• 1 蘭州軍區(qū)蘭州總醫(yī)院骨科中心（蘭州，710050）;;
  • 2 蘭州大學(xué)第二臨床醫(yī)學(xué)院臨床系;

目的 以去細(xì)胞肌肉生物支架（acellular muscle bioscaffolds，AMBS）接種BMSCs，同種異體移植修復(fù)大鼠脊髓半切損傷，觀察兩者聯(lián)合移植對(duì)脊髓損傷的修復(fù)作用。 方法取8周齡雌性SD大鼠，采用改良化學(xué)方法制備AMBS，并進(jìn)行復(fù)合冷滅菌；密度梯度離心法提取、貼壁法培養(yǎng)BMSCs，取第3代細(xì)胞用Hoechst 33342熒光標(biāo)記，采用注射法制備BMSCs-AMBS復(fù)合支架，14 d后掃描電鏡及熒光顯微鏡觀察其生物相容性。取成年雌性SD大鼠48只，制備T9～11脊髓半切損傷模型后隨機(jī)分為4組（n=12），A組于缺損處移植BMSCs-AMBS復(fù)合支架，B組單獨(dú)移植BMSCs，C組單獨(dú)移植AMBS，D組注射PBS作為空白對(duì)照組，分別于術(shù)后1、2、3、4周行運(yùn)動(dòng)功能評(píng)分，術(shù)后4周行HE染色觀察及免疫熒光檢測(cè)。 結(jié)果Masson染色及HE染色示AMBS內(nèi)部呈平行結(jié)構(gòu)，主要為膠原纖維，幾乎無(wú)肌纖維。BMSCs-AMBS復(fù)合培養(yǎng)14 d后熒光顯微鏡觀察示Hoechst 33342標(biāo)記BMSCs大量存活，掃描電鏡可見(jiàn)BMSCs貼附于支架內(nèi)表面生長(zhǎng)。術(shù)后2～4周，大鼠BBB評(píng)分A組均高于其余3組（P  lt; 0.05），D組明顯低于其余3組（P  lt; 0.05）；術(shù)后4周B組明顯高于C組（t=10.352，P=0.000）。術(shù)后4周，HE染色示脊髓空洞面積A組明顯小于其余3組，免疫熒光染色示A 組神經(jīng)絲蛋白200、巢蛋白陽(yáng)性細(xì)胞表達(dá)量高于其余3組，神經(jīng)膠質(zhì)原酸性蛋白（glial fibrillary acidic protein，GFAP）則明顯低表達(dá)。A、B組熒光示蹤的BMSCs移植到體內(nèi)后主要遷移至對(duì)側(cè)灰質(zhì)前角，部分分化為神經(jīng)元樣細(xì)胞。A、B、C、D組GFAP熒光半定量分析積分吸光度（IA）值分別為733.01 ± 202.04、926.42 ± 59.46、1 069.37 ± 33.42、1 469.46 ± 160.53，A組明顯低于其余3組，D組高于其余3組，差異均有統(tǒng)計(jì)學(xué)意義（P  lt; 0.05）。 結(jié)論AMBS具有相對(duì)規(guī)則的內(nèi)部結(jié)構(gòu)，與BMSCs有良好生物相容性，能抑制膠質(zhì)瘢痕，促進(jìn)BMSCs存活、遷徙、分化，是細(xì)胞移植理想的天然載體。兩者聯(lián)合移植修復(fù)大鼠脊髓損傷能發(fā)揮協(xié)同作用，促進(jìn)運(yùn)動(dòng)功能恢復(fù)。

引用本文： 魏祥科,文益民,張濤,李含. BMSCs聯(lián)合去細(xì)胞肌肉生物支架修復(fù)大鼠脊髓半切損傷的實(shí)驗(yàn)研究. 中國(guó)修復(fù)重建外科雜志, 2012, 26(11): 1362-1368. doi: 復(fù)制

1. Yang JT, Kuo YC, Chiu KH. Peptide-modified inverted colloidal crystal scaffolds with bone marrow stromal cells in the treatment for spinal cord injury. Colloids Surf B Biointerfaces, 2011, 84(1): 198-205.
2. Stokols S, Tuszynski MH. Freeze-dried agarose scaffolds with uniaxial channels stimulate and guide linear axonal growth following spinal cord injury. Biomaterials, 2006, 27(3): 443-451.
3. Zurita M, Otero L, Aguayo C, et al. Cell therapy for spinal cord repair: optimization of biologic scaffolds for survival and neural differentiation of human bone marrow stromal cells. Cytotherapy, 2010, 12(4): 522-537.
4. Gros T, Sakamoto JS, Blesch A, et al. Regeneration of long-tract axons through sites of spinal cord injury using templated agarose scaffolds. Biomaterials, 2010, 31(26): 6719-6729.
5. Itosaka H, Kuroda S, Shichinohe H, et al. Fibrin matrix provides a suitable scaffold for bone marrow stromal cells transplanted into injured spinal cord: a novel material for CNS tissue engineering. Neuropathology, 2009, 29(3): 248-257.
6. 汪大彬, 文益民, 藍(lán)旭, 等. 殼聚糖-藻酸鹽支架復(fù)合BMSCs修復(fù)急性脊髓損傷的實(shí)驗(yàn)研究. 中國(guó)修復(fù)重建外科雜志, 2010, 24(2): 190-196.
7. Arai T, Kanje M, Lundborg G, et al. Axonal outgrowth in muscle grafts made acellular by chemical extraction. Restor Neurol Neurosci, 2000, 17(4): 165-174.
8. Mligiliche N, Kitada M, Ide C. Grafting of detergent-denatured skeletal muscles provides effective conduits for extension of regenerating axons in the rat sciatic nerve. Arch Histol Cytol, 2001, 64(1): 29-36.
9. Carlson EC, Carlson BM. A method for preparing skeletal muscle fiber basal laminae. Anat Rec, 1991, 230(3): 325-331.
10. 汪大彬, 文益民, 藍(lán)旭, 等. 殼聚糖-藻酸鹽多通道支架材料細(xì)胞相容性研究. 中國(guó)矯形外科雜志, 2010, 18(10): 836-839.
11. Rivlin AS, Tator CH. Objective clinical assessment of motor function after experimental spinal cord injury in the rat. J Neurosurg, 1977, 47(4): 577-581.
12. Parr AM, Tator CH, Keating A. Bone marrow-derived mesenchymal stromal cells for the repair of central nervous system injury. Bone Marrow Transplant, 2007, 40(7): 609-619.
13. Lu P, Jones LL, Tuszynski MH. BDNF-expressing marrow stromal cells support extensive axonal growth at sites of spinal cord injury. Exp Neurol, 2005, 191(2): 344-360.
14. Sanes JR, Marshall LM, McMahan UJ. Reinnervation of muscle fiber basal lamina after removal of myofibers. Differentiation of regenerating axons at original synaptic sites. J Cell Biol, 1978, 78(1): 176-198.
15. 徐新智, 孫磊, 胡蘊(yùn)玉. 肌基膜管橋接周圍神經(jīng)缺損的電生理研究. 中華物理醫(yī)學(xué)與康復(fù)雜志, 2000, 22(2): 119-120.
16. Keilhoff G, Pratsch F, Wolf G, et al. Bridging extra large defects of peripheral nerves: possibilities and limitations of alternative biological grafts from acellular muscle and Schwann cells. Tissue Eng, 2005, 11(7-8): 1004-1014.
17. 張秀英, 薛輝, 孫皎, 等. 肌基膜管植入對(duì)大鼠脊髓半橫斷損傷模型的血管化作用. 吉林大學(xué)學(xué)報(bào): 醫(yī)學(xué)版, 2011, 37(5): 784-787.
18. 張秀英. 肌基膜管移植大鼠脊髓半切損傷模型血管生成情況的研究. 長(zhǎng)春: 吉林大學(xué), 2007.
19. Fansa H, Schneider W, Wolf G, et al. Host responses after acellular muscle basal lamina allografting used as a matrix for tissue engineered nerve grafts. Transplantation, 2002, 74(3): 381-387.
20. 宋宇, 劉佳梅, 薛輝, 等. 攜帶神經(jīng)干細(xì)胞肌基膜管組織工程支架中神經(jīng)干細(xì)胞的存活分化. 中國(guó)組織工程研究與臨床康復(fù), 2010, 14(47): 8751-8754.
21. 殷迪. 人羊膜上皮細(xì)胞與肌基膜管支架相容性的實(shí)驗(yàn)研究. 長(zhǎng)春: 吉林大學(xué), 2008.
22. 賈彬, 李杰, 賀西京, 等. 嗅黏膜嗅鞘細(xì)胞與肌基膜管聯(lián)合移植修復(fù)脊髓損傷. 中國(guó)組織工程研究與臨床康復(fù), 2008, 12(51): 10041-10044.
23. 王鴻飛, 鄭連杰, 張書(shū)琴, 等. 牛腦提取液與肌基膜管結(jié)合對(duì)脊髓損傷修復(fù)及再生的影響. 中國(guó)臨床康復(fù), 2003, 7(32): 4336-4337.
24. 李培建, 胥少汀. 神經(jīng)生長(zhǎng)因子及其結(jié)合肌基膜管移植修復(fù)脊髓橫斷性損傷的組織學(xué)觀察. 中華神經(jīng)外科雜志, 2000, 16(6): 367-370.
25. 張紅旭, 胥少汀, 吳霞, 等. 肌基膜管結(jié)合神經(jīng)生長(zhǎng)因子修復(fù)脊髓缺損的實(shí)驗(yàn)研究. 中華骨科雜志, 1995, 15(1): 32-35, T003.
  1. 1. Yang JT, Kuo YC, Chiu KH. Peptide-modified inverted colloidal crystal scaffolds with bone marrow stromal cells in the treatment for spinal cord injury. Colloids Surf B Biointerfaces, 2011, 84(1): 198-205.
  2. 2. Stokols S, Tuszynski MH. Freeze-dried agarose scaffolds with uniaxial channels stimulate and guide linear axonal growth following spinal cord injury. Biomaterials, 2006, 27(3): 443-451.
  3. 3. Zurita M, Otero L, Aguayo C, et al. Cell therapy for spinal cord repair: optimization of biologic scaffolds for survival and neural differentiation of human bone marrow stromal cells. Cytotherapy, 2010, 12(4): 522-537.
  4. 4. Gros T, Sakamoto JS, Blesch A, et al. Regeneration of long-tract axons through sites of spinal cord injury using templated agarose scaffolds. Biomaterials, 2010, 31(26): 6719-6729.
  5. 5. Itosaka H, Kuroda S, Shichinohe H, et al. Fibrin matrix provides a suitable scaffold for bone marrow stromal cells transplanted into injured spinal cord: a novel material for CNS tissue engineering. Neuropathology, 2009, 29(3): 248-257.
  6. 6. 汪大彬, 文益民, 藍(lán)旭, 等. 殼聚糖-藻酸鹽支架復(fù)合BMSCs修復(fù)急性脊髓損傷的實(shí)驗(yàn)研究. 中國(guó)修復(fù)重建外科雜志, 2010, 24(2): 190-196.
  7. 7. Arai T, Kanje M, Lundborg G, et al. Axonal outgrowth in muscle grafts made acellular by chemical extraction. Restor Neurol Neurosci, 2000, 17(4): 165-174.
  8. 8. Mligiliche N, Kitada M, Ide C. Grafting of detergent-denatured skeletal muscles provides effective conduits for extension of regenerating axons in the rat sciatic nerve. Arch Histol Cytol, 2001, 64(1): 29-36.
  9. 9. Carlson EC, Carlson BM. A method for preparing skeletal muscle fiber basal laminae. Anat Rec, 1991, 230(3): 325-331.
  10. 10. 汪大彬, 文益民, 藍(lán)旭, 等. 殼聚糖-藻酸鹽多通道支架材料細(xì)胞相容性研究. 中國(guó)矯形外科雜志, 2010, 18(10): 836-839.
  11. 11. Rivlin AS, Tator CH. Objective clinical assessment of motor function after experimental spinal cord injury in the rat. J Neurosurg, 1977, 47(4): 577-581.
  12. 12. Parr AM, Tator CH, Keating A. Bone marrow-derived mesenchymal stromal cells for the repair of central nervous system injury. Bone Marrow Transplant, 2007, 40(7): 609-619.
  13. 13. Lu P, Jones LL, Tuszynski MH. BDNF-expressing marrow stromal cells support extensive axonal growth at sites of spinal cord injury. Exp Neurol, 2005, 191(2): 344-360.
  14. 14. Sanes JR, Marshall LM, McMahan UJ. Reinnervation of muscle fiber basal lamina after removal of myofibers. Differentiation of regenerating axons at original synaptic sites. J Cell Biol, 1978, 78(1): 176-198.
  15. 15. 徐新智, 孫磊, 胡蘊(yùn)玉. 肌基膜管橋接周圍神經(jīng)缺損的電生理研究. 中華物理醫(yī)學(xué)與康復(fù)雜志, 2000, 22(2): 119-120.
  16. 16. Keilhoff G, Pratsch F, Wolf G, et al. Bridging extra large defects of peripheral nerves: possibilities and limitations of alternative biological grafts from acellular muscle and Schwann cells. Tissue Eng, 2005, 11(7-8): 1004-1014.
  17. 17. 張秀英, 薛輝, 孫皎, 等. 肌基膜管植入對(duì)大鼠脊髓半橫斷損傷模型的血管化作用. 吉林大學(xué)學(xué)報(bào): 醫(yī)學(xué)版, 2011, 37(5): 784-787.
  18. 18. 張秀英. 肌基膜管移植大鼠脊髓半切損傷模型血管生成情況的研究. 長(zhǎng)春: 吉林大學(xué), 2007.
  19. 19. Fansa H, Schneider W, Wolf G, et al. Host responses after acellular muscle basal lamina allografting used as a matrix for tissue engineered nerve grafts. Transplantation, 2002, 74(3): 381-387.
  20. 20. 宋宇, 劉佳梅, 薛輝, 等. 攜帶神經(jīng)干細(xì)胞肌基膜管組織工程支架中神經(jīng)干細(xì)胞的存活分化. 中國(guó)組織工程研究與臨床康復(fù), 2010, 14(47): 8751-8754.
  21. 21. 殷迪. 人羊膜上皮細(xì)胞與肌基膜管支架相容性的實(shí)驗(yàn)研究. 長(zhǎng)春: 吉林大學(xué), 2008.
  22. 22. 賈彬, 李杰, 賀西京, 等. 嗅黏膜嗅鞘細(xì)胞與肌基膜管聯(lián)合移植修復(fù)脊髓損傷. 中國(guó)組織工程研究與臨床康復(fù), 2008, 12(51): 10041-10044.
  23. 23. 王鴻飛, 鄭連杰, 張書(shū)琴, 等. 牛腦提取液與肌基膜管結(jié)合對(duì)脊髓損傷修復(fù)及再生的影響. 中國(guó)臨床康復(fù), 2003, 7(32): 4336-4337.
  24. 24. 李培建, 胥少汀. 神經(jīng)生長(zhǎng)因子及其結(jié)合肌基膜管移植修復(fù)脊髓橫斷性損傷的組織學(xué)觀察. 中華神經(jīng)外科雜志, 2000, 16(6): 367-370.
  25. 25. 張紅旭, 胥少汀, 吳霞, 等. 肌基膜管結(jié)合神經(jīng)生長(zhǎng)因子修復(fù)脊髓缺損的實(shí)驗(yàn)研究. 中華骨科雜志, 1995, 15(1): 32-35, T003.