RNA干扰技术的应用新进展

RNA干扰（RNA interference,RNAi）被发现十年来在生物研究领域已经得到了长足的发展,并且已经初步显示出了巨大的潜力。本文综述了介导SiRNA方法的研究及其在基础研究、疾病模型建立和药物研究中的最新应用进展。     

Characterization and Distribution of Ferritin Binding Sites in the Adult Mouse Brain

Abstract : Studies on iron uptake into the brain have traditionally focused on transport by transferrin. However, transferrin receptors are not found in all brain regions and are especially low in white matter tracts where high iron concentrations have been reported. Several lines of research suggest that a receptor for ferritin, the intracellular storage protein for iron, may exist. We present, herein, evidence for ferritin binding sites in the brains of adult mice. Autoradiographic studies using ¹²⁵I-recombinant human ferritin demonstrate that ferritin binding sites in brain are predominantly in white matter. Saturation binding analyses revealed a single class of binding sites with a dissociation constant ( K _D) of 4.65 × 10^-9 M and a binding site density ( B _max) of 17.9 fmol bound/μg of protein. Binding of radiolabeled ferritin can be competitively displaced by an excess of ferritin but not transferrin. Ferritin has previously been shown to affect cellular proliferation, protect cells from oxidative damage, and deliver iron. The significance of a cellular ferritin receptor is that ferritin is capable of delivering 2,000 times more iron per mole of protein than transferrin. The distribution of ferritin binding sites in brain vis-à-vis transferrin receptor distribution suggests distinct methods for iron delivery between gray and whi     

Developmental Regulation of Microtubule-Associated Protein 2 Expression in Regions of Mouse Brain

The relative levels of microtubule-associated protein 2(MAP2) were determined during postnatal development of the mouse in six different discrete brain regions: cerebellum, cortex, hippocampus, olfactory bulb, brainstem, and hypothalamus. Brain homogenates were electrophoresed on sodium dodecyl sulfate-containing gels and analyzed by immunoblotting with MAP2-specific antibodies. The levels of MAP2 in each region were determined using radiolabeled secondary antibodies and densitometric quantification of the autoradiograms over a range that was determined to have a linear response. The results indicated that in all regions and at all ages there was only one high-molecular-weight polypeptide of MAP2, which did not change in electrophoretic mobility after dephosphorylation. In most regions, the levels of MAP2 increased during the first 2 postnatal weeks. However, there were differences in the time course and relative levels of MAP2 between regions. In addition, all regions of the brain expressed the low-molecular-weight form of MAP2 (MAP2c) that was present at birth as a heterogeneous group of polypeptides with an apparent molecular weight of 70K. Most of the heterogeneity of MAP2c, however, was eliminated after dephosphorylation. The levels of MAP2c decreased dramatically after 2 weeks postnatally, except for the olfactory bulb, where the levels of MAP2c remained relatively high even in adults.     

Tet2 Rescues Age-Related Regenerative Decline and Enhances Cognitive Function in the Adult Mouse Brain

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RNA干扰NRP2基因表达对胃癌细胞SGC7901裸鼠移植瘤的抑制作用

目的:探讨特异性抑制NRP2基因表达对人胃癌细胞SGC7901裸鼠移植瘤生长及淋巴管新生的影响。方法:采用小RNA干扰方法,构建shNRP2质粒,稳定转染入SGC7901细胞株,Westen blot检测转染前后NRP2蛋白的表达。建立人胃癌细胞SGC7901裸鼠移植瘤模型,随机分为shNRP2组(实验组)、shCon组(HK阴性对照组)和正常对照组,观察移植瘤的生长情况。6周后处死裸鼠,免疫组化检测NRP2蛋白的表达及微淋巴管密度(Micro-vessel density,MLD)。结果:成功构建shNRP2质粒,与另两组比较,shNRP2组细胞NRP2蛋白表达明显降低,且移植瘤组织生长、NRP2表达及MLD明显受抑制。结论:抑制NRP2基因的表达,可以抑制胃癌裸鼠移植瘤的生长及淋巴管的形成,NRP2基因有可能成为一个潜在的胃癌生物治疗靶点。     

Developmental Expression of the Glycine Transporters GLYT1 and GLYT2 in Mouse Brain

Abstract: Using immunocytochemical localization, the distribution of the glycine transporters GLYT1 and GLYT2 in the developing mouse brain was studied. GLYT1 and GLYT2 immunoreactivity begins during the period of fiber outgrow and synaptogenesis. GLYT2 is first expressed in spinal and spinothalamic white matter and is followed by the expression of synaptophysin. In the postnatal stages, GLYT2 staining in the white matter disappears, and a punctuated pattern in the gray matter emerges. In contrast, in the fetal brain GLYT1 immunoreactivity coincides with gray matter neuropil and processes of radial glia. GLYT1 is distributed over a much wider area of the brain than GLYT2. However, the distribution of these two GLYTs implies that GLYT1 and GLYT2 operate in concert within the area where both are present. At the day 12 embryo stage, GLYT1 antibodies stain the liver, and later they also react with the pancreas and the gastroduodenal junction. No other organs exhibit significant GLYT1 immunoreactivity. We additionally observed the presence of GLYT1 in rat fetal cerebral cortex and hippocampus, which was not detected in fetal mouse brain. Moreover, GLYT1 immunoreactivity was found in the mouse floor plate and the ventral commissure but was not present in the same regions in rats. These findings suggest possible differences in the expression of GLYT1 between these two species.     

Isolation and Sequencing of a Genomic Clone for Mouse Brain Specific Small RNA

We isolated a mouse genomic clone that hybridized with small RNA present in the cytoplasm of the brain. The RNA was about 150 nucleotides long. This RNA seemed to be specific to the brain, since it was not found in the liver or kidney. The clone DNA contained a sequence homologous to 82-nucleotide "identifier" core sequence of cDNA clones of rat. The sequence contained a split promoter for RNA polymerase III and was flanked by a 12-nucleotide direct repeat (ATAAATAATTTA).     

RNA干扰在柯萨奇病毒致心肌炎小鼠模型中的抗病毒研究

为了研究慢病毒介导的shRNA(Short hairpin RNA,shRNA)在柯萨奇B组3型病毒(Coxsackievirus B3,CVB3)导致的心肌炎小鼠模型中的抗病毒作用,合成针对CVB3基因组3753～3771区域的慢病毒Lenti-sh3753,感染HeLa细胞后感染CVB3病毒,通过荧光显微镜观测shRNA的表达和病毒致细胞病变效应,并测定培养上清中的病毒滴度,将慢病毒Lenti-sh3753感染BALB/c小鼠后感染CVB3病毒,观察小鼠的存活率,心脏组织中的病毒滴度和病理变化。结果发现Lenti-sh3753能在HeLa细胞中表达shRNA,并能有效抑制细胞中病毒RNA的复制。在小鼠模型上,Lenti-sh3753能提高小鼠的存活率,降低心脏中的病毒含量,从而减轻病理反应。这些结果提示,Lenti-sh3753在细胞和动物模型中能针对性地降解CVB3病毒RNA,明显降低病毒滴度,有效控制病毒感染。     

RNA干扰在哺乳动物卵母细胞及早期胚胎发育研究中的应用

RNA干扰(RNAinterference,RNAi)是一种真核生物体内由特定双链RNA介导的转录后基因沉默现象。近年来,RNAi的作用机制已基本阐明,并广泛的应用于基因功能的研究。现对RNAi在哺乳动物卵母细胞及早期胚胎研究中的作用特点、应用情况、存在问题等几方面进行综述。     

RNAi在哺乳动物中的应用及研究进展

该文综述RNAi技术在哺乳动物中应用及研究方面的最新进展。     

Ang2-siRNA慢病毒干预裸鼠移植性恶性黑色素瘤的研究

目的研究血管生成素2小干扰RNA（Ang2-siRNA）对裸鼠移植性恶性黑色素瘤血管形成及其生长的影响。方法建立人恶性黑色素肿瘤裸鼠异种移植瘤模型。在体内使用pNL-EGFP—Ang2-siRNA慢病毒干扰裸鼠移植性恶性黑色素瘤Ang2基因的表达,观察统计各组肿瘤（空白组、空载组、实验组）生长情况。应用实时荧光定量RT—PCR检测肿瘤组织中Ang2基因的mRNA表达水平;CD34单克隆抗体作为血管内皮标志物,免疫组织化学法检测其表达,以评价肿瘤微血管密度。结果成功建立了恶性黑色素瘤裸鼠异种移植瘤模型,实验组肿瘤Ang2基因mRNA水平、微血管密度、肿瘤体积显著小于空白组和空载组（P〈0．01）,空白组和空载组之间无统计学意义（P〉0．05）。结论pNL—EGFP—Ang2-siRNA慢病毒能沉默Ang2的表达,显著抑制恶性黑色素瘤血管的形成和肿瘤的生长,可能为临床恶性黑色素肿瘤的基因治疗开辟新的途径。     

小鼠胚胎Sry基因的RNA干涉研究

为了研究Sry基因的调控网络,采用siRNA技术使Sry基因沉默,探讨了有效沉默Sry基因的途径和最佳条件.设计、合成针对小鼠Sry基因的发夹状寡核苷酸链,退火后连入真核表达载体pSilencer4.1-CMVneovector,构建以小鼠Sry基因为靶点的siRNA干涉载体pSilencer4.1/Sry217及pSilencer4.1/Sry565,通过尾静脉注射法将载体质粒导入妊娠小鼠体内,于小鼠妊娠第11.5天,即11.5dpc(dayspostcoitum,性交后天数)取出胚胎,采用双重PCR法对胚胎进行性别鉴定,鉴定为雄性的胚胎采用半定量RT-PCR法检测Sry基因的表达量,研究不同干扰序列、不同注射时间及注射剂量对Sry基因表达量的影响.研究结果,确定了质粒的最佳注射时间为9.5dpc,注射剂量为20μg,注射干扰质粒pSilencer4.1/Sry565对Sry基因的抑制效率达85%左右.结果表明,siRNA可以显著抑制雄性胚胎Sry基因的表达.     

Effect of Malnutrition and Subsequent Rehabilitation on the Development of Mouse Brain Myelin

Abstract: Malnutrition in mice from birth resulted in myelin of brain having higher than normal molar proportions of cholesterol and phospholipids relative to a molar unit of cerebroside + sulphatide. This was found at all ages between 20 and 60 days, and the molar ratio of these lipids in older animals was comparable to that in the younger controls. The phospholipid and the ganglioside patterns were also immature for age. The phospholipid composition was characterized by lower molar proportions of ethanolamine phosphoglyceride (EPG) and sphingomyelin (SPh) and higher proportion of choline phosphoglyceride (CPG), and the ganglioside pattern was characterized by higher molar proportions of the disialogangliosides G_Dla and G_Dlb and markedly lower proportion of the monosialoganglioside G_M1. Malnutrition imposed from 30 days of age did not affect the contents of the major lipids (and so their molar ratio), but within the phospholipids there was a small but significant deficit of SPh, which was compensated by a higher content of CPG. The ganglioside pattern was as if the animals were malnourished from birth. Nutritional rehabilitation up to 60 days of age subsequent to malnutrition for the first 30 days fully corrected the ganglioside pattern, but not the molar ratio, of the major lipids (because of persistent deficit in cerebroside + sulphatide) and the composition of the phospholipids (because of small but significant deficit of SPh). The results indicate that malnutrition instituted at any time during the entire programme of myelination can affect one or other aspect of myelin development, and nutritional rehabilitation of animals malnourished in early life cannot fully correct this developmental gap.     

Neuroanatomical Characterisation of the Expression of the Lipodystrophy and Motor-Neuropathy Gene Bscl2 in Adult Mouse Brain

The endoplasmic reticulum localised protein seipin, encoded by the gene Berardinelli–Seip congenital lipodystrophy type 2 (BSCL2), serves a critical but poorly defined function in the physiology of both adipose and neural tissue. In humans, BSCL2 loss-of-function mutations cause a severe form of lipodystrophy, whilst a distinct set of gain-of-toxic-function mutations are associated with a heterogeneous group of neuropathies. However, despite the importance of seipin dysfunction to the pathophysiology of these conditions, little is known about its physiological role in adipocytes or neurons. BSCL2 mRNA has previously been identified in human and mouse brain, yet no definitive assessment of its expression has been undertaken. Here we comprehensively characterised the neuroanatomical distribution of mouse Bscl2 using complementary in situ hybridisation histochemistry and immunohistochemistry techniques. Whilst Bscl2 was broadly expressed throughout the rostral-caudal extent of the mouse brain, it exhibited a discrete neuroanatomical profile. Bscl2 was most abundantly expressed in the hypothalamus and in particular regions associated with the regulation of energy balance including, the paraventricular, ventromedial, arcuate and dorsomedial nuclei. Bscl2 expression was also identified within the brainstem dorsal vagal complex, which together with the paraventricular nucleus of the hypothalamus represented the site of highest expression. Further neurochemical profiling of these two nuclei revealed Bscl2/seipin expression within energy balance related neuronal populations. Specifically, seipin was detected in oxytocin neurons of the paraventricular nucleus of the hypothalamus and in catecholamine neurons of the dorsal vagal complex. These data raise the possibility that in addition to its role in adipose tissue development, seipin may also be involved in the central regulation of energy balance.     

Brain Purines in a Genetic Mouse Model of Lesch-Nyhan Disease

Abstract: Mice carrying a mutation in the gene encoding the purine salvage enzyme hypoxanthine-guanine phosphoribosyltransferase (HPRT) have recently been produced to provide an animal model for Lesch-Nyhan disease. The current-studies were conducted to characterize the consequences of the mutation on the expression of HPRT and to characterize potential changes in brain purine content in these mutants. Our results indicate that the mutant animals have no detectable HPRT-immunoreactive material on western blots and no detectable HPRT enzyme activity in brain tissue homogenates, confirming that they are completely HPRT deficient (HPRT^-). Despite the absence of HPRT-mediated purine salvage, the animals have apparently normal brain purine content. However, de novo purine synthesis, as measured by [¹⁴C]formate incorporation into brain purines, is accelerated four- to fivefold in the mutant animals. This increase in the synthesis of purines may protect the HPRT^- mice from potential depletion of brain purines despite complete impairment of HPRT-mediated purine salvage.     

小鼠Dppa2基因shRNA载体的构建及干扰效果的检测

目的:构建有效的针对小鼠Dppa2基因的shRNA(short hairpin RNA)干扰载体。方法:设计合成2对针对小鼠Dppa2基因的shRNA序列以及1对与哺乳动物基因组无同源性的shRNA序列作为对照,构建pSUPER.Retro.puro干扰载体并进行PCR,酶切和测序验证。进一步将各干扰载体分别转染小鼠胚胎干细胞(embryonic stem cells,ESCs),RT-PCR检测干扰效率。结果:PCR,酶切和测序验证均表明各shRNA载体构建成功。将空载体及各重组载体分别转染小鼠ESCs发现,干扰组Dppa2基因表达水平相对于空载体对照组和阴性shRNA载体对照组明显下调。结论:成功构建了有效的针对小鼠Dppa2基因的shRNA干扰载体,为进一步研究Dppa2基因在维持小鼠ESCs不分化过程中的作用提供了基础。     

GAL4/UAS系统在果蝇Tis11基因RNA干扰中的应用

TTP在哺乳动物许多关键基因表达的转录后水平上起调控作用,Tis11是TTP蛋白在果蝇中的同源物.目前还没有现成的可用于研究Tis11功能的基因敲除或敲低的果蝇.为了获得肌动蛋白启动子或者热激蛋白启动子驱动表达Tis11 mRNA干扰序列的具有较高干扰效率的Tis11基因干扰果蝇,将肌动蛋白启动子或者热激启动子驱动表达的GAL4果蝇品系与融合有Tis11 mRNA干扰序列的UAS品系杂交,收集同时带有GAL4基因和UAS序列的子一代果蝇.提取所收集果蝇的总RNA,将其中的mRNA逆转录成cDNA,并设计检测Tis11基因的特异性引物,然后通过Real-time PCR检测Tis11 mRNA的表达情况.结果显示所收集的能表达Tis11基因干扰序列的子一代果蝇与不能表达Tis11基因干扰序列的对照果蝇相比,其体内Tis11 mRNA的表达水平下降明显.收集的果蝇其体内所表达的干扰序列对Tis11 mRNA干扰效果显著,我们成功获得了Tis11基因的RNA干扰果蝇.