脂质体介导法转染肿瘤细胞效率的优化

目的:研究优化影响脂质体转染效率的因素,以提高脂质体转染效率,为相关研究和应用提供参考.方法:以绿色荧光蛋白(GFP)作为报告基因,采用脂质体Lipofectamine 2000包裹pU6H1-GFP-FAK重组质粒转染Caco-2细胞,研究了细胞接种密度、DNA用量、脂质体与DNA的比例、脂质体-DNA复合物的形成时间、细胞与脂质体复合物的孵育时间、血清的有无及细胞的传代次数等因素对脂质体转染效率的影响.结果:2-5次细胞传代,2×105接种密度、4μg DNA用量、2.5:1的脂质体与DNA比例、30min脂质体-DNA复合物形成时间以及6h细胞与复合物孵育时间,转染效率最高.血清在本实验室条件下并不影响转染效率.结论:实验获得的优化条件可以明显提高脂质体对肿瘤细胞的转染效率,可作为有关研究或应用的参考.     

影响非洲绿猴肾细胞脂质体转染效率的因素

已有实验表明,细胞转染效率可能决定于ＤＮＡ－脂质体复合物的形成以及所转染的细胞种类。利用脂质体ＬｉｐｏｆｅｃｔＡＭＩＮＥ,研究了影响Ｖｅｒｏ细胞转染效率的参数如ＤＮＡ和脂质体的用量,转染的细胞数量以及细胞暴露于ＤＮＡ－脂质体复合物的时间长度。通过检测报告基因β－半乳糖苷酶（β－ｇａｌ）的表达,发现最高转染率只在一较窄范围内获得。β－ｇａｌ的表达随脂质体量增加而显著增加。在标准转染条件下,增加ＤＮＡ用量和延长细胞暴露时间反而使转染效率下降。转染细胞数量为４×１０４细胞／２４孔时,细胞转染效率最高。     

影响非洲猴肾细胞脂质体转染效率的因素

已有实验表明,细胞转染效率可能决定于ＤＮＡ－脂质体复合物的形成以及所转染的细胞种类。利用脂质体ＬｉｐｏｆｅｃｔＡＭＩＮＥ,研究了影响Ｖｅｒｏ细胞转染效率的参数如ＤＮＡ和脂质体的用量,转染的细胞数量以及细胞暴露子ＤＮＡ－脂质体复合物的时间长度。通过检测报告基因β－半乳糖苷酶（β－ｇａｌ）的表达,发现最高转染率在一较窄范围内获得。β－ｇａｌ的表达随脂质体量增加而显著增加。在标准转染条件下,增加ＤＮＡ用     

优化脂质体对肝癌细胞株HepG2细胞转染效率

摸索用Lipofectamine 2000(Lipo)转染质粒pEGFP-C1到肝癌细胞HepG2较为合适的转染条件。以HepG2细胞为研究对象,采用脂质体Lipofectamine 2000转染pEGFP-C1质粒,在24孔板先按每孔0.5μg固定pEGFP-C1质粒用量,摸索Lipo在1μL、1.5μL、2μL、2.5μL量上较为合适的用量,确定Lipo用量后,然后固定Lipo为1μL,摸索质粒用量0.5μg、1μg,确定脂质体与质粒的最佳比例。此外,对转染中培养基是否含血清,Lipofectamine 2000与pEGFP-C1质粒比例为1:0.5的基础上,扩大用量,即Lipofectamine 2000 2μL和pEGFP-C1质粒1μg,以及脂质体复合物孵育细胞时间进行优化。最后,采用荧光倒置显微镜观察细胞转染效率和方差分析及秩和检验的统计学方法进行统计分析。结果显示,pEGFP-C1质粒固定为0.5μg时,Lipo用量在1μL及1.5μL用量组转染效率最好,之后随着Lipo用量加大,转染效率下降;固定Lipo用量1μL,pEGFP-C1质粒0.5μg时转染效率最好(p0.05),比例不变,扩大Lipo和质粒用量,并不增加转染效率。转染后于3 h、6 h、8 h、12 h、24 h换成正常培养基培养,转染6 h后换液较好。此外,研究发现培养基是否含血清不影响转染效率。本研究表明在24孔板板中用Lipofectamine 2000转染HepG2细胞时较为合适的转染条件为每孔1μL Lipofectamine 2000和0.5μg质粒,脂质体与质粒的最佳比例为2:1,血清不影响转染效率,用含血清培养基转染后6 h换液培养。     

小鼠骨样细胞MLO-Y4转染方法的研究

为了建立质粒转染小鼠骨样细胞MLO-Y4的方法,分别采用阳离子脂质体法和电转染法将增强型绿色荧光蛋白（EGFP）质粒pEGFP-C1转染小鼠骨样细胞MLO-Y4,正常培养48h后检测并统计转染率和死亡率。结果显示,脂质体法转染,当质粒与脂质体比例为1∶4时,转染效率可达到（36.8±3.7）%,细胞死亡率为（18.4±1.9）%;电转染法转染,脉冲电压240 V,脉冲时间300μs,脉冲次数3次时,转染率最高,可达到（23.8±2.3）%,细胞死亡率为（14.1±1.1）%。而后MTT实验显示脂质体转染法相对于电转染法对MLO-Y4细胞的增殖有一定的抑制作用,但对后续实验研究影响不大。脂质体转染法转染小鼠骨样细胞MLO-Y4优于电转染法。     

电穿孔法转染293T细胞条件的优化

为筛选电穿孔转染人胚肾293T细胞的最优条件,将增强型绿色荧光蛋白(EGFP)基因克隆至启动子p CMV前,获得的重组质粒在不同电压、质粒浓度和电击次数条件下电穿孔转染293T细胞,继而在倒置荧光显微镜下观察转染细胞,根据EGFP表达情况评价转染效率。结果表明400 V电压、45 mg质粒电穿孔转染293T细胞2次时转染效率达到44%,与脂质体转染效率(51%)无统计学差异。     

小鼠精原干细胞不同转染方法效率的比较

本研究采用腺病毒感染、慢病毒感染、脂质体转染和电穿孔转化方法将含有绿色荧光蛋白（GFP）的质粒转入经过差异贴壁法初步分离纯化的小鼠精原干细胞(SSCs)中,转染48 h后通过流式细胞仪检测GFP阳性细胞比例比较4种方法在体外转染精原干细胞的效率.结果显示,脂质体转染效率最高仅为8.64%,不能满足对精原干细胞进一步实验的要求;电穿孔法效率最高达到25.27%,但转化后细胞大量死亡;腺病毒转染细胞的效率达到了32.4%;慢病毒转染效率最高,达到74.25%. 因此,慢病毒转染法是体外转染小鼠精原干细胞的有效方法.     

小鼠KGF基因毛囊特异性表达载体的构建及mESC脂质体悬浮转染条件的优化

旨在构建小鼠角质细胞生长因子(KGF)真核表达载体pCDsR-UKA,通过脂质体转染法转染小鼠胚胎干细胞( mESC),并进一步优化其转染条件,最终获得可以正常生长并稳定表达红色荧光蛋白的转KGF基因的ES细胞.利用RTPCR技术扩增小鼠KGF基因cDNA并构建表达载体pCDsR-UKA (6.6 kb),经鉴定正确的重组质粒DNA用脂质体包裹后转染mESC.从小鼠成纤维细胞cDNA扩增出891 bp的KGF基因片段与UHS启动子和BGH polyA序列成功重组到pCDsRed2载体中.经酶切和DNA测序验证,插入载体的DNA片段为KGF基因且方向正确.采用脂质体法优化转染条件,mESC最高转染效率达到(34.4±4.1)％.经G418筛选的转基因ES细胞通过PCR鉴定证实外源基因已整合在ES细胞基因组中.成功获得了小鼠KGF基因片段,以及真核表达载体pCDsR-UKA,经优化的脂质体悬浮法转染条件,在六孔板中当DNA与脂质体比例为3:10时,可获得最佳转染效率且不改变ES细胞的生长状态,经筛选获得了转基因ES细胞克隆.为下一步通过四倍体补偿技术获得ES小鼠提供了转基因ES细胞.     

阳离子脂质体介导RNA干扰的效果评价

考察自制的肽型阳离子脂质体CDO14作为RNA转染载体的细胞毒性及其运载si RNA进行RNA干扰的效果。通过MTT法检测脂质体对稳定表达荧光素酶的肺癌A549(Luc-A549)细胞的毒性。以脂质体为载体将荧光素酶si RNA(Luc-si RNA)转染至Luc-A549细胞内,用发光仪检测转染细胞内荧光素酶含量,BCA法检测细胞内总蛋白含量。在裸鼠腋下接种Luc-A549细胞,成瘤后尾静脉注射Luc-si RNA和脂质体的复合物,利用活体成像系统检测模型小鼠体内荧光素酶的表达量。细胞毒性实验表明,自制脂质体的毒性与商品脂质体DOTAP相近,低于商品脂质体Lipo2000;细胞转染实验表明自制脂质体作为基因转染载体的转染效率高于DOTAP;体内转染实验表明CDO14作为载体转染效果优于DOTAP。结果表明,肽型阳离子脂质体CDO14具有毒性小、转染效率高等优点,有望作为转染载体用于基因治疗。     

脂质体介导基因编辑载体质粒转染条件的优化

[目的]探讨脂质体介导基因编辑载体质粒转染人食管癌Eca-109细胞的最佳条件。[方法]以基于Cre/Lox P系统的p GT-A1B1和基于CRISPR/Cas9系统的p X458-sgRNA8两种基因编辑载体质粒为实验材料,将环状和线性质粒以不同DNA用量(0. 2、0. 4、0. 6和0. 8μg)和不同DNA与脂质体比例(1∶1、1∶1. 5、1∶2、1∶2. 5和1∶3)转染Eca-109细胞,计算转染效率。[结果]不同DNA用量条件下,p GT-A1B1在0. 4μg和0. 6μg时转染效率较高,p X458-sgRNA8在0. 6μg时转染效率较高,与其它DNA用量比较均有显著性差异(P 0. 05)。不同DNA与脂质体比例条件下,p GT-A1B1在比例为1∶1. 5和1∶2时转染效率较高,p X458-sgRNA8在比例为1∶2. 5时转染效率较高,与其它比例比较均有显著性差异(P 0. 05)。同一种质粒的环状和线性结构形态,在DNA用量或DNA与脂质体比例相同时,转染效率差异不显著(P 0. 05)。[结论]建立了脂质体介导基因编辑载体质粒转染Eca-109细胞的最佳转染条件,质粒p GT-A1B1和p X458-sgRNA8的DNA用量分别为0. 4～0. 6μg和0. 6μg,DNA与脂质体比例分别为1∶1. 5～1∶2和1∶2. 5。     

Recent advances in the study of Kaposi's sarcoma-associated herpesvirus replication and pathogenesis

It has now been over twenty years since a novel herpesviral genome was identified in Kaposi's sarcoma biopsies. Since then, the cumulative research effort by molecular biologists, virologists, clinicians, and epidemiologists alike has led to the extensive characterization of this tumor virus, Kaposi's sarcoma-associated herpesvirus(KSHV; also known as human herpesvirus 8(HHV-8)), and its associated diseases. Here we review the current knowledge of KSHV biology and pathogenesis, with a particular emphasis on new and exciting advances in the field of epigenetics. We also discuss the development and practicality of various cell culture and animal model systems to study KSHV replication and pathogenesis.     

The structure, reproduction and taxonomy of Vidalia and Osmundaria (Rhodophyta, Rhodomelaceae)

Comprises species occurring mostly in subtidal habitats in tropical, subtropical and warm-temperate areas of the world. An analysis of the type species, V. spiralis (Sonder) Lamouroux ex J. Agardh, a species from Australia, establishes basic characters for distinguishing species in the genus. These characters are (1) branching patterns of thalli, (2) flat blades that may be spiralled on their axis, (3) width of the blade, (4) primary or secondary derivation of sterile and fertile branchlets and (5) position of sterile and fertile branchlets on the thalli. Application of the latter two characters provides an important basic method for separation of species into three major groups. Osmundaria , a genus known only in southern Australia, was studied in relation to Vidalia , and its separation from the Vidalia assemblage is not accepted. Species of Vidalia therefore are transferred to the older genus name, Osmundaria. Two new species, Osmundaria papenfussii and Osmundaria oliveae are described from Natal. Confusion in the usage of the epithet, Vidalia fimbriala Brown ex Turner has been clarified, and Vidalia gregaria Falkenberg, described as an epiphyte on Osmundaria pro/ifera Lamouroux, is revealed to be young branches of the host, Osmundaria prolifera.     

New or rare chromosome counts in Artemisia L. (Asteraceae, Anthemideae) and related genera from Kazakhstan

Fifteen chromosome counts of six Artemisia taxa and one species of each of the genera Brachanthemum, Hippolytia, Kaschgaria, Lepidolopsis and Turaniphytum are reported from Kazakhstan. Three of them are new reports, two are not consistent with previous counts and the remainder are confirmations of very scarce (one to four) earlier records. All the populations studied have the same basic chromosome number, x = 9, with ploidy levels ranging from 2x to 6x. Some correlations between ploidy level, morphological characters and distribution are noted.     

肝癌中HBV和HCV基因和抗原的分布及意义

采用原位分子杂交方法检测HCV RNA及HBV X基因;采用免疫组织化学方法研究HCV核心抗原,非结构区C33c抗原及HBxAg在肝细胞肝癌中的定位及分布.结果表明(1)HCV RNA、HBV X基因在肝细胞肝癌组织检出率分别为40%(55/136)和82%(112/136).HCV RNA定位于癌细胞的胞浆内,阳性细胞呈散在、灶状及弥漫分布三种形式;HBV X基因在肝癌细胞中的分布呈胞浆型、核型及核浆型,阳性细胞也呈上述三种分布形式;(2)HCV C33c抗原、核心抗原在肝细胞肝癌中的阳性率为81%(133/164)及86%(141/164).C33c抗原定位于癌细胞及肝细胞的胞浆内;核心抗原既定位于癌细胞核中,又可定位于胞浆中.C33c抗原阳性细胞以灶状分布为主;而核心抗原阳性细     

Selection with two alleles and complete dominance

For a plant selection model with frequency-independent viabilities, fertilities and selfing rates, it is shown that apart from global fixation, for certain parameter combinations a protected polymorphism and facultative fixation (either allele may become fixed according to initial frequencies) may both occur. Facultative fixation requires different selling rates for the dominant and recessive type. Protection of the polymorphism requires resource allocation for male and female function. In this connection the problem of purely genetically caused population extinction is discussed.
For general frequency dependence and regular segregation, the chances for establishment of a completely recessive gene are compared to those of a completely dominant gene. It is proven that the process of establishment of the recessive gene, despite a fitness advantage, may be considerably endangered by drift effects if random mating prevails. The recessive gene may reach the same effectivity in establishment as a dominant gene, only if the recessive homozygote mates exclusively with its own type during the period of establishment.