新型联合基质桥接大鼠脊髓横断性损伤断端促进运动功能恢复实验研究  

A NEW COMPOSITE MATRIX BRIDGING BOTH STUMPS OF SPINAL CORD TRANSECTION IN RATS TO PROMOTE RECOVERY OF MOTOR FUNCTION

作　　者：梁洪生[1] 梁鹏[1] 高爱丽[2] 祁权[1] 刘恩重[1] 吴佳宁[1] 徐小平[1] 

机构地区：[1]哈尔滨医科大学第一临床医学院,哈尔滨150001 [2]东北农业大学生命科学学院

出　　处：《中国修复重建外科杂志》2009年第11期1376-1381,共6页Chinese Journal of Reparative and Reconstructive Surgery

基　　金：国家自然科学基金资助项目(30500517);黑龙江省青年科学技术专项基金(QC05C93);哈尔滨市科技创新人才专项资金资助项目(2008RFQXS101)~~

摘　　要：目的研究新型联合基质——BMSCs-去上皮细胞羊膜(denuded human amniotic membrane,DHAM)桥接大鼠脊髓横断性损伤断端,促进轴突再生及提高后肢运动功能的作用。方法取健康产妇正常剖宫产术后自愿捐献的羊膜,经联合消化法彻底去除表面上皮细胞制备DHAM,并行HE染色观察。健康4周龄雌性Wistar大鼠4只,取骨髓行BMSCs分离、培养,取第4代细胞以PKH26标记后,以1×105个/cm2密度接种于DHAM上制备联合基质(BMSCs-DHAM)。倒置相差显微镜和激光共聚焦显微镜观察BMSCs生长状态。另取8周龄雌性Wistar大鼠40只,体重200～220g,制备T9脊髓横断性损伤模型,随机分为4组,每组10只。A、C组分别用BMSCs-DHAM和单纯DHAM包裹脊髓的2个断端,B、D组分别于脊髓损伤中心注射10μL细胞浓度为1×104个/μL的BMSCs和等量生理盐水。术后12周采用BBB运动评分检测大鼠后肢运动功能恢复情况,行皮层运动诱发电位(motion evoked potential,MEP)测定;生物素化葡聚糖胺(biopinylated dext anamine,BDA)顺行示踪检测及神经丝蛋白200(neurofilament protein200,NF-200)免疫荧光染色观察。结果HE染色显示联合消化法成功制备DHAM。镜下观察到BMSCs在DHAM上生长状态良好。术后12周,A组BBB评分达(12.50±1.26)分,明显高于其余各组(P<0.05)。MEP测定显示,A组潜伏期(3.52±2.45)ms及波幅(480.68±18.41)μV,恢复效果优于其余各组(P<0.05)。BDA顺行示踪观察示,A组损伤平面以下BDA通过损伤部位的轴突再生率为54.12%±3.30%,均高于其余各组(P<0.05);且A组NF-200免疫荧光染色亦可见大量再生轴突通过损伤部位。结论新型联合基质BMSCs-DHAM一定程度上提高了脊髓横断性损伤大鼠的后肢运动功能,是一种具有广泛应用前景的基质材料。Objective To investigate a new composite matrix （BMSCs seeded on the denuded human amniotic membrane, BMSCs-DHAM） bridging the both stumps of spinal cord injury in rats to promote axon regeneration and improve motor function of hind limbs. Methods The human amniotic membrane （HAM） was voluntarily donated by the healthy pregnant women after a caesarean section. The cells on the HAM were completely removed with a tryptic and mechanical approach to prepare DHAM. The BMSCs were separated and cultured from 4-week-old female rats （n=4）, then the forth passage of BMSCs were labeled by PKH26 and seeded on DHAM （BMSCs-DHAM）. The growing state of BMSCs was observed under the microscopy. Moreover, 40 female rats （8-week-old, weighting 200-220 g） were made spinal cord injury models by transecting at T9 level, and were randomly divided into 4 groups （each group, n=10）. The both stumps were respectively wrapped by BMSCs- DHAM or simple DHAM in groups A and C, and the same dose of BMSCs or physiological saline were also respectively injected the central lesion in groups B and D. At 12 weeks after surgery, the functional recovery of the hindlimbs was evaluated by the BBB locomotor rating score, and other indexes were tested including cortical motion evoked potential （MEP）, anterograde biopinylated dextan amine （BDA） tracing, and immunoflnorescence of neurofilament protein 200 （NF-200）. Results HE staining proved that the DHAM was devoid of cellular components by this way, and BMSCs grew well on the substrate under the microscopy. At 12 weeks after operation, the BBB score （12.50 ± 1.26） in group A was significantly higher than those of other groups （P 〈 0.05）, and the recovery in latency （3.52 ± 2.45） ms and amplitude （480.68 ± 18.41） μV of MEP was also obviously improved in group A （P 〈 0.05） when compared with other groups. In addition, anterograde BDA tracing revealed that the rate of the positive BDA axons 54.12% ± 3.30% under the lesion level in group A was hi

关 键 词：组织工程 BMSCS 去上皮细胞羊膜 脊髓损伤 轴突再生 大鼠 

分 类 号：R651.2[医药卫生—外科学] R318.08[医药卫生—临床医学]