小鼠Islet-1基因慢病毒表达载体的构建及其诱导C_3H_(10)T1/2细胞向心肌样细胞特异性分化

目的研究Islet-1对干细胞分化的影响。方法用PCR钓取目的基因,将目的基因与pLenO-WPI载体连接,选取阳性质粒,与辅助质粒共同感染293T细胞生产出慢病毒载体。感染C3H10T1/2细胞,实时荧光定量PCR及Western blot检测Islet-1和心肌、肝脏、骨骼及神经各系统相关标志物的表达,免疫荧光检测心肌肌钙蛋白T(cTnT)表达部位。结果 PCR及测序显示目的片段正确插入,实验组有Islet-1表达;心肌早期发育相关基因GATA-4、MEF2C、NKx2.5在检测到荧光蛋白1周后升高,2周到达高峰,3周后可检测到心肌特异性蛋白cTnT(0.582±0.0576),其时序性表达呈随时间增强趋势;cTnT表达于胞质;肝脏系统特异性标志AFP及ALB、骨骼系统特异性标志BGP及BALP、神经系统特异性标志Nestin及GFAP均未表达。结论 Islet-1具有特异性促进干细胞向心肌样细胞分化的作用。     

pIRES2-EGFP-BCL11B真核表达载体体外表达的动态分析

目的: 研究真核表达载体pIRES2-EGFP-BCL11B(B细胞白血病/淋巴瘤11B基因)在K293(人胚肾细胞株)和Raji(人Burkitt 淋巴瘤细胞株)细胞中的表达情况。方法: 分别利用脂质体和核转染技术将真核表达载体pIRES2-EGFP-BCL11B(pBCL11B)转染至K293和Raji细胞中,并设立空质粒组(pI2)和空转染组(MOCK)作为对照。通过荧光显微镜和流式细胞仪检测转染后48 h各组EGFP的表达,确定转染效率。通过荧光实时定量PCR(Taqman)技术检测转染后24 h各组BCL11B mRNA的表达情况,通过Western blotting技术检测转染后48 h各组BCL11B蛋白的表达情况。高分辨率活细胞成像系统动态观察转染后36-72 h重组质粒在Raji细胞增殖过程中的表达。结果: K293细胞pBCL11B、pI2和MOCK组的转染效率分别为(71.23±4.62)%、(70.45±3.58)%和(0.30±0.10)%,而Raji细胞各组的转染效率分别为(23.12±5.94)%、(22.48±6.25)%和(0.30±0.10)%。实时定量PCR和Western blotting结果显示转染后pBCL11B组在mRNA和蛋白水平均能检测到 BCL11B 基因的有效表达,BCL11B的表达量均明显高于pI2组和MOCK组(P<0.05)。活细胞追踪显示转染后36-72 h重组质粒在细胞中可稳定表达且转染后的细胞可以正常分裂增殖。结论: 系统分析了真核表达载体pIRES2-EGFP-BCL11B转染至K293和Raji细胞的表达变化特点,转染后可以检测到该基因的有效上调,研究结果为下一步转染正常人T细胞奠定了基础。     

NK4基因慢病毒载体的构建及其在骨髓间充质干细胞中的表达

本研究构建携带人NK4基因的慢病毒载体,并将其转染人骨髓间充质干细胞(hBMSCs),明确转染后NK4表达情况。通过聚合酶链反应从HGFcDNA文库中克隆NK4基因,限制性内切酶酶切和基因重组构建慢病毒载体质粒pGC-FU-NK4,实时定量PCR检测病毒滴度。所获慢病毒(Lenti-NK4)转染hBMSCs后,荧光显微镜下观察荧光表达。ELISA检测培养上清中NK4表达。结果显示,所获NK4基因测序后与GeneBank报到序列完全一致,收集、浓缩病毒后测定其滴度为2×108TU/ml。Lenti-NK4转染hBMSCs后胞膜及胞浆有荧光分布,培养上清中NK4含量随感染时间延长而增加。提示携带NK4的慢病毒能安全、有效地转染hBMSCs,持续稳定表达。     

真核表达载体pIRES2-EGFP-NT3的构建及在豚鼠耳蜗成纤维细胞中的表达

目的 构建真核表达载体pIRES2-EGFP-NT3，并观察其在豚鼠耳蜗成纤维细胞中的表达及分布。方法 用RT－PCR法，从人肝脏组织中克隆NT3-cDNA基因，构建真核表达载体pIRES2-EGFP-NT3并经PCR及EcoR I/Bam H I双酶切鉴定。用脂质体介导的方法，将用真核表达载体IRES2-EGF-NT3瞬时转染豚鼠原代耳蜗成纤维细胞。转染48h后，分别在荧光显微镜下及用免疫组化法观察转染细胞中NT－3的表达。结果 人NT－3 cDNA的PCR产物约为744bp序列测定结果用BLAST软件进行同源性比较，同源性为100％。经Eco R I/Bam H I双酶切证实，NT－3 cDNA已成功地克隆到真核表达载体IRES2-EGFP中。用脂质体介导法，将pIRES2-EGFP-NT3瞬时转染豚鼠原代耳蜗成纤维细胞，在荧光显微镜下观察到呈绿色荧光的成纤维细胞。NT－3免疫组化染色结果显示，转染NT－3基因的成纤维细胞胞浆呈棕黄色着色；而转染空载体的成纤维细胞免疫组化染色呈阴性。结论 成功地构建真核表达载体pIRES2-EGFP-NT3并在豚鼠耳蜗原代成纤维细胞中表达，为NT－3基因转染治疗致聋豚鼠耳试验奠定了基础。     

大鼠ferroportin 1过表达慢病毒载体构建及其转染PC12细胞

目的:构建含大鼠ferroportin 1(FP1,膜铁转运蛋白1)基因和EGFP(绿色荧光蛋白)基因的过表达慢病毒载体并转染PC12细胞。方法:化学合成含有目的基因FP1的质粒,将酶切获得的目的基因连接入线性化慢病毒载体,构建重组质粒PGC-FU-FP1并进行测序鉴定;鉴定正确的阳性克隆采用Lipofectamine2000转染293T细胞,通过荧光显微镜和Western Blot检测FP1表达情况;在脂质体介导下将PGC-FU-FP1、pHelper1.0和pHelper2.0三质粒系统共转染入293T细胞,包装产生慢病毒,并通过实时荧光定量PCR法检测病毒滴度。以重组慢病毒载体质粒PGC-FU-FP1转染PC12细胞,荧光显微镜下观察EGFP的表达,实时荧光定量PCR和Western Blot法分别检测FP1mRNA和蛋白表达情况。结果:FP1基因序列经测序后与GeneBank报道的序列完全一致;重组质粒PGC-FU-FP1中携带有正确的FP1基因并能在293T细胞中表达;病毒滴度为2×108TU/ml。荧光显微镜、实时荧光定量PCR和Western Blot法证实目的基因FP1能被重组慢病毒高效导入PC12细胞并得到过表达。结论:成功构建携带FP1基因的慢病毒载体,并获得FP1基因修饰的PC12基因工程细胞。为进一步研究FP1在帕金森病中的作用及基因治疗奠定基础。     

Effects of berberine on 6-hydroxydopamine-induced neurotoxicity in PC12 cells and a rat model of Parkinson's disease

Protoberberine isoquinoline alkaloids including berberine inhibit dopamine biosynthesis and aggravate l-DOPA-induced cytotoxicity in PC12 cells. In this study, the effects of berberine on 6-hydroxydopamine (6-OHDA)-induced cytotoxicity in PC12 cells and on unilateral 6-OHDA-lesioned rats were investigated. In PC12 cells, berberine at 10 and 30 μM associated with 6-OHDA (10, 20, and 50 μM) enhanced cytotoxicity at 48 h compared to 6-OHDA alone, indicated by an increase in apoptotic cell death. In addition, treatment with berberine (5 and 30 mg/kg, i.p.) for 21 days in 6-OHDA-lesioned rats markedly depleted tyrosine hydroxylase-immunopositive cells in the substantia nigra as compared to berberine-untreated rats. Further, the levels of dopamine and norepinephrine were also significantly decreased by berberine administration (5 and 30 mg/kg) in the striatal regions of 6-OHDA-lesioned rats. These results suggested that berberine aggravated 6-OHDA-induced cytotoxicity in PC12 cells, and led to the degeneration of dopaminergic neuronal cells in the substantia nigra of 6-OHDA-lesioned rats. It is, therefore, suggested that the use of long-term l-DOPA therapy with isoquinoline derivatives including berberine may need to be examined for the presence of adverse symptoms.     

Pigment epithelium derived factor (PEDF) is neuroprotective in two in vitro models of Parkinson's disease

Transplantation of retinal pigment epithelial (RPE) cells in the basal ganglia has been proposed as a novel cell-based therapy for Parkinson's disease (PD), by providing a constant source of dopamine replacement via the melanin synthetic pathway enzyme tyrosinase. We have demonstrated previously that human RPE cells also produce a neurotrophic effect on primary cultures of rat striata mesencephalic (dopaminergic) neurons and showed that pigment epithelium derived factor (PEDF) accounted for a major portion of the neurotrophic effect. We now have also begun studies that demonstrate that the neurotrophic effect of PEDF corresponds to neuroprotection against toxins used to produce experimental PD. This was shown in (1) rotenone and (2) 6-hydroxydopamine (6-OHDA) in vitro models. The toxins were added at day 10 in culture, PEDF was added 1 h prior. The cultures were fixed and analyzed after tyrosine hydroxylase (TH) immunocytochemical staining. Cell count of TH+ neurons clearly shows the neuroprotective potential of PEDF in both neurotoxin models. The neurotoxic effect of rotenone (25 nM) on dopaminergic neurons is reversed by addition of PEDF. At a concentration of 1 ng/ml PEDF the neurotoxic effect of rotenone is completely counteracted. PEDF (1 ng/ml) has also a neuroprotective effect in the 6-OHDA midbrain in vitro model. The effect is most pronounced at concentrations of 25 μM and 50 μM 6-OHDA. We conclude that the neurotrophic factor PEDF, produced from RPE cells, can improve neuronal survival in models of PD, and plan to test if this effect can be observed using in vivo models of PD following RPE transplantation.     

含HIV-1 Tat基因重组反转录病毒表达载体构建与表达Tat蛋白功能检测

目的 构建含HIV-1 Tat基因重组反转录病毒表达载体及评价表达Tat蛋白的功能。方法 使用HindⅢ将HIV-1 Tatl0l蛋白编码基因从pEN质粒中切出，插入到表达质粒IZRSpBMN-Z中，构建成重组反转录病毒表达质粒IZRS-Tat101。采用磷酸钙转染法将重组质粒LZRS-Tat101转染进含反转录病毒env、gal和pol编码基因的包装细胞phoenix(φNX)中，嘌呤霉素筛选获得稳定细胞系。免疫组化(IHC)染色检查Tat在临时转染和稳定转染φNX细胞中的表达水平。收集临时转染和稳定转染包装细胞分泌的病毒上清，并分别感染293细胞，Western blot检测Tat在293中表达。与此同时，收集感染293细胞培养上清，加入到HL3T1细胞(HeLa-HIV-1-LTR／CAT报告基因)中，共培养72h后收集细胞，提取蛋白作CAT活性检测。结果 ①含Tat101基因重组反转录病毒表达质粒转染包装细胞后，Tat在临时转染φNX中表达水平显著高于在稳定转染中的水平；②临时转染病毒感染293细胞，Tat在感染细胞中得到了表达，且分泌至上清中的Tat蛋白能够激活靶细胞HL3T1中HIV-1的LTR启动子，使得其下游的CAT基因得到表达。结论 重组LZRS-Tat101反转录病毒能够在其感染靶细胞中表达Tat蛋白，且表达蛋白具有转录激活功能。     

小鼠pdx-1基因真核表达载体的构建及其在胚胎干细胞中的表达

目的： 克隆小鼠pdx-1基因，构建其真核表达载体，并在小鼠胚胎干细胞中表达，为糖尿病的细胞移植治疗奠定基础。方法： PCR扩增小鼠胰腺pdx-1基因 cDNA，酶切后和携带绿色荧光蛋白报告基因的真核表达载体pEGFP-N1重组，将pdx-1基因 cDNA片段连接到pEGFP-N1载体的多克隆位点，形成重组载体pEGFP/pdx-1，转化大肠杆菌DH5α菌株，构建成pdx-1基因真核表达载体质粒。扩增DH5α后抽提质粒DNA，Hind Ⅲ 和BamHⅠ酶切，电泳，DNA测序鉴定。鉴定正确的质粒DNA用脂质体包裹后转染小鼠胚胎干细胞MESPU13。结果： 从小鼠胰腺cDNA扩增出876 bp的DNA片段并成功重组到pEGFP-N1载体中。经酶切和DNA测序验证，插入载体的DNA片段为pdx-1基因，插入方向正确。重组质粒经脂质体转染胚胎干细胞MESPU13，24 h 后观察到绿色荧光蛋白报告基因和目的基因的pdx-1表达。结论： 小鼠pdx-1基因的克隆和真核表达载体构建获得成功，为进一步研究其功能奠定了基础。     

Fabry disease: polymorphic haplotypes and a novel missense mutation in the GLA gene

Fabry disease: polymorphic haplotypes and a novel missense mutation in the GLA gene. Fabry disease (FD) is an X-linked lysosomal storage disorder with a heterogeneous spectrum of clinical manifestations that are caused by the deficiency of α-galactosidase A (α-Gal-A) activity. Although useful for diagnosis in males, enzyme activity is not a reliable biochemical marker in heterozygous females due to random X-chromosome inactivation, thus rendering DNA sequencing of the α-Gal-A gene, alpha-galactosidase gene (GLA), the most reliable test for the confirmation of diagnosis in females. The spectrum of GLA mutations is highly heterogeneous. Many polymorphic GLA variants have been described, but it is unclear if haplotypes formed by combinations of such variants correlate with FD, thus complicating molecular diagnosis in females with normal α-Gal-A activity. We tested 67 female probands with clinical manifestations that may be associated with FD and 110 control males with normal α-Gal-A activity. Five different combinations of GLA polymorphic variants were identified in 14 of the 67 females, whereas clearcut pathogenetic alterations, p.Met51Ile and p.Met290Leu, were identified in two cases. The latter has not been reported so far, and both mutant forms were found to be responsive to the pharmacological chaperone deoxygalactonojirimycin (DGJ; migalastat hydrochloride). Analysis of the male control population, as well as male relatives of a suspected FD female proband, permitted the identification of seven different GLA gene haplotypes in strong linkage disequilibrium. The identification of haplotypes in control males provides evidence against their involvement in the development of FD phenotypic manifestations.     

Animal models in the study of Alzheimer's disease and Parkinson's disease: A historical perspective

Alzheimer's disease and Parkinson's disease are two of the most prevalent and disa-bling neurodegenerative diseases globally. Both are proteinopathic conditions and while occasionally inherited, are largely sporadic in nature. Although the advances in our understanding of the two have been significant, they are far from complete and neither diagnosis nor the current practices in treatment and rehabilitation is ad-equately helpful. Animal models have historically found application as testing beds for novel therapeutics and continue to be valuable aids in pharmacological research. This review chronicles the development of those models in the context of Alzheimer's and Parkinson's disease, and highlights the shifting paradigms in studying two human- specific conditions in non- human organisms.     

人血管内皮生长因子165基因真核表达载体的构建及表达

目的：构建人血管内皮生长因子165（hVEGF165)的真核表达载体，并在大鼠骨髓基质细胞中进行表达。方法：利用基因克隆技术，将原核克隆载体pSP73中的目的基因VEGF165用BamHI和XhoI双酶切后，再克隆到真核表达载体pcDNA3.1中，构建重组质粒pcDNA3.1-VEGF165。对重组质粒进行酶切分析和测序鉴定。通过脂质体介导，用重组质粒转染SD大鼠骨髓基质细胞，然后以G418筛选阳性克隆。用免疫细胞化学鉴定。结果：经酶切鉴定及基因测序证实，重组体中已插入目的基因片段VEGF165，免疫细胞化学证实，重组质粒转染的骨髓基质细胞中有VEGF165基因的表达。结论：成功地构建真核表达载体pcDNA3.1-VEGF165。并在骨髓基质细胞中得到表达，为将表达VEGF基因的骨髓基质细胞作为骨组织工程的种子细胞的可能性提供了实验依据。     

微小隐孢子虫CP15基因高效真核表达载体的构建及其在Hela细胞中的表达

目的 :构建隐孢子虫CP15真核表达载体pCR3 1 15 ,观察其在Hela细胞中的表达。方法 :用BglⅡ从pMD18 T 15中酶切得到CP15基因 ,将其插入真核表达载体pCR3 1(+)的BamHⅠ位点 ,构建CP15真核表达载体pCR3 1 15 ,脂质体介导法将其转染Hela细胞 ,并用G4 18加压筛选 ,用RT PCR方法检测外源CP15基因的转录 ,用ELISA法和间接免疫荧光法检测其活性。结果 :酶切鉴定表明已成功构建了重组真核表达载体pCR3 1 15 ;外源CP15基因能在转染细胞中有效转录 ;ELISA法和间接免疫荧光法实验结果表明表达产物具有良好的生物活性。结论 :构建的pCR3 1 15真核表达载体在Hela细胞中具有良好的表达活性。     

内皮抑素基因转移对乳腺癌细胞生长影响的体内和离体研究

目的：探讨内皮抑素(ES)基因转移在乳腺癌抗血管新生中的作用。 方法: 通过建立逆转录病毒介导的ES基因转移系统，用ES病毒转染人乳腺癌细胞系MDA-MB-231。以聚合酶链反应（PCR）、MTT法和裸鼠成瘤实验分析ES的生物学特性及其功能。 结果： 脂质体转染与交互感染策略获得ES病毒生产细胞；以ES病毒转染MDA-MB-231细胞后，经PCR分析显示其内有ES基因整合并持续表达，其分泌的ES能明显抑制内皮细胞EA.hy926的增殖(P<0.05)，但对肿瘤细胞的离体生长无明显影响（P>0.05）；裸鼠皮下移植瘤模型表明，ES基因表达可明显抑制MDA-MB-231细胞的生长(P<0.01)；实验组的肿瘤微血管密度（MVD）和血管内皮生长因子(VEGF)表达低于对照组(P<0.05)。 结论: 逆转录病毒载体介导的ES基因在乳腺癌细胞中可有效表达，能明显抑制血管内皮细胞生长，并通过旁分泌方式抑制血管新生，具有显著的抗肿瘤生长的作用。     

PRL-2基因转染对肝细胞增殖及细胞周期的影响

目的：研究PRL-2基因对肝细胞增殖和细胞周期的影响。方法:采用脂质体转染的方法将重组质粒稳定转染至正常永生化肝细胞系CL1中，G418筛选阳性克隆。应用实时荧光定量聚合酶链反应、Western印迹和免疫组化分析PRL-2在阳性细胞的表达及蛋白定位，MTT法检测细胞的群体倍增时间，流式细胞仪检测细胞周期变化，Western印迹分析细胞周期素A、D1、E以及周期蛋白依赖激酶抑制因子p16、p21WAF1及p27Kip1的变化,实时荧光定量聚合酶链反应检测p21WAF1mRNA的变化。结果: 成功构建PRL-2真核表达载体pcDNA3-PRL-2。脂质体转染细胞经过G418筛选后,获得稳定表达PRL-2的细胞亚系PRL-2-CL1。经实时荧光定量PCR、Western印迹和免疫组化证实,PRL-2-CL1细胞系PRL-2基因及蛋白表达水平高于对照组，流式细胞仪检测细胞周期S期细胞比例明显增高，MTT法检测细胞群体倍增时间缩短。Western印迹及实时荧光定量聚合酶链反应显示p21WAF1在转染后较对照组明显降低，细胞周期素A、D1、E及p16、p27Kip1无明显变化。结论: 重组PRL-2真核表达载体构建正确,并在永生化肝细胞中获得稳定、高效表达。PRL-2基因具有促进细胞增殖的作用，这种作用与其降低p21WAF1蛋白含量相关。