中华医学杂志
中華醫學雜誌
중화의학잡지
National Medical Journal of China
2010年
11期
768-771
,共4页
受体%肿瘤坏死因子%Ⅰ型%子宫内膜异位症
受體%腫瘤壞死因子%Ⅰ型%子宮內膜異位癥
수체%종류배사인자%Ⅰ형%자궁내막이위증
Receptors%Tumor necrosis factor%Type Ⅰ%Endometriosis
目的 构建LAP-MMP-hsTNFR Ⅰ-pcDNA3.1融合蛋白真核表达载体,检测融合蛋白的潜伏活性. 方法 将编码MMP分解部位的DNA序列经基因重组定向克隆至真核表达载体pcDNA3.1中,并将TGF-β1的LAP段和人可溶性肿瘤坏死因子受体(hsTNFR)1分别与MMP分解部位的N端和C端连接,构建LAP-MMP-hsTNFR Ⅰ-pcDNA3.1真核表达载体.经测序鉴定后转染COS-7细胞,通过RT-PCR及免疫印迹检测融合蛋白LAP-MMP-hsTNFR Ⅰ的表达.在融合蛋白被MMP或子宫内膜异位症患者腹腔液孵育的前后,用MTT法检测其对TNF-α细胞毒效应的抑制活性. 结果 成功构建了LAP-MMP-hsTNFR Ⅰ-pcDNA3-1重组载体,并在COS-7细胞得到了有效表达.生物学活性检测表明重组质粒LAP-MMP-hsTNFR Ⅰ转染组与空载体转染组的L929细胞死亡率差异无统计学意义(P>0.05).但在800 ng/L肿瘤坏死因子(TNF)-α作用下,重组质粒转染上清与MMP和子宫内膜异位症患者的腹腔液孵育后,L929细胞死亡率分别为(44.5±2.4)%和(33.8±1.9)%,显著低于孵育前(58.1±2.4)%,(P<0.05). 结论 LAP-MMP-hsTNFR Ⅰ重组融合蛋白的生物学活性可被LAP有效潜伏,并可被MMP和子宫内膜异位症患者的腹腔液激活,可应用于子宫内膜异位症的局部靶向治疗.
目的 構建LAP-MMP-hsTNFR Ⅰ-pcDNA3.1融閤蛋白真覈錶達載體,檢測融閤蛋白的潛伏活性. 方法 將編碼MMP分解部位的DNA序列經基因重組定嚮剋隆至真覈錶達載體pcDNA3.1中,併將TGF-β1的LAP段和人可溶性腫瘤壞死因子受體(hsTNFR)1分彆與MMP分解部位的N耑和C耑連接,構建LAP-MMP-hsTNFR Ⅰ-pcDNA3.1真覈錶達載體.經測序鑒定後轉染COS-7細胞,通過RT-PCR及免疫印跡檢測融閤蛋白LAP-MMP-hsTNFR Ⅰ的錶達.在融閤蛋白被MMP或子宮內膜異位癥患者腹腔液孵育的前後,用MTT法檢測其對TNF-α細胞毒效應的抑製活性. 結果 成功構建瞭LAP-MMP-hsTNFR Ⅰ-pcDNA3-1重組載體,併在COS-7細胞得到瞭有效錶達.生物學活性檢測錶明重組質粒LAP-MMP-hsTNFR Ⅰ轉染組與空載體轉染組的L929細胞死亡率差異無統計學意義(P>0.05).但在800 ng/L腫瘤壞死因子(TNF)-α作用下,重組質粒轉染上清與MMP和子宮內膜異位癥患者的腹腔液孵育後,L929細胞死亡率分彆為(44.5±2.4)%和(33.8±1.9)%,顯著低于孵育前(58.1±2.4)%,(P<0.05). 結論 LAP-MMP-hsTNFR Ⅰ重組融閤蛋白的生物學活性可被LAP有效潛伏,併可被MMP和子宮內膜異位癥患者的腹腔液激活,可應用于子宮內膜異位癥的跼部靶嚮治療.
목적 구건LAP-MMP-hsTNFR Ⅰ-pcDNA3.1융합단백진핵표체재체,검측융합단백적잠복활성. 방법 장편마MMP분해부위적DNA서렬경기인중조정향극륭지진핵표체재체pcDNA3.1중,병장TGF-β1적LAP단화인가용성종류배사인자수체(hsTNFR)1분별여MMP분해부위적N단화C단련접,구건LAP-MMP-hsTNFR Ⅰ-pcDNA3.1진핵표체재체.경측서감정후전염COS-7세포,통과RT-PCR급면역인적검측융합단백LAP-MMP-hsTNFR Ⅰ적표체.재융합단백피MMP혹자궁내막이위증환자복강액부육적전후,용MTT법검측기대TNF-α세포독효응적억제활성. 결과 성공구건료LAP-MMP-hsTNFR Ⅰ-pcDNA3-1중조재체,병재COS-7세포득도료유효표체.생물학활성검측표명중조질립LAP-MMP-hsTNFR Ⅰ전염조여공재체전염조적L929세포사망솔차이무통계학의의(P>0.05).단재800 ng/L종류배사인자(TNF)-α작용하,중조질립전염상청여MMP화자궁내막이위증환자적복강액부육후,L929세포사망솔분별위(44.5±2.4)%화(33.8±1.9)%,현저저우부육전(58.1±2.4)%,(P<0.05). 결론 LAP-MMP-hsTNFR Ⅰ중조융합단백적생물학활성가피LAP유효잠복,병가피MMP화자궁내막이위증환자적복강액격활,가응용우자궁내막이위증적국부파향치료.
Objective To construct a latent human soluble tumor necrosis factor receptor Ⅰ(hsTNFR Ⅰ) using the latency associated protein (LAP) of transforming growth factor-β1 (TGF-β1) fused via a matrix metalloproteinase (MMP) cleavage site to hsTNFR Ⅰ so as to detect the latent biological activity of LAP-MMP-hsTNFR Ⅰ fusion protein. Methods A double-stranded deoxyoligonucleotide coding for MMP cleavage site was cloned into plasmid pcDNA3.1. LAP and hsTNFR Ⅰ cDNA were then inserted into both two sides of MMP cleavage site. After being transferred by LAP-MMP-hsTNFR Ⅰ fusion gene with liposome, the expression of fusion protein in COS-7 cells was detected by RT-PCR and Western blot. The inhibitory effect of fusion protein upon cytotoxicity of TNF-α was detected by methyl thiazolyl tetrazolium (MTT) assay before and after the fusion protein incubated in MMP or peritoneal fluid from endometriosis patients. Results The recombinant plasmid LAP-MMP-hsTNFR Ⅰ -pcDNA3. 1 was constructed successfully and was expressed effectively in COS-7 cells. The MTT assay showed that there was no difference in the mortality rote of L929 cells between LAP-MMP-hsTNFR Ⅰ -pcDNA3.1 and empty vector transfection groups (P>0.05). The mortality rates of L929 cells with 800 ng/L TNF-α in LAP-MMP-hsTNFR Ⅰ -pcDNA3.1 transfection group after incubation with MMP or peritoneal fluid from endometriosis patients were(44.5 ±2.4) % and(33.8±1.9) % respectively. And it was lower than the pre-incubation period (58.1± 2.4) %(P<0.05). Conclusion The biological activity of LAP-MMP-hsTNFR Ⅰ fusion protein can be made latent by LAP and activated by peritoneal fluid from endometriosis. Thus a new method has been provided for a targeted therapy of endometriosis.
