In vitro cleavage of collagen mRNA in Ito cells by ribozyme

1. lto细胞的纯化方法：1.大鼠肝脏灌流，胶原酶消化后，用甲泛葡胺溶液离心分离lto细胞.目的RNA的合成将1型胶原α（1）的cDNA插入pBluescript中，用染料引物法测定其序列。证实该cDNA具有Gly-X-Y序列，对胶原基因具有特异性。通过使用[3 P]-UTP体外转录合成靶RNA<32>。核酶的合成选择了靶RNA中含有GUC序列的几个区域，设计了与这些区域互补的锤头状核酶。以化学合成的核苷酸为模板，以PCR产物为模板DNA，体外转录合成核酶.核酶对胶原mRNA的体外切割将靶RNA（胶原mRNA）与核酶在37 ℃孵育，通过电泳和放射自显影检测胶原mRNA的切割片段。从孵育后30分钟起检测到切割活性，并且最大切割约70%的胶原mRNA。最佳条件是靶/核酶比为1/1（摩尔基础）和MgCl_2浓度为10 mM。5.核酶对lto细胞胶原mRNA表达的影响将lto细胞与核酶共同孵育，用放射性标记的寡聚DNA进行北方印迹分析，以确定与胶原mRNA互补的寡聚DNA对lto细胞胶原mRNA表达的影响。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.