干扰串珠素表达对硬膜外瘢痕细胞中Ⅰ型胶原蛋白生成的影响

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

目的探讨干扰素表达对椎板切除后硬膜外瘢痕成纤维细胞中Ⅰ型胶原蛋白生成的影响。方法 选取30只2个月龄的SD雄性大鼠，行后路L_4-5腰椎全椎板切除术。术后第20天取每只大鼠硬膜后方的瘢痕组织，采用组织块贴壁法进行细胞培养。筛选第3代瘢良组织培养的成纤维细胞分别3组（n=6）：慢病毒介导小发夹RNA（shRNA）干扰组Ⅰ进行慢性病毒介导shRNA干扰串珠素表达实验1、纯慢病毒无shRNA组和空白组。通过逆转录-聚合酶链反应（RT-PCR）、Western-Blot检测各组Ⅰ型胶原蛋白的生成量，并进行比较。结果 最佳感染复数为100时，细胞形态正常，轮廓清晰，确定其为最佳感染复数值。RT-PCR测定慢病毒介导shRNA干扰组、纯慢病毒无shRNA组、空白组Ⅰ型胶原蛋白RNA目的条带吸光度与内参条带吸光度比值平均分别为0.043±0．003、0．473±0.011、0．475±0.013，Western Blot测定Ⅰ型胶原蛋白RNA目的条带吸光度与内参条带吸光度比值平均分别为0．190±0．005、2．116±0．012、2．122±0．009，3组间以上项目比较差异均有统计学意义（P＜0．05），其中慢病毒介导shRNA干扰组Ⅰ型胶原蛋白生成量较纯病毒无shRNA组及空白组明显减少，差异均有统计学意义（P〈0．05）。结论 干扰串珠素表达能有效减少椎板切除术后硬膜外瘢痕成纤维细胞生成Ⅰ型胶原蛋白，对抑制术后硬膜外瘢痕形成一定作用。

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 lumamr laminectomy on L_4-5. 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 perlecann 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 lo 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 cut-tured cells exhibited normal morphology and clear outline, RT-PCR showed Ihe 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 iu 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 medialed shRNA can reduce the expression of type Ⅰ collagen in epidural scar fibroblasts, it can be used to inhibit epidural sear fornation after laminectomy.