BMSCs移植联合应用IN-1抗体修复大鼠急性脊髓损伤的实验研究
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摘要
目的：研究骨髓间充质干细胞(Bone marrow-derived mesenchymal stem cells, BMSCs)移植联合髓鞘生长阻逆蛋白抗体IN-1(monoclonal antibody IN-1)对大鼠皮质脊髓束横断后脊髓损伤修复的影响及意义。
方法：采用密度梯度离心与贴壁筛选法相结合制备大鼠骨髓间充质干细胞,选用清洁级健康Sprague-Dawley(SD)大鼠180只,体重250-300g,雌雄不限,随机分成手术对照组(n=60)、BMSCs移植组(n=60)、BMSCs移植联合IN-1组(n=60),每组又按照手术后时间随机平均分为1d、4d、7d、14d、21d和28d六个小组,每个小组10只。所有大鼠首先行蛛网膜下腔置管,然后在相当于T8椎板水平用做好标记的显微剪刀剪断脊髓的背侧2/3,建立大鼠皮质脊髓束横断损伤模型。其中手术对照组通过置管每次注入生理盐水5μl,而BMSCs组每次注入骨髓间充质干细胞悬液(5μl),IN-1和BMSCs联合作用组,每次注入IN-12.5μl和骨髓间充质干细胞悬液2.5μl,每天分为早、中、晚三次注射,最长持续1 w。每组中取5只,在手术前及手术后1d、1w、2w、3w及4w,采用BBB (Basso, Beattle, Bresnahan)评分评价运动功能,然后取出脊髓组织检测胶质纤维酸性蛋白(glial fibrillry acidic protein, GFAP)表达的变化。其余对照组及BMSCs移植组、BMSCs移植联合IN-1组动物于损伤后1d、4d、7d、14d、21d和28d六个时间点取出伤段脊髓,分别行RT-PCR(reverse transcription -polymerase chain reaction)检测GAP-43基因表达的变化,所得数据采用单因素方差分析进行统计学处理,P<0.05结果有统计学差异。
结果：1、脊髓损伤后GAP-43 mRNA的表达呈现先升高后降低的趋势,其表达的高峰分别在脊髓损伤后7d、14d、21d时间点；2、移植BMSCs后三者mRNA的表达上调,联合应用IN-1后效果更加明显,各组相比有统计学意义(P<0.05)。3、脊髓损伤后双后肢瘫痪,BBB评分,2w、3w和4w点BMSCs移植组、BMSCs移植联合IN-1组移植组明显高于损伤对照组,有显著统计学差异(P<0.05)；BMSCs移植联合IN-1组高于BMSCs移植组,有统计学差异(P<0.05)。4、GFAP免疫组化结果显示,BMSCs移植联合IN-1组平均灰度值明显小于其他两组,有显著统计学差异(P<0.05)。
结论：(1)脊髓损伤后GFAP表达增加,而IN-1、BMSCs可以抑制GFAP表达,从而抑制胶质细胞的反应性增生。(2)IN-1、BMSCs可能通过上调GAP-43 mRNA的表达,从而对脊髓损伤后轴突再生起一定的促进作用。(3)BMSCs移植联合IN-1可促进损伤的脊髓神经的存活和再生,并互相协同作用,较单一应用BMSCs能更好的促进脊髓损伤修复和大鼠后肢功能恢复。
Objective:To study the bone marrow mesenchymal stem cell transplantation combined myelin growth IN-lon the rat corticospinal tract after transection of spinal cord injury repair and significance.
Methods:By density gradient centrifugation and adherent filter prepared by combining bone marrow mesenchymal stem cells, Use of clean healthy Sprague-Dawley (SD) rats were 180, weight 250-300g, male or female, were divided randomly into operated group (n= 60), BMSCs transplantation group (n= 60), BMSCs transplantation in combination with IN-1 group (n= 60), each group was in accordance with the time after surgery were randomly divided into the 1d,4d,7d,14d, 21d and 28d of six groups, each 10. All rats first line of subarachnoid catheter, then the equivalent of T8 vertebral level and marked with micro scissors cut the spinal cord dorsal 2/3 rat corticospinal tract transection injury model. Including operon control group injeaticted with normal saline through the catheter every 5μl, and BMSCs injected into each group of bone marrow mesenchymal stem cell suspension (5μl), IN-1 and BMSCs joint action group, each injection and bone marrow IN-12.5μl mesenchymal stem cell suspension 2.5μl, every day is divided into early, middle, late third injection, the longest 1 w. Taking five in each group, before surgery and after surgery 1d, 1w,2w,3w, and 4w, with BBB (Basso, Beattle, Bresnahan) score assessment of motor function, and then remove the spinal cord tissues of glial fibrillary acidic protein (glial fibrillry acidic protein, GFAP) expression changes. The remaining control group and BMSCs transplantation group, BMSCs transplantation combined IN-1 animals after injury 1d,4d,7d,14d,21d and 28d out of six time points of injured spinal cord, respectively row RT-PCR (reverse transcription-polymerase chain reaction) detection of GAP-43 gene expression data obtained by single factor analysis of variance for statistical analysis, P<0.05 significantly different results.
Results:1, spinal cord injury in GAP-43 mRNA expression increased firstly and then decreased, the peak of their expression after spinal cord injury at 7d,14d,21d time point; 2, after BMSCs transplantation increases the expression of three mRNA, combined with post-IN-1 was more obvious than in each group was statistically significant (P<0.05).3, paralysis of the posterior limbs after spinal cord injury, BBB score,2w,3w and 4w point BMSCs transplantation group, BMSCs transplantation and graft IN-1 group was significantly higher than the injured control group, there was a significant difference (P <0.05); BMSCs transplantation in combination with IN-1 group than in BMSCs transplantation group, significantly different (P<0.05).4, GFAP immunohistochemistry showed that, BMSCs transplantation in combination with IN-1 group, the average gray value lower than the other two groups, there was a significant difference (P<0.05).
Conclusions:(1) GFAP expression after spinal cord injury, while the IN-1, BMSCs can inhibit the expression of GFAP, which inhibit the proliferation of reactive glial cells. (2) IN-1, BMSCs upregulated GAP-43mRNA expression, and thus on axonal regeneration after spinal cord injury play a role in promoting. (3) BMSCs transplantation in combination with IN-1 can promote the survival of spinal cord injury and regeneration, and mutual synergies, better than the single application of BMSCs to promote spinal cord repair and functional recovery of rat hind limb.
方法：采用密度梯度离心与贴壁筛选法相结合制备大鼠骨髓间充质干细胞,选用清洁级健康Sprague-Dawley(SD)大鼠180只,体重250-300g,雌雄不限,随机分成手术对照组(n=60)、BMSCs移植组(n=60)、BMSCs移植联合IN-1组(n=60),每组又按照手术后时间随机平均分为1d、4d、7d、14d、21d和28d六个小组,每个小组10只。所有大鼠首先行蛛网膜下腔置管,然后在相当于T8椎板水平用做好标记的显微剪刀剪断脊髓的背侧2/3,建立大鼠皮质脊髓束横断损伤模型。其中手术对照组通过置管每次注入生理盐水5μl,而BMSCs组每次注入骨髓间充质干细胞悬液(5μl),IN-1和BMSCs联合作用组,每次注入IN-12.5μl和骨髓间充质干细胞悬液2.5μl,每天分为早、中、晚三次注射,最长持续1 w。每组中取5只,在手术前及手术后1d、1w、2w、3w及4w,采用BBB (Basso, Beattle, Bresnahan)评分评价运动功能,然后取出脊髓组织检测胶质纤维酸性蛋白(glial fibrillry acidic protein, GFAP)表达的变化。其余对照组及BMSCs移植组、BMSCs移植联合IN-1组动物于损伤后1d、4d、7d、14d、21d和28d六个时间点取出伤段脊髓,分别行RT-PCR(reverse transcription -polymerase chain reaction)检测GAP-43基因表达的变化,所得数据采用单因素方差分析进行统计学处理,P<0.05结果有统计学差异。
结果：1、脊髓损伤后GAP-43 mRNA的表达呈现先升高后降低的趋势,其表达的高峰分别在脊髓损伤后7d、14d、21d时间点；2、移植BMSCs后三者mRNA的表达上调,联合应用IN-1后效果更加明显,各组相比有统计学意义(P<0.05)。3、脊髓损伤后双后肢瘫痪,BBB评分,2w、3w和4w点BMSCs移植组、BMSCs移植联合IN-1组移植组明显高于损伤对照组,有显著统计学差异(P<0.05)；BMSCs移植联合IN-1组高于BMSCs移植组,有统计学差异(P<0.05)。4、GFAP免疫组化结果显示,BMSCs移植联合IN-1组平均灰度值明显小于其他两组,有显著统计学差异(P<0.05)。
结论：(1)脊髓损伤后GFAP表达增加,而IN-1、BMSCs可以抑制GFAP表达,从而抑制胶质细胞的反应性增生。(2)IN-1、BMSCs可能通过上调GAP-43 mRNA的表达,从而对脊髓损伤后轴突再生起一定的促进作用。(3)BMSCs移植联合IN-1可促进损伤的脊髓神经的存活和再生,并互相协同作用,较单一应用BMSCs能更好的促进脊髓损伤修复和大鼠后肢功能恢复。
Objective:To study the bone marrow mesenchymal stem cell transplantation combined myelin growth IN-lon the rat corticospinal tract after transection of spinal cord injury repair and significance.
Methods:By density gradient centrifugation and adherent filter prepared by combining bone marrow mesenchymal stem cells, Use of clean healthy Sprague-Dawley (SD) rats were 180, weight 250-300g, male or female, were divided randomly into operated group (n= 60), BMSCs transplantation group (n= 60), BMSCs transplantation in combination with IN-1 group (n= 60), each group was in accordance with the time after surgery were randomly divided into the 1d,4d,7d,14d, 21d and 28d of six groups, each 10. All rats first line of subarachnoid catheter, then the equivalent of T8 vertebral level and marked with micro scissors cut the spinal cord dorsal 2/3 rat corticospinal tract transection injury model. Including operon control group injeaticted with normal saline through the catheter every 5μl, and BMSCs injected into each group of bone marrow mesenchymal stem cell suspension (5μl), IN-1 and BMSCs joint action group, each injection and bone marrow IN-12.5μl mesenchymal stem cell suspension 2.5μl, every day is divided into early, middle, late third injection, the longest 1 w. Taking five in each group, before surgery and after surgery 1d, 1w,2w,3w, and 4w, with BBB (Basso, Beattle, Bresnahan) score assessment of motor function, and then remove the spinal cord tissues of glial fibrillary acidic protein (glial fibrillry acidic protein, GFAP) expression changes. The remaining control group and BMSCs transplantation group, BMSCs transplantation combined IN-1 animals after injury 1d,4d,7d,14d,21d and 28d out of six time points of injured spinal cord, respectively row RT-PCR (reverse transcription-polymerase chain reaction) detection of GAP-43 gene expression data obtained by single factor analysis of variance for statistical analysis, P<0.05 significantly different results.
Results:1, spinal cord injury in GAP-43 mRNA expression increased firstly and then decreased, the peak of their expression after spinal cord injury at 7d,14d,21d time point; 2, after BMSCs transplantation increases the expression of three mRNA, combined with post-IN-1 was more obvious than in each group was statistically significant (P<0.05).3, paralysis of the posterior limbs after spinal cord injury, BBB score,2w,3w and 4w point BMSCs transplantation group, BMSCs transplantation and graft IN-1 group was significantly higher than the injured control group, there was a significant difference (P <0.05); BMSCs transplantation in combination with IN-1 group than in BMSCs transplantation group, significantly different (P<0.05).4, GFAP immunohistochemistry showed that, BMSCs transplantation in combination with IN-1 group, the average gray value lower than the other two groups, there was a significant difference (P<0.05).
Conclusions:(1) GFAP expression after spinal cord injury, while the IN-1, BMSCs can inhibit the expression of GFAP, which inhibit the proliferation of reactive glial cells. (2) IN-1, BMSCs upregulated GAP-43mRNA expression, and thus on axonal regeneration after spinal cord injury play a role in promoting. (3) BMSCs transplantation in combination with IN-1 can promote the survival of spinal cord injury and regeneration, and mutual synergies, better than the single application of BMSCs to promote spinal cord repair and functional recovery of rat hind limb.
引文
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