CAJ | 학술논문

目的构建与鉴定沉默过氧化物酶体增殖子活化受体γ(PPARγ)同时表达降钙素基因相关肽(CGRP)基因重组载体.方法选用pGFP-V-RS载体,将PPARγ小干扰RNA(siRNA)寡核苷酸靶序列连接入pGFP-V-RS载体,酶切鉴定及测序正确后得到重组载体pGFP-V-RS-siPPARγ;将CGRP片段与Mlu Ⅰ酶切,并与末端羟基化的线性质粒pGFP-V-RS重组连接,得到重组载体pGFP-V-RS-exCGRP.将获取的CGRP基因片段连接入线性化重组载体pGFP-V-RS-siPPARγ,酶切鉴定及测序正确后得到重组载体pGFP-V-RS-siPPARγ-exCGRP.收集第3代HEK-293细胞,将细胞分为4组:CON组:转染空载体pGFP-V-RS质粒,作为对照；SI组:转染重组载体pGFP-V-RS-siPPARγ质粒；EX组:转染重组pGFP-V-RS-exCGRP质粒；DOU组:转染重组双基因载体pGFP-V-RS-siPPARγ-exCGRP质粒.每组电极杯中加入5x106个细胞,并分别加入上述质粒2.5μl,给予直流电低压脉冲1次,时间15 ms.培养48 h后收集各组细胞,用逆转录-聚合酶链反应(RT-PCR)与Western blot检测细胞内PPAR、CGRP mRNA及蛋白的表达.结果重组双基因载体pGFP-V-RS-siPPARγ-exCGRP经酶切鉴定及测序结果与设计完全一致.电转染HEK-293细胞后,DOU组细胞PPARγ基因呈低表达,PPARγmRNA与其蛋白的相对表达量为0.036±0.003、0.108 ±0.031,与EX组(0.156±0.014、0.778±0.103)及CON组(0.148±0.014、0.742 ±0.143)比较,差异有统计学意义(P＜0.05),与SI组(0.054±0.005、0.135 ±0.039)比较,差异无统计学意义(P＞0.05).DOU组细胞CGRP基因呈高表达,CGRP mRNA与其蛋白的相对表达量为1.144±0.106、1.244±0.184,与SI组(0.320±0.030、0.370±0.089)及CON组(0.444 ±0.041、0.274±0.062)比较,差异有统计学意义(P＜0.05),与EX组(1.021 ±0.095、1.221±0.181)比较,差异无统计学意义(P＞0.05).结论成功构建出沉默PPARγ基因并表达CGRP基因的重组载体pGFP-V-RS-siPPARγ-exCGRP,能够有效阻断PPARγ基因表达,同时促进CGRP基因表达. 目的構建與鑒定沉默過氧化物酶體增殖子活化受體γ(PPARγ)同時錶達降鈣素基因相關肽(CGRP)基因重組載體.方法選用pGFP-V-RS載體,將PPARγ小榦擾RNA(siRNA)寡覈苷痠靶序列連接入pGFP-V-RS載體,酶切鑒定及測序正確後得到重組載體pGFP-V-RS-siPPARγ;將CGRP片段與Mlu Ⅰ酶切,併與末耑羥基化的線性質粒pGFP-V-RS重組連接,得到重組載體pGFP-V-RS-exCGRP.將穫取的CGRP基因片段連接入線性化重組載體pGFP-V-RS-siPPARγ,酶切鑒定及測序正確後得到重組載體pGFP-V-RS-siPPARγ-exCGRP.收集第3代HEK-293細胞,將細胞分為4組:CON組:轉染空載體pGFP-V-RS質粒,作為對照；SI組:轉染重組載體pGFP-V-RS-siPPARγ質粒；EX組:轉染重組pGFP-V-RS-exCGRP質粒；DOU組:轉染重組雙基因載體pGFP-V-RS-siPPARγ-exCGRP質粒.每組電極杯中加入5x106箇細胞,併分彆加入上述質粒2.5μl,給予直流電低壓脈遲1次,時間15 ms.培養48 h後收集各組細胞,用逆轉錄-聚閤酶鏈反應(RT-PCR)與Western blot檢測細胞內PPAR、CGRP mRNA及蛋白的錶達.結果重組雙基因載體pGFP-V-RS-siPPARγ-exCGRP經酶切鑒定及測序結果與設計完全一緻.電轉染HEK-293細胞後,DOU組細胞PPARγ基因呈低錶達,PPARγmRNA與其蛋白的相對錶達量為0.036±0.003、0.108 ±0.031,與EX組(0.156±0.014、0.778±0.103)及CON組(0.148±0.014、0.742 ±0.143)比較,差異有統計學意義(P＜0.05),與SI組(0.054±0.005、0.135 ±0.039)比較,差異無統計學意義(P＞0.05).DOU組細胞CGRP基因呈高錶達,CGRP mRNA與其蛋白的相對錶達量為1.144±0.106、1.244±0.184,與SI組(0.320±0.030、0.370±0.089)及CON組(0.444 ±0.041、0.274±0.062)比較,差異有統計學意義(P＜0.05),與EX組(1.021 ±0.095、1.221±0.181)比較,差異無統計學意義(P＞0.05).結論成功構建齣沉默PPARγ基因併錶達CGRP基因的重組載體pGFP-V-RS-siPPARγ-exCGRP,能夠有效阻斷PPARγ基因錶達,同時促進CGRP基因錶達.
목적 구건여감정침묵과양화물매체증식자활화수체γ(PPARγ)동시표체강개소기인상관태(CGRP)기인중조재체.방법 선용pGFP-V-RS재체,장PPARγ소간우RNA(siRNA)과핵감산파서렬련접입pGFP-V-RS재체,매절감정급측서정학후득도중조재체pGFP-V-RS-siPPARγ;장CGRP편단여Mlu Ⅰ매절,병여말단간기화적선성질립pGFP-V-RS중조련접,득도중조재체pGFP-V-RS-exCGRP.장획취적CGRP기인편단련접입선성화중조재체pGFP-V-RS-siPPARγ,매절감정급측서정학후득도중조재체pGFP-V-RS-siPPARγ-exCGRP.수집제3대HEK-293세포,장세포분위4조:CON조:전염공재체pGFP-V-RS질립,작위대조；SI조:전염중조재체pGFP-V-RS-siPPARγ질립；EX조:전염중조pGFP-V-RS-exCGRP질립；DOU조:전염중조쌍기인재체pGFP-V-RS-siPPARγ-exCGRP질립.매조전겁배중가입5x106개세포,병분별가입상술질립2.5μl,급여직류전저압맥충1차,시간15 ms.배양48 h후수집각조세포,용역전록-취합매련반응(RT-PCR)여Western blot검측세포내PPAR、CGRP mRNA급단백적표체.결과 중조쌍기인재체pGFP-V-RS-siPPARγ-exCGRP경매절감정급측서결과여설계완전일치.전전염HEK-293세포후,DOU조세포PPARγ기인정저표체,PPARγmRNA여기단백적상대표체량위0.036±0.003、0.108 ±0.031,여EX조(0.156±0.014、0.778±0.103)급CON조(0.148±0.014、0.742 ±0.143)비교,차이유통계학의의(P＜0.05),여SI조(0.054±0.005、0.135 ±0.039)비교,차이무통계학의의(P＞0.05).DOU조세포CGRP기인정고표체,CGRP mRNA여기단백적상대표체량위1.144±0.106、1.244±0.184,여SI조(0.320±0.030、0.370±0.089)급CON조(0.444 ±0.041、0.274±0.062)비교,차이유통계학의의(P＜0.05),여EX조(1.021 ±0.095、1.221±0.181)비교,차이무통계학의의(P＞0.05).결론 성공구건출침묵PPARγ기인병표체CGRP기인적중조재체pGFP-V-RS-siPPARγ-exCGRP,능구유효조단PPARγ기인표체,동시촉진CGRP기인표체.
Objective To construct the recombinant vector silencing peroxisome proliferator-activated receptor-γ (PPARγ) and expressing calcitonin gene-related peptide (CGRP) gene.Methods The pGFP-V-RS vector we selected contains a small interfering RNA (siRNA) expressing unit,and also can express exogenous gene inserted.The PPARγ siRNA oligonucleotides sequence was ligated into pGFP-VRS vector after annealling,and then PPAR siRNA recombinant vector (pGFP-V-RS-siPPARγ) was obtained by restriction analysis and DNA sequencing.The CGRP gene fragment was ligated into linear plasmids pGFP-V-RS-siPPARγrecombinant vector,and then the recombinant vector pGFP-V-RS-siPPARγ-exCGRP was obtained by restriction analysis and DNA sequencing.The HEK-293 cells were collected and divided into 4 groups at random.In control group,the cells were only transfected with vector pGFP-V-RS plasmid.In SI group,the cells were transfected with recombinant vector pGFP-V-RS-siPPARγplasmid.In EX group,the cells were transfected with recombinant vector pGFP-V-RS-exCGRP plasmid.In DOU group,the cells were transfected with pGFP-V-RS-siPPARγ-exCGRP plasmid.There were 5 × 106 HEK-293 cells in electrode cup of each group,and 2.5 μl above-mentioned plasmids were added respectively.The cells were given DC voltage pulse one time (15 ms).The cells were harvested in each group after culture for 48 h,and CGRP and PPARγmRNA and protein expression levels were detected by using reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting.Results The results of restriction analysis and DNA sequencing were completely matched with those of the designed sequences after the PPARγsiRNA oligonucleotides sequence and CGRP gene fragment were cloned into pGFP-V-RS vector.After recombinant vector pGFP-V-RS-siPPARγ-exCGRP was transfected into HEK-293 cells,the expression levels of PPARγ mRNA and protein were reduced in DOU group DOU (0.036 ±0.003 and 0.108 ±0.031)as compared with EX group (0.156 ±0.014 and 0.778 ±0.103) and control group (0.148 ±0.014 and 0.742 ± 0.143) (P ＜ 0.05),but showed no significant difference in comparison to SI group (0.054 ± 0.005and 0.135±0.039) (P＞0.05).The expression level of CGRP mRNA and protein was significantly increased in DOU group (1.144 ±0.106 and 1.244 ±0.184) as compared with IS group (0.320 ±0.030 and 0.370 ±0.089) and control group (0.444 ±0.041 and 0.274 ±0.062) (P ＜0.05),but there was no significant difference in comparison to EX group (1.021 ±0.095 and 1.221 ±0.181) (P ＞0.05).Conclusion We successfully constructed the recombinant vector pGFP-V-RS-siPPARγ-exCGRP that silencing PPARγand expressing CGRP gene,and the recombinant vector can efficaciously suppress PPARγgene expression and promote CGRP gene expression simultaneously.