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1.
SUN Guo-fang DING Hao LI Ju-xiang HONG Kui Dong Jia-long WU Qing-hua CHENG Xiao-shu 《园艺学报》2011,27(12):2307-2312
AIM: To investigate the effects of Rho-associated coiled-coil protein kinase-1 (ROCK1) and ROCK2 on apoptosis induced by hypoxia in rat cardiomyocytes. METHODS: Rat cardiomyocytes were cultured primarily and identified using an antibody targeting α-actin of striated muscle. ROCK1-shRNA and ROCK2-shRNA were transiently transfected into the cells by liposome. After 48 h, these cells were subject to hypoxia for 6 h. The cells were divided into 5 groups: blank control group, hypoxia group, hypoxia+negative control shRNA group, hypoxia+ROCK1-shRNA group and hypoxia+ROCK2-shRNA group. The beating frequency and rhythm of the cardiomyocytes were assessed by microscopy. The activity of lactate dehydrogenase (LDH) in the cell culture supernatants was detected by automatic biochemical analyzer. The cell survival rate was analyzed by the method of MTT. The cell apoptotic rate was assessed by flow cytometry. Western blotting was used to determine the expression of ROCK1, ROCK2, caspase-3 and p-PI3K. RESULTS: The primary culture of the cardiomyocytes was successful. Western blotting results showed that the transfection of ROCK1-shRNA or ROCK2-shRNA decreased the expression of ROCK1 or ROCK2 in the cardiomyocytes. Hypoxia slowed down the beat frequency of the cardiomyocytes, also made the rhythm disorder. Hypoxia increased the release of LDH and decreased the cell survival rate. Flow cytometry results showed that hypoxia increased the cell apoptotic rate. Hypoxia increased the expression of caspase-3 and decreased the expression of p-PI3K. Transfection of ROCK1-shRNA and ROCK2-shRNA into the cardiomyocytes reduced all the effects of hypoxia mentioned above. CONCLUSION: Down-regulation of ROCK1 and ROCK2 expression suppresses the apoptosis of rat cardiomyocytes induced by hypoxia. The mechanism is associated with the inhibition of caspase-3 activation and the up-regulation of p-PI3K expression. 相似文献
2.
利用shRNA真核表达载体干涉雌性鸡胚性腺中芳香化酶基因(CYP19A1)的表达 总被引：1，自引：0，他引：1
本研究通过把构建的短发夹结构RNA(shRNA)真核表达载体导入鸡胚,检测鸡胚性腺分化期质粒载体在鸡胚体内代谢情况及雌性鸡胚性腺中芳香化酶基因(CYP19A1)mRNA表达效率,进而探讨利用该方法在鸡胚体内进行特定基因干涉的可行性.实验针对CYP19A1基因构建了4条特异性表达载体,一条非特异性表达载体.每个实验组选取45个新鲜种蛋作为实验材料进行胚盘下腔注射,并设立空白对照组.鸡胚发育12d时,检测各处理组鸡胚肝脏组织中质粒存在情况,并取其左侧性腺组织进行目标基因的荧光定量分析.研究结果表明,导入组在12日胚龄时所有鸡胚基因组中均可检测到绿色荧光蛋白基因(EGFP);荧光定量结果显示,导入特异性表达载体cyp-580、cyp-1083和cyp-1295后,对应雌性鸡胚性腺CYP19A1 mRNA表达效率显著低于空白对照组,干涉效率分别为:73％、52％和85％;特异性表达载体cyp-1403组CYP19A1mRNA表达效率与空白对照组相比有所降低但无显著性差异.本实验为诱导鸡胚性反转提供了新方法并建立了鸡胚发育期特定基因体内干涉新平台. 相似文献
3.
家兔Zfx基因shRNA干扰载体构建及验证 总被引：1，自引：0，他引：1
研究主要是为了构建出针对家兔Zfx基因的shRNA重组载体及效果验证。根据家兔Zfx基因mRNA序列特征,设计出3条shRNA片段,再与线性化的pLL 3.7载体进行连接重组,最后经过qRT-PCR检测Zfx基因mRNA表达量筛选出有效重组表达载体并通过体内试验进行效果验证。结果表明:试验组shRNA-c和shRNA-f干扰效率分别为74%和67%,与空白对照组相比差异极显著(P<0.01);试验组shRNA-e干扰效果为17%,与空白对照组相比差异不显著(P>0.05)。用shRNA-c和shRNA-f进行体内试验,结果显示,两试验组雄性率分别为44.92%(P>0.05)和33.16%(P<0.01)。本试验成功构建针对家兔Zfx基因的重组载体,体内试验显示子一代出现性别偏移,说明Zfx基因对动物的性别决定有一定作用,关于导致体内验证出现反转的原因仍需进一步验证。 相似文献
4.
端粒酶RNA亚基的shRNA对细胞端粒酶活性影响的研究 总被引：1，自引：1，他引：0
[目的]探讨端粒酶RNA亚基（chTR）的短发RNA（shRNA）shRNA对MDCC-MSB1细胞端粒酶活性的影响。[方法]设计合成chTRshRNA并构建表达载体,转染MDCC-MSB1细胞,应用改良TRAP法检测端粒酶活性。[结果]与对照组相比,转染24 h后,各组端粒酶活性变化不明显;转染48 h后,端粒酶活性均显著地下降,且针对模板区设计的干扰载体pSi-chTR-sh1转染后抑制效果最明显。[结论]chTRshRNA表达载体能够有效地抑制MDCC-MSB1细胞的端粒酶活性。 相似文献
5.
为验证腺病毒载体在鸡胚成纤维细胞(CEF)及鸡胚中递送小干扰RNA的可行性,本实验利用共转染技术将表达针对新城疫病毒(NDV)NP基因shRNA的重组质粒同源重组到pGSadeno腺病毒载体系统,用重组腺病毒感染CEF和鸡胚,通过红荧光报告基因的表达情况和荧光定量PCR检测NP基因mRNA的表达对其进行鉴定,并通过细胞形态观察、血凝价的测定评价其在CEF和鸡胚上对NDV增殖的影响。实验结果显示重组腺病毒能够感染CEF,感染CEF后6h、9h、12h与对照组比较NP基因mRNA的表达量分别降低了3.2倍,25.6倍,2.57倍,并能够推迟NDV致细胞病变效应,抑制NDV在鸡胚上的增殖,延缓鸡胚的死亡。本实验构建的重组腺病毒能够在CEF和鸡胚上递送特异的shRNA,为在CEF中的RNA干涉研究开辟了新的递送途径。 相似文献
6.
为了研究双特异性磷酸酶(dual-specificity phosphatase 1,dusp1)基因与鱼类低温诱导的细胞凋亡之间的关系,本研究经Eco RI和Age1酶切构建plko.1-dusp1-sh RNA1,plko.1-dusp1-shRNA2,plko.1-negative control(nc),plko.1-EGFP的重组质粒,并分别与慢病毒包装质粒pCMV-DR8.9.1、pCMV-VSVG共同转染至293T细胞中,经293T细胞包装的病毒,感染斑马鱼(Danio rerio)胚胎成纤维样细胞(zebrafish embryos fibroblast like cell line,ZF4),RT-PCR检测表明dusp1成功被敲降。经嘌呤霉素筛选获得稳定表达细胞系,将细胞于28℃培养(正常对照组)或经10℃低温处理10 d,使用annexin V-FITC/Propidium lodide(PI)双色标记流式细胞术检测ZF4细胞凋亡情况。结果显示:低温胁迫下dusp1敲降的细胞凋亡(dusp1-shRNA1敲降,dusp1-kd1;dusp1-shRNA2敲降,dusp1-kd2)比例较nc组显著上调,其中早期凋亡和晚期凋亡细胞比例dusp1-kd1显著高于nc组,并且dusp1-kd1活细胞数显著低于nc组(P0.05),进一步证明dusp1参与鱼类冷应激过程,并在低温情况下保护细胞。该研究为后期对斑马鱼细胞低温胁迫实验奠定了基础,其中dusp1基因沉默型斑马鱼细胞在10℃低温处理10 d凋亡数显著大于对照组,证明dusp1在细胞凋亡信号通路中起到重要调控作用,为低温下研究斑马鱼基因的分子机制提供新的思路。 相似文献
7.
Rmi Cousin Hugo Groult Chanez Manseur Romain Ferru-Clment Mario Gani Rachel Havret Claire Toucheteau Grgoire Prunier Batrice Colin Franck Morel Jean-Marie Piot Isabelle Lanneluc Kvin Baranger Thierry Maugard Ingrid Fruitier-Arnaudin 《Marine drugs》2021,19(10)
Sugar-based molecules such as heparins or natural heparan sulfate polysaccharides have been developed and widely studied for controlling heparanase (HPSE) enzymatic activity, a key player in extracellular matrix remodelling during cancer pathogenesis. However, non-enzymatic functions of HPSE have also been described in tumour mechanisms. Given their versatile properties, we hypothesized that sugar-based inhibitors may interfere with enzymatic but also non-enzymatic HPSE activities. In this work, we assessed the effects of an original marine λ-carrageenan derived oligosaccharide (λ-CO) we previously described, along with those of its native counterpart and heparins, on cell viability, proliferation, migration, and invasion of MDA-MB-231 breast cancer cells but also of sh-MDA-MB-231 cells, in which the expression of HPSE was selectively downregulated. We observed no cytotoxic and no anti-proliferative effects of our compounds but surprisingly λ-CO was the most efficient to reduce cell migration and invasion compared with heparins, and in a HPSE-dependent manner. We provided evidence that λ-CO tightly controlled a HPSE/MMP-14/MMP-2 axis, leading to reduced MMP-2 activity. Altogether, this study highlights λ-CO as a potent HPSE “modulator” capable of reducing not only the enzymatic activity of HPSE but also the functions controlled by the HPSE levels. 相似文献
8.
《中国兽医学报》2019,(1):14-20
RNAi技术可以特异性剔除或关闭特定基因的表达,使机体具有抗病毒的功效,本试验针对猪繁殖与呼吸综合征病毒(PRRSV)Nsp9基因设计多对shRNA序列,并从细胞水平验证其干扰效果,从中筛选可以使Nsp9基因沉默的优势shRNA序列。利用BLOCK-iTTM RNAi Designer软件设计并合成shRNA对应的DNA序列-ds Oligo,构建入门载体pENTR/U6/shRNA;采用共转染技术,经荧光显微镜观察、流式细胞仪检测及Real-time RT-PCR分析等方法筛选优势干扰序列。结果显示:成功构建的6对pENTR/U6/Nsp9-shRNA均具有抑制Nsp9基因表达的效果,其中pENTR/U6/Nsp9-4和pENTR/U6/Nsp9-6的抑制效果更为突出,同时成功构建1对无意义对照pENTR/U6/con-shRNA。结果表明:pENTR/U6/Nsp9-4和pENTR/U6/Nsp9-6两株优势干扰序列的成功筛选可为构建具有干扰PRRSV复制效果的稳定细胞系以及靶向PRRSV的siRNA的转基因动物提供素材。 相似文献
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试验旨在克隆猪SMYD3(SET and MYND domain-containing protein 3)基因并对其进行序列分析,研究其对猪成纤维细胞增殖的影响。首先克隆猪SMYD3基因,根据其他物种SMYD3基因siRNA和shRNA序列,经同源性比对分析,获得两条猪SMYD3基因shRNA序列,分别构建pSicoR-GFP-SMYD3 shRNA1/shRNA2表达载体,转染HEK293T细胞,利用实时荧光定量PCR分析干扰效率,筛选出抑制效率较好的shRNA,并构建pLVX-IRES-ZsGreen1-SMYD3及pSicoR-GFP-SMYD3 shRNA真核表达载体,同时分析SMYD3基因对猪成纤维细胞的增殖作用,检测细胞Nanog、DNMT1及DNMT3a基因表达情况。结果显示,试验克隆得到1 404 bp的猪SMYD3基因编码区序列,生物信息学分析发现,德保猪SMYD3基因与野猪、山羊和野耗牛相应氨基酸序列的同源性分别为99.5%、93.8%和92.9%。shRNA1/shRNA2均能显著抑制SMYD3基因表达(P<0.05),抑制效果分别是34%和54%,选择pSicoR-GFP-SMYD3 shRNA2进行后续研究。通过脂质体转染法将构建的pLVX-IRES-ZsGreen1-SMYD3及pSicoR-GFP-SMYD3 shRNA真核表达载体导入HEK293T细胞,均可观察到清晰的绿色荧光。慢病毒感染细胞及实时荧光定量PCR结果显示,与空白对照组及阴性对照组相比,过表达SMYD3基因促进猪成纤维细胞增殖,Nanog和DNMT1基因表达显著升高(P<0.05);抑制SMYD3基因表达,细胞增殖受到抑制,Nanog、DNMT1、DNMT3a基因表达显著降低((P<0.05),说明SMYD3基因的表达与猪成纤维细胞的增殖显著相关。 相似文献
10.
为降低RNAi技术的毒副作用，筛选高效抑制猪瘟病毒复制的shRNA序列，根据有关报道构建了荧光报告系统shRNA筛选平台。该平台以9种抑制效率分别为强、中、弱的shRNA筛选载体作为参照，结合荧光报告系统进一步量化shRNA的抑制效率。根据2种已知体外有效抑制猪瘟病毒复制的siRNA-N2、siRNA-NSSB，分别设计得到shRNA-N2、shRNA-NS5B表达栽体，继而采用高效、高灵敏度的shRNA筛选平台对这2种shRNA进行了检测，结果显示单拷贝情况下，shRNA-N2抑制靶序列效率较强，shRNA-NSSB抑制效率弱。本试验通过新策略大大提高了shRNA干扰效率检测的灵敏度，为采用shRNA转基因克隆动物进行抗病育种的研究提供了保障。 相似文献