【摘要】 目的:采用Brdu免疫组化标记技术观察经静脉移植人脐血间充质干细胞进入损伤脊髓组织的表达情况,探讨其促进损伤脊髓神经功能恢复的作用。方法:实验于2003-10/2005-03在苏州大学生物技术研究所干细胞实验室完成。①选取健康成年Wistar大鼠60只,随机数字表法分为假手术组、模型对照组、干细胞移植组,20只/组。脐血标本来自苏州大学附属第一医院产科,产妇及其家属均签署知情同意书。②自脐带抽取新鲜脐带血50～60mL,分离培养人脐血间充质干细胞,调整细胞浓度为5×109L-1待用。在细胞移植前48h,体外进行Brdu标记。③模型对照组、干细胞移植组大鼠采用重物压迫法建立脊髓损伤模型。麻醉后取俯卧位,于T11节段处行背后方正中切口,剥离椎旁肌,咬除T11棘突及椎板,显露4mm×5mm硬脊膜,将55g重砝码放置于硬脊膜表面1min,然后逐层缝合伤口。假手术组仅显露硬脊膜,然后缝合伤口。④造模后5d,干细胞移植组大鼠自后肢大隐静脉缓缓注入人脐血间充质干细胞悬液0.2mL～0.3mL(1×106个)。模型对照组、假手术组给予等量生理盐水。⑤分别于细胞移植后1d、1,2,3周,采用BassoBeatlieBresnahan(BBB)评分法和斜板试验法测定各组神经功能情况。于细胞移植后1,3周制作冰冻切片,检测各组脊髓组织内脐血间充质干细胞的分布情况。结果:①神经功能检测结果:脊髓损伤细胞移植后3周内,模型对照组、干细胞移植组大鼠后肢运动功能均有所恢复,但干细胞移植组恢复较快,于术后1周BBB评分和斜板试验结果即明显优于模型对照组[(10.71±6.24),(5.13±3.36)分,t=2.39,P<0.05;(51.67±5.22),(46.63±3.72)度,t=3.39,P<0.01],并维持至术后3周[(17.29±4.03),(11.25±5.01)分,t=3.89,P<0.01;(65.77±8.06),(57.05±4.61)度,t=4.07,P<0.01]。②脊髓组织中人脐血干细胞的鉴定:假手术组、模型对照组脊髓组织中未见Brdu阳性表达区。干细胞移植组于移植后1周脊髓损伤区可见大量棕褐色Brdu阳性表达的人脐血间充质干细胞存在,并持续至移植后3周,而非损伤区则始终无阳性表达。结论:Brdu对脐血间充质干细胞具有良好的标记作用,经静脉移植的人脐血间充质干细胞能够进入脊髓损伤区,并可以促进损伤脊髓神经功能的修复。更多还原

【Abstract】 AIM: To observe the vein translated human umbilical cord blood(hUCB) mesenchymal stem cells infiltrating into the injured spinal cord tissues by Brdu immunohistochemical labeled technique and explore its role of improving neurological function recovery. METHODS: The experiment was performed in Stem Cell Laboratory of the Biotechnology Institute of Soochow University from October 2003 to March 2005. ①Totally 60 healthy adult Wistar rats were selected randomly into sham operation group, model control group and stem cell transplantation group with 20 in each group. Cord blood preparation was provided by Department of Maternity, First Affiliated Hospital, Soochow University. Parturients and their family members signed the informed consent. ②50-60 mL fresh cord blood was obtained from cord blood. hUCB mesenchymal stem cells were isolated and cultured. Cells were adjusted to 5×109 L-1, which were marked by Brdu 48 hours before transplantation. ③Spinal cord injury models were established by compression method in rats of model control group and stem cell transplantation group. Rats were anesthetized at prone position and cut at middle back, striped paravertebral muscles, get rid of T11 spinal process and vertebral plate to expose 4 mm×5 mm spinal dura mater. 55 g counterweight was put on spinal dura mater for 1 minute, and then the wound was directly sewn. Only spinal dura mater was exposed in the sham operation group, and then the wound was sewn. ④5 days after establishing models, 0.2 mL-0.3 mL hUCB mesenchymal stem cells suspension (total 1×106) were slowly injected in great saphenous vein of the hind limb in the stem cell transplantation group. Those in the model control group and sham operation group received saline of the same volume. ⑤1 day, 1, 2, 3 weeks after cell transplantation, neurological function was assessed respectively by Basso Beatlie Bresnahan(BBB) and inclined plane test. 1 and 3 weeks after cell transplantation, frozen section was made to test the distribution of hUCB mesenchymal stem cells in spinal cord tissue in each group. RESULTS: ①neurological function determination: Within 3 weeks after spinal injury cell transplantation, the motor function of the hind limb in the model control group and the stem cell transplantation group was recovered, but the recovery was rapid in the stem cell transplantation group. The recovery determined by BBB score and inclined plane test was significantly improved as compared with the model control group [(10.71±6.24),(5.13±3.36) points,t =2.39,P < 0.05;(51.67±5.22),(46.63±3.72)°,t =3.39,P < 0.01], and maintained till the 3rd week after operation [(17.29±4.03),(11.25±5.01) points,t =3.89,P < 0.01;(65.77±8.06),(57.05±4.61)°,t =4.07,P < 0.01]. ②identification of hUCB mesenchymal stem cells in spinal cord tissue: There was no positive Brdu expression in the spinal cord tissue in sham operation group and model control group. A mass of positive brown Brdu expression hUCB mesenchymal stem cells appeared in injured spinal cord 1 week after transplantation in the stem cell transplantation group, and maintained till the 3rd week after transplantation while no Brdu positive cells could be seen in the other two groups. CONCLUSION: Brdu has a good labeled effect on hUCB mesenchymal stem cells. Via vein administered hUCB mesenchymal stem cells can infiltrate into the spinal cord injury region and improve neurological function.更多还原