成纤维细胞Ⅰ型胶原基因shRNA载体的构建及干扰效果

Construction of fibroblast human type Ⅰ collagen gene targeted short hairpin RNA expression vector and its interference efficacy

目的构建有效针对人成纤维细胞Ⅰ型胶原基因的shRNA干扰载体,检测RNA干扰后对病理性瘢痕成纤维细胞胶原合成的影响。方法设计合成4对针对人Ⅰ型胶原基因的shRNA靶序列（shRNA1、shRNA2、shRNA3、shRNA4）,将合成的shRNA片段通过重组技术克隆到真核表达载体pm U6,经酶切与测序验证表明成功构建针对人Ⅰ型胶原基因的shRNA干扰载体（Col-shRNA1、Col-shRNA2、Col-shRNA3、Col-shRNA4）,并将该干扰载体与空载体分别通过脂质体转染人的病理性瘢痕成纤维细胞（依次为Col-shRNA1组、Col-shRNA2组、Col-shRNA3组、Col-shRNA4组、空载体转染组）,另选未处理的成纤维细胞作为空白对照组,采用RT-PCR与羟脯氨酸含量测定试剂盒检测该干扰载体的干扰效应。结果酶切与测序结果表明成功构建了针对人Ⅰ型胶原基因的shRNA干扰载体。Ⅰ型胶原基因mRNA的相对表达水平在空白对照组与空载体转染组之间差异无统计学意义（P〉0.05）;而干扰组Col-shRNA（1-4）与空载体转染组之间比较,差异有统计学意义（P〈0.05,P〈0.01）。空白对照组与空载体转染组的羟脯氨酸含量及胶原合成差异无统计学意义,干扰组（Col-shRNA1、ColshRNA2、Col-shRNA3、Col-shRNA4）的羟脯氨酸含量和胶原合成与空载体转染组比较,差异有统计学意义（P〈0.05,P〈0.01）。结论成功构建了4组有效针对人Ⅰ型胶原基因的shRNA干扰载体,它们均能明显抑制病理性瘢痕成纤维细胞Ⅰ型胶原基因的表达与减少细胞胶原的合成,其中Col-shRNA1对成纤维细胞Ⅰ型胶原基因表达的干扰效果最强。

Objective To construct expression vectors of short hairpin RNA （shRNA） effectively targeting human type ] collagen gene and to detect their interference effects on collagen synthesis in the fibroblasts from pathological cicatrix. Methods Four pairs of shRNA sequences targeting human type ] collagen gene were designed and synthesized, and then followed by anneal and recombination into eukaryotie expression vector pmU6, which was evaluated and identified by enzyme digestion and sequencing. The shRNA recombinant vectors and control empty vectors were respective- ly transfected into fibroblasts from pathological cicatrix by liposome. Reverse transcription - polymerase chain reaction （ RT - PCR） and a detection kit for hydroxyproline were used to measure their interference effects. Results Restriction enzyme digestion and sequence analysis confirmed that the shRNA vectors targeting human type I collagen gene were suc- cessfully constructed. The results of RT - PCR detection indicated that there was no statistical significance of the relative expression level of type I collagen mRNA between blank control group and empty - vector transfeeted group （ P 〉 0. 05 ） , but there was apparently statistical significance between ColshRNA（ 1 -4） groups and empty -vector transfeeted group （P 〈 0. 01, P 〈 0. 05 ）. The results of hydroxyproline content determination demonstrated that there was no statistical significance for hydroxyproline content and collagen synthesis between blank control group and empty - vector transfected group （ P 〉 0. 05 ）, but obviously statistical significance was observed between empty - vector transfected group and the groups of Col - shRNA1, Col - shRNA2, Col - shRNA3, an Col - shRNA4 （ P 〈 0. 01, P 〈 0. 05）. Conclusion Four shRNA interference vectors targeting human type collagen gene are successfully constructed, which strongly inhibit the expression of type 1 collagen gene in fibroblasts from pathological cicatrix and reduce collagen synthesis. Col - shRNA1 vector provides the strongest efficacy inhibiting human type I collagen gene expression in fibroblasts.