摘要
目的研究L-精氨酸对人凝血因子Ⅷ（FⅧ）基因转录及表达的影响。方法将B结构域（氨基酸760～1639）缺失的人FⅧcDNA（BDDhFⅧcDNA）插入至质粒载体pcDNA6／V5-HisA，构建重组载体pcDNA6／V5-HisA-BDDhFⅧ，转染人脐静脉内皮细胞（HUVEC），加入L-精氨酸（终浓度10mmol／L）培养72h后，用ELISA方法检测细胞上清液中的人FⅧ抗原（FⅧ：Ag），一期凝血酶法检测FⅧ的促凝活性（FⅧ：C）。用Northern blot检测HUVEC中人FⅧ基因的转录。用PCR扩增BDDhFⅧcDNA的A1、A2、A3、C1和C2结构域基因，分别插入至pcDNA6／V5-HisA，构建五种载体pcDNA6／V5-HisA-BDDhFⅧ-A1、pcDNA6／V5-BDDhF Ⅷ-A2、pcDNA6／V5-HisA-BDDhF Ⅷ-A3、pcDNA6／V5-HisA-BDDhF Ⅷ-C1和pcDNA6／V5-HisA-BDDhF Ⅷ-C2，分别转染HUVEC后，加入L-精氨酸（终浓度10mmol／L）培养72h。膨胀裂解法分离细胞核，进行细胞核连缀（Run-on）反应，狭线印迹杂交检测A1、A2、A3、C1和C2结构域基因的转录。结果经L-精氨酸诱导后，HUVEC中人FⅧ基因表达明显增强[FⅧ：Ag为（146．08±4．78）ng／ml，10^6细胞诱导24h后FⅧ：C为（0．752±0．009）U／ml]，约是未经L-精氨酸诱导[FⅧ：Ag为（34．66±3．98）ng／ml，10^6细胞诱导24h后FⅧ：C为（0．171±0．006）U／ml]的4倍（P〈0．01）。Northern blot检测显示，加L-精氨酸培养之后，HUVEC中人BDDFⅧ mRNA的转录也较未加L-精氨酸显著增强，而只转染pcDNA6／V5-HisA的HUVEC中无人BDDFⅧ mRNA的转录存在。Run-on及狭线印迹杂交显示，加L-精氨酸培养后，A1和A2结构域基因的转录均较未经L-精氨酸诱导者明显增强，也显著强于A3、C1和C2结构域基因的转录，而A3、C1和C2结构域基因的转录在L-精氨酸诱导前后均无明显变化。结论L-精氨酸通过促进人FⅧ的A1和A2结构域基因的转录进而增强FⅧ在体外的转录和表达，因而在血友病A的基因治疗研究中，L-精氨酸是一个很有前途的诱导FⅧ转录和表达的物质。
Objective To investigate the effect of L-arginine on expression of human FⅧ gene. Methods Plasmid pcDNA6/VS-HisA-BDDhFⅧ containing B domain deleted human coagulant factor Ⅷ cDNA （BDDhF Ⅷ cDNA ） was constructed and transfected into human umbilical vein endothelial cells （HUVEC）. After 72 h incubation with L-arginine （final concentration was 10 mmol/L） , the supernant was collected for determining the antigen and clotting activity of human FⅧ （ FⅧ： Ag and FⅧ： C ） with ELISA and one stage clotting assay respectively. HUVECs were harvested for detecting human FⅧ mRNA by Northern blot analysis. The five functional domains of BDDhFⅧ cDNA including A1, A2, A3, C1 and C2 were amplified with PCR and inserted into pcDNA6/V5-HisA to construct the expression plasmids pcDNA6/V5-Hi- sA-BDDhFⅧ-A1, pcDNA6/V5-HisA-BDDhF Ⅷ-A2, pcDNA6/V5-HisA-BDDhF Ⅷ-A3, pcDNA6/V5-HisA-BDDhFⅧ-C1 and pcDNA6/V5-HisA-BDDhFⅧ-C2, respectively. HUVEC were transfected with the five plasmids respectively and incubated with L-arginine （at the final concentration of 10 mmol/L） for 72 h. Nueleoli were then isolated and underwent run-on assay. Results After 24 h incubation with L-arginine, FⅧ： Ag and FⅧ： C were increased markedly in the supernant of HUVEC [ FⅧ：Ag was （146.08 ±4.78 ） ng/ ml, and FⅧ： C （0. 752±0. 009） U/mL/10^6 cells · 24 h, while in control supernant without L-arginine, FⅧ： Agwas （34.66±3.98） ng/ml, and FⅧ： C （0. 171 ±0.006） U/mL/10^6 cell · 24 h, P〈0.01]. Northern blot analysis indicated that, after adding L-arginine, the transcription of human FⅧ mRNA was intensified remarkably in HUVEC transfected with pcDNA6/VS-HisA-BDDhFⅧ, but no any transcription in those transfected with pcDNA6/VS-HisA. Run-on assay demonstrated that with L-arginine induction, A1 and A2 domains transcription was increased obviously, while no change in A3, C1 and C2 domains transcription. Conclusion L-arginine increases expression of human FⅧ gene in HUVEC through enhancing its transcrip tion, particularly, domain A1 and A2 within FⅧ gene.
出处
《中华血液学杂志》
CAS
CSCD
北大核心
2007年第3期178-183,共6页
Chinese Journal of Hematology
基金
广东省自然科学基金资助项目（034622）
关键词
L-精氨酸
凝血因子Ⅷ
内皮细胞
脐静脉
人
基因表达
L-arginine
Coagulant factor Ⅷ
Endothelial cells, human umbilical vein
Gene expression
