抗端粒酶核酶抑制肝癌细胞裸鼠移植瘤生长的研究

设计针对端粒酶RNA组分的特异性核酶，研究该核酶对肝癌细胞裸鼠移植瘤生长的影响。构建含针对端粒酶RNA组分锤头状核酶基因的重组真核表达质粒pBBS212-RZ，以及建立人肝癌细胞株HepG2裸鼠移植瘤模型。将不同剂量的pBBS212-RZ用脂质体包裹后进行瘤体内多点注射，对照组注射生理盐水或空质粒载体pBBS212。连续注射2l天后，测量移植瘤体积和重量，检测瘤组织端粒酶活性。抗端粒酶特异性核酶使瘤组织端粒酶活性下降(抑制率为72％)，明显地抑制了肝癌细胞裸鼠移植瘤生长(抑制率为68％)，且存在剂量依赖性。抗端粒酶特异性核酶作为一种有效的端粒酶抑制剂，可抑制肝癌细胞的生长，有望在肝癌基因治疗中发挥作用。     

Effects of hammerhead ribozyme (RCP) on HBV P gene in vitro

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剪切HCV RNA的HDV核酶的设计及活性测定

目的：探讨丁型肝炎病毒（Hepatitis D virus,HDV）核酶用于抗丙型肝炎病毒（Hepatitis C virus,HCV）基因治疗的可能性。方法：以HDV基因组核酶的假结样结构为基础,优化其茎IV区,改建基底物结合区,获得3种针对HCV RNA的HDV核酶RzC1、RzC2和RzC3。体外转录获取含HCV RNA5‘－非编码区（5‘-noncoding region,5‘-NCR）及部分C区在内的底物RNA（HCV RNA 5‘－NCR－C）,并进行5‘端放射性标记。在pH7.5、37℃、Mg^2 20mmol/L和去离子甲酰胺2.5mol/L等条件下,将核酶和底物按摩尔比100：1混合,在不同的时间点观察剪切百分率。结论：RzC1、RzC2对底物的剪切百分率随时间延长而递增,90min分别达24.9％、20.3％;未观察到RzC3有剪切活性。结论：经过结构构优化的HDV基因组核酶在合适的位点能够剪切异源性RNA分子HCV RNA。     

切割bcl-2 mRNA核酶诱导人胆管癌细胞凋亡的形态学改变

研究切割ｂｃｌ－２ｍＲＮＡ核酶诱导胆管财亡的形态学改变。方法将合成的切割ｂｃｌ－２ｍＲＮＡ核酶民真核细胞表达载体重组,并用重组后的载体转染人胆管癌细胞,通过光镜及扫描和透射电镜手段,观察转染后的胆管癌细胞,通过光镜及扫描和透射电镜手段,观察传染后的胆管癌细胞凋亡的形态学改变。     

特异切割多药耐药基因(MDR1)的核酶设计与克隆

目的:设计与克隆特异切割多药耐药基因(MDR1) 的核酶。方法:针对人类MDR1 mRNA第Ⅶ外显子附近第196 密码子的GUC 序列,按照“锤头结构”模型,设计、合成核酶,并定向克隆入载体PGEM-3Zf(t) 中;通过PCR- SSCP法及限制性分析,确保扩增片断为外源插入的DNA序列,并经DNA测序证实。结果:重组质粒插入片断序列与设计序列一致。结论:设计、克隆核酶作用人类MDR1 mRNA是有效的     

tRNA~(val)-CTE复合核酶真核表达载体构建

目的构建tRNA^val—CTE复合核酶真核表达载体，为进一步研究打下基础。方法用RT—PCR法扩增CTE DNA；合成含tRNA^val启动子和HCV5-NCIK区195住的核酶以覆内切酶KpnⅠ和EcoRⅤ序列，通过退火连接到pPUR质粒的BamHⅠ和EcoRⅠ的酶切位点之间，构建质粒pPHCV—Rz；用KpnⅠ和EcoRⅤ双酶切后，将CTE cDNA定向插入表达载体pPHCV—Rz多克隆位点中，构建成重组表达质粒，并用酶切分析和序列测定进行了鉴定。结果酶切鉴定和测序证实tRNA^val启动子和HCV5-NCIK区195住的核酶基因以覆CTE cDNA被正确克隆入真核表达载体pPUK。结论tRNA^val-CTE复合核酶真核表达载体的成功构建，为开展利用tRNA^val-CTE复合核酶切割HCV RNA的研究奠定了基础。     

Encapsulation of ribozymes inside model protocells leads to faster evolutionary adaptation

Functional biomolecules, such as RNA, encapsulated inside a protocellular membrane are believed to have comprised a very early, critical stage in the evolution of life, since membrane vesicles allow selective permeability and create a unit of selection enabling cooperative phenotypes. The biophysical environment inside a protocell would differ fundamentally from bulk solution due to the microscopic confinement. However, the effect of the encapsulated environment on ribozyme evolution has not been previously studied experimentally. Here, we examine the effect of encapsulation inside model protocells on the self-aminoacylation activity of tens of thousands of RNA sequences using a high-throughput sequencing assay. We find that encapsulation of these ribozymes generally increases their activity, giving encapsulated sequences an advantage over nonencapsulated sequences in an amphiphile-rich environment. In addition, highly active ribozymes benefit disproportionately more from encapsulation. The asymmetry in fitness gain broadens the distribution of fitness in the system. Consistent with Fisher’s fundamental theorem of natural selection, encapsulation therefore leads to faster adaptation when the RNAs are encapsulated inside a protocell during in vitro selection. Thus, protocells would not only provide a compartmentalization function but also promote activity and evolutionary adaptation during the origin of life.

RNA is believed to have been a central constituent of early life (1–3). In the “RNA world” theory, functional RNAs (e.g., ribozymes) would both perform catalytic functions and store and transfer genetic information in a simple living system (4–6). Encapsulation of ribozymes in cell-like compartments, such as protocells, is thought to be an essential feature for the emergence of early life (7–11). In particular, compartmentalization would retain useful metabolites in the vicinity (12) and prevent a cooperative, self-replicating ribozyme system from collapsing under parasitization by selfish RNAs (13, 14). A major model of protocells is lipid vesicles, which consist of an aqueous interior surrounded by a semipermeable membrane (15, 16). However, while the ultimate advantages of compartmentalization may be clear, how encapsulation and confinement inside protocell vesicles would affect the activity and early evolution of ribozymes is not understood well.Confinement by lipid membranes presents a biophysical environment similar to macromolecular crowding (17). The effect of macromolecular crowding on the activity, function, and specificity of biomolecules (i.e., proteins and nucleic acids) has been examined extensively (18–23) using crowding agents such as dextran, polyethylene glycol, and Ficoll in vitro (24–29). In general, macromolecular crowding agents decrease the accessible volume for biomolecules, leading to the excluded-volume effect, in which the relative stability of compacted and folded structures is increased (30, 31). At the same time, chemical interactions between the crowding agents and the biomolecule can also stabilize or destabilize the folded structure, influencing catalytic activity (24, 32). While chemical interactions depend on the properties of the specific molecules under study, the excluded-volume effect resulting from spatial confinement inside vesicles is expected to be general. The effect of confinement can be studied while controlling for chemical interactions by comparing the encapsulated condition to the nonencapsulated but membrane-exposed condition. This comparison represents the prebiotic scenario in which RNAs would be present in the same milieu as lipids (33) and may become encapsulated or not. In this way, confinement inside vesicles was shown to increase the binding affinity of the malachite green RNA aptamer (34). Interestingly, spatial confinement inside a tetrahedral DNA framework has also been shown to increase thermodynamic stability and binding affinity of aptamers by facilitating folding (35).While these and other case studies (17, 25, 36–43) illustrate mechanisms by which RNA activity might be perturbed inside vesicles, understanding how encapsulation would affect evolution requires a broader scale of information. In particular, detailed knowledge of how encapsulation affects the sequence-activity relationship is required. This information is captured in the “fitness landscape,” or the function of fitness over sequence space, which embodies many important evolutionary features [e.g., fitness maxima, epistasis, and the viability of evolutionary trajectories (44–47)]. In practice, the fitness of a ribozyme can be considered to be its chemical activity for a particular function in the given environment (48–53).In the present work, we investigated how encapsulation inside model protocells would affect the catalytic activity and evolution of self-aminoacylating ribozymes. We studied tens of thousands of RNA sequences derived from five previously selected self-aminoacylating ribozyme families (53). These sequences were encapsulated in a mixed fatty acid/phospholipid vesicle system. Fatty acids mixed with phospholipids (1:1 molar ratio) have been used as model protocell membranes, as the vesicles tolerate Mg²⁺ concentrations needed for ribozyme activity and the membrane allows small, charged molecules to permeate while preserving large polynucleotides in the vesicle interior (54, 55). To study the biophysical effect of confinement rather than chemical interactions with the membrane, RNA activity inside vesicles was compared with RNA activity when exposed to the same vesicles without encapsulation. We show that ribozymes generally exhibit higher catalytic activity inside the vesicles and that more active sequences experience greater benefit. Using in vitro selection, we demonstrate that one of the evolutionary consequences of this trend is that encapsulation inside vesicles causes a greater rate of genotypic change due to natural selection.     

锤头状核酶对肝癌突变基因p53的抑制作用

目的 探讨p53核酶对肝癌细胞突变型抑癌基因p53的抑制作用。方法 应用计算机设计并合成针对突变型p53(249位密码子AGG→AGT)的锤头状核酶RZ，构建其体外转录和真核表达载体，检测核酶对突变型p53(mtp53)的体外切割作用，并在Lipofect AMINE^TM2000的介导下转染肝癌细胞MHCC97，应用逆转录聚合酶联反应(RT—PCR)检测核酶对肝癌细胞突变型p53的抑制作用。结果 测序证实核酶基因被正确克隆人体外转录载体pBSKU6和真核载体pEGFPC1中。体外切割效率为42％，而野生型p53(wtp53)没有被切割。在Lipofect AMINETM2000的介导下成功转染肝癌细胞MHCC97，RT—PCR检测证实突变型p53的mRNA水平明显下降，细胞内的切割效率为69％。结论 p53核酶可成功抑制肝癌细胞中突变型p53的表达，为肝癌的基因治疗提供了一个新的选择。