The effect of interference of perlecan expression on generation of type I collagen in epidural scar cells

Abstract

Objective To investigate the effect of interference of perlecan expression on generation of type Ⅰ collagen in epidural scar cells.Methods Thirty SD male rats,weighing 250 grams and 2 months of age,were subjected to complete posterior lumbar laminectomy on L4-5.The epidural scar tissues were sampled at 20 days postoperatively for cellular culture by adherence.The third passage fibroblasts derived from the cellular culture were randomized into 3 groups (6 samples in each group):group A subjected to interference of perlecan expression by lentivirus mediated shRNA,group B subjected to interference of perlecan expression by lentivirus without shRNA mediation and group C,the blank control.Then hematoxylin-eosin staining (HE) was done to observe the formation of epidural scar and the activity of fibroblasts and the quantity of collagen synthesis.Western Blot and RT-PCR were performed to quantify the expression of perlecan and generation of type Ⅰ collagen after interference by lentivirus mediated shRNA.Comparisons were done between the 3 groups.Results The multiplicity of infection of 100 was the optimal when the cultured cells exhibited normal morphology and clear outline.RT-PCR showed the ratio of absorbance of the target bands of the type Ⅰ collagen RNA to that of the reference bands was 0.043 ± 0.003 in group A,0.473 ±0.011 in group B and 0.475 ± 0.013 in group C.Western Blot showed the ratio of absorbance of the target bands of the type Ⅰ collagen RNA to that of the reference bands was 0.190±0.005 in group A,2.116±0.012 in group B and 2.122±0.009 in group C.There were significant differences between group A and groups B and C regarding the above indexes (P ＜ 0.05).The generation of type Ⅰ collagen in group A was significantly lower than in groups B and C (P ＜ 0.05).Conclusion Since the interference of perlecan expression by lentivirus mediated shRNA can reduce the expression of type Ⅰ collagen in epidural scar fibroblasts,it can be used to inhibit epidural scar formation after laminectomy. Key words: Laminectomy; Cicatrix; Collagen type Ⅰ; Fibroblasts; Perlecan