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目的探究TLR4突变对小鼠坐骨神经损伤修复产生的影响。方法选择TLR4基因突变型C3H/HeJ小鼠和野生型C3H/HeN小鼠作为实验动物,实验设C3H/HeJ夹伤组(HeJ)、C3H/HeN夹伤组(HeN)及C3H/HeN假手术组(control)。在无菌操作条件下,构建小鼠坐骨神经夹伤模型。分别于术后6、12、24和48 h,qPCR检测损伤神经IL-1β、IL-6、TNF-α因子的表达。术后12 h和24 h,Western blot检测损伤神经p75NTR蛋白的表达。术后1周、2周、4周和8周取小鼠坐骨神经,HE染色观察神经恢复情况,免疫组化检测GAP-43、p75NTR蛋白表达情况。坐骨神经功能指数(sciatic functional index,SFI)评价小鼠运动功能恢复情况。结果在6、12和24 h时,与control组相比,HeN组IL-1β和TNF-α的表达量显著升高(P<0.01),而HeJ组的变化不明显。HeN组与HeJ组之间TNF-α的表达差异显著(P<0.001)。与control组相比,在6 h时HeN组IL-6的表达显著上升(P<0.001)。Western blot结果显示,与HeN组相比,坐骨神经损伤后12 h或24 h时HeJ组小鼠p75NTR蛋白表达显著升高(P<0.001)。HE染色表明,与HeN组相比,HeJ组小鼠淋巴细胞表达较少,炎性反应较弱。免疫组化结果发现,与HeN组相比,HeJ组小鼠GAP43和p75NTR的表达显著升高(P<0.01)。SFI结果显示,与HeN组相比,HeJ组小鼠的SFI评分在术后同期相对较高,神经恢复更好。结论成功构建突变小鼠坐骨神经损伤模型,揭示出TLR4突变可减少炎性因子的释放而加快坐骨神经修复。为临床上治疗周围神经损伤提供了实验理论依据。
Objective To investigate the effect of TLR4 mutation on sciatic nerve injury in mice. Methods Mutant C3H / HeJ mice and wild-type C3H / HeN mice were selected as experimental animals. The C3H / HeJ group (HeJ), C3H / HeN group (HeN) and C3H / HeN sham group Control. Aseptic sciatic nerve injury models were constructed under aseptic conditions. The expressions of IL-1β, IL-6 and TNF-α in the injured nerve were detected by qPCR at 6, 12, 24 and 48 h after operation respectively. The expression of p75NTR protein in injured nerve was detected by Western blot at 12 h and 24 h after operation. After 1 week, 2 weeks, 4 weeks and 8 weeks after operation, the sciatic nerve of mice was taken out and the nerve recovery was observed by HE staining. The expression of GAP-43 and p75NTR protein was detected by immunohistochemistry. Sciatic nerve function index (sciatic functional index, SFI) evaluation of motor recovery in mice. Results At 6, 12 and 24 h, the expression of IL-1β and TNF-α in HeN group was significantly higher than that in control group (P <0.01), but not in HeJ group. The expression of TNF-α was significantly different between HeN and HeJ groups (P <0.001). Compared with control group, the expression of IL-6 in HeN group increased significantly at 6 h (P <0.001). Western blot results showed that compared with HeN group, the expression of p75NTR in HeJ group was significantly increased at 12 h or 24 h after sciatic nerve injury (P <0.001). HE staining showed that compared with HeN group, HeJ mice had less lymphocyte expression and weaker inflammatory response. The results of immunohistochemistry showed that the expression of GAP43 and p75NTR in HeJ group was significantly higher than that in HeN group (P <0.01). SFI results showed that compared with HeN group, the SFI score of HeJ group mice was relatively higher at the same period after surgery and nerve recovery was better. Conclusion The model of sciatic nerve injury was successfully established in mutant mice, which revealed that TLR4 mutation can reduce the release of inflammatory factors and accelerate the repair of sciatic nerve. It provides experimental theoretical basis for clinical treatment of peripheral nerve injury.