微囊化异种坐骨神经组织移植对脊髓损伤大鼠T淋巴细胞亚群的影响
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摘要
目的脊髓损伤(spinal cord injury,SCI)后,于损伤处移植周围神经组织能够促进神经元的存活、轴突的再生及功能恢复[1-3],海藻酸钡微囊的免疫隔离效应使得异种移植成为可能。本实验将预变性处理的家兔坐骨神经组织细胞微囊化后移植到SD大鼠SCI处,观察大鼠脊髓半横断损伤后植入微囊化异种坐骨神经组织细胞对宿主外周血中T淋巴细胞亚群的影响,初步探讨微囊化异种坐骨神经组织细胞移植治疗脊髓损伤修复的可能机制。
方法取健康成年家兔8只;健康成年Sprague-Dawley (SD)大鼠88只,随机将SD大鼠分为3组:A组(微囊化坐骨神经组织细胞移植组,n=40);B组(组织细胞悬液移植组,n=40);C组(正常对照,n=8)。分好组后对各组大鼠行右后肢运动功能BBB评分。取成年家兔预变性处理的双侧坐骨神经干制成组织细胞悬液,低速离心后与1.5%海藻酸钠溶液混合并喷入20 mmol/L的氯化钡溶液中制成微囊化坐骨神经组织细胞悬液。A、B组建立大鼠T10脊髓右半横断损伤模型,并分别在损伤处植入吸附10μl的微囊化组织细胞悬液的明胶海绵及组织细胞悬液的明胶海绵, C组未做任何处理。A组和B组随机取6只大鼠分别于术后第1、3、7、14、28d对右后肢运动功能进行BBB评分。A组和B组大鼠分别于术后的第1、3、7、14、28天各时相随机取8只大鼠断尾取血,另C组8只大鼠也行断尾取血术。流式细胞仪测定T淋巴细胞亚群CD4,CD8阳性细胞数。
结果脊髓损伤(SCI)后,细胞悬液组CD4~+ T细胞数于第3天较正常对照组有升高(P<0.05),第7、14和28天时,该差异具有显著性(P<0.01);该组CD8~+ T细胞数于第7、14和28天时较正常对照组有差异(P<0.05)。而微囊组各时相CD4~+ T细胞数和CD8~+ T细胞数较正常对照组均无差异(P>0.05)。第7、14和28天,细胞悬液组CD4~+ T细胞数较微囊组有明显升高(P<0.01),该组CD8~+ T细胞数于第7、14和28天时,较微囊组有升高。各组术前右后肢运动功能BBB评分均为21分, SCI后第1、3、7天时均较低且各组间无明显差别;第14天时A组大鼠右后肢功能恢复好于B、C组(P <0.05);第28天时A组大鼠右后肢BBB评分较B、C组大鼠右后肢BBB评分差异有显著性(P <0.01)。SCI后,各组大鼠右后肢运动功能均有不同程度的恢复,但以微囊组恢复最好。
结论微囊化异种坐骨神经组织移植可以有效地阻止大鼠CD4~+ T细胞和CD8~+T细胞的激活和增值,并能促进后肢运动功能恢复。
Objective Transplantation of peripheral nerve can enhance neuronal survival,axonal regeneration and functional rehabilitation after spinal cord injury (SCI). Barium-alginate microcapsules, which reveal an immunoisolation effect, make the xenotransplantation be possible. Here, the pretreated sciatic nerve tissue/cells from rabbit were microencapsulated and transplanted into the injured site of spinal cord in adult Sprague-Dawley (SD) rats. The alterations in T lymphocyte subsets of these SD rats were observed. And the possible mechanism of the rehabilitation induced by the transplantation of the microencapsulated xeno-sciatic nerve tissue/cells treated SCI were discussed.
Method Experiments were performed in 8 adult rabbits and 88 adult SD rats of either sex. All of the SD rats were randomly divided into three groups: Group A (transplantation of microencapsulated xeno-sciatic nerve tissue/cells, n=40), Group B (transplantation of xeno-tissue/cells suspension, n=40) and Group C (control group, n=8). After dividing, BBB score was enforced on right hindlimb of all rats. Bilateral sciatic nerves pretreated from rabbits were dissected under aseptic condition and made into nerve tissue/cells suspension. Following centrifugation at a low speed, the tissue/cells were mixed with 1.5% sodium alginate solution, which was then extruded into 20 mmol/L barium chloride solution by using a droplet generation device, forming microencapsulated sciatic nerve tissue/cells suspension. Group A and B were performed a right hemisection of spinal cord at T10 level and subsequently transplanted with the gelatin sponge sticking 10μl microencapsulated nerve tissue/cells suspension and the gelatin sponge sticking 10μl tissue/cells suspension respectively. On the 1st、3rd、7th、14th and 28th days postoperatively, 6 SD rats in group A and B, respectively and randomly, were subjected to behavioral testing using a Basso, Beattie and Bresnahan (BBB) scoring system. Peripheral blood were collected from 8 SD rats in group A and B, respectively and randomly, by tail cutting on the 1st、3rd、7th、14th and 28th days postoperatively and the number of CD4~+ and CD8~+ T lymphocyte subsets were taken account of by using the flow cytometer. Same operations were performed in the 8 rats of group C.
Result After operation, an increase was found in CD4~+ T cells of group B on the 3rd day postoperatively, comparing with group C (P<0.05). This increase became significant on the 7th、14th and 28th days postoperatively; the number of CD8~+ T cells increased comparing with group C on the 7th、14th and 28th days postoperatively(P<0.05). The difference of the number of CD4~+ T cells and CD8~+ T cells between group A and C does not possess statistic significance on each time phase. The difference of the number of CD4~+ T cells between group A and B was distinct on the 7th、14th and 28th days postoperatively, meanwhile the number of CD8~+ T cells of group B was higher than their counterpart in group A on the 7th、14th and 28th days postoperatively. BBB score of locomotive function was normal in group A, B and C before surgery, however it became low and no difference among each group on the 1st, 3rd, 7th days after SCI. Subsequently, the comparison between Group A and B existed a difference on the 14th days postoperatively (P <0.05). The score of Group A was significantly superior to that of Group B on the 28th days postoperatively, whose difference was remarkable (P <0.01), but them didn’t reach the normal level. The locomotive function of right hindlimbs of all rats got recovered to a greater or less extent, but Group A was the best.
Conclusion: microencapsulated sciatic nerve tissue can prevent the rat’s CD4~+ T and CD8~+ T cells from activation and proliferation and improve the rehabilitation of locomotive function of hind limb of the rats with SCI.
方法取健康成年家兔8只;健康成年Sprague-Dawley (SD)大鼠88只,随机将SD大鼠分为3组:A组(微囊化坐骨神经组织细胞移植组,n=40);B组(组织细胞悬液移植组,n=40);C组(正常对照,n=8)。分好组后对各组大鼠行右后肢运动功能BBB评分。取成年家兔预变性处理的双侧坐骨神经干制成组织细胞悬液,低速离心后与1.5%海藻酸钠溶液混合并喷入20 mmol/L的氯化钡溶液中制成微囊化坐骨神经组织细胞悬液。A、B组建立大鼠T10脊髓右半横断损伤模型,并分别在损伤处植入吸附10μl的微囊化组织细胞悬液的明胶海绵及组织细胞悬液的明胶海绵, C组未做任何处理。A组和B组随机取6只大鼠分别于术后第1、3、7、14、28d对右后肢运动功能进行BBB评分。A组和B组大鼠分别于术后的第1、3、7、14、28天各时相随机取8只大鼠断尾取血,另C组8只大鼠也行断尾取血术。流式细胞仪测定T淋巴细胞亚群CD4,CD8阳性细胞数。
结果脊髓损伤(SCI)后,细胞悬液组CD4~+ T细胞数于第3天较正常对照组有升高(P<0.05),第7、14和28天时,该差异具有显著性(P<0.01);该组CD8~+ T细胞数于第7、14和28天时较正常对照组有差异(P<0.05)。而微囊组各时相CD4~+ T细胞数和CD8~+ T细胞数较正常对照组均无差异(P>0.05)。第7、14和28天,细胞悬液组CD4~+ T细胞数较微囊组有明显升高(P<0.01),该组CD8~+ T细胞数于第7、14和28天时,较微囊组有升高。各组术前右后肢运动功能BBB评分均为21分, SCI后第1、3、7天时均较低且各组间无明显差别;第14天时A组大鼠右后肢功能恢复好于B、C组(P <0.05);第28天时A组大鼠右后肢BBB评分较B、C组大鼠右后肢BBB评分差异有显著性(P <0.01)。SCI后,各组大鼠右后肢运动功能均有不同程度的恢复,但以微囊组恢复最好。
结论微囊化异种坐骨神经组织移植可以有效地阻止大鼠CD4~+ T细胞和CD8~+T细胞的激活和增值,并能促进后肢运动功能恢复。
Objective Transplantation of peripheral nerve can enhance neuronal survival,axonal regeneration and functional rehabilitation after spinal cord injury (SCI). Barium-alginate microcapsules, which reveal an immunoisolation effect, make the xenotransplantation be possible. Here, the pretreated sciatic nerve tissue/cells from rabbit were microencapsulated and transplanted into the injured site of spinal cord in adult Sprague-Dawley (SD) rats. The alterations in T lymphocyte subsets of these SD rats were observed. And the possible mechanism of the rehabilitation induced by the transplantation of the microencapsulated xeno-sciatic nerve tissue/cells treated SCI were discussed.
Method Experiments were performed in 8 adult rabbits and 88 adult SD rats of either sex. All of the SD rats were randomly divided into three groups: Group A (transplantation of microencapsulated xeno-sciatic nerve tissue/cells, n=40), Group B (transplantation of xeno-tissue/cells suspension, n=40) and Group C (control group, n=8). After dividing, BBB score was enforced on right hindlimb of all rats. Bilateral sciatic nerves pretreated from rabbits were dissected under aseptic condition and made into nerve tissue/cells suspension. Following centrifugation at a low speed, the tissue/cells were mixed with 1.5% sodium alginate solution, which was then extruded into 20 mmol/L barium chloride solution by using a droplet generation device, forming microencapsulated sciatic nerve tissue/cells suspension. Group A and B were performed a right hemisection of spinal cord at T10 level and subsequently transplanted with the gelatin sponge sticking 10μl microencapsulated nerve tissue/cells suspension and the gelatin sponge sticking 10μl tissue/cells suspension respectively. On the 1st、3rd、7th、14th and 28th days postoperatively, 6 SD rats in group A and B, respectively and randomly, were subjected to behavioral testing using a Basso, Beattie and Bresnahan (BBB) scoring system. Peripheral blood were collected from 8 SD rats in group A and B, respectively and randomly, by tail cutting on the 1st、3rd、7th、14th and 28th days postoperatively and the number of CD4~+ and CD8~+ T lymphocyte subsets were taken account of by using the flow cytometer. Same operations were performed in the 8 rats of group C.
Result After operation, an increase was found in CD4~+ T cells of group B on the 3rd day postoperatively, comparing with group C (P<0.05). This increase became significant on the 7th、14th and 28th days postoperatively; the number of CD8~+ T cells increased comparing with group C on the 7th、14th and 28th days postoperatively(P<0.05). The difference of the number of CD4~+ T cells and CD8~+ T cells between group A and C does not possess statistic significance on each time phase. The difference of the number of CD4~+ T cells between group A and B was distinct on the 7th、14th and 28th days postoperatively, meanwhile the number of CD8~+ T cells of group B was higher than their counterpart in group A on the 7th、14th and 28th days postoperatively. BBB score of locomotive function was normal in group A, B and C before surgery, however it became low and no difference among each group on the 1st, 3rd, 7th days after SCI. Subsequently, the comparison between Group A and B existed a difference on the 14th days postoperatively (P <0.05). The score of Group A was significantly superior to that of Group B on the 28th days postoperatively, whose difference was remarkable (P <0.01), but them didn’t reach the normal level. The locomotive function of right hindlimbs of all rats got recovered to a greater or less extent, but Group A was the best.
Conclusion: microencapsulated sciatic nerve tissue can prevent the rat’s CD4~+ T and CD8~+ T cells from activation and proliferation and improve the rehabilitation of locomotive function of hind limb of the rats with SCI.
引文
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