不同时期终丝和生长因子对神经干细胞增殖及分化的影响
摘要
引言
先天性脊髓病变是严重影响患儿中枢神经系统发育的一类疾病,这类疾病包括脊髓拴系综合征(Tethered cord syndrome,TCS),脊髓纵裂(Split cord malformation, SCM),脊髓脊膜膨出(Myelomeningocele)等疾病。在上述疾病中尤其以脊髓拴系综合征(TCS)对患儿生长发育影响较大,TCS是由于终丝病变弹性降低,对脊髓圆锥末端产生持续牵拉作用,导致脊髓局部缺血缺氧改变,造成脊髓组织的神经元细胞线粒体氧化磷酸化功能障碍,导致神经纤维的传导功能受损,逐渐出现双下肢感觉运动障碍,双足畸形,大小便功能障碍等一系列神经损害临床表现的一种疾病。目前对于TCS主要治疗方式为病变终丝的切除和对脊髓圆锥末端粘连组织的松解,以达到对脊髓圆锥末尾端的充分松解,使其逐渐恢复到正常水平,进而终止或改善已出现的临床症状。但是目前这种治疗方式术后的效果并不理想。1992年Reynolds等从小鼠大脑组织(纹状体)中分离出在体外可以不断分裂增殖更新,具有向神经元细胞、星形胶质细胞和少突胶质细胞方向分化的多潜能的细胞群,提出了神经干细胞(neural stem cells,NSCs)的概念。NSCs概念的提出使人们改变了以往认为神经细胞是永久性细胞,损伤后不可再生的观念,为中枢及外周神经系统疾病的治疗和功能恢复带来了新的希望,同时也为神经系统疾病的治疗提供了一种新的治疗策略神经干细胞移植。以往对于脊髓中NSCs的研究认为其只存在于脊髓室管膜下,终丝组织不存在NSCs,对终丝组织的认识只是将其看作是从脊髓末端向下延续穿出硬脊膜连接尾骨的一结构简单的纤维血管复合物,但Varghese等分别在2009年和2010年利用人体终丝组织培养出具有NSCs增殖分化特点的细胞群,证明终丝组织中有NSCs的存在。这一发现使人们对于NSCs的认识又一次改变。目前国内对于终丝组织中NSCs研究未见有文献报道,此次实验利用胎儿终丝组织进行了NSCs的分离培养及鉴定的初步研究,这为终丝中NSCs的分离培养提供了一定的参考。
目的
1、通过原代悬浮培养的方法对胎儿终丝组织中NSCs进行分离培养并对培养出的细胞进行Nestin鉴定,表达阳性者为NSCs,从而证明终丝组织中存在NSCs。
2、通过对不同胎龄终丝及TCS患儿的病变终丝进行NSCs的分离培养来比较不同终丝组织中NSCs的增长和分化的特点。
3、通过对胎儿终丝组织中NSCs的体外分离培养的观察,分析表皮生长因子(EGF)、成纤维生长因子(bFGF)、白血病抑制因子(LIF)对NSCs增长及分化的影响。
材料和方法
1.收集2011.11-2012.8郑州大学第三附属医院妇产科12-28W流产胎儿15例,按胎龄大小分为3组：A组(12-17W),B组(18-23W),C组(24-28W),取胎儿终丝组织进行NSCs的原代分离培养,培养过程中用倒置显微镜定期观察NSCs的生长情况,计数神经球的数目比较三组胎儿终丝中NSCs的生长特点。取一定数量的NSCs进行Nestin鉴定,表达阳性者为NSCs,从而证实终丝组织中存在NSCs。
2.收集12-20W胎龄新鲜流产胎儿9例,根据在NSCs基础培养基DMEM/F12中添加物不同分为3组：A组(DMEM/F12+B27+EGF+bFGF),B组(DMEM/F12+B27+EGF+bFGF+LIF),C组(DMEM/F12+B27)为对照组,观察三组NSCs生长情况,之后用胎牛血清进行诱导分化,用免疫细胞化学的方法鉴定上述三组NSCs诱导分化后NSE和GFAP的分化比例,分析EGF、bFGF和LIF对NSCs增长和分化的影响。
3.收集2011.9-2012.5郑州大学第三附属医院小儿骨科收治的5例TCS患儿的术中切除的病变终丝,术前与家属沟通后征得其同意,上报医院伦理委员会批准同意后进行实验研究,通过观察病变和正常终丝组织中NSCs增长和分化的情况,比较不同类型终丝组织中NSCs的分离培养和分化的特点。
4.实验结果的统计学分析采用SPSS17.0统计软件包进行处理分析。实验结果以均数±标准差(x±s)表示,三组间NSCs数目、NSE和GFAP比例的比较采用单因素方差分析,组间NSCs数目、NSE和GFAP比例的两两比较采用LSD-t检验,以a=0.05为检验水准。
结果
1.对终丝中分离培养出的细胞在倒置显微镜下进行形态学观察,发现其具有NSCs形态特点,将培养出的细胞经Nestin鉴定其表达为阳性,说明培养出来的细胞为NSCs.
2.不同胎龄终丝组织中培养出的NSCs数量不同,对三组结果进行单因素方差分析(P<0.05),提示不同胎龄的终丝样本NSCs增长数目有显著性差异。对三组的NSCs数量进行两两比较,A组和B组比较P>0.05,差异无统计学意义；A组和C组比较、B组和C组比较,P均小于0.05,差异有统计学意义。
3.不同添加物组中NSCs增长和分化存在差异,对三组结果进行单因素方差分析(P(0.05),提示用含不同细胞因子的培养基分离培养NSCs,在增长数目、NSE和GFAP阳性细胞百分比方面均有显著性差异。在NSCs数目方面：三组两两比较P均小于0.05,差异有统计学意义；在NSCs分化方面：三组两两比较P均小于0.05,差异有统计学意义。
4.不同类型终丝组织中NSCs增长和分化存在差异,对二者差异进行单因素方差分析,P均小于0.05,差异有统计学意义,提示采用不同类型的终丝样本分离培养NSCs在增长和分化方面均有显著性差异。
结论
1.终丝组织在特定条件下可在体外分离培养出NSCs,胎龄大小与终丝组织中NSCs的数量有密切的关系,这可能与胎龄较小者NSCs的增长潜能较大有关。
2.不同的因子对终丝组织中NSCs的增长和分化有一定的影响,LIF在促进NSCs增长和抑制其分化方面作用较EGF和bFGF明显。
3.病变终丝与正常终丝均可以培养出NSCs,正常终丝组神经球的增长数量和分化后NSE所占的比例较病变终丝组明显增高,胎儿正常终丝组织更适宜作为NSCs的体外分离培养的组织来源。
Background
Congenital spinal cord lesion is a diseases with serious impact on children central nervous system development.It's Include tethered cord syndrome(TCS),Split cord malformation(SCM), myelomeningocele.Especially TCS with impact on children growth development.TCS is due to the lesions filum terminale to stretch the end of the conus, result in changes of spinal cord ischemia hypoxia, which led to the bowel and bladder dysfunction, deformity of the lower limbs and sensory dysfunction.At present TCS main treatment is resection the lesion filum terminale, release the end of the spinal cord,gradually returned to normal levels, and then terminate or improvement of clinical symptoms.But this treatment the results are unsatisfactory.In1992Reynolds isolated from mouse striatum in vitro proliferation of pluripotent cells, proposed the concept of neural stem cells (neural stem cells, NSCs).The concept of NSCs changed people in the past for the nerve cell is a permanent cell damage non-renewable understanding.Give a new hope of treatment diseases of the nervous system, as well as a new treatment strategy-neural stem cell transplantation for the treatment of central nervous system diseases. In the past spinal cord NSCs that exists only in the subependymal,there are no NSCs in the filum terminale.In the past the filum terminale is considered just a simple structure of fibrous tissue.Mercy and Varghese respective in2009and2010use of the human filum terminale tissue to culture the NSCs, then prove there is NSCs in human filum terminale tissue.This finding change people to understanding of the NSCs again, currently there is no reported on use of the human filum terminale tissue to culture NSCs in the domestic.In this study we use the fetal filum terminale tissue to culture NSCs preliminary study for isolating and identifying in the NSCs. This study provides some reference for isolation and culture of NSCs.
Objective
1. By primary cultured the NSCs with filum terminale tissue and identification the cells with Nestin.with positive expression is NSCs.Prove there are NSCs in the filum terminale tissue.
2. To compare the characteristics of growth number and differentiation of NSCs in different types of filum terminale tissue.
3. Observed the characteristics of proliferation and differentiation to NSCs, analysis epidermal growth factor (EGF), fibroblast growth factor (bFGF),and leukemia inhibitory factor (LIF) impact on NSCs in growth number and differentiation.
Materials and methods
1. Collect15cases of12to8w abortion Fetus in2011.11-2012.8provided by third affiliated hospital of zhengzhou university department of Obstetrics.Based on the gestational age divided into3groups:group A (12to17W), group B (18to23W), group C (24to28W). Primary cultured NSCs with the filum terminale tissue and periodical observe the growth of NSCs with inverted microscope.Count the number of neurospheres compare the growth characteristics of the three groups of NSCs.Identification the cells with Nestin.positive expression is NSCs.Prove there are NSCs in the filum terminale tissue.
2. Collect9cases of12to20w abortion Fetus.Based on additives different factors in NSCs basal medium DMEM/F12to divided into three groups:group A (DMEM/F12+B27+EGF+bFGF), B (DMEM/F12+B27+EGF+bFGF+LIF), C group(DMEM/F12+B27).C group is the control group.Observed three groups of NSCs growth,induced differentiation NSCs with fetal bovine serum.Identify three groups NSCs in NSE and GFAP with immunocytochemistry method,observed proportion of differentiation in NSCs.Analysis EGF,bFGF and LIF affect on growth number and differentiation of NSCs.
3. Collect5cases filum terminale tissue of TCS Children in2011.09-2012.05provided by third affiliated hospital of zhengzhou university department of pediatricorthopedics. Communication with parents of children with their agreement and consent hospital ethics committee agreement in preoperative, then to experimental study.Observe the Lesions and normal filum terminale tissue NSCs proliferation and differentiation,Compare characteristics of NSCs growth number and differentiation in different types of filum terminale tissue.
4. Statistical analyzes were performed using SPSS17.0statistical package for analysis. The experimental results expressed as(x±s), one-way ANOVA was used to compare the three groups of NSCs growth number,NSE and GFAP percentage.LSD-t test was used to compare the two groupsof NSCs growth number,NSE and GFAP percentage. Significant level being0.05.
Result
1. Observed cells morphology by inverted microscope, found those cells have NSCs morphological characteristics,those cells expression was positive by nestin identified, indicating that the cells are NSCs.
2. The number of NSCs cultured in different gestational age groups with differences, analysis results of the three groups with one-way ANOVA (p＜0.05).Prompt NSCs growth number in different gestational age groups had significant difference, each two comparison the results of three groups,compare group A and B(P＞0.05), no significant difference, compare group A and C,group B and C(p＜0.05), the difference was statistically significant.
3. NSCs growth number and differentiation have differences in different NSCs culture medium,analysis results of the three groups with one-way ANOVA(p＜0.05). Prompt used contain different cell factor medium to isolated and cultured NSCs, in growth number, NSE and GFAP-positive cell percentage were significant differences. In number of NSCs, compare three groups(P<0.05), the difference was statistically significant. In the differentiation of NSCs,compare three groups(p＜0.05), the difference was statistically significant.
4. NSCs growth number and differentiation have differences in different types of the filum terminale tissue, analysis results of the two groups with one-way ANOVA (p＜0.05), the difference was statistically significant.Prompt using different types of filum terminale tissue to isolated and cultured NSCs,in the growth number and differentiation were significantly different.
Conclusion
1. The filum terminale tissue under specific conditions can isolation and culture the NSCs,to prove there are NSCs existence in the final filament tissue. Gestational age and the NSCs they are closely related.This may be related to the proliferation potential of NSCs in the Young embryonic group.
2. Different growth factors influence the proliferation and differentiation of NSCs in the filum terminale tissue, LIF has obvious effect in promote NSCs proliferation and inhibit differentiation.
3. Lesions final filament and normal filum terminale both cultured NSCs,but the number of neurospheres and the NSE proportion in differentiation of neurospheres the normal filum terminale group was significantly higher compared with the lesions filum terminale group.The normal fetal filum terminale tissue is suitable as tissue-derived to isolation and culture NSCs in vitro.
先天性脊髓病变是严重影响患儿中枢神经系统发育的一类疾病,这类疾病包括脊髓拴系综合征(Tethered cord syndrome,TCS),脊髓纵裂(Split cord malformation, SCM),脊髓脊膜膨出(Myelomeningocele)等疾病。在上述疾病中尤其以脊髓拴系综合征(TCS)对患儿生长发育影响较大,TCS是由于终丝病变弹性降低,对脊髓圆锥末端产生持续牵拉作用,导致脊髓局部缺血缺氧改变,造成脊髓组织的神经元细胞线粒体氧化磷酸化功能障碍,导致神经纤维的传导功能受损,逐渐出现双下肢感觉运动障碍,双足畸形,大小便功能障碍等一系列神经损害临床表现的一种疾病。目前对于TCS主要治疗方式为病变终丝的切除和对脊髓圆锥末端粘连组织的松解,以达到对脊髓圆锥末尾端的充分松解,使其逐渐恢复到正常水平,进而终止或改善已出现的临床症状。但是目前这种治疗方式术后的效果并不理想。1992年Reynolds等从小鼠大脑组织(纹状体)中分离出在体外可以不断分裂增殖更新,具有向神经元细胞、星形胶质细胞和少突胶质细胞方向分化的多潜能的细胞群,提出了神经干细胞(neural stem cells,NSCs)的概念。NSCs概念的提出使人们改变了以往认为神经细胞是永久性细胞,损伤后不可再生的观念,为中枢及外周神经系统疾病的治疗和功能恢复带来了新的希望,同时也为神经系统疾病的治疗提供了一种新的治疗策略神经干细胞移植。以往对于脊髓中NSCs的研究认为其只存在于脊髓室管膜下,终丝组织不存在NSCs,对终丝组织的认识只是将其看作是从脊髓末端向下延续穿出硬脊膜连接尾骨的一结构简单的纤维血管复合物,但Varghese等分别在2009年和2010年利用人体终丝组织培养出具有NSCs增殖分化特点的细胞群,证明终丝组织中有NSCs的存在。这一发现使人们对于NSCs的认识又一次改变。目前国内对于终丝组织中NSCs研究未见有文献报道,此次实验利用胎儿终丝组织进行了NSCs的分离培养及鉴定的初步研究,这为终丝中NSCs的分离培养提供了一定的参考。
目的
1、通过原代悬浮培养的方法对胎儿终丝组织中NSCs进行分离培养并对培养出的细胞进行Nestin鉴定,表达阳性者为NSCs,从而证明终丝组织中存在NSCs。
2、通过对不同胎龄终丝及TCS患儿的病变终丝进行NSCs的分离培养来比较不同终丝组织中NSCs的增长和分化的特点。
3、通过对胎儿终丝组织中NSCs的体外分离培养的观察,分析表皮生长因子(EGF)、成纤维生长因子(bFGF)、白血病抑制因子(LIF)对NSCs增长及分化的影响。
材料和方法
1.收集2011.11-2012.8郑州大学第三附属医院妇产科12-28W流产胎儿15例,按胎龄大小分为3组：A组(12-17W),B组(18-23W),C组(24-28W),取胎儿终丝组织进行NSCs的原代分离培养,培养过程中用倒置显微镜定期观察NSCs的生长情况,计数神经球的数目比较三组胎儿终丝中NSCs的生长特点。取一定数量的NSCs进行Nestin鉴定,表达阳性者为NSCs,从而证实终丝组织中存在NSCs。
2.收集12-20W胎龄新鲜流产胎儿9例,根据在NSCs基础培养基DMEM/F12中添加物不同分为3组：A组(DMEM/F12+B27+EGF+bFGF),B组(DMEM/F12+B27+EGF+bFGF+LIF),C组(DMEM/F12+B27)为对照组,观察三组NSCs生长情况,之后用胎牛血清进行诱导分化,用免疫细胞化学的方法鉴定上述三组NSCs诱导分化后NSE和GFAP的分化比例,分析EGF、bFGF和LIF对NSCs增长和分化的影响。
3.收集2011.9-2012.5郑州大学第三附属医院小儿骨科收治的5例TCS患儿的术中切除的病变终丝,术前与家属沟通后征得其同意,上报医院伦理委员会批准同意后进行实验研究,通过观察病变和正常终丝组织中NSCs增长和分化的情况,比较不同类型终丝组织中NSCs的分离培养和分化的特点。
4.实验结果的统计学分析采用SPSS17.0统计软件包进行处理分析。实验结果以均数±标准差(x±s)表示,三组间NSCs数目、NSE和GFAP比例的比较采用单因素方差分析,组间NSCs数目、NSE和GFAP比例的两两比较采用LSD-t检验,以a=0.05为检验水准。
结果
1.对终丝中分离培养出的细胞在倒置显微镜下进行形态学观察,发现其具有NSCs形态特点,将培养出的细胞经Nestin鉴定其表达为阳性,说明培养出来的细胞为NSCs.
2.不同胎龄终丝组织中培养出的NSCs数量不同,对三组结果进行单因素方差分析(P<0.05),提示不同胎龄的终丝样本NSCs增长数目有显著性差异。对三组的NSCs数量进行两两比较,A组和B组比较P>0.05,差异无统计学意义；A组和C组比较、B组和C组比较,P均小于0.05,差异有统计学意义。
3.不同添加物组中NSCs增长和分化存在差异,对三组结果进行单因素方差分析(P(0.05),提示用含不同细胞因子的培养基分离培养NSCs,在增长数目、NSE和GFAP阳性细胞百分比方面均有显著性差异。在NSCs数目方面：三组两两比较P均小于0.05,差异有统计学意义；在NSCs分化方面：三组两两比较P均小于0.05,差异有统计学意义。
4.不同类型终丝组织中NSCs增长和分化存在差异,对二者差异进行单因素方差分析,P均小于0.05,差异有统计学意义,提示采用不同类型的终丝样本分离培养NSCs在增长和分化方面均有显著性差异。
结论
1.终丝组织在特定条件下可在体外分离培养出NSCs,胎龄大小与终丝组织中NSCs的数量有密切的关系,这可能与胎龄较小者NSCs的增长潜能较大有关。
2.不同的因子对终丝组织中NSCs的增长和分化有一定的影响,LIF在促进NSCs增长和抑制其分化方面作用较EGF和bFGF明显。
3.病变终丝与正常终丝均可以培养出NSCs,正常终丝组神经球的增长数量和分化后NSE所占的比例较病变终丝组明显增高,胎儿正常终丝组织更适宜作为NSCs的体外分离培养的组织来源。
Background
Congenital spinal cord lesion is a diseases with serious impact on children central nervous system development.It's Include tethered cord syndrome(TCS),Split cord malformation(SCM), myelomeningocele.Especially TCS with impact on children growth development.TCS is due to the lesions filum terminale to stretch the end of the conus, result in changes of spinal cord ischemia hypoxia, which led to the bowel and bladder dysfunction, deformity of the lower limbs and sensory dysfunction.At present TCS main treatment is resection the lesion filum terminale, release the end of the spinal cord,gradually returned to normal levels, and then terminate or improvement of clinical symptoms.But this treatment the results are unsatisfactory.In1992Reynolds isolated from mouse striatum in vitro proliferation of pluripotent cells, proposed the concept of neural stem cells (neural stem cells, NSCs).The concept of NSCs changed people in the past for the nerve cell is a permanent cell damage non-renewable understanding.Give a new hope of treatment diseases of the nervous system, as well as a new treatment strategy-neural stem cell transplantation for the treatment of central nervous system diseases. In the past spinal cord NSCs that exists only in the subependymal,there are no NSCs in the filum terminale.In the past the filum terminale is considered just a simple structure of fibrous tissue.Mercy and Varghese respective in2009and2010use of the human filum terminale tissue to culture the NSCs, then prove there is NSCs in human filum terminale tissue.This finding change people to understanding of the NSCs again, currently there is no reported on use of the human filum terminale tissue to culture NSCs in the domestic.In this study we use the fetal filum terminale tissue to culture NSCs preliminary study for isolating and identifying in the NSCs. This study provides some reference for isolation and culture of NSCs.
Objective
1. By primary cultured the NSCs with filum terminale tissue and identification the cells with Nestin.with positive expression is NSCs.Prove there are NSCs in the filum terminale tissue.
2. To compare the characteristics of growth number and differentiation of NSCs in different types of filum terminale tissue.
3. Observed the characteristics of proliferation and differentiation to NSCs, analysis epidermal growth factor (EGF), fibroblast growth factor (bFGF),and leukemia inhibitory factor (LIF) impact on NSCs in growth number and differentiation.
Materials and methods
1. Collect15cases of12to8w abortion Fetus in2011.11-2012.8provided by third affiliated hospital of zhengzhou university department of Obstetrics.Based on the gestational age divided into3groups:group A (12to17W), group B (18to23W), group C (24to28W). Primary cultured NSCs with the filum terminale tissue and periodical observe the growth of NSCs with inverted microscope.Count the number of neurospheres compare the growth characteristics of the three groups of NSCs.Identification the cells with Nestin.positive expression is NSCs.Prove there are NSCs in the filum terminale tissue.
2. Collect9cases of12to20w abortion Fetus.Based on additives different factors in NSCs basal medium DMEM/F12to divided into three groups:group A (DMEM/F12+B27+EGF+bFGF), B (DMEM/F12+B27+EGF+bFGF+LIF), C group(DMEM/F12+B27).C group is the control group.Observed three groups of NSCs growth,induced differentiation NSCs with fetal bovine serum.Identify three groups NSCs in NSE and GFAP with immunocytochemistry method,observed proportion of differentiation in NSCs.Analysis EGF,bFGF and LIF affect on growth number and differentiation of NSCs.
3. Collect5cases filum terminale tissue of TCS Children in2011.09-2012.05provided by third affiliated hospital of zhengzhou university department of pediatricorthopedics. Communication with parents of children with their agreement and consent hospital ethics committee agreement in preoperative, then to experimental study.Observe the Lesions and normal filum terminale tissue NSCs proliferation and differentiation,Compare characteristics of NSCs growth number and differentiation in different types of filum terminale tissue.
4. Statistical analyzes were performed using SPSS17.0statistical package for analysis. The experimental results expressed as(x±s), one-way ANOVA was used to compare the three groups of NSCs growth number,NSE and GFAP percentage.LSD-t test was used to compare the two groupsof NSCs growth number,NSE and GFAP percentage. Significant level being0.05.
Result
1. Observed cells morphology by inverted microscope, found those cells have NSCs morphological characteristics,those cells expression was positive by nestin identified, indicating that the cells are NSCs.
2. The number of NSCs cultured in different gestational age groups with differences, analysis results of the three groups with one-way ANOVA (p＜0.05).Prompt NSCs growth number in different gestational age groups had significant difference, each two comparison the results of three groups,compare group A and B(P＞0.05), no significant difference, compare group A and C,group B and C(p＜0.05), the difference was statistically significant.
3. NSCs growth number and differentiation have differences in different NSCs culture medium,analysis results of the three groups with one-way ANOVA(p＜0.05). Prompt used contain different cell factor medium to isolated and cultured NSCs, in growth number, NSE and GFAP-positive cell percentage were significant differences. In number of NSCs, compare three groups(P<0.05), the difference was statistically significant. In the differentiation of NSCs,compare three groups(p＜0.05), the difference was statistically significant.
4. NSCs growth number and differentiation have differences in different types of the filum terminale tissue, analysis results of the two groups with one-way ANOVA (p＜0.05), the difference was statistically significant.Prompt using different types of filum terminale tissue to isolated and cultured NSCs,in the growth number and differentiation were significantly different.
Conclusion
1. The filum terminale tissue under specific conditions can isolation and culture the NSCs,to prove there are NSCs existence in the final filament tissue. Gestational age and the NSCs they are closely related.This may be related to the proliferation potential of NSCs in the Young embryonic group.
2. Different growth factors influence the proliferation and differentiation of NSCs in the filum terminale tissue, LIF has obvious effect in promote NSCs proliferation and inhibit differentiation.
3. Lesions final filament and normal filum terminale both cultured NSCs,but the number of neurospheres and the NSE proportion in differentiation of neurospheres the normal filum terminale group was significantly higher compared with the lesions filum terminale group.The normal fetal filum terminale tissue is suitable as tissue-derived to isolation and culture NSCs in vitro.
引文
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