摘要目的 构建针对FMNL2基因的shRNA表达载体,以用于后续的功能学实验,并观察其对SW620细胞增殖的影响.方法 依据设计shRNA的原则,针对人FMNL2的mRNA序列,设计并合成编码的shRNA的两条寡核苷酸序列,构建重组质粒PGenesil-FMNL21和PGenesil-FMNL22,并设阴性对照PGenesil-HK.转染重组质粒至SW620中,转染48h后荧光显微镜下观察转染率,应用荧光定量PCR法检测PGenesil-FMNL21和PGenesil-FMNL22的瞬时干扰效果;应用MTT法检测转染48h和72h后对SW620增殖活性的影响.结果 酶切及测序证实重组质粒构建成功;转染48h荧光显微镜观察转染率:PGenesil-FMNL21为(60.8±2.8)%,PGenesil-FMNL22为(58.7±2.9)%,PGenesil-HK为(62.6±1.7)%,三者之间差异无统计学意义(P＞0.05);PGenesil-FMNL21对FMNL2基因的抑制效率最高为77.1%;转染12、24、48、72、96h后PGenesil-FMNL21对FMNL2的抑制率分别为13.5%、57.3%、80.3%、62.4%、33.2%;MTT检测在转染48 h和72 h后,实验组细胞的增殖活性小于对照组细胞的增殖活性(P＜0.05).结论 PGenesil-FMNL21和PGenesil-FMNL22均有沉默FMNL2基因的作用,其中PGenesil-FMNL21的沉默作用最大;FMNL2可能对促进细胞的增殖有一定的作用.

abstractsObjective To construct the specific small hairpin RNA(shRNA)plasmids for Formin-like 2(FMNL2)gene,and to observe its effect on proliferation of SW620.Methods Plasmids FMNL21 shRNA and plasmids FMNL22 shRNA complimentary to the sequence responsible for the function domain of human FMNL2 were constructed and transfected into SW620,respectively.The inhibition rates of FMNL2 expression were detected by re-al-time PCR after transfection with PGenesil-FMNL21 and PGenesil-FMNL22,respectively.The expression of FMNL2 was detected by real-time PCR at 12,24,48,72 and 96h after transfection.At 48 h and 72 h after transfection,the proliferation of SW620 was detected using MTT assay.Resuits The transfection rate in SW620 was(60.8±2.8)%for PGenesil-FMNL21,(58.7±2.9)%for PGenesil-FMNL22,and(62.6±1.7)%for PGenesil-HK(P＞0.05).The inhibition rate of FMNL2 expression after transfection with PGenesil-FMNL21 was 77.1%,which was the highest.The inhibition rates of FMNL2 expression were 13.5%,57.3%,80.3%,62.4%,and 33.2%at 12,24,48,72and 96h after transfection respectively.The proliferat capability of SW620 was obviously lower than that of control cells(P＜0.05).Conclusion Both plasmids FMNL21 shRNA and plasmids FMNL22 shRNA can inhibit the expression of FMNL2,of which Pgenesil-FMNL21 execs the greatest role.FMNL2 could be related to the proliferation of SW620.