慢病毒载体VP22-EGFP的构建及其对大鼠神经干细胞转染及表达的研究

目的 研究VP22穿梭蛋白对神经干细胞(neural stem cell,NSC)转染效率的影响.方法 通过酶切质粒pUL49ep获得VP22片段后克隆到含有增强型绿色荧光蛋白(enhanced greenfluorescent protein,EGFP)的慢病毒(Lentivirus)pHIV-EGFP质粒载体上,并采用PCR及基因测序技术对VP22基因进行鉴定.病毒包装采用三质粒系统共转染293T细胞后感染经体外原代培养、扩增的SD大鼠胎脑NSC,荧光显微镜下观察EGFP在NSC中的表达,流式细胞仪检测EGFP的表达,PCR检测基因组中VP22-EGFP基因的表达,MTT法测定转染前后单纯NSC、含有EGFP及含有VP22-EGFP的NSC的增殖活性.结果 PCR和DNA测序证实,外源基因VP22成功插入到质粒载体pHIV-EGFP的骨架结构中的启动子CMV和EGFP序列之间;荧光显微镜下均见有绿色荧光蛋白的表达;流式细胞仪测定结果:Lentivirus-EGFP及Lentivirus-VP22-EGFP转染的NSC的EGFP的表达率分别为(23.1±1.8)%及(34.9±2.9)%(P<0.01);PCR结果显示VP22整合入NSC的基因组DNA中;MTT结果显示转染前后单纯NSC、含有EGFP及含有VP22-EGFP的NSC的细胞生长曲线无明显改变.结论 VP22能够提高慢病毒对NSC的转染效率,且不影响其生长特性.表明VP22作为一种短链蛋白可以与编码的治疗基因形成融合蛋白增强基因治疗的疗效,在基因修饰的工程化干细胞中具有良好应用前景.     

平滑肌细胞纯化载体构建及在小鼠胚胎干细胞中的表达

背景：胚胎干细胞是平滑肌细胞重要的来源之一,但是胚胎干细胞分化细胞的异质性导致难以获得较纯的平滑肌细胞。 目的：为进一步纯化胚胎干细胞来源的平滑肌细胞,拟在体外构建平滑肌特异性SM22α启动子驱动的嘌呤霉素抗性(puromycin acetyltransferase,pac)基因与增强型绿色荧光蛋白(enhanced green fluorescence protein,EGFP)基因双表达载体,即pSM22-PAC-IRES2-EGFP载体,并在胚胎干细胞中检测其有效性及特异性。 设计、时间及地点：基因水平细胞观察实验,于2007-05/2008-09在解放军沈阳军区总医院全军心血管病研究所完成。 材料：小鼠胚胎干细胞系R1购自美国ATCC公司,编号SCRC-1011TM。pSM22α-EGFP载体由本实验室构建;pIRES2-EGFP载体、pSM2C载体、pSuper.basic载体购自Invitrogen公司。 方法：用聚合酶链反应方法从pSM22α-EGFP中扩增SM22α启动子,然后用该启动子替换pIRES2-EGFP载体中的CMV启动子,构建pSM22-IRES2-EGFP。再从pSM2C中用HindⅢ/ClaⅠ酶切获得pac基因,将pac基因片段亚克隆到pSuper.basic中,构建pSuper-PAC。最后BgⅢ/AccⅠ双酶切pSuper-PAC获得pac基因片段,将其插入到pSM22α-IRES2-EGFP,构建成pSM22α-PAC-IRES2-EGFP。将pSM22α-PAC-IRES2-EGFP用脂质体法转染胚胎干细胞,G418筛选阳性克隆。诱导胚胎干细胞阳性克隆分化,RT-PCR扩增pac基因鉴定阳性克隆。对分化细胞行平滑肌细胞标志物SM α-actin免疫荧光染色。 主要观察指标：①pSM22α-PAC-IRES2-EGFP测序结果。②pac基因扩增。③荧光显微镜下同时观察分化细胞EGFP的表达及SM α-actin染色情况。 结果：HindⅢ/ClaⅠ双酶切得到261 bp,664 bp,5 000 bp 3个片段,与预期结果一致,测序结果证实pSM22α- PAC-IRES2-EGFP构建成功。Pac基因扩增证实有4株胚胎干细胞克隆转染成功。转染成功的胚胎干细胞被诱导分化后,部分细胞表达EGFP,且这些细胞SM α-actin染色呈阳性。 结论：实验成功构建了平滑肌细胞筛选载体pSM22α-PAC-IRES2-EGFP。成功转染这一载体的胚胎干细胞表达pac基因及EGFP基因,且EGFP表达具有平滑肌特异性。     

腺病毒载体Ad-BDNF-EGFP构建及其在神经干细胞中的表达

摘要 目的 研究腺病毒载体Ad-BDNF -EGFP的构建及在神经干细胞（NSCs）中的表达。方法 通过RT-PCR从在大鼠的海马中获得BDNF基因,通过基因克隆以及HEK293包装,获得了含增强绿色荧光蛋白(EGFP)基因的重组腺病毒表达载体pAd-BDNF-EGFP,将其感染原代培养的神经干细胞,观察EGFP及BDNF两种基因的表达,镜下测定转染率,并检测RT-PCR产物,证实BDNF的存在。转染后的神经干细胞经G418筛选,抗性细胞传代扩增后获得成功转染BDNF基因的NSCs克隆。结果 荧光显微镜下可见感染后的NSCs表达EGFP而发出绿色荧光;通过RT-PCR证明感染后的NSCs具有表达BDNF的能力;用ELISA鉴定细胞上清中分泌的BDNF, 72h的含量达到最高值,为12.78ng/ml;证明通过构建病毒的感染可以使神经干细胞获得分泌BDNF的能力,且EGFP基因可作为神经干细胞移植研究中良好的示踪剂。结论 腺病毒病毒介导EGFP基因及BDNF基因在大鼠胚胎神经干细胞中成功表达,为应用以神经干细胞直接作为基因靶细胞,介导基因治疗中枢神经系统疾病莫定了基础。     

脂质体介导EGFP基因转染神经干细胞实验研究

目的用增强绿色荧光蛋白基因(EGFP)的表达载体pIRES2-EGFP转染神经干细胞(neural stem cells,NSCs),检测该真核表达载体对NSCs的转染效率及EGFP的表达状态,为用NSCs为载体基因治疗中枢神经系统疾病提供实验依据。方法体外扩增、酶切鉴定pIRES2-EGFP质粒;悬浮培养NSCs;阳离子脂质体lipofectam ineTM2000介导增强绿色荧光蛋白质粒转染NSCs;庆大霉素的氨基糖苷(G418)筛选重组子;荧光显微镜观察转染效率及表达情况;免疫细胞化学鉴定重组子。结果转染后6 h,荧光蛋白偶见表达,24 h表达量明显增加,48 h达到最高峰,1个月后抗性细胞有克隆球形成。结论脂质体介导报告基因—pIRES2-EGFP转染NSCs的方法简单、效率高、易成功,是一较为理想的基因转染方法。     

GDNF基因真核表达载体的构建及其在脐血CD34+干细胞的表达

目的构建人胶质细胞源性神经营养因子(GDNF)基因的新型真核表达载体,为进行GDNF基因修饰的脐血干细胞移植治疗脑梗死奠定基础。方法将GDNF基因克隆到增强型绿色荧光蛋白(EGFP)真核表达载体中,进行酶切鉴定及DNA测序分析,并将携带有GDNF基因的该真核表达载体,通过脂质体介导,转染人脐血CD34 细胞,荧光显微镜、ELISA检测转基因脐血干细胞GDNF的表达与释放。结果经双酶切鉴定和测序证实已将GDNF基因DNA片段正确插入到真核表达载体中,并能在脐血CD34 干细胞中表达、分泌有活性的GDNF。结论成功构建pEGFP/GDNF基因新型真核表达载体。     

腺相关病毒-2介导增强型绿色荧光蛋白基因转染神经干细胞的实验研究

目的探讨血清2型腺相关病毒(rAAV2)介导增强型绿色荧光蛋白(EGFP)基因转染神经干细胞后绿色荧光蛋白(GFP)的表达规律及转染对神经干细胞生物学特性的影响.方法应用荧光显微镜观察转染神经干细胞的绿色荧光蛋白表达情况.传代后第10天,比较转染组和对照组子代细胞形成的神经球数;分化后第14天行免疫组织化学检查,比较转染组和对照组神经元和胶质细胞的比例.结果转染后第3天,可观察到神经球发出绿色荧光,强度逐渐增强,并在第9天达到稳定状态(90%的神经球发出强弱不等的荧光).传代后第10天,转染组子代细胞每井形成的神经球数为(17.83±1.35)个,与对照组比较无显著性差别(P＞0.05).分化后第14天,转染组神经元、胶质细胞的胞体与纤细突起均可见绿色荧光,转染组神经元与胶质细胞比例分别为35.3%±3.1%和55.4%±7.3%,与对照组比较无显著性差别(P＞0.05).结论rAAV2-EGFP可以稳定、高效转染神经干细胞,且不影响神经干细胞的生物学特性,是神经干细胞理想的基因标记方法.     

大鼠κ—HA—pIRES2-EGFP的构建及表达

目的 构建带HA标签的大鼠κ型阿片受体的pIRES2-EGFP表达质粒(κ-HA-pIRES2-EGFP),并实现其在HEK293细胞中的表达.方法 提取大鼠脑组织总RNA,通过巢式RT-PCR扩增κ型阿片受体全长cDNA,将其克隆至pMD20-T载体中并进行测序鉴定,通过PCR引入HA标签,经酶切连接克隆入pIRES2-EGFP中,将获得的κ-HA-pIRES2-EGFP转染入HEK293细胞中,应用荧光显微镜观察EGFP表达情况,并采用细胞免疫荧光和蛋白印迹检测κ-HA表达.结果 测序及酶切结果表明成功获得带HA标签的κ基因,并构建入pIRES2-EGFP表达质粒中,在荧光显微镜下可观察到转染细胞内有绿色荧光,应用免疫荧光和蛋白印迹法可观察到目的 基因表达.结论 利用巢式RT-PCR等技术成功构建了κ-HA-pIRES2-EGFP表达质粒,并在HEK293细胞中实现了高效表达.     

重组质粒pcDNA3.1 his-hTH与pEGFP-C2共转染骨髓基质细胞源神经干细胞的实验研究

目的 构建携带人酪氨酸羟化酶(tyrosine hydroxylase，TH)目的基因片段的真核表达重组质粒-pcDNA3．1his-hTH，与pEGFP-C2共转染恒河猴骨髓源神经干细胞，观察hTH基因在骨髓源神经干细胞中的表达情况。方法 应用基因重组技术，将pWAV2-TH中TH基因亚克隆到pcDNA3．1his真核表达载体，以酶切和测序方法鉴定重组质粒pcDNA3．1his-hTH的正确性；将pcDNA3．1his-hTH和pEGFP-C2经电击穿孔法共转染恒河猴骨髓源神经干细胞，24h后观察EGFP瞬时表达情况，10d后进行hTH基因RT-PCR，以及hTH和6His单克隆抗体的免疫组化检测。结果 (1)酶切和测序结果均证实pcDNA3．1his-hTH的正确性；(2)细胞转染24h后，荧光显微镜下可观察到EGFP的表达，80％以上细胞发出绿色荧光；10d后RT-PCR检测到细胞内有hTH基因的表达，免疫组化结果显示细胞有hTH和6His抗原表达。结论 成功构建的pcDNA3．1．his-hTH和pEGFP-C2能够共转染恒河猴骨髓源神经干细胞，hTH、EGFP和6His基因在细胞内有效表达。该系统可以作为体外检测转染率、细胞移植治疗帕金森病活体跟踪移植细胞的技术平台。     

真核表达载体pcDNA3.1-3a的构建及其在神经母细胞瘤SK-N-SH细胞中的表达

目的在pcDNA3.1真核表达载体中构建表达融合myc/his标签的甲基化酶表达载体pcDNA3.1-3a,并在神经母细胞瘤细胞株SK-N-SH细胞中进行验证,以期获得可表达甲基化酶催化结构域的融合基因。方法以甲基化酶的pcDNA4.0-GBD-3a-myc/his质粒为模板,通过PCR的方法扩增获得融合有myc/his标签序列的目的区段--甲基化酶催化结构域3a,将其克隆入真核表达载体pcDNA3.1;以EcoRⅠ、EcoRⅤ双酶切和PCR方法对构建的表达载体进行鉴定,并通过测序证明载体构建正确,同时检测甲基化酶3a在SK-N-SH细胞中的表达。结果通过PCR方法获得了含有甲基化酶催化结构域的真核表达载体pcDNA3.1-3a,并通过免疫印记方法验证了在神经母细胞瘤细胞株SK-N-SH细胞样品中用抗his标签抗体可以特异性识别目的蛋白。结论正确构建了甲基化酶表达载体pcDNA3.1-3a,其可在神经母细胞瘤细胞株SK-N-SH细胞中表达。     

慢病毒介导人神经营养素3基因在许旺细胞中的表达

背景：神经营养素3是目前发现的在脊髓损伤修复中作用最强的神经营养因子,它能有效促进再生轴突穿越胶质瘢痕组织,从而修复脊髓损伤。 目的：构建含有人神经营养素3基因的重组慢病毒载体(LV-hNT3),观察转染后人神经营养素3基因在许旺细胞中的表达。 设计、时间及地点：观察性实验,于2007-06/2008-03在长海医院中心实验室完成。 材料：取新生3 d的SD乳鼠双侧坐骨神经,用于许旺细胞的培养及鉴定;慢病毒三质粒系统pGC-E1-EGFP,pHelper 1.0和pHelper 2.0为上海吉凯基因化学技术有限公司产品。 方法：通过双限制性内切酶消化和连接的方法构建pGC-E1-hNT3-EGFP质粒,接着该质粒转化感受态的大肠杆菌E.coli DH5α,通过PCR及基因测序鉴定阳性克隆,再经Lipofectamine 2000将pGC-E1-hNT3-EGFP,pHelper 1.0和pHelper 2.0三质粒系统共转染293 T细胞包装病毒,按照病毒感染复数=1,4,8,10,12加入预先混好重组病毒完全培养液2 mL,通过增强型绿色荧光蛋白的表达测定收集的病毒滴度。将慢病毒转染许旺细胞,以未经转染的许旺细胞和经空载的慢病毒转染的许旺细胞为对照组。 主要观察指标：慢病毒转染许旺细胞后,检测转染效率,通过实时荧光聚合酶链反应和蛋白免疫印迹检测人神经营养素3在许旺细胞中的表达。 结果：实验中重组质粒的外源基因序列与GeneBank中的人神经营养素3阅读框架序列完全一致;浓缩后病毒滴度为5×107 TU/L,LV-hNT3感染许旺细胞后,许旺细胞发出明亮的绿色荧光,当感染复数=10时转染效率最高达85%,实时荧光PCR检测表明人神经营养素3 mRNA在人神经营养素3-许旺细胞中高效表达,而对照组中未表达,蛋白免疫印迹证实人神经营养素3在许旺细胞中表达。 结论：实验构建的含有人神经营养素3基因的慢病毒载体能感染许旺细胞并且高效表达人神经营养素3。     

MicroRNA expression in mouse oligodendrocytes and regulation of proteolipid protein gene expression

Overexpression of the major myelin proteolipid protein (PLP) is detrimental to brain development and function and is the most common cause of Pelizaeus-Merzbacher disease. microRNA (miRNA), small, noncoding RNAs, have been shown to play critical roles in oligodendrocyte lineage. In this study, we sought to investigate whether miRNAs control PLP abundance. To identify candidate miRNAs involved in this regulation, we have examined differentiation-induced changes in the expression of miRNAs in the oligodendroglial cell line Oli-neu and in enhanced green fluorescent protein positive oligodendrocytes ex vivo. We have identified 145 miRNAs that are expressed in oligodendrocyte cell lineage progression. Dicer1 expression decreases in differentiated oligodendrocytes, and knock down of Dicer1 results in changes in miRNAs similar to those associated with differentiation. To identify miRNAs that control the PLP expression, we have selected miRNAs whose expression is lower in differentiated vs. undifferentiated Oli-neu cells and that have one or more binding site(s) in the PLP 3'-untranslated region (3'UTR). The PLP 3'UTR fused to the luciferase gene reduces the activity of the reporter, suggesting that it negatively regulates message stability or translation. Such suppression is relieved by knock down of miR-20a. Overexpression of miR-20a decreases expression of the endogenous PLP in primary oligodendrocytes and of the reporter gene. Deletion or mutation of the putative binding site for miR-20a in the PLP 3'UTR abrogated such effects. Our data indicate that miRNA expression is regulated by Dicer1 levels in differentiated oligodendrocytes and that miR-20a, a component of the cluster that controls oligodendrocyte cell number, regulates PLP gene expression through its 3'UTR.     

脊髓小脑性共济失调3型真核表达载体的构建及在PC12中的表达

目的构建以增强型绿色荧光蛋白(EGFP)作为报告基因、带有脊髓小脑性共济失调3型(SCA3)基因的真核细胞表达载体,并转染PC12细胞,观察外源基因的表达情况。方法将含有正常和突变SCA3基因的pcD-NA3．0-ataxin3Q28(基因内CAG重复28次)、pcDNA3．0-ataxin3Q84(基因内CAG重复84次)双酶切,水解片段插入pEGFP-C1,测序鉴定后脂质体转染法转染PC12细胞并优化转染条件,荧光显微镜观察EGFP的表达,Western印迹检测目的蛋白ataxin3的表达。结果真核表达载体pEGFPC1-ataxin3Q28、pEGFPC1-ataxin3Q84得以成功构建,EGFP及目的基因产物ataxin3Q28、ataxin3Q84顺利地在PC12细胞中表达,由此建立了SCA3的真核细胞表达模型。结论构建SCA3的真核表达载体并建立其细胞模型,对研究SCA3的发病机制有重要意义。     

靶向人端粒酶逆转录酶小片段干涉RNA表达载体的构建和表达

目的构建靶向人端粒酶逆转录酶（hTERT）的小片段RNA（siRNA）表达载体，为研究RNA干涉（RNAi）在哺乳动物细胞内抑制靶基因表达奠定基础。方法根据siRNA靶点设计的原则运用相关软件设计靶序列并合成其表达框，连接人质粒载体pRNAT-H1．1／Neo，转染293T细胞，荧光定量RT．PCR和Western blot法检测转染后293T细胞中hTERT表达。结果重组质粒经测序鉴定证明hTERTsiRNA转录模板完整、正确插入到pRNAT．H1．1／Neo质粒中，并筛选出一个抑制hTERT表达效率最高的序列，其hTERT表达仅达22．90％。结论利用基因重组和荧光定量PCR等技术能成功构建并筛选出一个抑制效率最好的靶向hTERT的siRNA表达载体，为以hTERT为靶点的肿瘤基因治疗的后续研究奠定基础。     

人脑胶质瘤中Fas及FasL的表达

目的　探讨Fas及FasL基因在人脑胶质瘤中的表达 ,与肿瘤的病理类型及恶性程度的关系。方法　应用Fas及FasL多抗和Ki 6 7单抗免疫组化染色检测 6例正常脑组织及 6 4例人脑胶质瘤。结果　Fas和FasL在胶质瘤中的总表达率分别为 83%和 75 % ,并随WHO分级升高而升高 ,与Ki 6 7LI呈正相关。Fas、FasL共同阳性率 (共表达率 )为 71% ,与肿瘤级别呈正相关。结论　脑胶质瘤中Fas及FasL表达与肿瘤病理类型及恶性程度密切相关     

Interleukin-6 expression and regulation in astrocytes

The physiological function of interleukin-6 (IL-6) within the central nervous system (CNS) is complex; IL-6 exerts neurotrophic and neuroprotective effects, and yet can also function as a mediator of inflammation, demyelination, and astrogliosis, depending on the cellular context. In the normal brain, IL-6 levels remain low. However, elevated expression occurs in injury, infection, stroke, and inflammation. Given the diverse biological functions of IL-6 and its expression in numerous CNS conditions, it is critical to understand its regulation in the brain in order to control its expression and ultimately its effects. Accumulating data demonstrate that the predominant CNS source of IL-6 is the activated astrocyte. Furthermore, a wide range of factors have been demonstrated to be involved in IL-6 regulation by astrocytes. In this review, we summarize information concerning IL-6 regulation in astrocytes, focusing on the role of proinflammatory factors, neurotransmitters, and second messengers.     

Prosodic comprehension and expression in schizophrenia.

Schizophrenics, manics, depressives and normal subjects (15 in each group) were tested for their ability to understand and express the prosodic quality of speech. Sentences in which one word was stressed (stress prosody comprehension) or in which a particular emotion was conveyed (emotional prosody comprehension) were taped and played to subjects to test their comprehension. Subjects were then asked to read out a list of sentences either stressing a nominated word (stress prosody expression) or conveying a nominated emotion (emotional prosody expression), and their efforts were rated by a panel of normal raters. The main results were 1) that schizophrenics were significantly inferior to the normal group, but equivalent to manics and depressives on emotional prosody comprehension; 2) schizophrenics were significantly inferior to all other groups on emotional prosody expression; and 3) all four groups were equivalently proficient on stress prosody comprehension and expression. The results are interpreted as lending support for the idea that there is an underlying right hemisphere dysfunction in schizophrenia.     

Astrocyte-targeted expression of interleukin-3 and interferon-alpha causes region-specific changes in metallothionein expression in the brain

Transgenic mice expressing IL-3 and IFN-alpha under the regulatory control of the GFAP gene promoter (GFAP-IL3 and GFAP-IFNalpha mice) exhibit a cytokine-specific, late-onset chronic-progressive neurological disorder which resemble many of the features of human diseases such as multiple sclerosis, Aicardi-Goutières syndrome, and some viral encephalopathies including HIV leukoencephalopathy. In this report we show that the metallothionein-I+II (MT-I+II) isoforms were upregulated in the brain of both GFAP-IL3 and GFAP-IFNalpha mice in accordance with the site and amount of expression of the cytokines. In the GFAP-IL3 mice, in situ hybridization analysis for MT-I RNA and radioimmunoassay results for MT-I+II protein revealed that a significant upregulation was observed in the cerebellum and medulla plus pons at the two ages studied, 1-3 and 6-10 months. Increased MT-I RNA levels occurred in the Purkinje and granular layers of the cerebellum, as well as in its white matter tracts. In contrast to the cerebellum and brain stem, MT-I+II were downregulated by IL-3 in the hippocampus and the remaining brain in the older mice. In situ hybridization for MT-III RNA revealed a modest increase in the cerebellum, which was confirmed by immunohistochemistry. MT-III immunoreactivity was present in cells that were mainly round or amoeboid monocytes/macrophages and in astrocytes. MT-I+II induction was more generalized in the GFAP-IFNalpha (GIFN12 and GIFN39 lines) mice, with significant increases in the cerebellum, thalamus, hippocampus, and cortex. In the high expressor line GIFN39, MT-III RNA levels were significantly increased in the cerebellum (Purkinje, granular, and molecular layers), thalamus, and hippocampus (CA2/CA3 and especially lacunosum molecular layers). Reactive astrocytes, activated rod-like microglia, and macrophages, but not the perivenular infiltrating cells, were identified as the cellular sources of the MT-I+II and MT-III proteins. The pattern of expression of the different MT isoforms in these transgenic mice differed substantially, demonstrating unique effects associated with the expression of each cytokine. The results indicate that the MT expression in the CNS is significantly affected by the cytokine-induced inflammatory response and support a major role of these proteins during CNS injury.     

Gene polymorphism and gene expression in schizophrenia

The author reviews relevant data on the neuropathology and molecular genetics of schizophrenia. Anatomical alterations are localized mainly in the hippocampus, dorsal thalamus and dorsolateral prefrontal cortex, and involve the morphology and molecular structure of the neurons and synapses. Several susceptibility genes [including COMT, dysbindin, neuregulin, DISCI, RGS4, GRM3, G72, PPP3CC, CHRNA7, PRODH2, Aktl, 5qGABA(A)] having physiological function in the brain have been identified and this supports the view of schizophrenia as a disorder of cerebral synaptic function. NMDA receptor-mediated glutamate transmission may be particularly involved, but disturbances of dopamine and GABA signalling seem to be linked as well. Based on recent data, an agreement is emerging between the roles of the genes on the molecular and synaptic levels and the understanding of the disorder at the neural systems level.     

真核表达载体pcDNA3-CTLA4Ig的构建及体内表达

背景： CTLA-4Ig的制备过程比较复杂，单克隆抗体的价格比较昂贵，而且应用CTLA-4Ig单克隆抗体注射本身存在着血浆药物浓度不稳定的问题。 目的：探讨Ctla4Ig表达质粒构建的可行性。 设计、时间及地点：基因水平观察实验，于2005/2006在中国医科大学中心实验室完成。 材料：雄性Wistar大鼠10只，近交系昆明小鼠10只。 方法：提取Wistar大鼠总RNA，反转录成cDNA第1链，PCR扩增CTLA4基因，将其和真核表达载体pcDNA3酶切、连接。转化感受态菌DH5α，培养后挑取阳性克隆、提取质粒，酶切鉴定重组子、测序，最后转染近交系昆明小鼠，应用Westen Blot法检测血清CTLA4Ig水平。 主要观察指标：构建的Ctla4Ig表达质粒基因序是否正确以及能否在小鼠体内进行蛋白表达。 结果：对含有pcDNA3-CTLA4质粒的LB菌液进行鉴定测序，目的基因片断大小为288 bp，与Genebank上公布的CTLA4序列完全一致，质粒构建正确。利用阳性脂质体载体包被pcDNA3-CTLA4Ig转染小鼠，有3只在第7天血清检测CTLA4Ig阳性，显示pcDNA3-CTLA4Ig能够在鼠肌细胞内表达。 结论：利用基因合成和重组技术成功构建了真核表达载体pcDNA3-CTLA4Ig，利用脂质体法成功的将pcDNA3-CTLA4Ig转染到鼠肌细胞内并进行表达。     

Induction and expression of salt appetite: effects on Fos expression in nucleus accumbens

Sodium depletion is a strong natural motivator that creates a pronounced sodium appetite and has been shown to activate neural regions associated with fluid and sodium balance. However, it is not known whether sodium appetite affects the mesolimbic circuitry associated with reward motivation. The present studies examined expression of the immediate early gene Fos in the nucleus accumbens (NAc) as a marker of neuronal activation following the induction and expression of furosemide-induced sodium appetite. During sodium appetite expression, sham-drinking and normal drinking were used to dissociate effects of NaCl taste stimulation from the repletion that follows absorption of sodium. These studies revealed that the combination of NaCl taste stimulation and persistent sodium depletion experienced by sham-drinking animals dramatically activates the NAc, while neither induction nor expression of sodium appetite alone is sufficient to increase Fos expression in this region. Results are discussed in terms of current theories of reward motivation.