激活态雪旺细胞临床应用安全性的实验研究

发布时间：2018-05-11 08:36

本文选题：雪旺细胞 + 预变性　；参考：《复旦大学》2010年博士论文

【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。
[Abstract]:Introduction : Tissue engineering provides a new method for the repair of peripheral nerve defect , which is expected to be used in clinic in the future . The activated Schwann cells obtained by exogenous stimulation of " active liquid " ( patent application ) are promising to be safe , reliable , non - carcinogenic and Teratogenic . The purpose of this study is to test whether activated Schwann cells are safe and reliable in vitro and in vitro .

Objective : To explore the culture technology of adult Schwann cells suitable for clinical application by transverse comparison .

Methods : Adult SD rats were divided into 4 groups , 10 rats in each group and Schwann cells were cultured from bilateral sciatic nerve .
Group B adopts in vivo pre - degeneration method + slow enzyme digestion method ;
Group C was subjected to an in vitro pre - denaturation method and a rapid enzymatic digestion method ;
( 1 ) In vivo , in vitro pre - degeneration increases the number of Schwann cells in the material nerve .
( 2 ) fast and slow enzyme digestion method to obtain the efficiency of Schwann cells from the nerve ;
( 3 ) The efficiency of cold - injection passage method and pancreatic enzyme digestion and passage method . Screening makes animals less painful , while the number of Schwann cells is much , the purity is the most suitable method for clinical application .

Results : In vitro pre - degeneration and in vivo pre - degeneration could significantly increase the number of Schwann cells in the nerve of the material , but the in vitro predenaturation reduced the pain of the patients . The rapid enzyme digestion and the slow enzyme digestion method were similar to the number of Schwann cells obtained at 1 week .
The pancreatic enzyme digestion and subculture method has no purification function and is low in technical requirements of operators .

Conclusion : In vitro predenaturation reduces the pain of patients and is more suitable for clinical application .
There was no significant difference between the quick and slow enzymatic digestion methods , and the application was also considered .
The cold - injection passage method can be used in combination with trypsin digestion and passaging , the former is applied when the purity of Schwann cells is low , and the latter is applied when the purity of Schwann cells is high .

Objective : To study the biological characteristics of activated Schwann cells in vitro , compare with benign and malignant cells , and analyze the characteristics of malignant cells .

Methods : The activated Schwann cells were isolated from the sciatic nerve of adult rats and cultured in vitro . The biological characteristics of activated Schwann cells were assessed by continuous observation of cell morphology , changes of growth pattern , identification of immunological markers , identification of immunological markers , detection of proliferation and purity , chromosome karyotype analysis , whole genome expression profiling gene chip and cytokine antibody chip .

Results : The cultured Schwann cells were cultured in vitro and had no changes of heterogeneity . The proliferation ability was increased and the effect of density inhibition was decreased . The immune markers , such as S100 , P75LNGFR , GFAP and other immunological markers were not lost in the continuous passage .

Conclusion : The activated Schwann cells isolated from peripheral nerve of adult rats and cultured in vitro have no biological characteristics of malignant cells .

Objective : To explore the clinical application of activated Schwann cells in vivo and to explore whether tumors could be formed .

Methods : A sufficient amount of activated Schwann cells were cultured from bilateral sciatic nerve of adult rats , and compared with benign and malignant cells . 50 adult rats were divided into three groups : group A : activated Schwann cells , 1 脳 107 cells in autologous transplantation , 20 rats in rats ;
B : normal Schwann cells , the number of autologous transplantation cells was 1 脳 107 , the rats were 20 rats ;
Group C , C6 cells of malignant rat glioma , 2 脳 106 cells in transplanted cells , 10 rats in rats , 0.5 months , 1 month , 2 months , 4 months and 6 months after transplantation of cells .

Results : In the rats ( group C ) injected with malignant cells , the tumor was isolated from 0.5 to 1 month . The activated Schwann cells ( group A ) and normal Schwann cells ( group B ) did not form tumors in vivo , and the process was similar in vivo . At least 2 months were viable under the axilla , and the immunological markers were expressed .

Conclusion : Activated Schwann cells can survive at least 2 months in the abnormal microenvironment of the body and express the immunological markers and do not form tumors .

Objective : To explore the effect of autologous transplantation of activated Schwann cells on offspring .

Methods : A sufficient amount of activated Schwann cells were cultured from the sciatic nerve of adult female animals . The biological characters of offspring were compared with those in male rats . The results showed that the biological characters of offspring were compared with those of simple sciatic nerve , such as pregnancy rate , number of live births , number of stillbirths , weight , length of life , tail length , number of chromosomes , development of neonatal rats at 4 weeks , basic behavior , cognition of injury , etc .

Results : No significant difference was found in the number of live births , the number of dead fetuses , the weight , the length of the body , the tail length , the development of neonatal mice , the basic behavior and the cognition of the injury , regardless of whether the activated Schwann cells were transplanted , the number of dead fetuses , the body weight , the body length , the tail length , the development status , the basic behavior and the cognition of the injury .

Conclusion : Autotransplantation of activated Schwann cells has no significant effect on the biological characters of offspring of adult females .

【学位授予单位】：复旦大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R329

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