激活态雪旺细胞临床应用安全性的实验研究
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摘要
引言：组织工程为周围神经缺损的修复提供了新的方法,填充了雪旺细胞的可吸收人工神经导管有望在将来替代自体神经移植应用于临床。通过“激活液”(专利申请中)外源性刺激获得的激活态雪旺细胞,在增殖能力和分泌活性都明显超过正常的雪旺细胞,而且促进神经再生的效果也明显增强。体外培养、扩增的激活态雪旺细胞应用于临床的一个必要前提是安全可靠,无致癌性、致畸性。本研究的目的是联合体内、体外多个实验,检测激活态雪旺细胞是否安全,有无应用于临床的可能。本研究共分为4个部分：
目的：通过横向比较,探索适合临床应用的成年雪旺细胞培养技术。
方法：成年SD大鼠,分4组,每组10只,从双侧坐骨神经取材培养雪旺细胞。A组采用体内预变性法+快速酶消化法；B组采用体内预变性法+慢速酶消化法；C组采用体外预变性法+快速酶消化法；D组采用体外预变性法+慢速酶消化法。细胞传代使用冷喷射传代法和胰酶消化传代法。比较(1)体内、体外预变性增加取材神经中雪旺细胞的数目有无差别；(2)快速、慢速酶消化法从神经中获取雪旺细胞的效率；(3)冷喷射传代法和胰酶消化传代法的效率。筛选使动物痛苦小、而雪旺细胞数量多、纯度高等最适合临床应用的方法。
结果：体外预变性和体内预变性都能明显增加取材神经中的雪旺细胞数目,无统计学差异,但体外预变性减轻了患者的痛苦。快速酶消化法和慢速酶消化法在培养1周时获得的雪旺细胞数目相似,无统计学差异。冷喷射传代法兼有纯化雪旺细胞的作用,会损失一定数量的雪旺细胞,对操作者技术要求高；胰酶消化传代法没有纯化的作用,对操作者技术要求低。
结论：体外预变性减轻患者痛苦,更适合临床应用；快速、慢速酶消化法效果无明显差异,均可考虑应用；冷喷射传代法可以和胰酶消化传代法有机结合使用,前者在雪旺细胞纯度较低时应用,后者在雪旺细胞纯度较高时应用。
目的：体外检测激活态雪旺细胞的生物学特性,与良性、恶性细胞对比,分析其是否有恶性细胞的特点。
方法：从成年大鼠坐骨神经中分离并体外培养激活态雪旺细胞,通过对细胞形态的连续观察、生长方式的变化、免疫学标志物鉴定、增殖率和纯度检测、迁移能力和侵袭能力检测、染色体核型分析,及全基因组表达谱基因芯片、细胞因子抗体芯片检测,对激活态雪旺细胞的生物学特性进行综合评估。
结果：激活态雪旺细胞在体外培养形态均一、无异质性变化。增殖能力增加,受密度抑制影响减小。连续传代不丢失S100、P75LNGFR、GFAP等免疫学标识物,迁移能力、侵袭能力略有增加,但明显弱于恶性细胞。染色体仍为2倍体。基因和蛋白表达存在动态平衡,第2、3代总体表达最高。
结论：从成年大鼠周围神经分离并体外培养获得的激活态雪旺细胞,基本不具备恶性细胞的生物学特性。
目的：模拟临床应用,探索激活态雪旺细胞的体内变化过程,是否会形成肿瘤。
方法：从成年大鼠双侧坐骨神经培养足量的激活态雪旺细胞,移植到自体腋窝下,与良性、恶性细胞移植对比。50只成年大鼠分三组：A组：激活态雪旺细胞,自体移植细胞数目1×107个,大鼠20只；B：正常雪旺细胞,自体移植细胞数目1×107个,大鼠20只；C组,恶性大鼠胶质瘤C6细胞,移植细胞数目2×106个,大鼠10只。分别在移植细胞后0.5个月、1个月、2个月、4个月、6个月对大鼠腋窝注射细胞处取材,观察大体标本上是否有细胞团或肿瘤形成,解剖局部及内脏观察有无肿瘤转移灶的形成。将局部的细胞团块或肿瘤做免疫组织化学鉴定,并查找病理性核分裂相。
结果：注射恶性细胞的大鼠(C组),均在0.5-1个月长出肿瘤。而激活态雪旺细胞组(A组)和正常雪旺细胞组(B组)在体内不形成肿瘤,体内的过程相似,至少可以在腋窝下存活2个月,并表达免疫学标志物。
结论：激活态雪旺细胞在体内非正常微环境下至少可以存活2个月,并表达免疫学标志物,不形成肿瘤。
目的：探索激活态雪旺细胞自体移植是否对后代产生影响。
方法：从成年雌性动物坐骨神经培养出足量的激活态雪旺细胞后自体移植,然后与雄性大鼠同笼喂养3个月。与单纯坐骨神经切除的动物对比后代的生物学性状,如怀孕率,新生鼠的活胎数、死胎数、体重、体长、尾长的比较,染色体数目的检测。新生鼠4周时的发育、基本行为能力、对伤害的认知等。
结果：无论是否移植激活态雪旺细胞,单侧坐骨神经切除后的成年雌性大鼠,3个月内怀孕率均为40%。实验组和对照组的新生鼠的活胎数、死胎数、体重、体长、尾长无统计学差异,新生鼠发育状况、基本行为能力、对伤害的认知等均未见明显异常。新生鼠的染色体仍为21对。
结论：激活态雪旺细胞的自体移植对成年雌性动物的后代的生物学性状不产生明显的影响。
Introduction:
Tissue engineering supplies a new approach in repairing peripheral nerve defects. The absorbable artificial nerve conduits filled with Schwann cells could be an alternative substitute of peripheral nerve autograft. Activated Schwann cells, which are acquired through stimulating Schwann cells with "activation liquid" (patent in applying), have higher reproductive activity and secrete more growth factors. Furthermore, activated Schwann cells appear to facilitate much more peripheral nerve regeneration. An important prerequisite of activated Schwann cells utilizing in clinical application is absolute safeness, that is, no carcinogenicity and no teratogenicity. The aim of this study was to detect the malignant potential of activated Schwann cells in vitro and in vivo. This study includes four parts:
Objective:We aim to find some clinically applicable techniques in adult Schwann cell culture through comparative study.
Methods:Fourty adult rats were randomly divided into 4 groups. Schwann cells were harvested from bilateral sciatic nerves. Group A:Predegeneration in vivo+Fast enzyme digestion; Group B:Predegeneration in vivo+Slow enzyme digestion; Group C:Predegeneration in vitro+Fast enzyme digestion; Group:Pregegeneration in vitro +Slow enzyme digestion. Cells were passaged with cold jet or trypsinization. Comparative studies include:(1) The difference of predegeneration between in vitro and in vivo in increasing the number of Schwann cells within peripheral nerves; (2) The efficiency of harvesting Schwann cells between fast enzyme digestion and slow enzyme digestion; (3) The efficiency of cold jet and trypsinization in passage. Through comparative studies, the most suitable methods were found to culture Schwann cells in clinical application.
Results:Predegeneration, both in vivo and in vitro were effective in increasing the number of Schwann cells within peripheral nerves. The number of Schwann cells expanded in vitro for 1 week, were similar with either fast enzyme digestion or slow enzyme digestion. No statistical significance was found among the four groups. Both cold jet and trypsinization were effective in passage. The differences were that cold jet could purify Schwann cells during passage, but needs skillful operators and losses a certain number of cells; and trypsinization does the opposite. Conclusion:Predegeneration in vitro alleviates patients'suffering, and more applicable in clinical medicine. Both fast and slow enzyme digestion could be use in harvesting Schwann cells from peripheral nerves. Cold jet and trypsinization should be combined in passaging Schwann cells.
Objective:We aim to assess the malignant potential of activated Schwann cells in vitro through comparison with both benign and malignant cells.
Methods:After dissociation from adult rat sciatic nerves, activated Schwann cells were cultured in vitro. We detected the biological characteristics of activated Schwann cells in vitro through a serial of experiments, such as successive cell morphology and growth pattern observations, immunological marker identifications, proliferation index and purity detections, wound healing assay and Transwell invasion assay, karyotype analysis and detections of gene expression profiling chips and cytokine antibody chips. And the characteristics of activated Schwann cells were compared with normal Schwann cells and malignant cells.
Results:Morphology of activated Schwann cells was the same with normal ones. Proliferation activity was increased, and density inhibition was decreased in activated Schwann cells. Activation and successive passages did not influence the expression of immunological markers of Schwann cells. Proliferation rate of activated Schwann cells was higher than normal ones. After activation, the abilities of migration and invasion of Schwann cells were a little enhanced, however, much lower than malignant cells. The karyotype of activated Schwann cells was normal. The expressions of genes and proteins were under dynamic balance, and the highest viability was in the second and third passages.
Conclusion:Activated Schwann cells, which were dissociated from rat peripheral nerves and expanded in vitro, have no biological characteristics of malignant cells.
Objective:This study was to detect whether activated Schwann cells have carcinogenicity in vivo or not.
Methods:Sufficient activated Schwann cells were harvested from rat bilateral sciatic nerves. Then the cells were were autotransplanted into axillary fossa to see whether they could produce tumors in vivo or not. Normal Schwann cells were used as a negative control, and malignant rat C6 glioma cells were used as a positive control. Fifty rats were randomly divided into 3 groups. Group A:A total of 1×107 activated Schwann cells were autotransplanted into right axillary fossa of 20 rats; Group B:A total of 1×107normal Schwann cells were autotransplanted into right axillary fossa of 20 rats; Group C:A total of 2×106 rat glioma cells were transplanted into right axillary fossa of 10 rats. Equivalent rats were sacrificed at 0.5 months,1 months,2 months,4 months and 6 months after cell transplantation, respectively. The complete gross necropsies of adjacent organs were done to find any potential hypercellular area, which were further examined histologically. The origin of cell mass was identified through immunohistochemistry. Karyokinesis was observed in sections from all cell masses after H&E staining.
Results:All the 10 rats receiving C6 cell transplantation developed tumors during 0.5 to 1 month. And all rats which were autotransplanted with Schwann cells, whether activated or normal Schwann cells, were found no tumor formation. And Schwann cells could survive at axillary fossa for at least 2 months and express immunological markers.
Conclusion:Activated Schwann cells could survive and express immunological markers at least 2 months in abnormal micro-enviroment in vivo, and produce no tumors.
Objective:This study was to detect whether autotransplantation of activated Schwann cells have influences on descendants.
Methods:Sufficient activated Schwann cells were harvested from sciatic nerves of adult female rats. Adult female rats simply resected sciatic nerves were used as controls. After autotransplantation, all rats were caged with male rats. Items of comparative detection include:pregnancy rate, the amount of foeti, body weight, body length, tail length and chromosome number. It was also observed that growth, fundamental behavioral capacity and cognition of injury till four weeks.
Result:The pregnancy rates of adult female rats which were resected of unilateral sciatic nerve were 40% in 3 months. No difference was seen between autotransplantation and control group. Similarly, no significant difference was seen in parameters of newly born rats, such as live feoti, dead feoti, body weight, body length, tail length, et al. No abnormalities were found in growth, fundamental behavioral capacity and cognition of injury of newly born rats four weeks later. The chromosome number of newly born rats was 42.
Conclusion:Autotransplation of activated Schwann cells did not influence the biological characteristics of descendants of adult female rats.
目的：通过横向比较,探索适合临床应用的成年雪旺细胞培养技术。
方法：成年SD大鼠,分4组,每组10只,从双侧坐骨神经取材培养雪旺细胞。A组采用体内预变性法+快速酶消化法；B组采用体内预变性法+慢速酶消化法；C组采用体外预变性法+快速酶消化法；D组采用体外预变性法+慢速酶消化法。细胞传代使用冷喷射传代法和胰酶消化传代法。比较(1)体内、体外预变性增加取材神经中雪旺细胞的数目有无差别；(2)快速、慢速酶消化法从神经中获取雪旺细胞的效率；(3)冷喷射传代法和胰酶消化传代法的效率。筛选使动物痛苦小、而雪旺细胞数量多、纯度高等最适合临床应用的方法。
结果：体外预变性和体内预变性都能明显增加取材神经中的雪旺细胞数目,无统计学差异,但体外预变性减轻了患者的痛苦。快速酶消化法和慢速酶消化法在培养1周时获得的雪旺细胞数目相似,无统计学差异。冷喷射传代法兼有纯化雪旺细胞的作用,会损失一定数量的雪旺细胞,对操作者技术要求高；胰酶消化传代法没有纯化的作用,对操作者技术要求低。
结论：体外预变性减轻患者痛苦,更适合临床应用；快速、慢速酶消化法效果无明显差异,均可考虑应用；冷喷射传代法可以和胰酶消化传代法有机结合使用,前者在雪旺细胞纯度较低时应用,后者在雪旺细胞纯度较高时应用。
目的：体外检测激活态雪旺细胞的生物学特性,与良性、恶性细胞对比,分析其是否有恶性细胞的特点。
方法：从成年大鼠坐骨神经中分离并体外培养激活态雪旺细胞,通过对细胞形态的连续观察、生长方式的变化、免疫学标志物鉴定、增殖率和纯度检测、迁移能力和侵袭能力检测、染色体核型分析,及全基因组表达谱基因芯片、细胞因子抗体芯片检测,对激活态雪旺细胞的生物学特性进行综合评估。
结果：激活态雪旺细胞在体外培养形态均一、无异质性变化。增殖能力增加,受密度抑制影响减小。连续传代不丢失S100、P75LNGFR、GFAP等免疫学标识物,迁移能力、侵袭能力略有增加,但明显弱于恶性细胞。染色体仍为2倍体。基因和蛋白表达存在动态平衡,第2、3代总体表达最高。
结论：从成年大鼠周围神经分离并体外培养获得的激活态雪旺细胞,基本不具备恶性细胞的生物学特性。
目的：模拟临床应用,探索激活态雪旺细胞的体内变化过程,是否会形成肿瘤。
方法：从成年大鼠双侧坐骨神经培养足量的激活态雪旺细胞,移植到自体腋窝下,与良性、恶性细胞移植对比。50只成年大鼠分三组：A组：激活态雪旺细胞,自体移植细胞数目1×107个,大鼠20只；B：正常雪旺细胞,自体移植细胞数目1×107个,大鼠20只；C组,恶性大鼠胶质瘤C6细胞,移植细胞数目2×106个,大鼠10只。分别在移植细胞后0.5个月、1个月、2个月、4个月、6个月对大鼠腋窝注射细胞处取材,观察大体标本上是否有细胞团或肿瘤形成,解剖局部及内脏观察有无肿瘤转移灶的形成。将局部的细胞团块或肿瘤做免疫组织化学鉴定,并查找病理性核分裂相。
结果：注射恶性细胞的大鼠(C组),均在0.5-1个月长出肿瘤。而激活态雪旺细胞组(A组)和正常雪旺细胞组(B组)在体内不形成肿瘤,体内的过程相似,至少可以在腋窝下存活2个月,并表达免疫学标志物。
结论：激活态雪旺细胞在体内非正常微环境下至少可以存活2个月,并表达免疫学标志物,不形成肿瘤。
目的：探索激活态雪旺细胞自体移植是否对后代产生影响。
方法：从成年雌性动物坐骨神经培养出足量的激活态雪旺细胞后自体移植,然后与雄性大鼠同笼喂养3个月。与单纯坐骨神经切除的动物对比后代的生物学性状,如怀孕率,新生鼠的活胎数、死胎数、体重、体长、尾长的比较,染色体数目的检测。新生鼠4周时的发育、基本行为能力、对伤害的认知等。
结果：无论是否移植激活态雪旺细胞,单侧坐骨神经切除后的成年雌性大鼠,3个月内怀孕率均为40%。实验组和对照组的新生鼠的活胎数、死胎数、体重、体长、尾长无统计学差异,新生鼠发育状况、基本行为能力、对伤害的认知等均未见明显异常。新生鼠的染色体仍为21对。
结论：激活态雪旺细胞的自体移植对成年雌性动物的后代的生物学性状不产生明显的影响。
Introduction:
Tissue engineering supplies a new approach in repairing peripheral nerve defects. The absorbable artificial nerve conduits filled with Schwann cells could be an alternative substitute of peripheral nerve autograft. Activated Schwann cells, which are acquired through stimulating Schwann cells with "activation liquid" (patent in applying), have higher reproductive activity and secrete more growth factors. Furthermore, activated Schwann cells appear to facilitate much more peripheral nerve regeneration. An important prerequisite of activated Schwann cells utilizing in clinical application is absolute safeness, that is, no carcinogenicity and no teratogenicity. The aim of this study was to detect the malignant potential of activated Schwann cells in vitro and in vivo. This study includes four parts:
Objective:We aim to find some clinically applicable techniques in adult Schwann cell culture through comparative study.
Methods:Fourty adult rats were randomly divided into 4 groups. Schwann cells were harvested from bilateral sciatic nerves. Group A:Predegeneration in vivo+Fast enzyme digestion; Group B:Predegeneration in vivo+Slow enzyme digestion; Group C:Predegeneration in vitro+Fast enzyme digestion; Group:Pregegeneration in vitro +Slow enzyme digestion. Cells were passaged with cold jet or trypsinization. Comparative studies include:(1) The difference of predegeneration between in vitro and in vivo in increasing the number of Schwann cells within peripheral nerves; (2) The efficiency of harvesting Schwann cells between fast enzyme digestion and slow enzyme digestion; (3) The efficiency of cold jet and trypsinization in passage. Through comparative studies, the most suitable methods were found to culture Schwann cells in clinical application.
Results:Predegeneration, both in vivo and in vitro were effective in increasing the number of Schwann cells within peripheral nerves. The number of Schwann cells expanded in vitro for 1 week, were similar with either fast enzyme digestion or slow enzyme digestion. No statistical significance was found among the four groups. Both cold jet and trypsinization were effective in passage. The differences were that cold jet could purify Schwann cells during passage, but needs skillful operators and losses a certain number of cells; and trypsinization does the opposite. Conclusion:Predegeneration in vitro alleviates patients'suffering, and more applicable in clinical medicine. Both fast and slow enzyme digestion could be use in harvesting Schwann cells from peripheral nerves. Cold jet and trypsinization should be combined in passaging Schwann cells.
Objective:We aim to assess the malignant potential of activated Schwann cells in vitro through comparison with both benign and malignant cells.
Methods:After dissociation from adult rat sciatic nerves, activated Schwann cells were cultured in vitro. We detected the biological characteristics of activated Schwann cells in vitro through a serial of experiments, such as successive cell morphology and growth pattern observations, immunological marker identifications, proliferation index and purity detections, wound healing assay and Transwell invasion assay, karyotype analysis and detections of gene expression profiling chips and cytokine antibody chips. And the characteristics of activated Schwann cells were compared with normal Schwann cells and malignant cells.
Results:Morphology of activated Schwann cells was the same with normal ones. Proliferation activity was increased, and density inhibition was decreased in activated Schwann cells. Activation and successive passages did not influence the expression of immunological markers of Schwann cells. Proliferation rate of activated Schwann cells was higher than normal ones. After activation, the abilities of migration and invasion of Schwann cells were a little enhanced, however, much lower than malignant cells. The karyotype of activated Schwann cells was normal. The expressions of genes and proteins were under dynamic balance, and the highest viability was in the second and third passages.
Conclusion:Activated Schwann cells, which were dissociated from rat peripheral nerves and expanded in vitro, have no biological characteristics of malignant cells.
Objective:This study was to detect whether activated Schwann cells have carcinogenicity in vivo or not.
Methods:Sufficient activated Schwann cells were harvested from rat bilateral sciatic nerves. Then the cells were were autotransplanted into axillary fossa to see whether they could produce tumors in vivo or not. Normal Schwann cells were used as a negative control, and malignant rat C6 glioma cells were used as a positive control. Fifty rats were randomly divided into 3 groups. Group A:A total of 1×107 activated Schwann cells were autotransplanted into right axillary fossa of 20 rats; Group B:A total of 1×107normal Schwann cells were autotransplanted into right axillary fossa of 20 rats; Group C:A total of 2×106 rat glioma cells were transplanted into right axillary fossa of 10 rats. Equivalent rats were sacrificed at 0.5 months,1 months,2 months,4 months and 6 months after cell transplantation, respectively. The complete gross necropsies of adjacent organs were done to find any potential hypercellular area, which were further examined histologically. The origin of cell mass was identified through immunohistochemistry. Karyokinesis was observed in sections from all cell masses after H&E staining.
Results:All the 10 rats receiving C6 cell transplantation developed tumors during 0.5 to 1 month. And all rats which were autotransplanted with Schwann cells, whether activated or normal Schwann cells, were found no tumor formation. And Schwann cells could survive at axillary fossa for at least 2 months and express immunological markers.
Conclusion:Activated Schwann cells could survive and express immunological markers at least 2 months in abnormal micro-enviroment in vivo, and produce no tumors.
Objective:This study was to detect whether autotransplantation of activated Schwann cells have influences on descendants.
Methods:Sufficient activated Schwann cells were harvested from sciatic nerves of adult female rats. Adult female rats simply resected sciatic nerves were used as controls. After autotransplantation, all rats were caged with male rats. Items of comparative detection include:pregnancy rate, the amount of foeti, body weight, body length, tail length and chromosome number. It was also observed that growth, fundamental behavioral capacity and cognition of injury till four weeks.
Result:The pregnancy rates of adult female rats which were resected of unilateral sciatic nerve were 40% in 3 months. No difference was seen between autotransplantation and control group. Similarly, no significant difference was seen in parameters of newly born rats, such as live feoti, dead feoti, body weight, body length, tail length, et al. No abnormalities were found in growth, fundamental behavioral capacity and cognition of injury of newly born rats four weeks later. The chromosome number of newly born rats was 42.
Conclusion:Autotransplation of activated Schwann cells did not influence the biological characteristics of descendants of adult female rats.
引文
[1]Brenner M J, Hess J R, Myckatyn T M, et al. Repair of motor nerve gaps with sensory nerve inhibits regeneration in rats.[J]. Laryngoscope,2006,116(9):1685-1692.
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