CAJ | 학술논문

目的观察Rho/ROCK Ⅱ特异抑制性小分子多肽在脊髓损伤(SCI)微环境下对新生大鼠背根节神经元(DRGNs)轴突生长的影响. 方法取健康雌性SD成年大鼠5只,按WD法制成T9平面以下截瘫模型,术后7d取T8-10节段脊髓制作截瘫大鼠脊髓提取液.取新生SD大鼠背根神经节经酶解消化、机械吹打、离心、重悬、纯化,进行原代培养观察.DRGNs体外培养5d后随机分组加入不同物质共同培养:A组:DRGNs +60 μL PBS;B组:DRGNs+60 μL截瘫大鼠脊髓提取液；C组:DRGNs+60 μL截瘫大鼠脊髓提取液+20 μL脂质体；D组:DRGNs+ 60μL截瘫大鼠脊髓提取液+20 μL脂质体+不同量多肽(2、4、6、8、10、12 μg).不同环境下共同培养2d后行免疫荧光,测量神经轴突长度和轴突远端平均荧光密度. 结果 B、C组平均轴突长度和荧光密度均小于其他组,差异有统计学意义(P＜0.05),而B、C组间差异无统计学意义(P＞0.05).8μg多肽组平均轴突长度增长最明显,平均荧光密度最大,与其他多肽组比较差异均有统计学意义(P＜0.05);2 μg多肽组与4μg多肽组的平均轴突长度和荧光密度比较差异均无统计学意义(P＞0.05)；6μg多肽组、10 μg多肽组、12μg多肽组的平均轴突长度和荧光密度比较差异均无统计学意义(P＞0.05). 结论 Rho/ROCKⅡ特异抑制性小分子多肽能促进SCI微环境中DRGNs轴突生长,当多肽含量为8μg时作用最明显.
목적 관찰Rho/ROCK Ⅱ특이억제성소분자다태재척수손상(SCI)미배경하대신생대서배근절신경원(DRGNs)축돌생장적영향. 방법 취건강자성SD성년대서5지,안WD법제성T9평면이하절탄모형,술후7d취T8-10절단척수제작절탄대서척수제취액.취신생SD대서배근신경절경매해소화、궤계취타、리심、중현、순화,진행원대배양관찰.DRGNs체외배양5d후수궤분조가입불동물질공동배양:A조:DRGNs +60 μL PBS;B조:DRGNs+60 μL절탄대서척수제취액；C조:DRGNs+60 μL절탄대서척수제취액+20 μL지질체；D조:DRGNs+ 60μL절탄대서척수제취액+20 μL지질체+불동량다태(2、4、6、8、10、12 μg).불동배경하공동배양2d후행면역형광,측량신경축돌장도화축돌원단평균형광밀도. 결과 B、C조평균축돌장도화형광밀도균소우기타조,차이유통계학의의(P＜0.05),이B、C조간차이무통계학의의(P＞0.05).8μg다태조평균축돌장도증장최명현,평균형광밀도최대,여기타다태조비교차이균유통계학의의(P＜0.05);2 μg다태조여4μg다태조적평균축돌장도화형광밀도비교차이균무통계학의의(P＞0.05)；6μg다태조、10 μg다태조、12μg다태조적평균축돌장도화형광밀도비교차이균무통계학의의(P＞0.05). 결론 Rho/ROCKⅡ특이억제성소분자다태능촉진SCI미배경중DRGNs축돌생장,당다태함량위8μg시작용최명현.
Objective To explore the effect of micromolecular polypeptide of Rho/ROCK Ⅱ inhibitors on the axonal growth of dorsal root ganglia neurons (DRGNs) after spinal cord injury in neogenetic rats.Methods Five healthy adult female SD rats were subjected to weight-drop impact which caused complete paraplegia at T9 level.The T8-10 spinal cord extracts (SCE) were harvested at day 7 after spinal cord injury.All thoracolumbar DRGNs (20 to 25 DRGNs per rat) were harvested from neogenetic SD rats (＜5d) under the stereopsis microscopy before they were digested,centrifuged,cultured,purified and indentified.The experiment was conducted in 4 different mediums:DRGNs + 60 μL PBS (group A),DRGNs +60 μL SCE (group B),DRGNs + 60 μL SCE + 20 μL liposome (group C),and DRGNs + 60 μL SCE +20 μL liposome + polypeptide (of 2,4,6,8,10 and 12 μg respectively) (group D).The axonal length,positive expression of Tubulin β Ⅲ and mean fluorescence density at the axonal distal end of DRGNs were observed with phase-contrast microscopy and immunofluorescence after co-culture for 2 days.Results The axonal length and mean fluorescence density at the axonal distal end in groups B and C were significantly smaller than in the other 2 groups (P ＜ 0.05) while there were no significant differences between groups B and C (P ＞ 0.05).In the 8 μg polypeptide group the average axonal length and expression of tubulin β Ⅲ at the axonal distal end were significantly greater than in other polypeptide groups (P ＜ 0.05); there were no significant differences between the 2 μg and 4 μg polypeptide groups regarding the average axonal length and expression of tubulin β Ⅲ at the axonal distal end (P ＞ 0.05); there were no significant differences either between the 6 μg,10 μg and 12 μg polypeptide groups (P ＞ 0.05).Conclusion The micromolecular polypeptide of Rho/ROCK Ⅱ inhibitors can promote the axonal growth of DRGNs after spinal cord injury in neogenetic rats,particularly in the medium with 8μg polypeptide.