大鼠Toll样受体2基因siRNA重组腺病毒载体的构建及鉴定

目的:构建特异性抑制大鼠TLR2基因的重组腺病毒载体,并在大鼠嗜铬细胞瘤PC12细胞中进行功能鉴定。方法:体外合成3对大鼠siTLR2的双链DNA序列,经退火定向克隆到穿梭质粒pSES-HUS中获得pSES-HUS-siTLR2质粒,Pme I线性化后在BJ5183细菌中与pAdEasy-1骨架质粒进行同源重组获得pAd-siTLR2质粒,脂质体转染HEK293细胞,包装获得Ad-siTLR2腺病毒颗粒,继而感染PC12细胞,通过Real-time PCR和Western blot方法检测3对siRNA对TLR2基因的抑制效率。结果:PCR凝胶电泳和测序结果均证实目的基因正确克隆到腺病毒载体中;Real-time PCR和Western blot结果显示:携带siTLR2的病毒颗粒能够在mRNA和蛋白水平有效抑制PC12细胞中TLR2的表达。结论:成功构建了Ad-siTLR2重组腺病毒载体,并在HEK293细胞中包装成重组腺病毒,转染PC12细胞后能有效抑制TLR2基因的表达,为进一步研究TLR2在不同疾病中的免疫调节机制奠定重要基础。     

大鼠GLUT1真核表达载体的构建及其在293细胞中的表达

目的：构建大鼠葡萄糖转运体1（GLUT1）的真核表达载体。 方法： 以RT-PCR方法从大鼠脑组织中获取GLUT1全长cDNA片段，将其克隆至真核表达质粒pcDNA3.1(+)中，构建重组真核表达质粒pcDNA3.1(+)-Glut1，随后用lipofectamineTM 2000介导转染HEK293细胞，以RT-PCR法检测重组质粒在mRNA水平的表达，以免疫组化的方法检测重组质粒在蛋白水平的表达。 结果： 以构建的重组真核表达质粒pcDNA3.1(+)-Glut1转染293细胞后，在基因及蛋白表达水平均检测到了葡萄糖转运体1的表达。 结论： 成功构建了携带大鼠葡萄糖转运体1的真核表达载体pcDNA3.1(+)-Glut1，且证实其可在293细胞中成功表达目的基因，为进一步研究外源性GLUT1表达对缺血缺氧脑细胞的保护作用奠定了基础。     

绿色荧光蛋白标记的人血管内皮生长因子165基因重组腺病毒载体的构建与鉴定

目的构建与鉴定携带绿色荧光蛋白标记的人血管内皮生长因子165(hVEGF165)基因的重组腺病毒载体Ad5-hVEGF165-GFP。方法将hVEGF165基因进行扩增,并同源重组入表达质粒pAV-MCMV-HA-P2A-GFP,转化入DH5a感受态细胞,得到的转化子经菌落聚合酶链反应(PCR)鉴定和测序鉴定后,通过Ad Max腺病毒包装系统转染HEK293细胞,进行病毒的包装和扩增。包装后镜下观察HEK293细胞,测定腺病毒滴度,Western blot检测重组腺病毒的表达。结果成功构建了重组腺病毒载体Ad5-hVEGF165-GFP,重组腺病毒转染的HEK293细胞出现了明显的细胞病变效应;获得重组腺病毒的滴度为1.106×10~8 IFU/mL;Western blot检测重组腺病毒表达在23 000左右。结论成功获得重组腺病毒载体Ad5-hVEGF165-GFP,为进一步研究基因治疗脓毒症/感染性休克奠定了实验基础。     

Ubc9基因重组腺病毒的构建及其在HeLa细胞中的表达

目的构建携带Ubc9基因的重组腺病毒质粒pAdEasy-1/Ubc9,制备含Ubc9基因的重组腺病毒Ad-Ubc9,并使Ubc9在HeLa细胞中高效表达。方法 PCR法扩增目的Ubc9基因;用pemI酶切将穿梭质粒pAdTrack-CMV-Ubc9线性化;将线性化的穿梭质粒pAdTrack-CMV-Ubc9与腺病毒骨架质粒pAdeasy-1在感受态BJ5183菌内进行同源重组,筛选阳性重组子pAdEasy-1/Ubc9;再用PacI酶切pAdEasy-1/Ubc9使之线性化,线性化的重组腺病毒质粒经脂质体转染HEK293细胞,进行重组腺病毒的包装和扩增。收集重组腺病毒,感染HeLa细胞,用Westernblot方法检测Ubc9在HeLa细胞中的表达。结果通过PacI酶切证实携带Ubc9基因的腺病毒载体构建成功,包装出携带Ubc9基因的腺病毒能有效感染HeLa细胞。结论利用细菌内同源重组方法成功地构建了携带Ubc9的重组腺病毒载体,并能在HeLa细胞中高效表达。     

携带EphrinA1-caspase-3基因的重组腺病毒载体的构建和包装

目的:构建携带ephrinA1-caspase-3基因的重组腺病毒载体,通过包装、扩增、鉴定,为后续开展目的基因靶向治疗乳腺癌研究奠定基础。方法:以pMD18T-Casp3、pMD18T-EphrinA1为模板扩增caspase-3、EphrinA1基因,以pET28a为载体,通过双酶切、连接、转化、测序鉴定,构建pET28a-EphrinA1-Casp3。然后以质粒pEC3.1(+)为载体,获得pEC3.1-EphrinA1-Casp3。采用LR体外同源重组,构建pAd-EphrinA1-Casp3,以HEK293包装和放大培养。结果:成功将PCR扩增的EphrinA1胞外端基因和人活性型caspase-3基因定向克隆入质粒pET28a,并将EphrinA1-caspase-3融合基因转移到穿梭质粒pEC3.1(+),获得pEC3.1-EphrinA1-Casp3载体;经过体外同源重组,成功构建携带EphrinA1-caspase-3基因的重组腺病毒载体pAd-EphrinA1-Casp3;并在HEK 293细胞中完成包装和扩增;获得重组腺病毒rAd-EphrinA1-Casp3。结论:利用GatewayTM技术成功构建重组腺病毒载体pAd-EphrinA1-Casp3,经HEK293细胞包装,获得重组腺病毒rAdEphrinA1-Casp3。     

重组腺病毒HIV-1 vpr基因的构建和表达

目的 构建携带HIV-1 vpr基因的重组腺病毒,使CD4 T淋巴细胞C8166内源性的高表达Vpr蛋白.方法 利用AdEasy-1系统, 通过将含有目的 基因片段的穿梭载体pAdTrack-CMV-vpr和骨架质粒pAdEasy-1在BJ5183细菌内同源重组的方法构建重组腺病毒质粒Ad-vpr, 用脂质体法将重组质粒转染至HEK293A细胞包装, 获得重组腺病毒Ad-vpr, 荧光显微镜观察Ad-vpr感染C8166细胞GFP的表达 , Western blotting鉴定Vpr在C8166细胞内的特异性表达,流式细胞术检测Ad-vpr感染 C8166细胞的效率.结果 成功构建携带HIV-1 vpr基因的重组腺病毒 ,Western blotting结果表明重组腺病毒Ad-vpr感染的C8166细胞内源性的高表达Vpr 蛋白,流式细胞术检测结果表明Ad-vpr感染C8166细胞效率高(44.07±3.62)%.结论 成功构建出携带HIV-1 vpr基因的重组腺病毒,使C8166细胞内源性的高表达Vpr蛋白.     

HAX1和EGFP共表达重组腺病毒载体的构建、鉴定

目的 构建和鉴定HAX1和EGFP双基因共表达重组腺病毒载体.方法 采用DNA重组技术,将目的 基因HAX1克隆至含有报告基因EGFP的穿梭质粒pAdTrack-CMV中,并转化于大肠埃希菌DH5α;筛选出重组质粒pAdTrack-CMV- HAX1,并在BJ5183细菌中与pAdEasy-1质粒进行同源重组,产生重组腺病毒载体;用lipofectamine将其转染HEK293细胞,包装携带全长HAX1的重组复制缺陷型腺病毒pAd-HAX1-EGFP,酶切和序列测定鉴定;用制备好的Ad-HAX1-EGFP感染HEK293细胞,流式细胞术检测其感染效率,RT-PCR、Western 印迹鉴定外源基因HAX1的表达.BrdU检测感染了Ad-HAX1-EGFP的HEK293细胞增殖情况.结果 pAdTrack-CMV-HAX1重组质粒构建成功.pAdTrack-CMV-HAX1 质粒与pAdEasy-1质粒同源重组后与预期结果相符.构建好的Ad-HAX1-EGFP能有效感染HEK293细胞;外源基因能在239细胞中有效表达.HAX1高表达的HEK293细胞其增殖率得以提高.结论 成功构建了表达HAX1和EGFP共表达的重组腺病毒载体,HAX1能够促进结肠癌细胞HEK293细胞的增殖.     

过表达大鼠TIPE2基因重组腺病毒载体的构建

背景：TIPE2抗炎蛋白,通过对T细胞受体(TCR)和T细胞TOLL样受体信号途径实行负向调节,从而对适应性免疫和固有免疫起到负性调控作用,有效地维持机体内环境的稳定。目的：使用人工合成腺病毒载体构建能过表达大鼠TIPE2基因的重组腺病毒。　方法：利用RT-PCR的方法,从大鼠淋巴细胞中扩增出大鼠TIPE2基因,克隆到穿梭质粒pShuttle-clontech的表达框中,然后将包含TIPE2的完整表达框,进一步亚克隆到黑猩猩来源的腺病毒包装载体AdC68,转染HEK 293A细胞,包装出重组腺病毒。并以其感染HEK293A细胞,采用western blotting方法检测TIPE2基因的表达水平。　结果与结论：PCR扩增、酶切鉴定和测序结果表明,所获取的cDNA为TIPE2的蛋白编码基因,western blotting结果表明,重组腺病毒能可高效表达TIPE2基因。说明成功完成AdC68-TIPE2重组腺病毒的构建,该腺病毒载体,能稳定表达大鼠TIPE2基因。 中国组织工程研究杂志出版内容重点：组织构建;骨细胞;软骨细胞;细胞培养;成纤维细胞;血管内皮细胞;骨质疏松;组织工程全文链接：     

大鼠白细胞介素-6重组腺病毒构建及初步鉴定

目的构建携带大鼠白细胞介素-6(interleukin 6,IL-6)基因的重组腺病毒,为研究IL-6在神经损伤中的生物学作用提供技术手段。方法体外扩增大鼠IL-6基因,定向克隆到pAdTrace-TOX腺病毒穿梭质粒中,构建重组pAdTrace-IL-6过表达腺病毒质粒,并与骨架质粒pAd-Easy-1在BJ5183菌中发生同源重组,获得pAd-IL-6腺病毒载体,经PacⅠ酶切后转染HEK293细胞进行包装扩增。通过Ad-IL-6腺病毒感染PC12细胞,Real-time PCR和Western blot检测PC12细胞中IL-6及STAT3的表达。结果 PCR电泳及酶切、测序鉴定均证实目的基因IL-6正确克隆至腺病毒载体中,所构建的腺病毒Ad-IL-6可感染PC12细胞,有效增加IL-6的表达水平,促进IL-6信号通路中关键蛋白磷酸化STAT3的表达。结论成功构建携带IL-6的重组腺病毒载体,该载体可显著增高PC12细胞中IL-6基因和蛋白表达水平,并上调IL-6相关信号通路。     

人KLK1和EGFP双顺反子重组腺病毒构建及其在血管平滑肌细胞中的表达

目的：构建携EGFP为报告基因和人组织激肽释放酶1(hKLK1)基因双顺反子的重组腺病毒（Ad-hKLK1-IRES-EGFP）,并观察在自发性高血压大鼠（SHR）血管平滑肌细胞（VSMCs）中的表达变化。方法:通过双酶切质粒pBluescritII KS-hKLK1，将hKLK1基因定向克隆至带有IRES-EGFP的腺病毒穿梭质粒pDC316中，构建成重组穿梭质粒 ，将其与腺病毒骨架质粒BHGloxE1，3Cre共转染293A细胞，经包装并获得重组腺病毒，用PCR、酶切及测序方法对其进行鉴定，测定病毒滴度；用重组腺病毒感染VSMCs，用荧光显微镜下观察到的绿色荧光来测定感染率，用RT－PCR和Western blotting法测定hKLK1基因在VSMCs中的表达。结果:PCR、酶切和测序表明携EGFP的重组穿梭质粒pDC316-hKLK1-IRES-EGFP构建正确，并与骨架质粒在293A细胞中成功包装出重组腺病毒Ad-hKLK1-IRES-EGFP，其滴度为4.5×10¹¹/L。重组腺病毒感染VSMCs后，在荧光显微镜下观察到明亮绿色荧光蛋白表达，感染率高达90％以上，可检测到hKLK1基因的mRNA及蛋白表达，并与感染时间(1 d-7 d) 有显著依赖关系，峰值感染复数为100 MOI。结论:成功构建并包装了携EGFP和hKLK1基因的双顺反子重组腺病毒载体系统Ad-hKLK1-IRES-EGFP；感染VSMCs可检测到基因hKLK1和EGFP的独立共同高表达，该系统为一直观、安全、高效的基因转移系统。     

GLUT1 and GLUT8 in endometrium and endometrial adenocarcinoma.

Glucose is provided to cells by a family of glucose transport facilitators known as GLUTs. These transporters are expressed in a tissue specific manner and are overexpressed in many primary tumors of these tissues. Regulation of glucose transport facilitator expression has been demonstrated in endometrial tissue and endometrial adenocarcinoma. The following experiments were conducted to quantify and localize the expression of GLUT1 and GLUT8 in benign endometrium and compare this expression to endometrial cancer. Endometrial tissue samples were obtained from random hysterectomy specimens of patients with benign indications for surgery and endometrial cancer. Immunoblot and immunolocatization studies were performed using GLUT1 and GLUT8 specific antisera. Endometrial samples from 65 women who had undergone hysterectomy were examined (n=38 benign, n=27 malignant). A 44 and a 35.4 kDa immunoreacive species was demonstrated in endometrium and endometrial cancer for GLUT1 and GLUT8, respectively. Upregulation of GLUT1 expression was demonstrated with increasing grade of tumors (P<0.002). GLUT8 expression was increased in all tumor subtypes compared to atrophic endometrium (P<0.001). Apical localization by GLUT1 and GLUT8 was demonstrated in endometrial glands. GLUT1 and GLUT8 demonstrated diffuse intracellular localization in the cancer subtypes. GLUT1 and GLUT8 are expressed in both human endometrium and endometrial cancer. There appears to be a step-wise progression in GLUT1 and GLUT8 expression as tumor histopathology worsens. GLUT1 and GLUT8 may be important markers in tumor differentiation, as well as providing energy to rapidly dividing tumor cells.     

Autosomal dominant transmission of GLUT1 deficiency

GLUT1 deficiency is caused by a defect in the facilitative glucose transporter GLUT1. Impaired glucose transport across brain tissue barriers is reflected by hypoglycorrhachia and results in an epileptic encephalopathy with developmental delay and motor disorders. Recently heterozygous mutations in the GLUT1 gene (1p35-31.3) have been reported in sporadic patients. Parents and siblings carried the GLUT1 wild-type, suggesting a de novo, autosomal dominant condition resulting from GLUT1 haploinsufficiency. We report a father and two children from separate marriages affected by GLUT1 deficiency and carrying a novel heterozygous missense mutation (G272A) in the GLUT1 gene. Mutations were identified by polymerase chain reaction and DNA sequencing and confirmed by restriction fragment digest. The predicted amino acid change (Gly91Asp) affects an Arg-X-Gly-Arg-Arg motif between helices 2 and 3 that represents a cytoplasmic anchor point and is highly conserved among transporters of the major facilitator superfamily down to yeast and bacteria. GLUT1 immunoreactivity was normal, but 3-O-methyl-D-glucose uptake into erythrocytes was significantly reduced, suggesting a quantitatively normal, but functionally impaired, GLUT1 protein at the cell membrane. This is the first report of autosomal dominant transmission of GLUT1 deficiency, confirming that this condition is the result of haploinsufficiency. The Gly-->Asp mutation within a highly conserved sequence highlights its importance for GLUT1 function. GLUT1 deficiency should be considered in patients with epilepsy, mental retardation and motor disorders. Our observations have bearing on the identification of this treatable disorder in pediatric and adult patients, will modify current biochemical protocols which use parental controls and will enable genetic counseling of affected families.     

LPS上调大鼠HIF-1和GLUT表达

目的 探讨脂多糖(LPS)致大鼠内毒素血症早期,葡萄糖转运体(GLUT)家族在脑、心和肝组织等表达水平及低氧诱导因子-1(HIF-1)调控的相关性.方法 雄性SD大鼠分为对照组(腹腔注射0.9％氯化钠注射液)和给药组(腹腔注射2 mg/kg LPS),每组6只.测定给药后0～24h体温.ELISA法检测血清IL-1水平.RT-PCR法测定GLUT家族mRNA表达.Western blot法检测GLUT1和HIF-1蛋白表达.结果 在大鼠脑、心及肝等组织中,GLUT1和GLUT4均有表达;GLUT2在脑和肝组织中有表达;GLUT3仅在脑组织特异性表达.LPS作用24h可引起大鼠体温升高,血清IL-1水平上调(P＜0.05).LPS可上调脑组织GLUT1 mRNA水平和蛋白翻译水平(P＜0.01);同时LPS可促进脑组织HIF-1蛋白稳定表达(P＜0.001).结论 LPS促进大鼠HIF-1稳定表达,上调GLUT1表达及调控糖代谢.     

Developmental regulation of glucose transporters GLUT3, GLUT4 and GLUT8 in the mouse cerebellar cortex

Glucose uptake into the mammalian nervous system is mediated by the family of facilitative glucose transporter proteins (GLUT). In this work we investigate how the expression of the main neuronal glucose transporters (GLUT3, GLUT4 and GLUT8) is modified during cerebellar cortex maturation. Our results reveal that the levels of the three transporters increase during the postnatal development of the cerebellum. GLUT3 localizes in the growing molecular layer and in the internal granule cell layer. However, the external granule cell layer, Purkinje cell cytoplasm and cytoplasm of the other cerebellar cells lack GLUT3 expression. GLUT4 and GLUT8 have partially overlapping patterns, which are detected in the cytoplasm and dendrites of Purkinje cells, and also in the internal granule cell layer where GLUT8 displays a more diffuse pattern. The differential localization of the transporters suggests that they play different roles in the cerebellum, although GLUT4 and GLUT8 could also perform some compensatory or redundant functions. In addition, the increase in the levels and the area expressing the three transporters suggests that these roles become more important as development advances. Interestingly, the external granule cells, which have been shown to express the monocarboxylate transporter MCT2, express none of the three main neuronal GLUTs. However, when these cells migrate inwardly to differentiate in the internal granule cells, they begin to produce GLUT3, GLUT4 and GLUT8, suggesting that the maturation of the cerebellar granule cells involves a switch in their metabolism in such a way that they start using glucose as they mature.     

Mutational analysis of GLUT1 (SLC2A1) in Glut-1 deficiency syndrome

Fifteen children presenting with infantile seizures, acquired microcephaly, and developmental delay were found to have novel heterozygous mutations in the GLUT1 (SLC2A1). We refer to this condition as the Glut-1 Deficiency Syndrome (Glut-1 DS). The encoded protein (Glut-1), which has 12 transmembrane domains, is the major glucose transporter in the mammalian blood-brain barrier. The presence of GLUT1 mutations correlates with reduced cerebrospinal fluid glucose concentrations (hypoglycorrhachia) and reduced erythrocyte glucose transporter activities in the patients. We used Florescence in situ hybridization, PCR, single-stranded DNA conformational polymorphism, and DNA sequencing to identify novel GLUT1 mutations in 15 patients. These abnormalities include one large-scale deletion (hemizygosity), five missense mutations (S66F, R126L, E146K, K256V, R333W), three deletions (266delC, 267A>T; 904delA; 1086delG), three insertions (368-369 insTCCTGCCCACCACGCTCACCACG, 741-742insC, 888-889insG), three splice site mutations (197+1G>A; 1151+1G>T; 857T>G, 858G>A, 858+1del10), and one nonsense mutation (R330X). In addition, six silent mutations were identified in exons 2, 4, 5, 9, and 10. The K256V missense mutation involved the maternally derived allele in the patient and one allele in his mother. A spontaneous R126L missense mutation also was present in the paternally derived allele of the patient. The apparent pathogenicity of these mutations is discussed in relation to the functional domains of Glut-1.     

GLUT1 is Highly Expressed in Cementoblasts but not in Osteoblasts

Cementum is a specialized mineralized tissue covering root surface of the tooth. Although the tissue's composition resembles bone, there are distinct structural and functional differences between the two mineralized tissues. In this study, the genes that are differentially expressed in putative cementoblasts (human cementum-derived cells [HCDCs]) compared with preosteoblastic cells (human bone marrow stromal cells [BMSCs]) were screened by two independent microarray systems, and some of the selected genes were further analyzed by quantitative real-time RT-PCR. The gene encoding glucose transporter 1 [GLUT1], which showed the greatest difference between the two groups by the latter analysis, was subjected to further analyses. High levels of the GLUT1 protein in HCDCs, but not in BMSCs, were detected by Western blotting and immunocytochemistry. Furthermore, intense immunoreactivities for GLUT1 were observed in cementoblasts and cementocytes but not in osteoblasts or osteocytes in human periodontal tissues. These results indicate that GLUT1 may play a role in cementogenesis and could serve as a biomarker to differentiate between cells of cementoblastic and osteoblastic lineage.     

GLUT1 is highly expressed in cementoblasts but not in osteoblasts

Cementum is a specialized mineralized tissue covering root surface of the tooth. Although the tissue's composition resembles bone, there are distinct structural and functional differences between the two mineralized tissues. In this study, the genes that are differentially expressed in putative cementoblasts (human cementum-derived cells [HCDCs]) compared with preosteoblastic cells (human bone marrow stromal cells [BMSCs]) were screened by two independent microarray systems, and some of the selected genes were further analyzed by quantitative real-time RT-PCR. The gene encoding glucose transporter 1 [GLUT1], which showed the greatest difference between the two groups by the latter analysis, was subjected to further analyses. High levels of the GLUT1 protein in HCDCs, but not in BMSCs, were detected by Western blotting and immunocytochemistry. Furthermore, intense immunoreactivities for GLUT1 were observed in cementoblasts and cementocytes but not in osteoblasts or osteocytes in human periodontal tissues. These results indicate that GLUT1 may play a role in cementogenesis and could serve as a biomarker to differentiate between cells of cementoblastic and osteoblastic lineage.     

GLUT4研究进展

葡萄糖转运蛋白 4 (GLUT4 )是脂肪细胞和骨骼肌细胞协助葡萄糖转运的主要蛋白质 ,基础状态时分布于细胞内 ,在胰岛素刺激或运动等刺激下转位至细胞膜上。对GLUT4表达的调节在转录水平和转录后水平都存在。GLUT4转位涉及胰岛素信号传导途径和一磷酸腺苷激活的蛋白激酶 (AMPK)途径。GLUT4分子内部结构变化也可影响葡萄糖的转运。     

Inheritance of erythrocyte GLUT1 and age-dependent change in Japanese Shiba dogs

Three families of Shiba dogs were investigated for GLUT1 erythrocyte inheritance. Of 22 adult dogs, 14 contained both GLUT1 and GLUT4 (GLUT1 type) while others had only GLUT4 (normal). Erythrocytes with GLUT1 showed greater capacity for ascorbic acid (AA) recycling than those with only GLUT4. Puppies at 1 month of age presented uniformly high levels of GLUT1 and AA recycling capacity. Eight puppies were examined for postnatal changes until differences between types were apparent. Two puppies completely lost GLUT1 after 6 months of age and had only GLUT4, indicating normal type. Six others including an offspring of a normal pair showed persistent GLUT1, and were eventually GLUT1 type. GLUT4 was invariably present in all dogs. Hence, GLUT1 in erythrocytes of Shiba dogs is inherited in an autosomal recessive mode. Expression and function of GLUT1 are not associated with Na,K-ATPase or stomatin in Shiba dog erythrocytes.