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Experimental research about the treatment for postoperative pathological changes : control of endothelial activation using antisense adenovirus vector

反义腺病毒载体控制内皮细胞活化治疗术后病变的实验研究

基本信息

批准号：
08671385
负责人：
YAMAGUCHI Masahiko
金额：
$ 1.41万
依托单位：
Showa University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1996
资助国家：
日本
起止时间：
1996 至 1997
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-08671385/
关键词：
Antisense therapy Adenovirus vector ICAM-1 Vascular endothelial cells PDGF-A鎖

项目摘要

Activated vascular endothelial cells (ECs) produce various factors to contribute to the development of postoperative pathological changes. In this study, we investigated whether antisense adenovirus vector sgsist ICAM-1 mRNA could suppress ICAM-1 expression on activated ECs.DNAs complementary to ICAM-1, VCAM-1, E-selectin, and PDGF-A chain mRNAs were produced by RT/PCR of total cellular RNA from TNF-_<alpha> activated human umbilical vein ECs using respective sense and antisense primers. cDNAs were subcloned in plasmids and checked their sequences. Checked cDNAs were reversely ligated into the specific cosmids for generation of adenovirus vector. Antisense adenovirus vectors against ICAM-1, VCAM-1, and E-selection mRNAs was generated in 293 cells, resulting from the homologous recombination between cDNAs-reversely-ligated cosmids and adenovirus DNA.The adenovirus vectors were transfected into ECs and their transcripts were checked by RT/PCR.ICAM-1 pression was analyzed by cell-ELISA on lipopolysaccharide-activated ECs which was tranfected with antisense adenovirus vector against ICAM-1 mRNA.However, ICAM-1 expression inbuced by lipopolysaccharide could not be reduced by transfection of the antisense adenovirus vector. We considered that the reasons why the vector failed to reduced the ICAM-1 expression might be due the fact that the expression cassette of the vector included a short cDNA sequence as 143bp and a long mismatchcd sequence as 200bp. Moreover, we have experienced another failure of antisense adenovirus vector. Antisense adenovirus vector against insulin failed to reduce insulin secretion from the ECs transfected with insulin adenovirus vector. Since the vector contained 334 bp-reversed cDNA and 200bp-mismatched sequence, it is suggested that mismatched sequence may really inhibit the binding of antisense mRNA to sense mRNA or that antisense treatment may not be so effective as it has been reported.

活化的血管内皮细胞（ECs）产生多种因素促进术后病理改变的发展。在本研究中，我们研究了反义腺病毒载体sgsist ICAM-1 mRNA是否能抑制ICAM-1在活化的内皮细胞上的表达。利用TNF-_< α >激活的人脐静脉内皮细胞，分别用正义和反义引物对细胞总RNA进行RT/PCR，得到与ICAM-1、VCAM-1、E-selectin和PDGF-A链mrna互补的dna。在质粒中亚克隆cdna并检查其序列。将检查过的cdna反向连接到特定的细胞中，以产生腺病毒载体。在293细胞中，通过反向连接的cdna与腺病毒DNA的同源重组，生成了针对ICAM-1、VCAM-1和e -选择mrna的反义腺病毒载体。将腺病毒载体转染到ECs中，RT/PCR检测其转录产物。用细胞酶联免疫吸附法（elisa）分析脂多糖激活的内皮细胞对ICAM-1 mRNA的表达。然而，转染反义腺病毒载体不能降低脂多糖诱导的ICAM-1表达。我们认为，载体未能降低ICAM-1表达的原因可能是载体的表达盒中含有143bp的短cDNA序列和200bp的长mismatchcd序列。此外，我们还经历了另一次反义腺病毒载体的失败。抗胰岛素的反义腺病毒载体不能减少转染胰岛素腺病毒载体的内皮细胞的胰岛素分泌。由于载体含有334个bp反向的cDNA和200个bp错配序列，因此我们认为错配序列可能确实抑制了反义mRNA与义mRNA的结合，或者反义治疗可能不像报道的那样有效。