NSC和OEC联合移植对SCT大鼠大脑皮质运动区神经元的作用及相关机制研究
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摘要
【目的】探讨NSC和OEC联合移植对脊髓全横断损伤大鼠的运动功能及大脑皮质运动区神经元的保护作用,并探讨其作用机制。
【方法】采用细胞培养、免疫组织化学、TUNEL法、蛋白质印迹法和逆转录聚合酶链式反应等技术,结合动物行为学观察(BBB评分及运动诱发电位检测),研究全横断脊髓损伤及NSC和OEC联合移植后,损伤大鼠运动功能的变化、大脑皮质运动区神经元的存活与凋亡、相关凋亡基因表达变化及其信号转导分子的调节。
【结果】
1.T_9全横断脊髓损伤后,大鼠后肢运动功能完全丧失,随时间延长,有一定的(甚至无)自发性恢复,但此种自发性恢复能力非常有限。在脊髓全横断损伤后的1～4周的各个时间点,手术组大鼠BBB评分明显低于假手术组(P＜0.001)。术后4周,手术组80%的大鼠未检测到MEP,20%的大鼠检测到波幅极低的MEP波形;假手术组MEP检出率为100%,波幅、潜伏期正常。细胞凋亡TUNEL检测显示:术后2周,手术组大脑皮质V层TUNEL阳性细胞数较假手术组增多(P＜0.05)。免疫组化结果表明:术后4周,手术组大脑皮质V层NeuN阳性细胞数较假手术组减少(P＜0.05);同时,手术组Bax和Caspase-3的阳性细胞数较假手术组明显增多(P＜0.05),而Bcl-2两组相比无统计学差异(P＞0.05)。RT-PCR结果显示:大脑皮质运动区Bax的表达在手术组各时间点与假手术组比较均无差异(P＞0.05);而Bcl-2的表达术后14天和28天较假手术组明显减少(P＜0.05);Caspase-3则在术后3天较假手术组表达高(P＜0.05),其他时间点接近假手术组水平。
2.从细胞移植术后第3周末开始,NSC和OEC联合移植组大鼠后肢运动功能的恢复明显优于单纯全横断组(P＜0.05);三个细胞移植组大鼠的BBB评分在整个恢复过程中均有明显的提高,其中NSC和OEC联合移植组的BBB分值在第3周末高于NSC移植组和OEC移植组,在第4周末高于NSC移植组(P＜0.05);NSC移植组和OEC移植组的BBB评分在移植术后第4周末高于单纯全横断组(P＜0.05)。细胞移植术后4周,各细胞移植组均有不同程度的MEP波幅,但均明显低于假手术组(P＜0.01);NSC和OEC联合移植组的所有受试动物均检测到MEP波形,检出率达100%,NSC和OEC单独移植组MEP检出率均为50%。细胞移植后2周,假手术组和各细胞移植组大鼠大脑皮质V层TUNEL阳性染色细胞较单纯全横断手术组少(P＜0.05);联合移植组较OEC移植组细胞凋亡数少(P＜0.05)。细胞移植术后4周,免疫组化结果显示:尽管全横断损伤组、NSC和OEC单独移植组的NeuN阳性细胞数较假手术组明显减少(P＜0.05),而NSC和OEC联合移植组与假手术组相比却无明显差异(P＞0.05),说明细胞联合移植可减轻皮质运动神经元的凋亡。结果还发现:联合移植组大脑皮质V层抗凋亡因子Bcl-2阳性细胞数较各对照组多(P＜0.05);而联合或单细胞移植组促凋亡基因Bax阳性细胞数均较手术组减少(P＜0.05);Caspase-3阳性细胞数仅联合移植组和OEC移植组较手术组减少,且联合移植组较两种细胞单一移植组减少明显(P＜0.05)。RT-PCR检测发现:NSC和OEC联合移植后3天,Bax mRNA的表达较NSC移植组增多,但较OEC移植组降低(P＜0.05);而在联合移植后14天和21天,Bcl-2的表达较OEC移植组增高(P＜0.05);Caspase-3的表达在移植后3天和7天较手术组表达降低(P＜0.05),移植后14天,较NSC移植组表达降低(P＜0.05)。
3.信号通路分子RT-PCR检测显示:细胞移植术后3天,联合移植组、OEC移植组和手术组大脑皮质运动区PKB mRNA的表达均明显高于NSC移植组(P＜0.05),手术组还较假手术组表达高(P＜0.05);而术后14天,NSC移植组PKB mRNA的表达明显回升且显著高于OEC移植组(P＜0.05);移植术后21天,NSC和OEC联合移植组PKB mRNA的表达较NSC移植组和OEC移植组高(P＜0.05),其中,OEC移植组还低于手术组和假手术组(P＜0.05)。术后7天和28天,各组比较无差异(P＞0.05)。比较之,单独和联合移植术后ERK1mRNA的表达在各时间点均较假手术组和手术组明显减低(P＜0.01);且术后3天,NSC移植组高于OEC移植组和联合移植组(P＜0.01)。Stat-3 mRNA的表达则表现为细胞移植术后3天,联合移植组和OEC移植组较假手术组及手术组表达高(P＜0.05);移植术后14天手术组较假手术组降低(P＜0.05),而各细胞移植组均较手术组明显升高(P＜0.05);在细胞移植术后21天,各细胞移植组Stat-3的表达均较假手术组降低(P＜0.05);而在移植术后28天,NSC移植组Star-3较假手术组和联合移植组表达增高(P＜0.05)。进而,Western-blot检测结果发现:移植术后14天,联合移植组Akt非磷酸化蛋白、ERK1/2磷酸化蛋白的表达均比手术组和假手术组高,且Akt非磷酸化蛋白在NSC移植组比手术组表达高(P＜0.05)。联合移植组非磷酸化Stat-3的表达较假手术组、手术组、NSC移植组表达高(P＜0.05);Akt磷酸化蛋白、Stat-3磷酸化蛋白和ERK1/2非磷酸化蛋白的表达各组间比较均无差异(P＞0.05)。
4.体外共培养显示:NSC和OEC共培养7天,联合培养组NeuN阳性细胞数较NSC单独培养组多(P＜0.05),而未分化的Nestin阳性细胞较单独培养组低(P＜0.05);GFAP和APC阳性细胞分化率两组比较无差异(P＞0.05)。提示OEC可能促进NSC向神经细胞分化。
【结论】
1.脊髓全横断损伤后可能有部分自主运动功能恢复,但非常有限,需要寻找有效的治疗措施;脊髓全横断损伤引发大脑皮质运动区神经元凋亡基因表达失控,进而导致细胞凋亡,因此,脊髓损伤的研究必须考虑到脊髓自身以外的损害,特别是大脑皮质。
2.NSC和OEC作为两种有用的“种子”细胞,其单独和联合移植能有效促进脊髓损伤大鼠运动功能部分恢复,且联合移植优于单细胞移植。
3.NSC和OEC联合移植发挥作用的机制可能涉及:①OEC能促进NSC的增殖和向神经元分化;②减少大脑皮质运动区神经元凋亡;③促进抗凋亡因子Bcl-2的基因和蛋白质表达;④调节ERK1/2信号转导通路。
Objective To explore the effects of neural stem cells (NSC) and olfactory ensheathing cells (OEC) co-transplantation on the neurons of primary motor cortex and the locomotor functions in the rats subjected to spinal cord transection (SCT). And elucidate the underlying molecular mechanisms.
Method The cells culture, Immunohistochemistry (IHC), Terminal deoxynucleotidyl transferase mediated dUTP Nick end labeling (TUNEL), Western-blot, Reverse Transcription polymerase Chain Reaction (RT-PCR) and Behavioral test (BBB locomotor score and Motor evoked potential) were employed to test the hindlimb locomotor functions, the surviving or dying of the neurons of primary motor cortex , the expression of apoptosis associated factors and the molecular signal transduction molecular in the rats subjected to SCT and received NSC and OEC co-transplantation.
Result
1. Rats subjected to SCT at spinal level T9, which hindlimbs showed flaccid paralysis, indicating that the transection of the cord was complete. There was spontaneous, howbeit incomplete, recovery of the hindlimb locomotor functions after SCT. The BBB score of operation group was significant lower than sham operation group at all of the different time points (P<0.05). At 4 weeks after SCT, transcranial magnetic motor-evoked potentials (MEP) were not recorded in 80% experiment rats. Whereas all of the sham operation rats had the successful MEP records. At 2 weeks after transection, the TUNEL staining showed the operation group rats had more positive staining cells in the V layer of cortex than the sham operation group rats (P<0.05). Immunohistochemical staining revealed that the operation group rats expressed specific neuronal marker (Neun) in the V layer of cortex was significantly less than sham operation group rats at 4 weeks post operation (P<0.05). And the quantitative analysis demonstrated that the number of Bax and Caspase-3 immunoreactive products were more in the operation group rats than sham operation group rats in the V layer of cortex (P<0.05). Whereas the Bcl-2 immunoreactive products have no statistical difference between the operation group and sham operation group (P>0.05). The result of RT-PCR showed as follows: Compared with sham-operation group, the expression of Bcl-2 mRNA in the primary motor cortex decreased significantly in operation group at 14 and 21 days post operation (dpo), while a significant increase on the expression of Caspase-3 mRNA was found at 3dpo in the operation group rats (P<0.05). The expression of Bax, however, had no statistical difference between two groups (P>0.05).
2. The rats received NSC and OEC co-transplantation had a better recovery of hindlimb locomotor functions than operation group rats from 3 weeks after transplantation (P<0.05). All of the rats received cell transplantation had a significant improvement of BBB locomotor score. The rats received NSC and OEC co-transplantation had the highest BBB score among three cell transplantation groups at 3 weeks after operation (P<0.05) . At 4 weeks post operation, NSC and OEC co-transplantation group rats had higher BBB score than NSC transplantation group rats (P<0.05). The BBB score of NSC transplantation group and OEC transplantation group was higher than operation group at 4 weeks post operation, respectively (P<0.05). At 4 weeks post transplantation, all of the rats received NSC and OEC co-transplantation had the successful MEP records. However, the amplitudes were lower than sham operation group (P<0.01). There was only one half rats had the MEP records in NSC transplantation group and OEC transplantation group. Both had lower amplitudes than sham operation group rats (P<0.01). The latencies of OEC transplantation group rats were the longest among all the experimental rats (P<0.05). Two weeks after cell transplantation, the TUNEL staining revealed the operation group rats had the most numbers of TUNEL positive staining cells in the V layer of motor cortex among all the experimental animals (P<0.05). The TUNEL staining cells of NSC and OEC co-transplantation rats were less than OEC transplantation group rats (P<0.05). Four weeks following cell transplantation, the statistical result showed the sham operation group rats had the most number of NeuN immunoreactivity in the V layer of motor cortex among all of the experiment rats except for NSC and OEC co-transplantation group (P<0.05). There was, however, no statistical difference of NeuN immunoreactive between sham operation group and co-transplantation group (P>0.05). Which suggested NSC and OEC co-transplantation have the potential to rescue the neurons of primary motor cortex. Significantly, the number of the Bcl-2 immunoreactive products in NSC and OEC co-transplantation group were the most among all of the experimental group rats (P<0.05). In the V layer of motor cortex, all of the cell transplantation groups rats had less Bax immunoreactive products than the operation group rats (P<0.05). NSC and OEC co-transplantation group and OEC transplantation group had less Caspase-3 immunoreactive products than operation group (P<0.05). Moreover, NSC and OEC co-transplantation group had the least number of Caspase-3 immunoreactive products among three cell transplantation groups (P<0.05). The result of RT-PCR showed that the expression of Bax mRNA in co-transplantation group was increased compared with NSC transplantation group, whereas decreased compared with OEC transplantation group at 3dpo (P<0.05). The expression of Bcl-2 mRNA in the NSC and OEC co-transplantation group was increased at 14dpo and 21dpo compared with OEC transplantation group and increased at 21 dpo compared with operation group (P<0.05). Compared with operation group, the expression of Caspase-3 in the motor cortex of NSC and OEC co-transplantation group rats were decreased at 3 and 7dpo (P<0.05). And lower than NSC transplantation group at 14dpo (P<0.05).
3.The result of RT-PCR showed that the expression of PKB mRNA in the motor cortex of NSC transplantation group rats was lower than co-transplantation group, OEC transplantation group and operation group at 3 dpo (P<0.05). At the same time point, operation group was lower than sham operation group (P<0.05). At 14dpo, the expression of PKB mRNA in NSC transplantation group was reversed and was higher than OEC transplantation group (P<0.05). NSC and OEC co-transplantation group rats had the highest expression of PKB mRNA among three cell transplantation groups (P<0.05). And OEC transplantation group was lower than operation group and sham operation group (P<0.05) .There was no statistical difference in all of the experimental rats at 7 dpo and 28 dpo (P>0.05). Conversely, the expression of ERK1 mRNA was higher in the motor cortex of sham operation group and operation group than three cell transplantation groups (i.e. NSC transplantation group, OEC transplantation group and co-transplantation group) at all of the time points (P<0.01). Moreover, the expression of ERK mRNA was the highest in the motor cortex of NSC transplantation group among three cell transplantation groups at 3dpo (P<0.01). At 3dpo, the expression of Stat-3 mRNA in the motor cortex of co-transplantation group and OEC transplantation group was higher than operation group and sham operation group (P<0.05) . At 14 dpo, operation group was less than sham operation group (P <0.01). And each cell transplantation groups was higher than operation group, respectively (P<0.05) . At 21 dpo, the expression of Stat-3 mRNA of every cell transplantation group was less than sham operation group (P<0.05). NSC transplantation group showed a significant upregulation at 28 dpo compared with sham operation group and NSC and OEC co-transplantation group (P <0.05) . At 14 dpo, Western-blot analysis showed that the expression of Akt non-phosphorylated protein and ERK 1/2 phosphorylated protein in the motor cortex of NSC and OEC co-transplantation group was increased compared with operation group and sham-operation group (P<0.05). And the expression of Akt non-phosphorylated protein in NSC transplantation group was higher than operation group (P<0.05). Compared with sham operation group, operation group and NSC transplantation group, there was a significant upregulation of Stat-3 non-phosphorylated protein in the motor cortex of co-transplantation group (P<0.05). The result also showed there was no difference on the expressions of Akt phosphorylated protein, ERK1/2 non-phosphorylated protein and Stat-3 phosphorylated protein in all of the experiment animals (P>0.05).
4. Immunocytochemical analysis showed that there was more NeuN immunoreactivity neurospheres and less Nestin-positive cells in the co-culture medium than NSC single culture medium at 7 days (P<0.05). There was, however, no statistical difference in the numbers of GFAP-positive cells and APC-positive cells between co-culture and single culture medium (P>0.05). Which suggested OEC are capable of promoting NSC differentiate into neurons.
Conclusion
1. There was spontaneous, howbeit very limited, recovery of the hindlimb locomotor functions after SCT. It is urgent to find an optional and effective measure to improve the motor functions after spinal cord injury. The neurons of motor cortex in the rats suffered to apoptosis and death after SCT. The abnormal expression of apoptosis genes in the motor cortex would be responsible for the result, which was triggered by SCT. Therefore, it is prerequisite to play light on the changes of cerebral cortex after SCI.
2. NSC and OEC as two types of 'seed' cells. Both of them single or combinated engrafted into the rats subjected to SCT could effectively promote the recovery of the hindlimb locomotor functions. Whereas, co-transplantation had the better effects than single transplantation.
3. The underlying mechanism of NSC and OEC co-transplantation maybe as follows: The first, OEC have the potential to promote NSC profileration and differentiate into neurons. The second, co-transplantation prevented the neurons of motor cortex from apoptosis in the rats subjected to SCT. The third, NSC and OEC co-transplantation promoted the expression of Bcl-2 mRNA and protein level. And the effect was associated with the activated of ERK signal transduction molecular.
【方法】采用细胞培养、免疫组织化学、TUNEL法、蛋白质印迹法和逆转录聚合酶链式反应等技术,结合动物行为学观察(BBB评分及运动诱发电位检测),研究全横断脊髓损伤及NSC和OEC联合移植后,损伤大鼠运动功能的变化、大脑皮质运动区神经元的存活与凋亡、相关凋亡基因表达变化及其信号转导分子的调节。
【结果】
1.T_9全横断脊髓损伤后,大鼠后肢运动功能完全丧失,随时间延长,有一定的(甚至无)自发性恢复,但此种自发性恢复能力非常有限。在脊髓全横断损伤后的1～4周的各个时间点,手术组大鼠BBB评分明显低于假手术组(P＜0.001)。术后4周,手术组80%的大鼠未检测到MEP,20%的大鼠检测到波幅极低的MEP波形;假手术组MEP检出率为100%,波幅、潜伏期正常。细胞凋亡TUNEL检测显示:术后2周,手术组大脑皮质V层TUNEL阳性细胞数较假手术组增多(P＜0.05)。免疫组化结果表明:术后4周,手术组大脑皮质V层NeuN阳性细胞数较假手术组减少(P＜0.05);同时,手术组Bax和Caspase-3的阳性细胞数较假手术组明显增多(P＜0.05),而Bcl-2两组相比无统计学差异(P＞0.05)。RT-PCR结果显示:大脑皮质运动区Bax的表达在手术组各时间点与假手术组比较均无差异(P＞0.05);而Bcl-2的表达术后14天和28天较假手术组明显减少(P＜0.05);Caspase-3则在术后3天较假手术组表达高(P＜0.05),其他时间点接近假手术组水平。
2.从细胞移植术后第3周末开始,NSC和OEC联合移植组大鼠后肢运动功能的恢复明显优于单纯全横断组(P＜0.05);三个细胞移植组大鼠的BBB评分在整个恢复过程中均有明显的提高,其中NSC和OEC联合移植组的BBB分值在第3周末高于NSC移植组和OEC移植组,在第4周末高于NSC移植组(P＜0.05);NSC移植组和OEC移植组的BBB评分在移植术后第4周末高于单纯全横断组(P＜0.05)。细胞移植术后4周,各细胞移植组均有不同程度的MEP波幅,但均明显低于假手术组(P＜0.01);NSC和OEC联合移植组的所有受试动物均检测到MEP波形,检出率达100%,NSC和OEC单独移植组MEP检出率均为50%。细胞移植后2周,假手术组和各细胞移植组大鼠大脑皮质V层TUNEL阳性染色细胞较单纯全横断手术组少(P＜0.05);联合移植组较OEC移植组细胞凋亡数少(P＜0.05)。细胞移植术后4周,免疫组化结果显示:尽管全横断损伤组、NSC和OEC单独移植组的NeuN阳性细胞数较假手术组明显减少(P＜0.05),而NSC和OEC联合移植组与假手术组相比却无明显差异(P＞0.05),说明细胞联合移植可减轻皮质运动神经元的凋亡。结果还发现:联合移植组大脑皮质V层抗凋亡因子Bcl-2阳性细胞数较各对照组多(P＜0.05);而联合或单细胞移植组促凋亡基因Bax阳性细胞数均较手术组减少(P＜0.05);Caspase-3阳性细胞数仅联合移植组和OEC移植组较手术组减少,且联合移植组较两种细胞单一移植组减少明显(P＜0.05)。RT-PCR检测发现:NSC和OEC联合移植后3天,Bax mRNA的表达较NSC移植组增多,但较OEC移植组降低(P＜0.05);而在联合移植后14天和21天,Bcl-2的表达较OEC移植组增高(P＜0.05);Caspase-3的表达在移植后3天和7天较手术组表达降低(P＜0.05),移植后14天,较NSC移植组表达降低(P＜0.05)。
3.信号通路分子RT-PCR检测显示:细胞移植术后3天,联合移植组、OEC移植组和手术组大脑皮质运动区PKB mRNA的表达均明显高于NSC移植组(P＜0.05),手术组还较假手术组表达高(P＜0.05);而术后14天,NSC移植组PKB mRNA的表达明显回升且显著高于OEC移植组(P＜0.05);移植术后21天,NSC和OEC联合移植组PKB mRNA的表达较NSC移植组和OEC移植组高(P＜0.05),其中,OEC移植组还低于手术组和假手术组(P＜0.05)。术后7天和28天,各组比较无差异(P＞0.05)。比较之,单独和联合移植术后ERK1mRNA的表达在各时间点均较假手术组和手术组明显减低(P＜0.01);且术后3天,NSC移植组高于OEC移植组和联合移植组(P＜0.01)。Stat-3 mRNA的表达则表现为细胞移植术后3天,联合移植组和OEC移植组较假手术组及手术组表达高(P＜0.05);移植术后14天手术组较假手术组降低(P＜0.05),而各细胞移植组均较手术组明显升高(P＜0.05);在细胞移植术后21天,各细胞移植组Stat-3的表达均较假手术组降低(P＜0.05);而在移植术后28天,NSC移植组Star-3较假手术组和联合移植组表达增高(P＜0.05)。进而,Western-blot检测结果发现:移植术后14天,联合移植组Akt非磷酸化蛋白、ERK1/2磷酸化蛋白的表达均比手术组和假手术组高,且Akt非磷酸化蛋白在NSC移植组比手术组表达高(P＜0.05)。联合移植组非磷酸化Stat-3的表达较假手术组、手术组、NSC移植组表达高(P＜0.05);Akt磷酸化蛋白、Stat-3磷酸化蛋白和ERK1/2非磷酸化蛋白的表达各组间比较均无差异(P＞0.05)。
4.体外共培养显示:NSC和OEC共培养7天,联合培养组NeuN阳性细胞数较NSC单独培养组多(P＜0.05),而未分化的Nestin阳性细胞较单独培养组低(P＜0.05);GFAP和APC阳性细胞分化率两组比较无差异(P＞0.05)。提示OEC可能促进NSC向神经细胞分化。
【结论】
1.脊髓全横断损伤后可能有部分自主运动功能恢复,但非常有限,需要寻找有效的治疗措施;脊髓全横断损伤引发大脑皮质运动区神经元凋亡基因表达失控,进而导致细胞凋亡,因此,脊髓损伤的研究必须考虑到脊髓自身以外的损害,特别是大脑皮质。
2.NSC和OEC作为两种有用的“种子”细胞,其单独和联合移植能有效促进脊髓损伤大鼠运动功能部分恢复,且联合移植优于单细胞移植。
3.NSC和OEC联合移植发挥作用的机制可能涉及:①OEC能促进NSC的增殖和向神经元分化;②减少大脑皮质运动区神经元凋亡;③促进抗凋亡因子Bcl-2的基因和蛋白质表达;④调节ERK1/2信号转导通路。
Objective To explore the effects of neural stem cells (NSC) and olfactory ensheathing cells (OEC) co-transplantation on the neurons of primary motor cortex and the locomotor functions in the rats subjected to spinal cord transection (SCT). And elucidate the underlying molecular mechanisms.
Method The cells culture, Immunohistochemistry (IHC), Terminal deoxynucleotidyl transferase mediated dUTP Nick end labeling (TUNEL), Western-blot, Reverse Transcription polymerase Chain Reaction (RT-PCR) and Behavioral test (BBB locomotor score and Motor evoked potential) were employed to test the hindlimb locomotor functions, the surviving or dying of the neurons of primary motor cortex , the expression of apoptosis associated factors and the molecular signal transduction molecular in the rats subjected to SCT and received NSC and OEC co-transplantation.
Result
1. Rats subjected to SCT at spinal level T9, which hindlimbs showed flaccid paralysis, indicating that the transection of the cord was complete. There was spontaneous, howbeit incomplete, recovery of the hindlimb locomotor functions after SCT. The BBB score of operation group was significant lower than sham operation group at all of the different time points (P<0.05). At 4 weeks after SCT, transcranial magnetic motor-evoked potentials (MEP) were not recorded in 80% experiment rats. Whereas all of the sham operation rats had the successful MEP records. At 2 weeks after transection, the TUNEL staining showed the operation group rats had more positive staining cells in the V layer of cortex than the sham operation group rats (P<0.05). Immunohistochemical staining revealed that the operation group rats expressed specific neuronal marker (Neun) in the V layer of cortex was significantly less than sham operation group rats at 4 weeks post operation (P<0.05). And the quantitative analysis demonstrated that the number of Bax and Caspase-3 immunoreactive products were more in the operation group rats than sham operation group rats in the V layer of cortex (P<0.05). Whereas the Bcl-2 immunoreactive products have no statistical difference between the operation group and sham operation group (P>0.05). The result of RT-PCR showed as follows: Compared with sham-operation group, the expression of Bcl-2 mRNA in the primary motor cortex decreased significantly in operation group at 14 and 21 days post operation (dpo), while a significant increase on the expression of Caspase-3 mRNA was found at 3dpo in the operation group rats (P<0.05). The expression of Bax, however, had no statistical difference between two groups (P>0.05).
2. The rats received NSC and OEC co-transplantation had a better recovery of hindlimb locomotor functions than operation group rats from 3 weeks after transplantation (P<0.05). All of the rats received cell transplantation had a significant improvement of BBB locomotor score. The rats received NSC and OEC co-transplantation had the highest BBB score among three cell transplantation groups at 3 weeks after operation (P<0.05) . At 4 weeks post operation, NSC and OEC co-transplantation group rats had higher BBB score than NSC transplantation group rats (P<0.05). The BBB score of NSC transplantation group and OEC transplantation group was higher than operation group at 4 weeks post operation, respectively (P<0.05). At 4 weeks post transplantation, all of the rats received NSC and OEC co-transplantation had the successful MEP records. However, the amplitudes were lower than sham operation group (P<0.01). There was only one half rats had the MEP records in NSC transplantation group and OEC transplantation group. Both had lower amplitudes than sham operation group rats (P<0.01). The latencies of OEC transplantation group rats were the longest among all the experimental rats (P<0.05). Two weeks after cell transplantation, the TUNEL staining revealed the operation group rats had the most numbers of TUNEL positive staining cells in the V layer of motor cortex among all the experimental animals (P<0.05). The TUNEL staining cells of NSC and OEC co-transplantation rats were less than OEC transplantation group rats (P<0.05). Four weeks following cell transplantation, the statistical result showed the sham operation group rats had the most number of NeuN immunoreactivity in the V layer of motor cortex among all of the experiment rats except for NSC and OEC co-transplantation group (P<0.05). There was, however, no statistical difference of NeuN immunoreactive between sham operation group and co-transplantation group (P>0.05). Which suggested NSC and OEC co-transplantation have the potential to rescue the neurons of primary motor cortex. Significantly, the number of the Bcl-2 immunoreactive products in NSC and OEC co-transplantation group were the most among all of the experimental group rats (P<0.05). In the V layer of motor cortex, all of the cell transplantation groups rats had less Bax immunoreactive products than the operation group rats (P<0.05). NSC and OEC co-transplantation group and OEC transplantation group had less Caspase-3 immunoreactive products than operation group (P<0.05). Moreover, NSC and OEC co-transplantation group had the least number of Caspase-3 immunoreactive products among three cell transplantation groups (P<0.05). The result of RT-PCR showed that the expression of Bax mRNA in co-transplantation group was increased compared with NSC transplantation group, whereas decreased compared with OEC transplantation group at 3dpo (P<0.05). The expression of Bcl-2 mRNA in the NSC and OEC co-transplantation group was increased at 14dpo and 21dpo compared with OEC transplantation group and increased at 21 dpo compared with operation group (P<0.05). Compared with operation group, the expression of Caspase-3 in the motor cortex of NSC and OEC co-transplantation group rats were decreased at 3 and 7dpo (P<0.05). And lower than NSC transplantation group at 14dpo (P<0.05).
3.The result of RT-PCR showed that the expression of PKB mRNA in the motor cortex of NSC transplantation group rats was lower than co-transplantation group, OEC transplantation group and operation group at 3 dpo (P<0.05). At the same time point, operation group was lower than sham operation group (P<0.05). At 14dpo, the expression of PKB mRNA in NSC transplantation group was reversed and was higher than OEC transplantation group (P<0.05). NSC and OEC co-transplantation group rats had the highest expression of PKB mRNA among three cell transplantation groups (P<0.05). And OEC transplantation group was lower than operation group and sham operation group (P<0.05) .There was no statistical difference in all of the experimental rats at 7 dpo and 28 dpo (P>0.05). Conversely, the expression of ERK1 mRNA was higher in the motor cortex of sham operation group and operation group than three cell transplantation groups (i.e. NSC transplantation group, OEC transplantation group and co-transplantation group) at all of the time points (P<0.01). Moreover, the expression of ERK mRNA was the highest in the motor cortex of NSC transplantation group among three cell transplantation groups at 3dpo (P<0.01). At 3dpo, the expression of Stat-3 mRNA in the motor cortex of co-transplantation group and OEC transplantation group was higher than operation group and sham operation group (P<0.05) . At 14 dpo, operation group was less than sham operation group (P <0.01). And each cell transplantation groups was higher than operation group, respectively (P<0.05) . At 21 dpo, the expression of Stat-3 mRNA of every cell transplantation group was less than sham operation group (P<0.05). NSC transplantation group showed a significant upregulation at 28 dpo compared with sham operation group and NSC and OEC co-transplantation group (P <0.05) . At 14 dpo, Western-blot analysis showed that the expression of Akt non-phosphorylated protein and ERK 1/2 phosphorylated protein in the motor cortex of NSC and OEC co-transplantation group was increased compared with operation group and sham-operation group (P<0.05). And the expression of Akt non-phosphorylated protein in NSC transplantation group was higher than operation group (P<0.05). Compared with sham operation group, operation group and NSC transplantation group, there was a significant upregulation of Stat-3 non-phosphorylated protein in the motor cortex of co-transplantation group (P<0.05). The result also showed there was no difference on the expressions of Akt phosphorylated protein, ERK1/2 non-phosphorylated protein and Stat-3 phosphorylated protein in all of the experiment animals (P>0.05).
4. Immunocytochemical analysis showed that there was more NeuN immunoreactivity neurospheres and less Nestin-positive cells in the co-culture medium than NSC single culture medium at 7 days (P<0.05). There was, however, no statistical difference in the numbers of GFAP-positive cells and APC-positive cells between co-culture and single culture medium (P>0.05). Which suggested OEC are capable of promoting NSC differentiate into neurons.
Conclusion
1. There was spontaneous, howbeit very limited, recovery of the hindlimb locomotor functions after SCT. It is urgent to find an optional and effective measure to improve the motor functions after spinal cord injury. The neurons of motor cortex in the rats suffered to apoptosis and death after SCT. The abnormal expression of apoptosis genes in the motor cortex would be responsible for the result, which was triggered by SCT. Therefore, it is prerequisite to play light on the changes of cerebral cortex after SCI.
2. NSC and OEC as two types of 'seed' cells. Both of them single or combinated engrafted into the rats subjected to SCT could effectively promote the recovery of the hindlimb locomotor functions. Whereas, co-transplantation had the better effects than single transplantation.
3. The underlying mechanism of NSC and OEC co-transplantation maybe as follows: The first, OEC have the potential to promote NSC profileration and differentiate into neurons. The second, co-transplantation prevented the neurons of motor cortex from apoptosis in the rats subjected to SCT. The third, NSC and OEC co-transplantation promoted the expression of Bcl-2 mRNA and protein level. And the effect was associated with the activated of ERK signal transduction molecular.
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