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

激活的血管内皮细胞（ECs）产生多种因子参与术后病理变化的发生。本研究以TNF-α激活的人脐静脉内皮细胞总RNA为模板，分别用正、反义引物进行RT/PCR，扩增出与ICAM-1、VCAM-1、E-选择素和PDGF-A链互补的cDNA<alpha>。将cDNA亚克隆到质粒中并检查其序列。将重组腺病毒的cDNA与特异性载体连接，制备腺病毒载体。在293细胞中产生针对ICAM-1、VCAM-1和E-选择mRNA的反义腺病毒载体，将重组腺病毒载体转染内皮细胞，用RT/PCR方法检测ICAM-1的转录水平，用细胞ELISA法检测ICAM-1反义腺病毒载体转染内皮细胞后，经脂多糖激活的内皮细胞ICAM-1的表达。转染反义腺病毒载体不能降低脂多糖诱导的ICAM-1表达。我们认为该载体不能降低ICAM-1表达的原因可能是由于该载体的表达盒中含有一个143 bp的短cDNA序列和一个200 bp的长错配序列。而且，我们又经历了一次反义腺病毒载体的失败。胰岛素反义腺病毒载体不能降低转染胰岛素腺病毒载体的内皮细胞的胰岛素分泌。由于该载体含有334 bp的反向cDNA和200 bp的错配序列，因此，错配序列可能确实抑制了反义mRNA与正义mRNA的结合，或者反义处理可能不如报道的那样有效。

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.