Ki67反义肽核酸、反义寡核酸对肾癌细胞系增殖及凋亡影响的研究

目的探讨肿瘤增殖相关基因Ki67反义肽核酸(PNAs)、反义寡核酸(ASODNs)对人肾癌细胞增殖及凋亡的调控。寻找肾癌反义治疗的合适药物。方法将PNAs转染人肾癌786-0细胞系,采用免疫组化、Westernblot技术检测Ki67表达,细胞生长曲线、3H-thymidine掺入试验检测肾癌细胞增殖,TUNEL法检测癌细胞凋亡。并与相同浓度的ASODNs进行对比。结果PNAs处理组(10μmol/L)786-0细胞Ki67表达阳性率(%)(16.9±0.7)降低,Ki67蛋白(%)(42.1±2.2)降低,与ASODNs处理组(28.6±0.4)(83.6±1.4)比较差异有显著性(P<0.01,P<0.01)。PNAs处理组3H-thymidine掺入率(%)(20.7±1.5)减少,与ASODNs处理组(58.6±1.4)比较差异有显著性(P<0.01)。PNAs处理组凋亡细胞阳性率(%)(28.7±2.3)增加,与ASODNs处理组(13.8±1.0)比较差异有显著性(P<0.01)。结论PNAs可在反义及反基因二个环节发挥抗肿瘤作用,与ASODNs相比,PNAs有更强的阻抑人肾癌Ki67基因表达、增殖及促进凋亡作用,是一种有前途的基因治疗药物。

HIF-1α反义寡核苷酸脂质体的制备和对视网膜血管内皮细胞HIF-1α表达的影响(英文)

目的应用HIF-1αASODN处理牛眼视网膜微血管内皮细胞,观察细胞摄取ASODN的情况和对缺氧时细胞HIF-1α表达的影响。方法对分离的牛视网膜血管内皮细胞进行HIF-1α反义寡核苷酸的转染,CoCl2模拟缺氧培养,采用免疫组化检测不同缺氧时间HIF-1α的表达。结果硫代化修饰的标有FAM的HIF-1α反义寡核苷酸能被Lipofectin转染进BREC细胞。转染6h后,在荧光显微镜下可观察到细胞内的绿色荧光斑。计算细胞转染率为(93.8±3.4)%。未转染组开始时HIF-1α有极低表达,在缺氧1h时表达量显著上升,4h达到高峰,16h后下降。而ASODN转染组HIF-1α的表达始终在极低水平,两组间有显著性差异(P<0.01)。结论以脂质体为载体能高效率地将HIF-1αASODN转染至视网膜血管内皮细胞内,明显抑制HIF-1α蛋白质的表达,这可能为HIF-1αASODN用于视网膜新生血管的治疗提供理论依据。

bcl-2反义寡核苷酸增强K562白血病细胞对As_2O_3药物敏感性

采用细胞培养法 ,免疫组化实验技术和流式细胞仪等项技术 ,研究了bcl 2基因反义核酸和砷剂单独及联合应用对K5 6 2细胞的生物效应 ,DNA及bcl 2蛋白的变化。结果显示 ,As2 O3(2 .0 μmol L) +bcl 2ASODN(10 .0 μmol L)联合作用比单用As2 O3 (2 .0 μmol L)或bcl 2ASODN(10 .0 μmol L)显著增强诱导细胞凋亡的作用 (P <0 .0 1) ;流式细胞仪检测显示 ,联合作用引起的bcl 2蛋白表达亦明显减少 (P <0 .1)。结论表明 ,bcl 2基因反义核酸能明显提高和显著增强K5 6 2白血病细胞对As2 O3

hTERT反义寡核苷酸对子宫内膜癌细胞增殖的影响

目的探讨人端粒酶催化亚基(human telomerase reverse transcriptase,hTERT)反义寡核苷酸对子宫内膜癌细胞增殖的影响。方法反义寡核苷酸转染ishikawa细胞系,采用TRAP-ELISA法检测转染后细胞端粒酶的活性,观察其前后的变化。MTT法检测细胞活性,流式细胞仪观察细胞周期及细胞凋亡率变化。结果转染细胞的端粒酶活性及细胞生长都明显受到抑制。流式细胞仪检测到细胞凋亡峰,细胞阻滞于使细胞停滞在G_0～G_1期,并诱导细胞凋亡,正义寡核苷酸则无此作用。结论端粒酶hTERT为靶点的反义寡核苷酸可明显抑制子宫内膜癌细胞的增殖,且具有促凋亡作用。

miR-30d在舌鳞状细胞癌组织中表达及反义miR-30d对细胞增殖、侵袭能力的影响

目的:探讨miR-30d在舌鳞状细胞癌组织中表达及反义miR-30d对细胞增殖、侵袭能力的影响。方法:选取2013年3月~2015年4月在我院行根治性手术治疗的TSCC患者68例,实时荧光定量PCR检测TSCC组织和癌旁正常组织中miR-30d基因表达,培养舌癌SCC-4细胞,分为ASO-miR-30d组、ASO-阴性对照组和空白对照组,检测各组细胞中miR-30d基因表达,MTT法检测各组细胞增殖能力,Transwell法检测各组细胞迁移和侵袭能力。结果:TSCC组织中miR-30d相对表达量(1.89±0.21),显著高于癌旁正常组织的(1.31±0.16),差异有统计学意义(P=0.000);TSCC组织中miR-30d相对表达量与TNM分期、分化程度和颈淋巴结转移有关(P<0.05);与空白对照组和ASO-阴性对照组比较,ASO-miR-30d组细胞中miR-30d相对表达量降低,差异有统计学意义(P<0.05);MTT实验结果显示,与空白对照组和ASO-阴性对照组比较,ASO-miR-30d组24h、48h、72h和96h时细胞A值均降低,差异均有统计学意义(P<0.05);与空白对照组和ASO-阴性对照组比较,ASO-miR-30d组迁移细胞数和侵袭细胞数均减少,差异均有统计学意义(P<0.05)。结论:miR-30d在TSCC组织中呈高表达,反义miR-30d可有效减少TSCC细胞增殖,抑制细胞迁移和侵袭能力。

反义miR-18a对乳腺癌细胞侵袭迁移的影响

目的:分析miR-18a在乳腺癌组织中的表达情况;体外研究miR-18a反义寡核酸(antisense oligonucleotide,ASO)对乳腺癌细胞侵袭和迁移的影响。方法:运用荧光定量PCR检测108例乳腺癌组织及对应癌旁乳腺组织中miR-18a的表达;通过miR-18a ASO降低乳腺癌细胞中miR-18a的表达,采用Transwell实验和划痕实验观察miR-18a ASO对乳腺癌细胞侵袭和迁移能力的影响,同时运用Western blot的方法检测侵袭相关蛋白的表达变化。结果:在108例乳腺癌病例中,50.93%(55/108)的乳腺癌组织miR-18a表达明显高于对应正常组织(t=28.68,P=0.000);与空白对照组和转染随机ASO组相比,miR-18a ASO可以明显降低miR-18a的表达(F=321.67,P=0.001;F=563.28,P=0.000);Transwell实验和划痕实验结果显示转染miR-18a ASO后,乳腺癌细胞的侵袭迁移能力明显下降(P=0.000),同时相关侵袭蛋白基质金属蛋白酶(matrix metalloproteinase,MMP)2和MMP9表达下降(P=0.000)。结论:miR-18a在乳腺癌组织中表达上调,降低miR-18a的表达,可有效抑制乳腺癌细胞的侵袭和迁移。miR-18a有可能成为乳腺癌侵袭转移调控的新靶点。

反义抑制miRNA-155对皮肤鳞状细胞癌A431细胞增殖、凋亡及侵袭力的影响

目的观察转染反义微小核糖核酸(miRNA)-155对人皮肤鳞状细胞癌A431细胞增殖、凋亡、迁移和侵袭力的影响。方法采用脂质体介导对皮肤鳞状细胞癌A431细胞转染miRNA-155无义序列(阴性对照组)、转染反义miRNA-155(转染组),空白对照组细胞不作处理。实时定量聚合酶链式反应(qRT-PCR)检测转染后A431细胞miRNA-155的相对表达水平;采用四甲基偶氨唑盐(MTT)检测转染后皮肤鳞状细胞癌A431细胞增殖活性;采用流式细胞术(FCM)检测转染后皮肤鳞状细胞癌A431细胞周期改变和凋亡变化;采用Transwell法检测侵袭力与细胞迁移能力。结果转染组A431细胞中miRNA-155 mRNA的相对表达水平低于空白对照组和阴性对照组(F=634.57,P<0.01),但空白对照组和阴性对照组比较差异无统计学意义。转染72 h后,转染组较空白对照组和阴性对照组细胞存活率明显降低,转染120 h降低最为明显(P<0.05)。转染组G_0/G_1期细胞增多,S期细胞减少,整体细胞增殖指数降低,A431细胞凋亡率、细胞迁移和侵袭力升高(P<0.05),但各组G_2/M期细胞周期变化、后2组A431细胞凋亡率、细胞迁移和侵袭力差异无统计学意义。结论皮肤鳞状细胞癌A431细胞转染反义miRNA-155可减少miRNA-155的表达,有效抑制A431细胞增殖,促进其凋亡。miRNA-155可能成为治疗皮肤鳞状细胞癌基因表达调控的新靶点。

针对HBV调节基因ENⅡ的ASON抑制HBV复制表达的体外研究

目的 :筛选出高效特异而无毒的抗HBV反义寡核苷酸片段。方法 :以 2 2 15细胞为模型 ,设计合成针对HBVENⅡ的反义硫代寡苷酸 (ASON) ,ELISA法检测ASON对宿主细胞分泌表达HBsAg和HBeAg的抑制作用 ,MTT法检测ASON对细胞增殖代谢的影响。结果 :ASON对HBsAg和HBeAg的抑制率分别为 92 %和 75 % ,与非互补序列对照组 (抑制率均为 11% )相比差异有显著性意义 (P <0 0 0 1) ,用药前后MTT比色结果和活细胞比率差异无显著性意义 (P >0 0 5 )。结论

基因沉默策略及其应用研究进展

基因沉默(Gene silencing)是生物体中特定基因由于各种原因不表达或表达减少的现象,是通过表观遗传控制基因表达的重要机制,通过基因沉默技术探索疑难疾病的治疗方法是当下的研究热点.随着基因工程的迅速发展,基因沉默技术不断有新技术出现取代已有的技术.对基因沉默技术所包括的核酶技术、反义寡核酸技术、基因敲除、RNA干扰技术及其应用进行介绍.

Efficacy of Antisense Oligodeoxynucleotide of Neuropeptide Y Y5 Receptor on Treating of Hyperleptinemia by Intraventricular Administration in Diet-induced Obese Rats

Objective: To study the efficacy of antisense oligonucleotide of neuropeptide Y (NPY) Y5 receptor on treating hyperleptinemia by intracerebral ventricular administration in diet-induced obese rats.Methods: The obese rats were prepared by feeding a high-nutritive diet for 7 weeks. The lateral ventricle of obese rats was cannulated. Either 10 μl of different neuropeptide Y Y5 receptor oligodeoxynucleotide, including antisense, sense and missense oligodeoxynucleotide (5 g/L) or 10 μl saline was administered into the ventricle through cannula three times per day in every rat. Two days later the rats were slaughtered .The weights of both retroperitoneal and epididymal adipose tissues were measured, and the serum insulin and leptin were detected by radioimmunoassay method and the murine leptin ELISA kit respectively. Results: ①The level of serum was significantly higher in experimental rats than that in normal rats. Similarly, the level of serum insulin and the weights of both retroperitoneal and epididymal adipose tissues were increased in experimental rats. ②After the diet-induced obese rats were intraventricularly administered with NPY Y5 receptor antisense oligodeoxynucleotide, the levels of serum leptin and insulin were significantly decreased and combined with the reduction of weight in retroperitoneal adipose tissue. There was, however, no significant difference in the weight of epidymal adipose tissue between pre-treated and post-treated duration. ③There was significant positive correlation among the level of serum leptin, the level of serum insulin and the weight of retroperritoneal adipose tissue in diet-induced obese rats. Conclusion: Intracerebral ventricular administration of antisense oligodeoxynucleotide of neuropeptide Y Y5 receptor may alleviate hyperleptinemia in diet-induced obese rats and decrease the weight of retroperitoneal adipose tissue and the level of serum insulin.

Effects of Ghrelin Antisense Inhibition on VEGF and Its Receptor Flt-1 mRNA Expression

[ Objective] This study was to investigate the effect of VEGF and its receptor Fit-1 mRNA expression in Mongolia sheep umbilical vein endothelial cells by ghrelin antisense inhibition. [ Method] Experiments were divided into 4 groups： group Ⅰ （blank control group） ; group Ⅱ （liposome group） ; group Ⅲ （SCON group： 20 μmol/L sense oligonucleotide） ; group Ⅳ （ASCON： 20 μmol/L antisense oligonucleotide）. VEGF and its receptor Fit-1 mRNA expression changes were detected by using real-time fluorescence quantitative detection after 24, 36 and 48 h. [ Result] The expression of VEGF mRNA in group Ⅰ, group Ⅱ were insignificantly different at higher expression levels, and did not change significantly with the time; the expression of VEGF mRNA in group Ⅲ assumed a slight decrease, but there were no significant differences between group I and group Ⅱ （P 〉0.05）, the expression of VEGF mRNA in group Ⅳ（antisense oligonucleotide group ） decreased significantly （P 〈 0.05） ; the expression of VEGF receptor FLT-1 mRNA was similar to that of VEGF. [ Conclusion] Antisense inhibition ghrelin has a downward effect to the expression of VEGF and its receptor Fit-1 the mRNA.

Selection of Effective Bcl-2 Antisense Oligodeoxynucleotides with Computer Software and Experimental Assay

Objective： To explore and investigate the selection of effective antisense oligodeoxynuleotides with the help of computer and RNAstructure folding software. Methods： Bcl-2 gene was used as the target gene and five antisense oligodeoxynuleotides were designed to be bound to Bcl-2 mRNA optimal secondary structure regions that were predicted free from intramolecular fold or instability of free energy. The five antisense oligodeoxynucleotides were studied with experimental assay of leukemia cells, including cell grow assay with tropan blue exclusion, expression of Bcl-2 protein detected with immunochemistry and flowcytometry, Bcl-2 mRNA content detected with RT-PCR technique, as well as apoptosis observed and determined with morphonological method, electrophoresis and flowcytometry. Results： The results showed that two of the five antisense oligodeoxynucleotides were effective antisense oligodeoxynucleotides, which were able to inhibit cell growth in leukemia, to decrease the level of Bcl-2 mRNA and protein, to induce apoptosis of leukemia cells significantly. Conclusion： The computational prediction of antisense efficacy is faster than other methods and more efficient, which can potentially speed the development of sequences for both research and clinical applications.

Antisense oligonucleotide targeting midkine suppresses in vivo angiogenesis

AIM: To evaluate the effect of antisense oligonucleotide targeting midkine (MK-AS) on angiogenesis in chick chorioallantoic membrane (CAM) and in situ human hepatocellular carcinoma (HCC). METHODS: An in situ human hepatocellular carcinoma (HCC) model and CAM assay were used in this experiment. The effect of MK-AS on angiogenesis was evaluated by cell proliferation assay and hematoxylin- eosin (HE) staining. RESULTS: MK-AS significantly inhibited human umbilical vein endothelial cells (HUVEC) and in situ human HCC growth. At the same time, MK-AS suppressed the angiogenesis both in human hepatocellular carcinoma cell line (HEPG2)-induced CAM and in situ human HCC tissues. CONCLUSION: MK-AS is an effective antiangiogenesis agent in vivo.

Enhanced therapeutic effects of combined chemotherapeutic drugs and midkine antisense oligonucleotides for hepatocellular carcinoma

AIM: To evaluate the effect of combined antisense oligonucleotides targeting midkine (MK-AS) and chemotherapeutic drugs [cisplatin(DDP), 5-fluorouracil (5-FU) and adriamycin (ADM)] on inhibition of HepG2 cell proliferation, and to analyze the efficacy of MK-AS used in combined ADM in in situ human hepatocellular carcinoma (HCC) model. METHODS: HepG2 cells were treated with MK-AS and/or chemotherapeutic drugs mediated by Lipofectin, and cell growth activity was determined by MTS assay. An in situ HCC model was used in this experiment. MK- AS, ADM and MK-AS + ADM were given intravenously for 20 d, respectively. The animal body weight and their tumor weight were measured to assess the effect of the combined therapy in vivo. RESULTS: Combined treatment with MK-AS reduced the IC50 of DDP, 5-FU and ADM in HepG2 cells. MK-AS significantly increased the inhibition rate of DDP, 5-FU and ADM. Additionally, synergism (Q 1.15) occurred at a lower concentration of ADM, 5-FU and DDP with combined MK-AS. Combined treatment with MK-AS and ADM resulted in the more growth inhibition on in situ human HCC model compared with treatment with chemotherapeutic drugs alone. CONCLUSION: MK-AS increases the chemosensitivity in HepG2 cells and in situ human HCC model, and thecombination of MK-AS and ADM has a much better in vitro and in vivo synergism.

The Role of β3-adrenergic Receptor Gene in Neuropeptide Y Y5Receptor Antisense Gene Therapy of Diet-induced Obese Rats

Objective: To study the role of β3-adrenergic receptor gene in neuropeptide Y(NPY) Y5 receptor antisense gene therapy of diet-induced obese rats.Methods: The diet-induced obese rats were prepared by feeding a high-nutrition diet. Lateral ventricular was cannulated in obese rats which then received an intraventricular injection of either 5 μg/μl NPY Y5 receptor antisense or 10 μl missense oligodeoxynucleotide or saline of 10 μl respectively in every rat. When the rats were killed, the wet weight of abdominal adipose tissue, the level of serum lipid and lipoprotein were measured. Total RNA from the retroperitoneal adipose tissue was extracted and the level of β3-adrenergic receptor gene mRNA expression was evaluated by RT-PCR.Results: ①The wet weight of abdominal adipose tissue, the levels of serum lipids were greatly higher in diet-induced obese rats than those in normal rats. However, there were much lower β3-adrenergic receptor gene mRNA expression levels in retroperitoneal adipose tissue in diet-induced obese rats as compared with those in normal rats. ②After the diet-induced obese rats were intraventricularly administered with NPY Y5 receptor antisense oligodeoxynucleotide, the levels of β3-adrenergic receptor gene mRNA expression in retroperitoneal adipose tissue of diet-induced obese rats were strikingly up-regulated, whereas the wet weight of abdominal adipose tissue, the levels of serum lipids were markedly reduced.Conclusion: Intraventricular administration of antisense oligodeoxynucleotide to NPY Y5 receptor could significantly reduce the abdominal adipose tissue and the levels of serum lipids in diet-induced obese rats by up-regulating the level of β3-adrenergic receptor gene mRNA expression in retroperitoneal adipose tissue.

In vitro and in vivo suppression of hepatocellular carcinoma growth by midkine-antisense oligonucleotide-loaded nanoparticles

AIM:To synthesize antisense oligonucleotides (ASODNs) of midkine (MK), package the ASODNs with nanoparticles, and to inhibit hepatocellular carcinoma (HCC) growth using these nanoparticles.METHODS: HepG2 cell proliferation was analyzed in vitro using the 3-(4,5-dimethythiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2Htetrazolium, inner salt assay. The in vivo activity of nanoparticles delivering the MK-ASODNs was analyzed by histopathological and immunohistochemical staining and quantitative real time polymerase chain reaction (PCR). RESULTS: The in vitro proliferation of HepG2 cells was signif icantly inhibited by the nanoparticles packaged with MK-ASODNs (NANO-ASODNs). Furthermore, the NANO- ASODNs signif icantly inhibited the growth of HCC in the mouse model. CONCLUSION: NANO-ASODNs can significantly suppress the growth of HCC in vitro and in vivo.

Role of Smad7 in inflammatory bowel diseases

Crohn's disease and ulcerative colitis,the major forms of inflammatory bowel diseases(IBD) in man,are complex diseases in which genetic and environmental factors interact to promote an excessive mucosal immune response directed against normal components of the bacterial microflora.There is also evidence that the pathologic process is due to defects in counterregulatory mechanisms,such as those involving the immunosuppressive cytokine transforming growth factor(TGF)-1.Indeed,studies in human IBD tissues and murine models of colitis have documented a disruption of TGF-1 signalling marked by a block in the phosphorylation of Smad3,a signalling molecule associated with the activated TGF-receptor,due to up-regulation of Smad7,an intracellular inhibitor of Smad3 phosphorylation.Knock-down of Smad7 with a specific antisense oligonucleotide restores TGF-1/Smad3 signalling,thus resulting in a marked suppression of inflammatory cytokine production and attenuation of murine colitis.These findings together with the demonstration that Smad7 antisense oligonucleotide is not toxic when administered in mice have paved the way for the development of a Smad7 antisense oligonucleotidebased pharmaceutical compound that is now ready to enter the clinics.In this article we review the available data supporting the pathogenic role of Smad7 in IBD and discuss whether and how Smad7 antisense therapy could help dampen the ongoing inflammation in IBD.

Suppression of intracranial glioma tumorigenesis with vascular endothelial growth factor antisense oligonucleotide in rats

Objective: To observe the inhibition of intracranial glioma tumorigenesis by vascular endothelial growth factor (VEGF) antisense oligodeoxynucleotide (ODN) in rats. Methods: Totally 20 μ1 Hank's liquid containing 1×106 C6 glioma cells was seeded into rat right caudate putaraen in high-flow microinfusion with stereotactic technique. VEGF antisense ODN was simultaneously used with glioma cell. Each rat of the treated group Ⅰ and the treated group Ⅱ was treated with 1 000 μmol/L VEGF antisense ODN. Each rat of the treated group Ⅲ and the treated group Ⅳ was treated with 2 000 μmol/L VEGF antisense ODN. The experimental periods of the treated group Ⅰ , the treated group Ⅲ and the control group Ⅰ were 2 weeks, those of the treated group Ⅱ , the treated group Ⅳ and the control group Ⅱ were 3 weeks. Before sacrifice, MRI was performed on each rat. Tumor magnitude and pathologic examination were detected after samples were dissected. Results: The survival state of all treated rats was better, and that of the control rats was in severe danger. The tumor volumes of the treated group Ⅰ and the treated group Ⅱ were remarkably lessened. Tumor tissue could not be found macroscopically in the brain samples of the treated group Ⅲ and the treated group Ⅳ, but tumor nest could be found with microscopy. Tumors of the treated group I and the treated group Ⅱ had weak expressions of VEGF mRNA and VEGF, while normal brains and the samples of the treated group Ⅲ and the treated group Ⅳ had negative expressions, but tumors of the control groups had strong expressions. Conclusion: VEGF antisense ODN used early in situ can suppress angiogenesis and growth of rat intracranial glioma to retard tumorigenesis.

Antisense oligonucleotide to insulin-like growth factorⅡ induces apoptosis in human ovarian cancer AO cellline

The effects of antisense oligonucleotide to insulin-like growth factor 11 (IGFII) to induce apoptosis in human ovarian cancer cells were evaluated. Antiproliferation effects of antisense to IGFII in ovarian cancer AO cells were determined by 3H-thymidine incorporation. Apoptosis of the IGFll antisense-treated cells was quantitated by both nuclear condensation and flow cytometry after cells were stained with propidium iodide. IGFII antisense (4.5μM)treatment of 48 h maximally inhibited proliferation of AO cells. More than 25% of IGFII antisense-treated cells (4.5PM for 24 h) had undergone apoptosis, whereas less than 3% of the cells were apoptotic in either IGFII sense-treatedcells or untreated cells. Antisense oligonucleotide to IGFII significantly inhibited cell proliferation and induced apoptosis in human ovarian cancer AO cell. These data suggest that IGFII may be a potential target in treatment of ovarian cancer and antisense oligonucleotide to IGFⅡmay serve as a therapeutic approach.