined by using real-time PCR. Insulin-stimulated glucose uptake was measured by 2-deoxy-D-[SH] glucose uptake assay. Chemokine/cytokine expression and glucose uptake in adipocytes transfected with small interfering RNA （siRNA） targeting NOD 1 upon fatty acids treatment were analyzed. Results Oleate/palmitate mixture activated the NF-κB pathway and induced interleukin-6, tumor necrosis factor-R, and monocyte chemoattractant protein-1 mRNA expressions in adipocytes from mice deficient in Toll-like receptor 4, and these effects were blocked by siRNA targeting NOD1. Furthermore, saturated fatty acids decreased the ability of insulin-stimulated glucose uptake. Importantly, siRNA targeting NOD 1 partially reversed saturated fatty acid-induced suppression of insulin-induced glucose uptake. Conclusion NOD1 might play an important role in saturated fatty acid-induced insulin resistance in adipocytes, suggesting a mechanism by which reduced NOD1 activity confers beneficial effects on insulin action.     

siRNA干扰胰岛新生相关蛋白表达对胰岛细胞株INS-1细胞增殖的抑制作用

目的 探讨靶向胰岛新生相关蛋白(INGAP)基因RNAi对胰岛细胞增殖的抑制效应.方法 设计合成siRNA,通过脂质体转入胰岛细胞株INS-1中,研究其对靶基因的抑制效应,采用逆转录.聚合酶链反应(RT-PCR)、流式细胞仪、Western-blot法分别检测转染后的INS-1细胞中INGAP mRNA和蛋白表达的变化,用噻唑蓝(MTT)法来检测细胞的增殖.结果 结果显示siRNA6序列对细胞增殖有明显的抑制效应,INGAP mRNA及蛋白表达明显降低,INS-1细胞增殖受到抑制,P<0.05.结论 干扰INGAP基因的表达能有效抑制胰岛细胞的增殖,INGAP有可能成为治疗β细胞严重减少或损害的重要新靶点.

Abstract：

Objective To investigate the effect of small interfering RNA (siRNA)-mediated islet neogenesis associated protein (INGAP) gene silencing on the proliferation of islet cells. Methods Different siRNAs targeting INGAP gene were designed and transfected into INS-1 islet cells, and the expression levels of INGAP mRNA and protein following the transfection were detected using RT-PCR, flow cytometry and Western blotting. The proliferation of the transfected INS-1 cells was evaluated using MTT assay. Results Compared with those in the irrelevant siRNA, empty vector control, and un-transfected groups, the expression levels of INGAP mRNA and protein in the cells transfected with siRNA6 were reduced significantly. The cell proliferation rate significantly increased after transfection with siRNA6 (P<0.05). Conclusion siRNA targeting INGAP can effectively down-regulate INGAP expression and inhibit the proliferation of INS-1 cells.     

Female Fertility: Is it Safe to “Freeze?”

Objective:To evaluate the safety and risk of cryopreservation in female fertility preservation.Data sources:The data analyzed in this review were the English articles from 1980 to 2013 from journal dat...     

PEG10基因siRNA真核表达载体的构建及其对肝癌HepG2细胞凋亡的影响

目的:构建针对遗传印记基因PEG10的siRNA真核表达载体,体外观察其对肝癌HepG2细胞凋亡的影响. 方法:设计针对PEG10基因的3个siRNA靶序列,构建真核表达载体siRNA1,siRNA2和siRNA3,脂质体分别转染HepG2细胞后实时定量PCR,检测其对HepG2细胞PEG10基因表达的影响,MTT法、流式细胞仪和激光共聚焦检测转染siRNA后的HepG2细胞的生长活性及凋亡. 结果:成功构建了针对PEG10基因的3个特异性siRNA表达载体. 它们不同程度地抑制了HepG2细胞PEG10基因的表达,以siRNA2抑制效率最高,可达93.99%. HepG2细胞的生长也明显受到抑制(P＜0.05),大量HepG2细胞凋亡,siRNA2凋亡率最高,为66.91%. 结论:靶向PEG10基因的siRNA表达载体可抑制肝癌HepG2细胞的生长,诱导细胞凋亡,该作用与降低PEG10基因的表达有关.     

Fertility Preservation for Female

Introduction Over the past 30 years, the incidence of cancer in female has been increased by up to 20% while mortality rates have declined remarkably due to progress in cancer treatment. With aggressive chemotherapy and/or radiotherapy coupled with bone m…     

PAX2特异性siRNA在诱导HEC-1A细胞增殖及凋亡中的作用

目的研究靶向全长配对盒基因-2(PAX2)siRNA在诱导子宫内膜癌细胞增殖、凋亡中的作用。方法慢病毒载体介导针对PAX2基因的siRNA转染HEC-1A细胞,Western blot、激光共聚焦显微镜、Annexin-V/PI双标法及四甲基偶氮唑蓝(MTT)分别检测PAX2蛋白表达、细胞凋亡形态学改变、凋亡率及细胞增殖变化。结果 PAX2-siRNA转染后,HEC-1A细胞的PAX2蛋白表达明显降低(P<0.05);细胞早期凋亡率显著增加(22.07±2.17)%,与空载体转染组(1.12±0.37)%及未转染组(0.89±0.52)%比较,差异均具有统计学意义(均P<0.01);细胞生长速率显著减慢,细胞经Hoechst 33258荧光染色,见转染后细胞的胞核呈浓集固缩改变,而空载体转染组和未转染组细胞的胞核无凋亡形态学改变。结论 PAX2特异性siRNA能明显抑制PAX2蛋白在子宫内膜癌HEC-1A细胞中的表达,抑制细胞增殖并诱导HEC-1A细胞凋亡。     

唾液酸酶NEU1 siRNA对人卵巢癌OVCAR3细胞增殖、凋亡和侵袭的影响

目的 探讨唾液酸酶1(neuraminidase,NEU1)小干扰RNA (siRNA)对人卵巢癌细胞增殖、凋亡和侵袭的影响。 方法 将人类卵巢癌细胞OVCAR3接种于无抗生素的培养基后进行NEU1 RNA沉默处理。对照组转染加入250 μl Opti-mem无血清培养基,模型组(MOCK)转染加入245 μl Opti-mem无血清培养基和5 μl MOCK siRNA (100 pmol),实验组转染加入245 μl Opti-mem无血清培养基和5 μl NEU1 siRNA (100 pmol)。处理48 h后,收集被转染的细胞,并进行免疫印迹,细胞增殖、细胞周期、细胞凋亡和细胞侵袭试验。 结果 细胞增殖试验和凋亡试验(流式细胞术)结果表明,与模型组相比,NEU1 siRNA能有效抑制癌细胞增殖,阻滞细胞周期G₀/G₁期,并且诱导细胞凋亡。Transwell实验的结果表明,经NEU1 siRNA处理过的OVCAR3细胞侵袭力被显著抑制。此外,Western blot结果表明NEU1 siRNA可抑制Cln3和Cln5的表达以及降低ATP5B和ATP5J的表达水平。 结论 NEU1 siRNA能有效抑制人类卵巢癌OVCAR3细胞的增殖、凋亡和侵袭,为卵巢癌的靶向治疗提供了一个新方向。      

siRNA干预CTGF和TIMP-1对大鼠肝星状细胞胶原分泌的影响

目的研究小干扰RNA(small interfering RNA,siRNA)沉默结缔组织生长因子(connective tissue growthfactor,CTGF)和组织金属蛋白酶抑制剂-1(tissue inhibitor of matrix metalloproteinases,TIMP-1)对肝星状细胞(hepaticstellate cell,HSC)CTGF和TIMP-1基因表达以及对Ⅰ、Ⅲ型胶原分泌的影响。方法根据已筛选出的对CTGF和TIMP-1基因最有效的RNA干扰靶位,将化学合成siRNA CTGF和siRNA TIMP-1以脂质体LipofectamineTM2000介导,瞬时转染HSC-T6细胞,分别设siRNA CTGF组、siRNA TIMP-1组、siRNA CTGF和siRNA TIMP-1联合组、脂质体组及非特异性(negative control,NC)siRNA组,抽提转染24、48h细胞mRNA和蛋白,并收集细胞上清液。应用RT-PCR鉴定CTGF和TIMP-1mRNA的表达;Western blot检测其蛋白表达;ELISA法检测Ⅰ、Ⅲ型胶原的分泌。结果各干预组转染24、48h后分别与脂质体组和NC siRNA组比较,HSC-T6细胞CTGF及TIMP-1mRNA和蛋白表达均明显下调(均P<0.05),且干预组HSC-T6培养上清液中Ⅰ、Ⅲ型胶原含量明显减少(均P<0.05)。siRNA联合组分别与siRNA CTGF组和siRNA TIMP-1组比较,于转染48hsiRNA联合组较siRNA TIMP-1组抑制率高,细胞上清液中Ⅰ、Ⅲ型胶原量比siRNA TIMP-1组少,且差异具有统计学意义(均P<0.05);而与siRNA CTGF组比较,其CTGF mR-NA和蛋白抑制率增高,细胞上清液中Ⅰ、Ⅲ型胶原量较siRNA联合组减少,但差异无统计学意义(均P>0.05)。结论针对HSC-T6CTGF mRNA基因全长943位点和TIMP-1mRNA 304位点化学合成的siRNA,对靶基因mRNA和蛋白表达有较好的抑制效果,CTGF和TIMP-1沉默可显著减少细胞上清液中Ⅰ、Ⅲ型胶原含量;CTGF和TIMP-1两种基因同时沉默,有增强抗肝纤维化效果的可能。     

联合应用以MRP和bcl-2为靶标的siRNA对鼻咽癌细胞耐药和凋亡的调节

[摘要] 目的 研究联合转染以MRP和bcl－2为靶标的siRNA后,鼻咽癌细胞株CNE2和耐药细胞CNE2/DDP的药物敏感性和凋亡率的改变。方法 以MRP和bcl－2为靶标的siRNA(均为100 umol/L)分别或联合转入顺铂处理的CNE2和CNE2/DDP以单纯化疗处理组和未处理组为对照。转染后24,48,72 h MTT法检测各组生长抑制率,计算IC50值。RT-PCR检测转染12 h各组细胞相应靶基因mRNA表达水平,流式细胞仪检测转染48 h各组MRP和bcl－2蛋白表达率和细胞凋亡率。结果 细胞分别转染以MRP和 bcl－2为靶标的siRNA后,相应靶基因的mRNA及蛋白的表达明显减低,细胞的凋亡率生高,IC50均较单纯化疗组低,有统计学意义（P<0.05）,两者间也有统计学差异（P<0.05）;联合转染两种siRNA后细胞的IC50进一步显著下降,细胞凋亡率显著升高,与各组相比均有统计学差异（P<0.01）。结论 联合以MRP和bcl－2基因为靶标的siRNA,通过降低靶基因蛋白表达,产生协同作用,明显增加耐药和亲本肿瘤细胞药物敏感性和凋亡率。 [关键词] Bcl-2 MRP siRNA 鼻咽肿瘤     

C-myc小干扰RNA对HL-60细胞凋亡的影响

目的:研究c-myc小干扰RNA对白血病细胞凋亡的影响,探寻白血病基因治疗的新方法。方法:应用针对c-myc的小干扰RNA转染HL-60细胞48 h后,倒置显微镜下观察细胞形态学变化,琼脂糖凝胶电泳法检测DNA梯形带和流式细胞仪Annexin V-FITC/PI法检测细胞凋亡。结果:c-myc小干扰RNA作用后,HL-60细胞生长受抑,可见细胞核固缩,胞桨内出现凋亡小体。阴性对照组和空白对照组没有明显变化。DNA电泳干扰组出现典型的梯状条形带,对照组未出现明显凋亡梯形带。流式细胞仪分析干扰组细胞凋亡率可达26.21±0.75%,明显高于阴性对照组的4.58±0.48%和空白对照组的3.76±0.30%。结论:c-myc小干扰RNA对HL-60细胞有明显的抑制作用,可抑制细胞增殖,导致细胞凋亡,有望成为白血病靶向基因治疗的新工具。