目的 探讨聚左旋丙交酯纳米纤维联合骨髓间充质干细胞移植修复大鼠脊髓损伤的效果。方法 在体外,制备材料和提取干细胞后,将骨髓间充质干细胞种植于聚左旋丙交酯纳米纤维材料上,通过CCK-8法观察材料上干细胞的增殖情况,在维甲酸的诱导分化后,通过光镜和免疫组织化学方法观察材料上干细胞的神经分化。在体内,制备成大鼠脊髓损伤的模型后,分别移植有干细胞黏附的聚左旋丙交酯纳米纤维膜(联合组)、聚左旋丙交酯纳米纤维膜(纤维膜组)、骨髓间充质干细胞(干细胞组),以及空白植入(空白对照组)。术后2、4、8周对大鼠进行BBB功能评分,观察大鼠脊髓损伤修复的效果。结果 体外研究观察发现,在聚左旋丙交酯纳米纤维材料上的干细胞与普通培养皿中的相比,第1、4天未见明显差异,第7天材料上的干细胞增殖好于普通培养皿中的。在纤维材料上的干细胞向神经样细胞的诱导分化也好于普通培养皿中的。体内实验发现,术后4周,联合组、纤维膜组与干细胞组之间未见明显差异,均优于空白对照组,术后8周,联合组优于纤维膜组及干细胞组,且均优于空白对照组。结论 聚左旋丙交酯纳米纤维联合骨髓间充质干细胞移植修复大鼠脊髓损伤效果良好。
Abstract
Objective To explore the effect of implantation of PLLA nanofibers and BMSCs on spinal cord injury of rats.Methods In vitro, PLLA nanofibers were fabricated and BMSCs were isolated before BMSCs were implanted on the PLLA nanofibers.The Cell Counting Kit-8 was applied to detect the proliferation of BMSCs.The neural differentiation of BMSCs on the PLLA nanofibers induced by RA(retinoic acid) was observed using immunohistochemistry and light microscopy.After the spinal cord injury(SCI) model was prepared, forty-eight rats were randomly and equally assigned to four groups(n=12): the PLLA nanofibers+BMSCs group, PLLA nanofibers group, BMSCs group, and the blank control group.The motor functional recovery of rats was evaluated with the BBB score at 2 w, 4 w, 8 w after operation.Results There was no significant change in the proliferation of BMSCs on PLLA nanofibers compared with culture dishes at 1-day and 4-day.The proliferation of BMSCs on PLLA nanofibers was better than in culture dishes at 7-day.The neural differentiation of BMSCs on PLLA nanofibers was better than in culture dishes.In vivo, there was no significant difference in the rats’ functional recovery after SCI between the PLLA nanofibers+BMSCs group, the PLLA nanofibers group and the BMSCs group at 4-week after operation.The rats’ functional recovery of the PLLA nanofibers+BMSCs group was better than that of the PLLA nanofibers group and the BMSCs group at 8-week after operation.The rats’ functional recovery of the above three groups was better than that of the blank control group at 4-week and 8-week after operation.Conclusions The implantation of PLLA nanofibers and BMSCs can promote the recovery of spinal cord injury of rats.
关键词
聚左旋丙交酯纳米纤维 /
骨髓间充质干细胞 /
脊髓损伤 /
神经分化
Key words
PLLA nanofibers /
BMSCs /
spinal cord injury /
neural differentiation
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参考文献
[1] Metin U, Anup D.Sharma, et al.Development of multifunctional films for peripheral nerve regeneration[J].Acta Biomaterialia, 2017(56): 141-152.
[2] 张燕青, 曾园山, 何留民, 等.聚左旋丙交酯纳米纤维支架与大鼠脊髓组织相容性的研究[J].组织工程与重建外科杂志, 2009, 5(3): 143-147.
[3] Lü L X, Zhang X F, Wang Y Y, et al.Effects of hydroxyapatite-containing composite nanofibers on osteogenesis of mesenchymal stem cells in vitro and bone regeneration in vivo[J].ACS Appl Mater Interfaces, 2013,5(2):319-330.
[4] 高 锋, 张晓峰, 段艳伟, 等.经骨髓间充质干细胞诱导分化的神经样细胞移植对大鼠脊髓损伤的修复作用[J].武警医学, 2016, 27(3): 285-288.
[5] Khan T, Havey R M, Sayers S T, et al.Animal models of spinal cord contusion injuries[J].Lab Anim Sci, 1999, 49(2):161-172.
[6] Basso D M, Beattie M S, Bresnahan J C.A sensitive and reliable locomotor rating scale for open-field testing in rats[J].J Neurotrauma, 1995, 12(1):1-21.
[7] Gregory ERutkowski, Carole A.Heath Development of a Bioartificial Nerve Graft.II.Nerve Regeneration in vitro Biotechnol[J].Prog, 2002, 18(2): 373-379.
[8] 张燕青, 曾园山, 何留民, 等.两种高分子支架对骨髓间充质干细胞生长的影响[J].组织工程与重建外科杂志, 2007, 3(6): 305-308.
[9] Kabiri M,Oraee-Yazdani S,Dodel M,et al.Cytocompatibility of a conductive nanofibrous carbon nanotube/poly (L-Lactic acid) composite scaffold intended for nerve tissue engineering[J].EXCLI J,2015,14:851-860.
[10] Guarino V, Causa F, Taddei P.Polylactic acid fibre-reinforced polycaprolactone scaffolds for bone tissue engineering[J].Biomaterials, 2008, 29(27): 3662-3670.
[11] Zhang N, Yah H, Wen X, et al.Tissue-engineering approaches for axonal guidance[J].Brain res rev, 2005, 49(1): 48-64.
[12] Matsumoto M, Chosa E, Nabeshima K, et al.Influence of bioresorhable,unaintered hydroxyapatite/poly-L-lactide composite films on spinalcord, nerve roots, and epidural space[J].J Biomed Mater Res,2002,60(1):101-109.
[13] Li J, Shi R.Fabrication of patterned multi-walled poly-L-lactic acid conduits for nerve regeneration[J].J Neurosci Methods, 2007, 165(2): 257-264.
[14] Ma P X,Zhang R.Synthetic nano-scale fibrous extracellular matrix[J].J Biomed Mater Res, 1999, 46(1): 60-72.
[15] 林成楷, 戎利民, 刘 斌.多壁碳纳米管/聚左旋乳酸复合纳米纤维支架材料与小鼠神经干细胞的生物相容性[J].中国组织工程研究,2017,21(6): 940-945.
[16] Alexanian A R.Epigenetic modifiers promote efficient Generation of neural-like cells from bone marrow-derived mesenchymal cells grown in neural environment[J].J Cell Biochem, 2007, 100(2): 362-371.
[17] Zhang W, Zeng Y S, Zhang X B, et al.Combination of adenoviral vector-mediated neurotrophin-3 gene transfer and retinoic acid promotes adult bone marrow cells to differentiate into neuronal phenotypes[J].Neurosci Lett, 2006, 408(2):98-103.
[18] Bao C, Wang Y, Min H, et al.Combination of Ginsenoside Rg1 and Bone Marrow Mesenchymal Stem Cell Transplantation in the Treatment of Cerebral Ischemia Reperfusion Injury in Rats[J].Cell Physiol Bio-chem, 2015, 37(3): 901-910.
[19] Wei C C, Lin A B, Hung S C.Mesenchymal stem cells in regenerative medicine for musculoskeletal diseases: bench, bedside, and industry[J].Cell Transplant, 2014, 23(4-5): 505-512.
[20] 李 昂, 李 波, 魏志坚, 等.骨髓间充质干细胞治疗脊髓损伤的研究进展[J].中国脊柱脊髓杂志, 2016, 26(1): 90-94.
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