中华生物医学工程杂志
中華生物醫學工程雜誌
중화생물의학공정잡지
CHINESE JOURNAL OF BIOMEDICAL ENGINEERING
2011年
2期
128-133
,共6页
莫红缨%黎毅敏%肖正伦%黄红川%杨淳%何为群%刘晓清
莫紅纓%黎毅敏%肖正倫%黃紅川%楊淳%何為群%劉曉清
막홍영%려의민%초정륜%황홍천%양순%하위군%류효청
髓样细胞表达的激发受体1%短发夹RNA%质粒%RNA干扰%基因表达
髓樣細胞錶達的激髮受體1%短髮夾RNA%質粒%RNA榦擾%基因錶達
수양세포표체적격발수체1%단발협RNA%질립%RNA간우%기인표체
Triggering receptor expressed on myeloid cells 1%Short hairpin RNA%Plasmids%RNA interference%Gene expression
目的 构建编码髓样细胞表达的激发受体1(TREM-1)基因短发夹RNA(shRNA)真核质粒表达载体,并筛选出基因沉默效果最明显的shRNA质粒表达载体.方法 针对TREM-1的靶位点设计含shRNA的靶序列,分别构建pGCsi-TREM-1 shRNA1和pGCsi-TREM-1 shRNA2的质粒表达载体和pGCsi-NegshRNA阴性质粒表达载体,通过酶切及测序进行鉴定.鉴定后以脂质体包裹转染肺泡上皮A549细胞,分为未转染对照组(A组)、转染空质粒载体pGCsi对照组(B组)、转染阴性shRNA质粒表达载体pGCsi-NegshRNA对照组(C组)、转染TREM-1 shRNA1质粒表达载体组(D组)和转染TREM-1shRNA2质粒表达载体组(E组),48 h后观察转染细胞绿色荧光蛋白表达效果.转染24、48、72 h后,以A组为对照采用MTT法测定B、C、D、E组细胞的存活率.转染48 h后,通过荧光定量PCR检测各组TREM-1 mRNA的表达水平并与转染前相比较.结果 经酶切和测序鉴定证实,目的 TREM-1基因shRNA1和shRNA2片段已被克隆到pGCsi载体中.荧光显微镜下,B、C、D、E组A549细胞有明显的绿色荧光蛋白表达.各时间点B、C、D、E组细胞存活率均达90%以上.A、B、C 3组转染前后TREM-1mRNA的表达水平差异无统计学意义,D、E组转染的重组质粒均能有效抑制A549细胞的TREM-1mRNA的表达([(1.945±0.252)×105比(1.010±0.194)×105,(1.933±0.216)×105比(1.202±0.171)×105,均P＜0.05],抑制率分别为48.07%和37.82%.结论 成功构建了靶向TREM-1基因的shRNA质粒表达载体,可有效抑制细胞中TREM-1目的 基因的表达,为后续脓毒症的基因治疗提供了依据.
目的 構建編碼髓樣細胞錶達的激髮受體1(TREM-1)基因短髮夾RNA(shRNA)真覈質粒錶達載體,併篩選齣基因沉默效果最明顯的shRNA質粒錶達載體.方法 針對TREM-1的靶位點設計含shRNA的靶序列,分彆構建pGCsi-TREM-1 shRNA1和pGCsi-TREM-1 shRNA2的質粒錶達載體和pGCsi-NegshRNA陰性質粒錶達載體,通過酶切及測序進行鑒定.鑒定後以脂質體包裹轉染肺泡上皮A549細胞,分為未轉染對照組(A組)、轉染空質粒載體pGCsi對照組(B組)、轉染陰性shRNA質粒錶達載體pGCsi-NegshRNA對照組(C組)、轉染TREM-1 shRNA1質粒錶達載體組(D組)和轉染TREM-1shRNA2質粒錶達載體組(E組),48 h後觀察轉染細胞綠色熒光蛋白錶達效果.轉染24、48、72 h後,以A組為對照採用MTT法測定B、C、D、E組細胞的存活率.轉染48 h後,通過熒光定量PCR檢測各組TREM-1 mRNA的錶達水平併與轉染前相比較.結果 經酶切和測序鑒定證實,目的 TREM-1基因shRNA1和shRNA2片段已被剋隆到pGCsi載體中.熒光顯微鏡下,B、C、D、E組A549細胞有明顯的綠色熒光蛋白錶達.各時間點B、C、D、E組細胞存活率均達90%以上.A、B、C 3組轉染前後TREM-1mRNA的錶達水平差異無統計學意義,D、E組轉染的重組質粒均能有效抑製A549細胞的TREM-1mRNA的錶達([(1.945±0.252)×105比(1.010±0.194)×105,(1.933±0.216)×105比(1.202±0.171)×105,均P＜0.05],抑製率分彆為48.07%和37.82%.結論 成功構建瞭靶嚮TREM-1基因的shRNA質粒錶達載體,可有效抑製細胞中TREM-1目的 基因的錶達,為後續膿毒癥的基因治療提供瞭依據.
목적 구건편마수양세포표체적격발수체1(TREM-1)기인단발협RNA(shRNA)진핵질립표체재체,병사선출기인침묵효과최명현적shRNA질립표체재체.방법 침대TREM-1적파위점설계함shRNA적파서렬,분별구건pGCsi-TREM-1 shRNA1화pGCsi-TREM-1 shRNA2적질립표체재체화pGCsi-NegshRNA음성질립표체재체,통과매절급측서진행감정.감정후이지질체포과전염폐포상피A549세포,분위미전염대조조(A조)、전염공질립재체pGCsi대조조(B조)、전염음성shRNA질립표체재체pGCsi-NegshRNA대조조(C조)、전염TREM-1 shRNA1질립표체재체조(D조)화전염TREM-1shRNA2질립표체재체조(E조),48 h후관찰전염세포록색형광단백표체효과.전염24、48、72 h후,이A조위대조채용MTT법측정B、C、D、E조세포적존활솔.전염48 h후,통과형광정량PCR검측각조TREM-1 mRNA적표체수평병여전염전상비교.결과 경매절화측서감정증실,목적 TREM-1기인shRNA1화shRNA2편단이피극륭도pGCsi재체중.형광현미경하,B、C、D、E조A549세포유명현적록색형광단백표체.각시간점B、C、D、E조세포존활솔균체90%이상.A、B、C 3조전염전후TREM-1mRNA적표체수평차이무통계학의의,D、E조전염적중조질립균능유효억제A549세포적TREM-1mRNA적표체([(1.945±0.252)×105비(1.010±0.194)×105,(1.933±0.216)×105비(1.202±0.171)×105,균P＜0.05],억제솔분별위48.07%화37.82%.결론 성공구건료파향TREM-1기인적shRNA질립표체재체,가유효억제세포중TREM-1목적 기인적표체,위후속농독증적기인치료제공료의거.
Objective To construct eukaryotic plasmid expression vectors encoding the short hairpin RNA(shRNA)targeting triggering receptor expressed on myeloid cells 1(TREM-1)mRNA,and to screen the shRNA plasmid expression vectors with most obvious effects for silencing TREM-1 gene.Methods According to the target site of TREM-1 gene,shRNA target sequence was designed.Two plasmid expression vectors of pGCsi-TREM-1 shRNA1 and pGCsi-TREM-1 shRNA2,and negative control plasmid expression vectors of pGCsi-NegshRNA were constructed respectively.After being identified by restriction endonuclease digestion and nucleotide sequencing, the recombinant plasmids with TransFectin lipid were transfected into alveolar epithelial cells A549.A549 cells were allocated into the un-transfected group(group A), pGCsi transfected group (group B),pGCsi-NegshRNA transfected group (group C) , TREM-1 shRNA1 transfected group (group D) and TREM-1 shRNA2 transfected group(group E). The effects of green fluorescent protein expression in transfected cells were studied at 48 h later.Cell viability was assessed by MTT at 24, 48 and 72 h after transfection with group A as reference. The expression of TREM-1 mRNA was detected by quantitative real-time PCR and compared with the level before transfection. Results Restriction endonuclease digestion and nucleotide sequencing showed that TREM-1 shRNA1 and shRNA2 oligonucleotide fragments were correctly inserted into pGCsi vector.Under fluorescence microscopy, A549 cells in groups B, C, D and E clearly showed expression of green fluorescent protein. At all the time spots, over 90% of cells in these groups were viable. There was no change of TREM-1 mRNA expression in groups A, B and C from pre-transfection. The recombinant plasmids showed effective inhibition of TREM-1 mRNA expression in 48.07% and 37.82% of A549 cells in groups D and E respectively[(1.945±0.252)×105 vs (1.010±0.194)×l05, (1.933±0.216)×l05 vs (1.202±0.171)×105, all P＜0.05). Conclusion The expression vector of TREM-1 shRNA is successfully constructed and can inhibit TREM-1 mRNA expression in A549 cells, which provides evidences for subsequent studies on gene therapy for sepsis.
