细胞因子(IL-6、TNF-α)在损伤时间中原位杂交法研究

STUDY ON EXPRESSION OF CYTOKINES(IL-6,TNF-α)BY IN SITU HYBRIDIZATION FOR TIMING OF INJURIES

采用原位杂交法，研究大鼠切削皮肤中细胞团子IL－6、TNF－αmRNA表达量，旨在探讨IL－6、TNF－α推断法医损伤时间的应用价值以及其在损伤生活反应中的分子机制。研究结果表明，根据大鼠损伤皮肤中TNF－αmRNA表达量能够区别生前伤与死后伤，并可以利用TNF－αmRNA表达量改变准确区别60min内和60min后损伤时间，但IL－6mRNA表达量在研究组内均未见阳性反应，不能推断损伤时间。

In situ hybridization was developed to study expressions of IL-6 mRNA and TNF-a mRNA onratts skin incised wounds in order to find out the roles of IL-6 and TNF-α in estimation of time of in-juries. Rat's IL-6 cDNA PMT,T plasrnid and TNF-a cDNA PBR322 plasmid were treated with restric-tion enzymes EcoR I or Pst I. IL,-6 cDNA (560bp) and TNF-α cDNA (800bp) were extracted.Probes were labelled by digoxigenin random primer method. In site hybridization was carried out bythe dot-blotting. 48 Sprague Dawley rats were randomly divided into five groups according to survivaltime after injuries: 15min, 30min, 60min, 90min and 120min groups. 3 rats each group. Master groupsinclude antemortem wound, postmortem wound, wound made under anesthesia and normal groups. ln-cised wounds were made on ratls dorsum. Skin from edge of wounds were taken as sample,After hy-bridization, image analysis and statistical calculation were performed. The results were as follows:There was positive reaction of TNF-a mRNA in samples from antemortetri wound and wounds madein anesthesia groups. In samples of postmortem or normal skin groups,negative react0n was observed.So that the expression of TNF-a mRNA in injuried tissues could be used for distinguishing ante-mortem frorn postmortern wounds- Expression of TNF-a mRNA showed no linear relationship withthe clape of the time after injury- However, in the antemortern, incised group,a significant difference ofexpression of TNF mRNA was observed 60min and 60min aftor inflicting wounds. As for the expres-sion of IL 6 mRNA,no positive reaction was observed in all samples.