Objective To construct the recombinant plasmid vector that codes calcitonin generelated peptide (CGRP) of rat and investigate whether it can release cerebral vasospasm after subarachnoid hemorrhage (SAH) in rat model.Methods The purpose gene was extracted from the brain tissue of rat with Trizol reagent.Recombinant colonies were constructed and identified by restriction enzyme digestion and DNA sequencing.We then transfected the recombinant plasmid vector pLXSN-CGRP into 293T cell in vitro.Real-time quantitative polymerase chain reaction (Q-PCR) and Western-blotting were used to detect the expression of CGRP.SAH model was introduced by injecting autologous blood into cisterna magna.Animals were randomly divided into 3 groups (saline group,pLSXN group and CGRP gene treated group) by the intraventricular injection of saline or plasmid after the SAH model.The degree of cerebral vasospasm and the expression level of CGRP were detected.Results (1) Identification of the recombinant colony:the digested products presented 2 specific bands which coincided with the sizes of pLXSN and rCGRPcDNA respectively.The acquired sequence was demonstrated to be completely identical with the coding region sequence of rCGRP according to the analysis of basic local alignment search tool.We detected the expression of CGRP in vitro through Q-PCR and Western-blotting after the transfection of the recombinant plasmid vector into 293T cell.(2) The pathological damage of basal artery(μm) was much lighter(609.7 ±47.9) and the degree of cerebral vasospasm (μm) was decreased(14.35 ± 1.87) in CGRP gene treated group(all P ＜ 0.01),compared with saline(460.1 ±52.5,t =7.053;23.91 ± 1.96,t =11.812) and pLSXN group (446.2 ±55.3,t =7.713 ; 24.55 ± 2.11,t =12.602).(3) The expression of CGRP (ng/L) was significantly increased in CGRP gene treated group (173.4 ± 6.3),compared with saline(102.7 ± 5.7) and pLSXN group(94.3 ±6.1 ; t-=5.004,5.601,both P ＜0.01 respectively).Conclusions The recombinant plasmid vector pLXSN-CGRP is constructed successfully.Our study demonstrate that intraventricular infusion of CGRP gene can release cerebral vasospasm in the rat SAH model.%目的 构建重组真核表达载体pLXSN-降钙素基因相关肽(CGRP),探讨CGRP对蛛网膜下腔出血(SAH)后脑血管痉挛(CVS)的治疗作用.方法 从SD大鼠中提取目的基因,构建重组质粒.将36只SD大鼠按照随机数字表法随机分成3组,均采用枕大池1次注血法制成SAH模型,于SAH后立体定向经侧脑室分别注入生理盐水、空载质粒pLXSN、重组质粒pLXSN-CGRP各20μl.分别检测SAH后24h基底动脉形态学改变及血浆中CGRP的表达水平.结果 (1)重组质粒鉴定:双酶切后电泳可见2条特异条带,分别与pLXSN和rCGRPcDNA大小相吻合；测序结果经basic local alignment search tool分析,与rCGRP基因编码区全长序列基本完全一致；体外转染293-T细胞,Q-PCR及蛋白质印迹检测确认CGRP有表达.(2)基底动脉形态学检测:生理盐水组、空载质粒组、pLXSN-CGRP组血管管腔内周长(μm)分别为:460.1 ±52.5、446.2±55.3、609.7±47.9,生理盐水组与空载质粒组相比差异无统计学意义；生理盐水组、空载质粒组周长明显短于pLXSN-CGRP组(t=7.053、7.713,均P＜0.01).3组血管壁厚度(μm)分别为:23.91±1.96、24.55 ±2.11、14.35±1.87,生理盐水组与空载质粒组差异无统计学意义；与pLXSN-CGRP组相比,生理盐水组、空载质粒组血管壁厚度明显增加(t=11.812、12.602,均P＜0.01).pLXSN-CGRP组基底动脉痉挛程度较轻,表现为血管管腔内周长增加,血管壁厚度减小.(3)血浆中CGRP浓度(ng/L)检测:生理盐水组为102.7 ±5.7、空载质粒组为94.3±6.1、pLXSN-CGRP组为173.4 ±6.3,前两组相比差异无统计学意义；较pLXSN-CGRP组浓度明显降低(t=5.004、5.601,均P＜0.01).治疗组血浆中CGRP表达量增大.结论 成功构建重组质粒pLXSN-CGRP.枕大池1次注血法建立大鼠SAH后CVS模型,经侧脑室注入重组质粒导入CGRP基因,在SAH早期就可以缓解CVS,起到脑保护作用.
展开▼
