In vitro cleavage of collagen mRNA in Ito cells by ribozyme

1. 1 .大鼠lto细胞经肝灌注、胶原酶消化后，用甲硝唑胺溶液离心分离。将目标RNARat型胶原α (1)cDNA插入pBluescript中，用染料引物法测序。证实该cDNA具有胶原蛋白基因特异性的Gly-X-Y序列。目的RNA通过体外转录[^<32>P]-UTP.3合成。核酶的合成选择目标RNA中具有GUC序列的几个区域，设计与这些区域互补的锤头核酶。该核糖酶由模板DNA体外转录合成，模板DNA由化学合成的寡核苷酸聚合酶链反应产物合成。体外核酶对胶原mRNA的裂解目的RNA（胶原mRNA）与核酶在37℃的温度下孵育，通过电泳和放射自显影检测胶原mRNA的裂解片段。孵育30 min后检测裂解活性，最高可裂解约70%的胶原mRNA。最佳条件为靶核酶比为1/1（摩尔基），MgCl_2浓度为10 mM.5。核酶对lto细胞胶原mRNA表达的影响核酶对lto细胞中胶原mRNA表达的影响核酶对lto细胞中胶原mRNA表达的影响核酶对lto细胞中胶原mRNA表达的影响通过Northern blotting检测胶原mRNA表达。裂解活性检测不足。我们正计划用腺病毒载体将核酶转移到lto细胞中。

1. Purification of lto cellsRat lto cells were separated by centrifugation using metrizamide solution after perfusion and digestion of the liver with collagenase.2. Synthesis of target RNARat type l collagen alpha (1)cDNA was inserted into pBluescript and sequenced by dye primer method. It was confirmed that this cDNA had Gly-X-Y sequence, specific for collagen genes. The target RNA was synthesized by in vitro transcriprtion using [^<32>P]-UTP.3. Synthesis of ribozymeThe several regions with GUC sequence in target RNA were selected and hammerhead ribozyme complimentary to such regions was designed. The ribozyme was synthesized by in vitro transcription from template DNA,which was made from PCR products using chemically synthesized oligonucleotides.4. In vitro cleavage of collagenmRNA by ribozymeThe target RNA (collagen mRNA) was incubated with ribozyme at 37ﾟC and the cleaved fragments of collagen mRNA were detected by electrophoresis and autoradiograph. The cleavage activity was detected since 30 min after incubation and approximately 70% of collagen mRNA was cleaved at maximum. The optimal conditions were a target/ribozyme ratio of 1/1 (molar basis) and a MgCl_2 concentration of 10 mM.5. Effect of ribozyme on expression of collagen mRNA in lto cellsIto cells were incubated with the addition of ribozyme and the expression of collagen mRNA was determined by Northern blotting using radiolabeled oligo DNA prove complimentary to collagen mRNA.The cleavage activity was not sufficiently detected. We are planning the transfer of ribozyme into lto cells using adenovirus vectors.