Ⅰ、Ⅲ型前胶原基因第2外显子核酶对靶RNA的体外切割活性的研究

In vitro cleavage activity of the ribozymes constructed specifically for the second exons of pro alpha Ⅰand Ⅲ collagen genes

目的体外研究锤头型α1 Ⅰ型及Ⅲ型前胶原基因第2外显子片段核酶对各自靶RNA分子的切割活性及反应条件.同时观察两反义核酶对瘢痕中成纤维细胞胶原合成的影响.方法将含α1 Ⅰ型及Ⅲ型前胶原基因第2外显子片段的重组质粒(pT-Ⅰ、pT-Ⅲ),经体外32p标记转录后形成产物靶RNA.同时将含特异性核酶基因的重组质粒(pT-g Ⅰ、pT-gⅢ)进行非标记的体外转录,产物(核酶)与各自的32P-靶RNA按不同的条件混和反应,电泳后放射自显影观察结果.将构建好的核酶以脂质体包裹后导入培养的成纤维细胞内,采用图像分析法观察核酶对成纤维细胞Ⅰ型、Ⅲ型胶原蛋白mRNA合成的影响.结果两种核酶在37℃、42℃均能有效切割各自的靶RNA,对Mg2+浓度的要求范围较宽(10～20mmol/L);反应温度从65℃逐渐降至并维持在37℃的条件下核酶切割活性显著提高.Ⅰ、Ⅲ型胶原蛋白mRNA的表达明显降低,胶原蛋白生成降低,胶原生成明显受抑制.结论针对α1 Ⅰ型及Ⅲ型前胶原基因第2外显子片段的核酶能有效地在体外对靶RNA进行切割并能有效地抑制瘢痕中成纤维细胞胶原的合成.

Objective To study the in vitro cleavage activity and the reaction conditions of the hammerhead ribozymes for the second exons of pro alpha 1Ⅰand Ⅲcollagen genes, and observe the effect of the two ribozymes on collagen synthesis by the fibroblasts in scar tissue. Methods The fragments of the second exons of pro alpha 1Ⅰand Ⅲcollagen genes were cloned in-to the plasmids pT-Ⅰand pT-Ⅲand labeled with 32 P during transcription to obtain the target RNA. The transcription products of the plasmids pT-gⅠand pT-gⅢcontaining specific ribozymes were incubated with the label target RNAs, respectively, un-der various conditions, and the results observed by electrophoresis autoradiography. The constructed ribozymes were trans-duced into cultured fibroblasts via liposomes for investigation of their effects on mRNA synthesis of type Ⅰand Ⅲcollagen protein by image analysis. Results The two ribozymes (rgⅠand rg Ⅲ) could efficiently cleave their target RNAs at both 37 ℃and 42 ℃, in the presence of Mg 2+ within a relatively wide concentration range form 10 to 20 mmol/L. When the temperature was lowered from 65 ℃to 37 ℃and maintained at 37 ℃, the cleavage activity of the ribozymes reached the maximum. The expression of mRNA of Ⅰand Ⅲcollagen decreased accompanied by reduced collagen synthesis. Conclusion Hammerhead ribozymes for the fragments of the second exons of pro alpha 1Ⅰand Ⅲcollagen genes can cleave its target RNAs in vitro and effectively inhibit the collagen synthesis by the scar-derived fibroblasts.