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21.
We investigated the regulation of the rat neuron-specific enolase gene using a transient transfection approach. Recent transgenic
mouse studies have shown that a 1.8-kb segment of the ratNSE gene 5′ flanking region, including the first (noncoding) exon but not the first intron, is able to drive expression of a
reporter gene in parallel with endogenousNSE. These data suggest thatcis-acting elements responsible for the spatial and temporal pattern ofNSE gene expression are located within the proximal 1.8 kb of the 5′ flanking sequence. To further investigate this region, we
joined the 1.8-kb regulatory cassette to thecat reporter gene and generated a number of constructs in which the flanking sequence was progressively deleted from the 5′ end.
These constructs were tested by transient transfection into neuronal and nonneuronal cells, followed by an assay for CAT activity.
We found that as little as 255 bp of 5′ flanking sequence was able to confer cell type-specificity on the reporter gene. Further
truncation to 120 bp of 5′ sequence resulted in a sharp downregulation of reporter activity in PC12 cells but a significant
rise in both Neuro-2A neuroblastoma cells and nonneuronal Ltk- cells, indicating thatcis-acting elements controlling the regulation ofNSE in Ltk-, Neuro-2A, and PC12 cells may lie within the 135 bp region covered by this deletion. This region contains an AP-2
site and an element similar in sequence and position to a motif identified in the proximal promoter region of the neuron-specific
peripherin gene. Reduction to 95 bp of 5′ sequence resulted in a slight downregulation of CAT activity in all cell lines tested,
and further truncation to 65 bp of 5′ sequence caused a universal reduction to background levels of CAT activity, concomitant
with the disruption of the basalNSE promoter. Our results show that the 5′ flanking region of theNSE gene is capable of conferring cell type-specificity on a heterologous gene in transfected cells and that elements responsible
for this are located within the proximal 255 bp. 相似文献
22.
重组逆转录病毒pLNCX2-GDNF转染许旺细胞及其表达 总被引:1,自引:1,他引:0
目的 利用基因转染技术将胶质细胞源性神经营养因子(GDNF)基因转入许旺细胞(SC)以提高其分泌该因子的水平。方法 应用逆转录聚合酶链反应(RT—PCR)方法合成GDNFcD—NA片段,以逆转录病毒pLNCX2为载体导入许旺细胞内,并运用RT—PCR、免疫细胞化学染色、ELISA及运动神经元联合培养法检测外源性基因在mRNA和蛋白水平的表达及其产物的活性。结果 RT—PCR检测结果示GDNF—SCs表达GDNFmRNA的水平明显优于SCs;免疫细胞化学检测发现GDNF—SCs抗GDNF蛋白染色呈强阳性,正常SCs呈弱阳性;ELISA法检测结果示转染后4周GDNF—SCs分泌GD—NF蛋白的水平升至正常SCs的5.1倍;运动神经元联合培养法结果显示GDNF—SCs分泌的外源性基因产物具有生物学活性。结论 GDNF基因转染SC在mRNA和蛋白水平表达GDNF明显优于正常SCs,外源性基因表达产物具有神经生物学活性。 相似文献
23.
目的 :在小鼠NIH3T3细胞转染表达人天然GPI锚固型CD5 5和重组跨膜型CD5 5 TM分子 ,观察比较它们对人补体溶破异源细胞的抑制功能。方法 :将带有CD5 5cDNA、CD5 5 TMcDNA的重组逆病毒表达质粒CD5 5 pLXSN、CD5 5TM pLXSN经脂质体法转染PA317细胞 ,用病毒上清感染小鼠成纤维母细胞NIH3T3。经G418加压筛选 ,利用FACS检测获得表达CD5 5和CD5 5 TM分子的阳性细胞克隆 ,通过MTT比色法比较两种分子对人血清补体溶破细胞的抑制功能有无差别。结果 :细胞转染筛选获得多个表达跨膜型人CD5 5分子的NIH3T3细胞克隆 ,补体杀伤试验证实其具有抑制人补体溶破的功能 ,且两种分子的补体抑制功能无明显差异。结论 :成功地建立了稳定表达天然CD5 5、跨膜型CD5 5分子的小鼠NIH3T3细胞 ,证实其表达的GPI型CD5 5分子和CD5 5TM分子均具有抑制人补体溶破细胞的功能 ,为进一步探讨应用跨膜型的CD5 5分子对PNH进行基因治疗奠定了基础。 相似文献
24.
25.
目的:探讨视网膜母细胞瘤(Rb)的发生与Rb基因(Rb1)缺失、失活等异常的关系。方法:用逆转录病毒载体pDOR与全长4.7kb野生型Rb1cDNA构建逆转录病毒表达载体pDOR-Rb1+。用脂质体介导法将pDOR-Rb1+转入CRIP包装细胞系。结果:实验产生了0.5X105Cfu具有一次感染能力的重组逆转病毒。利用该病毒感染SO-Rb50,经G418筛选,获得了抗G418细胞群体。运用PCR及Southern杂交技术对转染细胞进行检测,结果表明该抗G418细胞中有完整的外源Rb1存在。Northern杂交发现其Rb1mRNA表达水平有所提高。对细胞群体生长速率、软琼脂集落形成能力的测定表明,外源Rb1的表达使SO-Rb50在软琼脂中集落形成能力降低,而群体生长速率无明显影响。结论:外源Rb1对SO-Rb50恶性表型有一定影响。 相似文献
26.
It is documented that alkaline phosphatase (AP) plays an important role in bone mineralization. Considering that TN-AP is
expressed in periodontal ligament fibroblasts, renal epithelial cells, and vascular endothelial cells, and that TN-AP is both
a calcium-/phosphate-binding protein and a phosphohydrolytic enzyme, we hypothesize that membrane-bound AP also plays an important
role in the initiation of physiological and pathological mineralizations in tissues other than bone and cartilage. To test
this hypothesis, nonosteoblast cell lines, including a fibroblast line, a renal epithelial line, and a capillary endothelial
line, were stably transfected to express high levels of rat bone AP on their cell surfaces. These rat bone AP-expressing cells
were then cultured on filter membranes in the presence or absence of β-glycerol phosphate. von Kossa staining for calcium
phosphate and transmission electron microscopy with electron diffraction analysis for minerals were employed to investigate
the effect of membrane AP on extracellular calcium phosphate mineralization. Our results indicated that AP expression on these
nonosteoblast-like cell surfaces have induced extracellular hydroxyapatite (HAP) mineralization. Our findings support the
concept that membrane-bound AP contributes to extracellular apatitic mineralization by mechanisms that do not necessarily
involve its hydrolase activity. They also suggest that AP might be important for the initiation of pathological mineralization
in nonosteogenic tissues.
Received: 11 January 1996 / Accepted: 31 October 1996 相似文献
27.
人β-防御素3基因在COS-7细胞中的表达 总被引:3,自引:0,他引:3
目的:构建人β—防御素3(hBD3)的真核表达载体,建立稳定表达hBD3的细胞株。方法:用EcoR Ⅰ酶切合有hBD3全长基因的pGEM-hBD3重组质粒,获得其编码区全长序列,将其连接入EcoR Ⅰ预处理过的pcDNA3中。转化大肠杆菌,酶切鉴定筛选出插入方向正确的转化子。采用脂质体转染法将重组pcDNA3-hBD3真核表达载体导入COS-7细胞,用G418进行抗性筛选,用RT-PCR和Western印迹检测目的基因hBD3的mRNA和蛋白表达水平。结果与结论:构建的pcDNA3-hBD3真核表达载体转染COS-7细胞后可稳定表达hBD3的mRNA和蛋白,且蛋白主要以分泌形式存在于培养上清中。 相似文献
28.
反义核苷酸抑制人高迁移率族蛋白1表达对胰腺癌细胞的影响 总被引:3,自引:0,他引:3
目的 构建高迁移率族蛋白 1(HMGB1)基因的反义真核表达载体 ,寻找胰腺癌基因治疗新途径。方法应用分子克隆技术构建HMGB1基因反义真核表达载体 pcDNA3 1/antisense HMGB1,转染胰腺癌细胞株PANC 1,通过逆转录 聚合酶链反应 (RT PCR)、免疫印迹法 (Westernblot)、噻唑蓝 (MTT)比色法检测转染 4 8h后胰腺癌细胞HMGB1基因表达和体外增殖活性的变化。结果 成功构建 pcDNA3 1/antisense HMGB1反义真核表达载体。所获反义表达载体 pcDNA3 1/antisense HMGB1转染可使PANC 1细胞HMGB1mRNA和蛋白表达水平显著降低 (P <0 0 1)。反义 pcDNA3 1/antisense HMGB1的导入能有效抑制PANC 1增殖活性 (P <0 0 1)。 结论 应用反义RNA技术阻断HMGB1基因的表达 ,能有效抑制癌细胞的体外增殖活性 ,为基因治疗提供了新思路 相似文献
29.
Wong Merng Koon 《中华创伤骨科杂志》2006,8(10):944-948
Objective Intra-articular fractures of the distal radius in young adults comprise a distinet fracture pattern that is diffficuh to manage and associated with a high frequency of post-traumatic arthritis.Restoration of articular congruency and alignment should improve the outcome.Methods In this study we prospectively re- viewed the results of 21 consecutive cases of dorsally displaced intra-articular distal radius fractures which were treated with internal fixation after failing to achieve articular congruency with closed reduction.Results 3 patients were lost to follow-up.For the rest of 18 patients,follow-up time ranges from 18 to 75 weeks the fractures had healed with highly satisfactory radiographic and functional results.The final volar tilt averaged 4.9°;radial inclination 23.9°;radial length 14mm;and articular incongruity,0.1 mm.Wrist motion at final follow-up examination aver- aged flexion 62°,extension 60°,radial deviation 16°,ulnar deviation 27°,pronation 77°and supination 74°.Grip strength averaged 83% of the uninjured side.The overall outcome of 18 patients(94.4%)had a good or excellent result according to the system of Gartland and Werley and 18 patients(72.2%)had a good result according to the modified system of Green and O'Brien at the most recent evaluation.The only complication in this series was a superficial pin tract infection,which was rapidly resolved with removal of pins at 5th week of external fixation. Conclusion Thus restoration of articular congruency and alignment is possible with minimal complication using modern non-angular stable methods of internal fixation. 相似文献
30.
Stable isotope methodologies offer a number of possibilities for the nutritional assessment of many different processes and metabolic pathways. The application of stable isotopes has been boosted by the development of new mass spectrometers, lower costs of probes, and the risks associated with radioactive tracers. The use of 13 C as a tracer offers all the advantages of stable isotopes and has been widely applied for measuring various types of metabolic processes. This review is focused on clinical and nutritional assessments using 13 C breath tests. 相似文献