中华麻醉学杂志
中華痳醉學雜誌
중화마취학잡지
CHINESE JOURNAL OF ANESTHESIOLOGY
2010年
8期
988-990
,共3页
颜学滔%王焱林%王成夭%何祥虎%饶艳
顏學滔%王焱林%王成夭%何祥虎%饒豔
안학도%왕염림%왕성요%하상호%요염
重组融合蛋白质类%转导,遗传%血红素加氧酶-1%肌细胞,心脏%细胞低氧%氧
重組融閤蛋白質類%轉導,遺傳%血紅素加氧酶-1%肌細胞,心髒%細胞低氧%氧
중조융합단백질류%전도,유전%혈홍소가양매-1%기세포,심장%세포저양%양
Recombinant fusion proteins%Transduction,genetic%Heme oxygenase-1%Myocytes,cardiac%Cell hypoxia%Oxygen
目的 探讨PEP-1-血红素加氧酶(HO)-1融合蛋白转导对大鼠H9c2心肌细胞缺氧复氧损伤的影响.方法 构建含人HO-1基因的原核表达质粒pETl5b-PEP1-hHO-1,质粒转化后诱导目的 蛋白PEP-1-HO-1表达.用含15%胎牛血清高糖DMEM培养基培养H9c2心肌细胞,随机分为4组(n=4):正常对照组(C组)常规培养;缺氧复氧组(H/R组)细胞缺氧22 h,复氧8 h;低浓度融合蛋白组(L-HO组)缺氧前用终浓度为1.0 μmol/L PEP-1-HO-1融合蛋白孵育细胞;高浓度融合蛋白组(H-HO组)缺氧前即刻用终浓度为2.0 μmol/L PEP-1-HO-1融合蛋白孵育细胞.复氧结束后收集细胞及培养液上清,采用2,4-二硝基苯肼显色法检测培养液乳酸脱氢酶(LDH)活性,硫代巴比妥酸比色法检测细胞MDA含量,黄嘌呤氧化酶法检测细胞SOD活性.结果 与C组比较,H/R组、L-HO组、H-HO组心肌细胞SOD活性降低,MDA含量升高,培养液LDH活性升高(P＜0.05);与H/R组比较,L-HO组和H-HO组心肌细胞SOD活性升高,MDA含量降低,培养液LDH活性降低(P＜0.05);与L-HO组比较,H-HO组心肌细胞SOD活性升高,MDA含量降低,培养液LDH活性降低(P＜0.05).结论 PEP-1-HO-1融合蛋白转导入大鼠H9c2心肌细胞可减轻细胞缺氧复氧损伤.
目的 探討PEP-1-血紅素加氧酶(HO)-1融閤蛋白轉導對大鼠H9c2心肌細胞缺氧複氧損傷的影響.方法 構建含人HO-1基因的原覈錶達質粒pETl5b-PEP1-hHO-1,質粒轉化後誘導目的 蛋白PEP-1-HO-1錶達.用含15%胎牛血清高糖DMEM培養基培養H9c2心肌細胞,隨機分為4組(n=4):正常對照組(C組)常規培養;缺氧複氧組(H/R組)細胞缺氧22 h,複氧8 h;低濃度融閤蛋白組(L-HO組)缺氧前用終濃度為1.0 μmol/L PEP-1-HO-1融閤蛋白孵育細胞;高濃度融閤蛋白組(H-HO組)缺氧前即刻用終濃度為2.0 μmol/L PEP-1-HO-1融閤蛋白孵育細胞.複氧結束後收集細胞及培養液上清,採用2,4-二硝基苯肼顯色法檢測培養液乳痠脫氫酶(LDH)活性,硫代巴比妥痠比色法檢測細胞MDA含量,黃嘌呤氧化酶法檢測細胞SOD活性.結果 與C組比較,H/R組、L-HO組、H-HO組心肌細胞SOD活性降低,MDA含量升高,培養液LDH活性升高(P＜0.05);與H/R組比較,L-HO組和H-HO組心肌細胞SOD活性升高,MDA含量降低,培養液LDH活性降低(P＜0.05);與L-HO組比較,H-HO組心肌細胞SOD活性升高,MDA含量降低,培養液LDH活性降低(P＜0.05).結論 PEP-1-HO-1融閤蛋白轉導入大鼠H9c2心肌細胞可減輕細胞缺氧複氧損傷.
목적 탐토PEP-1-혈홍소가양매(HO)-1융합단백전도대대서H9c2심기세포결양복양손상적영향.방법 구건함인HO-1기인적원핵표체질립pETl5b-PEP1-hHO-1,질립전화후유도목적 단백PEP-1-HO-1표체.용함15%태우혈청고당DMEM배양기배양H9c2심기세포,수궤분위4조(n=4):정상대조조(C조)상규배양;결양복양조(H/R조)세포결양22 h,복양8 h;저농도융합단백조(L-HO조)결양전용종농도위1.0 μmol/L PEP-1-HO-1융합단백부육세포;고농도융합단백조(H-HO조)결양전즉각용종농도위2.0 μmol/L PEP-1-HO-1융합단백부육세포.복양결속후수집세포급배양액상청,채용2,4-이초기분정현색법검측배양액유산탈경매(LDH)활성,류대파비타산비색법검측세포MDA함량,황표령양화매법검측세포SOD활성.결과 여C조비교,H/R조、L-HO조、H-HO조심기세포SOD활성강저,MDA함량승고,배양액LDH활성승고(P＜0.05);여H/R조비교,L-HO조화H-HO조심기세포SOD활성승고,MDA함량강저,배양액LDH활성강저(P＜0.05);여L-HO조비교,H-HO조심기세포SOD활성승고,MDA함량강저,배양액LDH활성강저(P＜0.05).결론 PEP-1-HO-1융합단백전도입대서H9c2심기세포가감경세포결양복양손상.
Objective To investigate the effect of PEP-1-heme oxygenase-1 (PEP-1-HO-1) fusion protein transduction on hypoxia-reoxygenation (H/R) injury in rat H9c2 cells. Methods After construction of the prokaryotic expression plasmid pET15b-PEP-1-hHO-1 containing the human heme oxygenase-1 gene, it was then transformed to make PEP-1-HO-1 fusion protein express. The H9c2 cells were cultured in high-glucose Dulbecco's modified Eagle's medium (DMEM) supplemented with 15% fetal bovine serum and randomly divided into 4 groups (n = 4 each): control group (group C), H/R group, low-concentration fusion protein group (group L-HO), and high-concentration fusion protein group (group H-HO). The cells were exposed to 22 h of hypoxia followed by 8 h of reoxygenation. PEP-1-HO-1 fusion protein was added to the culture medium with a final concentration of 1.0 μ mol/L (group L-HO) or 2.0 μmol/L (group H-HO) before hypoxia. The cells and supernatant of the culture medium were collected after reoxygenation to determine the activity of lactate dehydrogenase (LDH) in the supernatant and the content of malondialdehyde (MDA) and activity of superoxide dismutase (SOD) in the cells. Results The SOD activity was significantly lower, while the MDA content and LDH activity were significantly higher in group H/R, L-HO and H-HO than in group C (P ＜0.05). The SOD activity was significantly higher, while MDA content and LDH activity were significantly lower in group L-HO and H-HO than in group H/R, and in group H-HO than in group L-HO ( P ＜ 0.05). Conclusion PEP-1-HO-1 fusion protein transdution can protect H9c2 cells against H/R injury in rats.
