脂质体包裹人促甲状腺激素受体胞外段基因真核表达质粒的构建

目的:构建阳离子脂质体包裹的人促甲状腺激素受体胞外段基因真核表达质粒。方法:PCR扩增穿梭质粒PHMCMVTSHR289目的基因并连接于真核表达质粒pcD NA3.1+上,重组质粒pcD NA3.1+/TSHR289采用酶切及测序法鉴定。阳离子脂质体包裹重组质粒pcD NA3.1+/TSHR289。结果:重组质粒pcD NA3.1+/TSHR289用Hind III酶切后产物经0.8%琼脂糖凝胶电泳检测显示出现512bp条带。正向测序发现AAC突变为AAT,为同义突变。反向测序发现GCG突变为GCT,亦为同义突变。阳离子脂质体与重组质粒的体积质量比例为3∶1。结论:酶切及测序鉴定重组质粒pcD NA3.1+/TSHR289构建成功。     

蛋白酶体β5亚单位基因真核表达载体的构建

目的:构建蛋白酶体亚单位β5基因真核表达质粒。方法:从人晶状体上皮细胞株SRA01/04中提取总RNA,经RT-PCR扩增获得β5亚单位的全长cDNA片段,将其克隆至真核表达载体pcDNA3.1上。结果:RT-PCR法扩增出约792bp的β5亚单位基因全部编码序列的片段。酶切鉴定和测序分析证实所插入的β5亚单位的基因序列完全正确。结论:成功构建了蛋白酶体β5亚单位基因真核表达重组质粒。     

癌钙调蛋白／鱼精蛋白截短体融合基因在原核表达载体中的构建及鉴定

目的在原核表达载体中构建癌钙调蛋白（OM）／鱼精蛋白截短体（tp）融合基因，进行基因测序鉴定。方法提取SD大鼠腹腔巨噬细胞，并进行细胞计数，经巨噬细胞特异性抗体CD68鉴定。设计引物扩增OM基因，并在其3’端引入印的编码基因，经PCR扩增获得OM／tp融合基因，通过中间载体PUC57进行目的基因克隆，将阳性克隆质粒酶切，与原核表达载体PET32a连接，转化大肠杆菌感受态细胞，筛选阳性克隆，小量提取质粒，送样经公司测序鉴定。结果琼脂糖凝胶电泳获得OM／tp目的基因，与预期的目的基因大小一致，回收目的基因与中间载体PUC57连接转化大肠杆菌感受态细胞DH5α后，菌检PCR获得约500bp的阳性克隆，与预期大小一致。抽提质粒PUC57-OM／tp，经测序鉴定后与预期的序列一致。质粒PUC57-OM／tp酶切后与载体PET32a连接转化大肠杆菌感受态细胞DH5α，菌检PCR获得约500bp的阳性克隆，与预期大小一致。抽提质粒PET32a—OM／tp经测序后与预期的序列一致。结论OM／tp融合基因的成功构建，为该融合蛋白促进视神经损伤后再生的研究奠定了基础。     

大鼠癌钙调蛋白基因原核表达载体的构建及表达

目的 在原核表达载体中构建癌钙调蛋白（OM）基因,并对构建的原核表达质粒进行表达和鉴定.方法 取SD大鼠6只,提取腹腔巨噬细胞并活化,提取总RNA,设计引物对OM基因进行RT-PCR扩增.通过中间载体pUC57进行目的 基因克隆,将阳性克隆质粒酶切,与原核表达载体pET-22b（＋）连接,转化入大肠杆菌感受态细胞DH5α,菌检PCR筛选阳性克隆后小量提取质粒,送样行核苷酸序列分析鉴定.成功构建的原核表达质粒,在大肠杆菌BL21中经异丙基-β-D-硫代半乳糖苷（IPTG）诱导重组体的表达,并用Western blot鉴定表达产物.结果 琼脂糖凝胶电泳获得目的 基因OM,大小为350 bp左右,与预期的目的 基因大小一致.构建的重组质粒pET-22b（＋）/OM菌检PCR获得长度为500 bp左右的阳性克隆,与预期大小一致.抽提质粒经核苷酸序列分析后表明,克隆的片段与OM基因序列一致.在IPTG的诱导下,重组体表达出分子量约11.7 kD的表达产物,Western blot结果证实其为目的 蛋白.结论 实验成功构建了OM基因原核表达载体及其表达,为该蛋白促进视神经损伤后再生的研究奠定了基础.     

siRNA-LEDGFp52真核表达载体的构建和鉴定

目的构建晶状体来源的上皮生长因子p52（lens epithelium deftved growth factor p52，LEDGFp52）基因RNA干扰（RNA interference，RNAi）的真核细胞表达载体。方法以LEDGVpS2为靶基因，以pCensil-1质粒为载体，设计构建重组体，根据GenBank数据库提供的LEDGFp52基因核苷酸序列，按照Tuschl设计原则，选择设计2条带发夹结构的核苷酸序列，克隆到空载体pGensil-1中，转化DH50t菌株，提取质粒，进行限制性内切酶酶切鉴定和测序分析。结果经酶切鉴定筛选出的重组体测序结果与目的序列完全一致，重组载体构建成功，重组质粒转染HeLa细胞48h，Western blotting检测到LEDGFp52蛋白表达的改变。结论利用RNAi技术可成功构建抑制LEDGFp52表达的小干扰RNA重组体。[眼科新进展2,007；27（3）：184．187]     

分泌人内皮抑素的微囊化基因工程细胞系的构建

目的 建立能稳定分泌人内皮抑素(hES)的基因工程细胞系,观察内皮抑素(ES)蛋白和hES的表达.方法 以hES重组质粒pcDNA3.0(pcDNA3-Endo)为模板,通过聚合酶链反应(PCR)扩增获得hES基因片段,且在基因前加信号肽序列,将其定向插入真核表达载体绿色荧光蛋白(pEGFP-N1)质粒中,获得重组质粒pEGFP-N1-Es;利用阳离子脂质体介导将其转染到人胚胎肾细胞(HeK-293)细胞中,G418筛选后得到阳性克隆hES/293,采用蛋白免疫印迹(Western blot)法检测转染细胞上清中ES蛋白的表达;用海澡酸钠壳聚糖(ACA)微囊包裹hES/293细胞,分别收集培养3、7、21、35 d的ACA微囊化hES/293细胞的培养上清液,Western blot法检测包裹后培养液上清中hES的表达.结果 重组质粒pEGFP-N1-ES经限制性核对内切酶HindⅢ和限制性核酸内切酶BamH Ⅰ双酶切得到4700碱基对(bp)和600 bp 2条带;PCR扩增出600 bp条带;测序结果与NCBI上序列比对软件(BLAST)比对,同源性达到100%.pEGFP-N1-ES转染HeK-293细胞,经G418筛选后获得阳性克隆,选取筛选的10株单克隆细胞培养上清液进行Western blot分析,在相对分子质量为20×103处出现蛋白条带.在ACA微囊内hES/293细胞随着培养时间的延长,细胞团逐渐长大,充满整个囊内空间.培养3、7、21、35 d时,在相对分子质量为20×103处出现蛋白条带.结论 重组pEGFP-N1-ES真核表达载体构建正确,转染HeK-293细胞后可有效的表达hES蛋白,并能分泌到细胞外;微囊化hES/293细胞产生的ES蛋白可以自由扩散出微囊膜外,并呈持续性表达.     

LEDGFp52-siRNA真核表达载体的构建和鉴定

目的: 构建 LEDGFp52 基因 RNA 干扰(RNAi)的真核细胞表达载体。方法: 以 LEDGFp52 为靶基因, 以 pGenSil-l 质粒为载体, 设计构建重组体, 根据 GenBank 数据库提供的LEDGFp52 基因核苷酸序列, 按照 Tuschl 设计原则, 选择设计两条带发夹结构的核苷酸序列, 克隆到空载体pGenSil-l 中, 转化 DH5α菌株, 提取质粒, 进行限制性内切酶酶切鉴定和测序分析。结果: 经酶切鉴定筛选出的重组体测序结果与目的序列完全一致, 重组载体构建成功,重组质粒转染 HeLa 细胞48h, Western blotting 检测到 LEDGFp52 蛋白表达的改变。结论: 利用 RNAi 技术可成功构建抑制 LEDGFp52 表达的小干扰 RNA 重组体。     

单纯疱疹病毒I型截短糖蛋白B基因序列的克隆及鉴定

目的构建单纯疱疹病毒I型（herpes simplex virusI，HSV—I）SM44株截短糖蛋白B基因序列，为研制联合基因疫苗奠定基础，用于角膜炎的防治。方法利用聚合酶链反应（polymerase chain response，PCR）技术从感染HSV—I SM44株的vero细胞中扩增出HSV-gBt编码基因，经双酶切鉴定后将目的基因定向插入真核表达质粒pcDNA，载体中，构建出重组真核表达质粒pcDNA3-gBt，并对其进行酶切分析和测序鉴定。结果对pcDNA3-gBt双酶切后，电泳可见目的基因（1．5kb）和线性质粒pcDNA3（5．4kb）两条带；测序结果表明，克隆基因插入方向正确，与基因库中登录的HSV-1 F株gB基因序列比较，同源性达99．5％。结论经证实成功地构建了重组真核表达质粒pcDNA3-gBt，为进一步研究其免疫学效应及构建病毒糖蛋白联合疫苗并最终用于单疱病毒角膜炎的预防和治疗奠定理论基础.     

hTERT启动的双自杀基因CDTK载体的构建

目的:构建hTERT启动子启动的双自杀基因载体CDTK(pc-ChCDTK)。方法:利用PCR扩增CMV增强子、hTERT启动子、yCD及TKgly4种元件,并构建过渡载体T-CMV,T-hTERT,T-yCD,T-TKgly,连至pc-DNA3.1(-)质粒载体上构建成双自杀基因载体pc-DNA3.1-CMV-hTERT-CDTK(pc-ChCDTK);对各个元件、过渡载体及最终载体进行琼脂糖凝胶电泳、酶切和测序鉴定。结果:实验所构建的最终载体的酶切产物琼脂糖凝胶电泳所得片段大小分别为6773bp和288bp,与预期片段一致;测序证实最终载体与pc-ChCDTK序列一致。结论:成功构建了含有hTERT启动子的肿瘤特异性双自杀基因载体pc-ChCDTK。     

Strabisme Alternant - Etude clinique - traitement

The author defines motor and sensory alternation: the term alternation should not be used in isolation, it should always be accompanied by the name of the parameter concerned. Sensory alternation is always found together with motor alternation but the reverse is not true.The examining criteria for a diagnosis of sensory alternation are given, sensory alternation must not be confused with alternating inhibition. Working from clinical observations of cases of motor alternating strabismus, the author selects 2 types of binocular sensory relations which allow one to differentiate between:- cases of primary alternating strabismus- cases of secondary alternating strabismusThese forms will develop in different ways; in both cases a cure is possible providing that the right treatment is prescribed and once prescribed carefully followed, etc. It is always a case of serious forms of strabismus whose developmental period is spread over several years.According to the authors, the frequency of cases of true primary strabismus is from 1–3%, the frequency of cases of secondary alternating strabismus varies according to the type of therapy practised on cases of monocular strabismus with amblyopia. These latter will become cases of alternating strabismus under the influence of certain types of therapy carried out over several years (penalization, rocking, alternated occlusion, etc...).Experimental data on kittens confirm clinical data; kittens placed in abnormal environments during the sensitive period will show modification in the distribution of cortical cells and the absence of binocular cells (either because the excitation of the two eyes was not simultaneous, or not identical: artificial strabismus, occlusion, opaque glasses). This disturbances become irreversible after a certain period of exposure (a function of age, length of exposure, etc...).It is thus necessary to bear in mind: 1) the iatrogenic risks of certain orthoptic treatments, 2) the necessity for a binocular form of treatment as soon as possible, as once a certain stage is passed, cortical plasticity diminishes and the elaboration of normal binocular relations becomes impossible.

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Effects of Adenosine Agonists on Intraocular Pressure and Aqueous Humor Dynamics in Cynomolgus Monkeys

The effects of single or multiple topical doses of the relatively selective A₁adenosine receptor agonists (R)-phenylisopropyladenosine (R-PIA) and N⁶-cyclohexyladenosine (CHA) on intraocular pressure (IOP), aqueous humor flow (AHF) and outflow facility were investigated in ocular normotensive cynomolgus monkeys. IOP and AHF were determined, under ketamine anesthesia, by Goldmann applanation tonometry and fluorophotometry, respectively. Total outflow facility was determined by anterior chamber perfusion under pentobarbital anesthesia. A single unilateral topical application of R-PIA (20–250 μg) or CHA (20–500 μg) produced ocular hypertension (maximum rise=4.9 or 3.5 mmHg) within 30 min, followed by ocular hypotension (maximum fall=2.1 or 3.6 mmHg) from 2–6 hr. The relatively selective adenosine A₂antagonist 3,7-dimethyl-1-propargylxanthine (DMPX, 320 μg) inhibited the early hypertension, without influencing the hypotension. Neither 100 μg R-PIA nor 500 μg CHA clearly altered AHF. Total outflow facility was increased by 71% 3 hr after 100 μg R-PIA. In conclusion, the early ocular hypertension produced by topical adenosine agonists in cynomolgus monkeys is associated with the activation of adenosine A₂receptors, while the subsequent hypotension appears to be mediated by adenosine A₁receptors and results primarily from increased outflow facility.