Ihh/Glil通路对大鼠急性脊髓损伤后内源性神经干细胞增殖分化的调控及川芎嗪干预作用研究
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摘要
目的：观察大鼠急性脊髓损伤后IhhmRNA. GlilmRNA在受损脊髓的分布及不同时间点的表达变化,分析Ihh/Glil信号通路对脊髓内源性神经干细胞的调控作用,明确川芎嗪治疗脊髓损伤的分子机制。
方法：将SD大鼠随机分为空白组(A组6只)、假手术组(B组36只)、模型组(C组36只),甲强龙对照组(D组36只),川芎嗪治疗组(E组36只)。用改良Allen's法建立大鼠急性脊髓损伤模型。E组在造模后每日腹腔注射盐酸川芎嗪注射液,400mg/kg/d,首次给药在造模后30min,共给药10天；D组造模后按照甲强龙大剂量冲击疗法于造模后30分钟腹腔注射甲强龙187.5mg/kg;伤后1小时即开始按33.75mg/kg计算23小时总量,23小时内分四次腹腔注射。A、B、C组在同一时间给予腹腔注射等量生理盐水,共10天,D组于造模后第二天给予腹腔注射等量生理盐水共9天。于造模后第8小时、1天、3天、7天、14天、28天进行斜板实验、BBB评分神经功能行为学评价。于造模后第8小时、1天、3天、7天、14天、28天每组分别处死6只大鼠,以损伤脊髓为中心上下各5mm取大鼠的脊髓进行检测,3只用于HE.尼氏染色、免疫组织化学及原位杂交检测,3只用于Real-time PCR检测。用HE、尼氏染色观察脊髓的病理形态学变化,用免疫组化法检测代表脊髓内源性神经干细胞的Brdu+细胞和Nestin+细胞的表达,用原位杂交和Real-time PCR检测IhhmRNA和GlilmRNA在大鼠脊髓中的分布和表达变化情况。对检测结果进行统计分析,并对Brdu+和Nestin+细胞、IhhmRNA和GlilmRNA在脊髓中的表达进行相关性分析。
结果：
1、大鼠造模损伤脊髓后,C组、D组、E组大鼠的后肢运动功能显著降低,脊髓功能严重损伤,大鼠在斜板上维持的最大角度与BBB评分均明显降低,与A组比较有显著性差异(P<0.01)；E组大鼠治疗后在斜板上维持的最大角度呈明显上升趋势,在14天,28天与模型对照组对比有显著差异(P<0.05)；D组优于E组,在治疗后第3天至28天时两组比较有显著差异(P<0.01)。E组大鼠在治疗后BBB评分逐渐增加,在3天、14天、28天与C组比较有显著差异(P<0.05或P<0.01),但BBB评分不及D组(P<0.05或P<0.01)；
2、通过大鼠脊髓组织切片进行HE,尼氏染色观察各组大鼠脊髓组织病理学变化。脊髓损伤后可见脊髓水肿,局灶性出血,神经元胞体皱缩、核固缩,尼氏小体开始减少和消失,神经元变性,3天后出现脊髓水肿加重,局灶性出血范围增大,炎性细胞浸润明显,灰质部分组织溶解形成空泡；至7天、14天坏死范围进一步扩大,炎症水肿逐渐减轻,至28天,坏死区周围瘢痕增生,向坏死区填充。川芎嗪能明显减轻脊髓损伤后水肿、炎性细胞浸润及出血程度,保护神经细胞,促进脊髓组织修复；
3、脊髓损伤后,Brdu+细胞被激活,在脊髓中的表达明显增多,在脊髓白质、灰质及中央管室管膜区均有表达。在第7天达到峰值,随后逐渐减少。川芎嗪能明显促进Brdu+细胞增殖,在损伤后7天、14天、28天,与C组比较有显著差异(P<0.05或P<0.01),而甲强龙则抑制Brdu+细胞增殖,与C组比较有显著性差异(P<0.05或P<0.01)。川芎嗪能显著促进Nestin+细胞表达,在第7天达到峰值,在损伤后7天、14天、28天与C、D组比较有显著性差异(P<0.05或P<0.01),而甲强龙则抑制Nestin+细胞表达;
4、在正常成年大鼠脊髓中IhhmRNA主要分布在白质区,在室管膜细胞也有少量表达；而GlilmRNA主要表达在胶质细胞的胞质和灰质区神经元除核仁以外的细胞核。脊髓损伤后IhhmRNA与GlilmRNA表达减少,C组与A组在各时间点比较有显著性差异(P<0.05或P<0.01),第3至7天将至最低点,后又逐渐增多,但仍低于正常值。C组、D组和E组在各时间点比较无明显差异(P<0.05)。
5、经对IhhmRNA、GlilmRNA、Nestin+细胞和Brdu+细胞在脊髓中的表达量进行相关性分析,IhhmRNA与GlilmRNA表达呈正相关,IhhmRNA与Nestin+细胞表达呈负相关。
结论：1、Ihh在正常成年大鼠的脊髓中有分布,并且主要分布在脊髓的白质区,中央管仅有微量表达；Glil主要表达在神经元核仁以外的细胞核和胶质细胞的胞质中,中央管周围未见表达。2、Ihh/glil信号转导通路对脊髓内源性干细胞的增殖分化起负性调控作用；3、川芎嗪能够对脊髓损伤后内源性神经干细胞增殖分化有促进作用；4、川芎嗪通对脊髓损伤后脊髓内源性神经干细胞增殖分化的影响可能不是通过对Ihh/Glil信号通路的干预达到的。
Objective:To observe the variation of expression and distribution of IhhmRNA and GlilmRNA in the lesion areas after acute injury of spinal cord and to analyze the regulation of Ihh/Glil signal pathways on endogenous neural stem cells to ensure the molecule treatment mechanism of Ligustrazine for spinal cord.
Methods:The SD (Sprague Dawley)rats were randomly divided into gap group (group A), pseudo surgery group (group B), model group (group C), Methylprednisolone control group (group D) and Ligustrazine treatment group (group E). The rat models of acute spinal cord injury were established by modified Allen's method in the experiment. The rats of group E after molding were given abdominal Ligustrazine Hydrochloride Injection for400mg/kg per day, and the first injection was given after30min after the operation. Adapting Methylprednisolone high dose impulsive therapy, the rats in group D were given187.5mg/kg Methylprednisolone injection in abdomen after30min surgery. After one hour, the total amount of23hours with33.75mg/kg per hour was worked out to averagely inject four times in23hours for the rats. The group A, B and C were given the same amount of physiological saline in abdomen at the same time for ten days while group D was given the same mount with the former three groups in the following nine days after molding on the second day. Oblique board test and BBB evaluation on nerve function were applied to test the postoperative results on the8th hour,1st day,7th day,14th day, and28th day respectively.6rats in each group were sacrificed on the8th hour,1st day,7th day,14th day, and28th day after spinal cord injury. Then the injuried spinal cord about the5mm right and left of it was cut out to be detected,3of them for the HE, Nissl staining, immunohistochemistry and in situ hybridization, and3of them only for Real-time PCR detection. Meanwhile, the experiment adapted HE and Nissl staining methods to observe the morphological changes of spinal cord. Immunohistochemistry was applied to test the expression of Brdu+and Nestin+cells. Besides, in-situ hybridization and Real-time PCR were used to test the distribution and expression of IhhmRNA and GlilmRNA in the spinal cord. All of the test results were statistically analysis, and the correlation analysis were made among the expression of Brdu+and Nestin+cells, IhhmRNA and GlilmRNA in the spinal cord.
Results:
Firstly, motor function of hind limbs and the function of spinal cord of the rats decreased significantly after spinal cord injury; The maximum angles the rats maintained on oblique board in the Ligustrazine treatment group were on the uprising trend and there was significant difference compared with model control group on the14th day and28th day(p<0.05); There was significant difference between Methylprednisolone control group and Ligustrazine treatment group (P<0.01), and Methylprednisolone control group is better than Ligustrazine group from3th to28th after spinal cord injury. There was significant difference compared with model control group on the3th day,14th day and28th day according to the BBB score(P<0.05or P<0.01). However, the impact was declined compared with Methylprednisolone control group (P<0.05or P<0.01)
Secondly, Ligustrazine could remarkably reduce edema, inflammatory cell infiltration and blooding and promote the repair of spinal cord tissues after injury.
Thirdly, there was significant difference (P<0.01) on the7th day,14th day and28th day after spinal cord injury since Ligustrazine enabled to increase the proliferation of Brdu+cells (compared with group C P<0.05or P<0.01) while Methylprednisolone restrained its proliferation(compared with group C P<0.05or P<0.01). Ligustrazine could conspicuously enhance the expression of Nestin+cells and reach its summit on the7th day and there was significant difference compared with group C and group D on the7th day,14th day and28th day after spinal cord injury(P<0.05or P<0.01) while Methylprednisolone control group revealed minor expression (compared with group C P<0.05or P<0.01)
Fourthly, there were IhlimRNA and GlilmRNA in the spinal cord of normal adult rats. IhhmRNA mainly distribute in the whiter matter as well as minor expression in ependymocytes. While GlilmRNA are mainly expressed in the cytoplasm of glial cells and the cell nucleolus of gray matter neurons in addition to outside the plasmosome. In the experiment, the expression of IhhmRNA and GlilmRNA decreased to the lowest on the3th day and7th day, and then gradually increased, but still lower than the normal value. There is no obvious difference between among Ligustrazine treatment group, Methylprednisolone control group and model control group at each time point (P>0.05). Fifthly, IhhmRNA is positively correlated with the expression of GlilmRNA and is negatively related to the Nestin+cells according to the partial correlation analysis.
Finally, The correlation analysis was performed on the expression of IhhmRNA, GlilmRNA, Nestin+cells and Brdu+cells in the spinal cord. IhhmRNA positively correlated with the expression of GlilmRNA, and is negatively related to Nestin+expression.
Conclusion:Firstly, Ihh distributes in the spinal cord of normal adult rats with main distribution in the white matter and minor in the ventricular zone. Gli1mRNA are mainly expressed in the cytoplasm of glial cells and the cell nucleolus of gray matter neurons in addition to outside the plasmosome. Secondly, Ihh/glil signal transduction pathway negatively regulates the proliferation and differentiation of endogenous neural stem cells. Thirdly, Ligustrazine could promote the proliferation and differentiation of endogenous neural stem cells after spinal cord injury. Fourthly, the influence Ligustrazine exerted on proliferation and differentiation of endogenous neural stem cells may not realize by the intervention of Ihh/Glil signal pathways.
方法：将SD大鼠随机分为空白组(A组6只)、假手术组(B组36只)、模型组(C组36只),甲强龙对照组(D组36只),川芎嗪治疗组(E组36只)。用改良Allen's法建立大鼠急性脊髓损伤模型。E组在造模后每日腹腔注射盐酸川芎嗪注射液,400mg/kg/d,首次给药在造模后30min,共给药10天；D组造模后按照甲强龙大剂量冲击疗法于造模后30分钟腹腔注射甲强龙187.5mg/kg;伤后1小时即开始按33.75mg/kg计算23小时总量,23小时内分四次腹腔注射。A、B、C组在同一时间给予腹腔注射等量生理盐水,共10天,D组于造模后第二天给予腹腔注射等量生理盐水共9天。于造模后第8小时、1天、3天、7天、14天、28天进行斜板实验、BBB评分神经功能行为学评价。于造模后第8小时、1天、3天、7天、14天、28天每组分别处死6只大鼠,以损伤脊髓为中心上下各5mm取大鼠的脊髓进行检测,3只用于HE.尼氏染色、免疫组织化学及原位杂交检测,3只用于Real-time PCR检测。用HE、尼氏染色观察脊髓的病理形态学变化,用免疫组化法检测代表脊髓内源性神经干细胞的Brdu+细胞和Nestin+细胞的表达,用原位杂交和Real-time PCR检测IhhmRNA和GlilmRNA在大鼠脊髓中的分布和表达变化情况。对检测结果进行统计分析,并对Brdu+和Nestin+细胞、IhhmRNA和GlilmRNA在脊髓中的表达进行相关性分析。
结果：
1、大鼠造模损伤脊髓后,C组、D组、E组大鼠的后肢运动功能显著降低,脊髓功能严重损伤,大鼠在斜板上维持的最大角度与BBB评分均明显降低,与A组比较有显著性差异(P<0.01)；E组大鼠治疗后在斜板上维持的最大角度呈明显上升趋势,在14天,28天与模型对照组对比有显著差异(P<0.05)；D组优于E组,在治疗后第3天至28天时两组比较有显著差异(P<0.01)。E组大鼠在治疗后BBB评分逐渐增加,在3天、14天、28天与C组比较有显著差异(P<0.05或P<0.01),但BBB评分不及D组(P<0.05或P<0.01)；
2、通过大鼠脊髓组织切片进行HE,尼氏染色观察各组大鼠脊髓组织病理学变化。脊髓损伤后可见脊髓水肿,局灶性出血,神经元胞体皱缩、核固缩,尼氏小体开始减少和消失,神经元变性,3天后出现脊髓水肿加重,局灶性出血范围增大,炎性细胞浸润明显,灰质部分组织溶解形成空泡；至7天、14天坏死范围进一步扩大,炎症水肿逐渐减轻,至28天,坏死区周围瘢痕增生,向坏死区填充。川芎嗪能明显减轻脊髓损伤后水肿、炎性细胞浸润及出血程度,保护神经细胞,促进脊髓组织修复；
3、脊髓损伤后,Brdu+细胞被激活,在脊髓中的表达明显增多,在脊髓白质、灰质及中央管室管膜区均有表达。在第7天达到峰值,随后逐渐减少。川芎嗪能明显促进Brdu+细胞增殖,在损伤后7天、14天、28天,与C组比较有显著差异(P<0.05或P<0.01),而甲强龙则抑制Brdu+细胞增殖,与C组比较有显著性差异(P<0.05或P<0.01)。川芎嗪能显著促进Nestin+细胞表达,在第7天达到峰值,在损伤后7天、14天、28天与C、D组比较有显著性差异(P<0.05或P<0.01),而甲强龙则抑制Nestin+细胞表达;
4、在正常成年大鼠脊髓中IhhmRNA主要分布在白质区,在室管膜细胞也有少量表达；而GlilmRNA主要表达在胶质细胞的胞质和灰质区神经元除核仁以外的细胞核。脊髓损伤后IhhmRNA与GlilmRNA表达减少,C组与A组在各时间点比较有显著性差异(P<0.05或P<0.01),第3至7天将至最低点,后又逐渐增多,但仍低于正常值。C组、D组和E组在各时间点比较无明显差异(P<0.05)。
5、经对IhhmRNA、GlilmRNA、Nestin+细胞和Brdu+细胞在脊髓中的表达量进行相关性分析,IhhmRNA与GlilmRNA表达呈正相关,IhhmRNA与Nestin+细胞表达呈负相关。
结论：1、Ihh在正常成年大鼠的脊髓中有分布,并且主要分布在脊髓的白质区,中央管仅有微量表达；Glil主要表达在神经元核仁以外的细胞核和胶质细胞的胞质中,中央管周围未见表达。2、Ihh/glil信号转导通路对脊髓内源性干细胞的增殖分化起负性调控作用；3、川芎嗪能够对脊髓损伤后内源性神经干细胞增殖分化有促进作用；4、川芎嗪通对脊髓损伤后脊髓内源性神经干细胞增殖分化的影响可能不是通过对Ihh/Glil信号通路的干预达到的。
Objective:To observe the variation of expression and distribution of IhhmRNA and GlilmRNA in the lesion areas after acute injury of spinal cord and to analyze the regulation of Ihh/Glil signal pathways on endogenous neural stem cells to ensure the molecule treatment mechanism of Ligustrazine for spinal cord.
Methods:The SD (Sprague Dawley)rats were randomly divided into gap group (group A), pseudo surgery group (group B), model group (group C), Methylprednisolone control group (group D) and Ligustrazine treatment group (group E). The rat models of acute spinal cord injury were established by modified Allen's method in the experiment. The rats of group E after molding were given abdominal Ligustrazine Hydrochloride Injection for400mg/kg per day, and the first injection was given after30min after the operation. Adapting Methylprednisolone high dose impulsive therapy, the rats in group D were given187.5mg/kg Methylprednisolone injection in abdomen after30min surgery. After one hour, the total amount of23hours with33.75mg/kg per hour was worked out to averagely inject four times in23hours for the rats. The group A, B and C were given the same amount of physiological saline in abdomen at the same time for ten days while group D was given the same mount with the former three groups in the following nine days after molding on the second day. Oblique board test and BBB evaluation on nerve function were applied to test the postoperative results on the8th hour,1st day,7th day,14th day, and28th day respectively.6rats in each group were sacrificed on the8th hour,1st day,7th day,14th day, and28th day after spinal cord injury. Then the injuried spinal cord about the5mm right and left of it was cut out to be detected,3of them for the HE, Nissl staining, immunohistochemistry and in situ hybridization, and3of them only for Real-time PCR detection. Meanwhile, the experiment adapted HE and Nissl staining methods to observe the morphological changes of spinal cord. Immunohistochemistry was applied to test the expression of Brdu+and Nestin+cells. Besides, in-situ hybridization and Real-time PCR were used to test the distribution and expression of IhhmRNA and GlilmRNA in the spinal cord. All of the test results were statistically analysis, and the correlation analysis were made among the expression of Brdu+and Nestin+cells, IhhmRNA and GlilmRNA in the spinal cord.
Results:
Firstly, motor function of hind limbs and the function of spinal cord of the rats decreased significantly after spinal cord injury; The maximum angles the rats maintained on oblique board in the Ligustrazine treatment group were on the uprising trend and there was significant difference compared with model control group on the14th day and28th day(p<0.05); There was significant difference between Methylprednisolone control group and Ligustrazine treatment group (P<0.01), and Methylprednisolone control group is better than Ligustrazine group from3th to28th after spinal cord injury. There was significant difference compared with model control group on the3th day,14th day and28th day according to the BBB score(P<0.05or P<0.01). However, the impact was declined compared with Methylprednisolone control group (P<0.05or P<0.01)
Secondly, Ligustrazine could remarkably reduce edema, inflammatory cell infiltration and blooding and promote the repair of spinal cord tissues after injury.
Thirdly, there was significant difference (P<0.01) on the7th day,14th day and28th day after spinal cord injury since Ligustrazine enabled to increase the proliferation of Brdu+cells (compared with group C P<0.05or P<0.01) while Methylprednisolone restrained its proliferation(compared with group C P<0.05or P<0.01). Ligustrazine could conspicuously enhance the expression of Nestin+cells and reach its summit on the7th day and there was significant difference compared with group C and group D on the7th day,14th day and28th day after spinal cord injury(P<0.05or P<0.01) while Methylprednisolone control group revealed minor expression (compared with group C P<0.05or P<0.01)
Fourthly, there were IhlimRNA and GlilmRNA in the spinal cord of normal adult rats. IhhmRNA mainly distribute in the whiter matter as well as minor expression in ependymocytes. While GlilmRNA are mainly expressed in the cytoplasm of glial cells and the cell nucleolus of gray matter neurons in addition to outside the plasmosome. In the experiment, the expression of IhhmRNA and GlilmRNA decreased to the lowest on the3th day and7th day, and then gradually increased, but still lower than the normal value. There is no obvious difference between among Ligustrazine treatment group, Methylprednisolone control group and model control group at each time point (P>0.05). Fifthly, IhhmRNA is positively correlated with the expression of GlilmRNA and is negatively related to the Nestin+cells according to the partial correlation analysis.
Finally, The correlation analysis was performed on the expression of IhhmRNA, GlilmRNA, Nestin+cells and Brdu+cells in the spinal cord. IhhmRNA positively correlated with the expression of GlilmRNA, and is negatively related to Nestin+expression.
Conclusion:Firstly, Ihh distributes in the spinal cord of normal adult rats with main distribution in the white matter and minor in the ventricular zone. Gli1mRNA are mainly expressed in the cytoplasm of glial cells and the cell nucleolus of gray matter neurons in addition to outside the plasmosome. Secondly, Ihh/glil signal transduction pathway negatively regulates the proliferation and differentiation of endogenous neural stem cells. Thirdly, Ligustrazine could promote the proliferation and differentiation of endogenous neural stem cells after spinal cord injury. Fourthly, the influence Ligustrazine exerted on proliferation and differentiation of endogenous neural stem cells may not realize by the intervention of Ihh/Glil signal pathways.
引文
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