不同部位取材的雪旺细胞培养、纯化及其与丝素蛋白纳米纤维材料的相容性研究
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摘要
目的:建立能够获得高活性、高纯度和足够数量雪旺细胞(schwann cells,SCs)的培养方法,为周围神经缺损的修复提供优质、足量的种子细胞。寻找支持SCs的粘附、生长的材料,提供可引导其生长和具有良好生物相容性的三维生物支架。
方法:1.选用6只5 d龄SD大鼠,切取背根神经节(实验组)和坐骨神经(对照组)。采用联合酶消化加机械吹打法分离培养SCs,并进行纯化及传代培养。取第1代SCs,用计数法绘制培养8 d内SCs生长曲线,MTT法检测8 d内SCs增殖情况,抗S-100免疫细胞化学法检测SCs纯度,酶联免疫吸附试验(enzyme-linked immunosorbent assay,ELISA)检测脑源性神经营养因子(brain-derived neurotrophic factor,BDNF)浓度。2.采用静电纺丝技术制备1000 nm直径的丝素蛋白纳米纤维。3.将从新生SD大鼠背根神经节分离的SCs经改良差速贴壁纯化培养后,取第1代细胞分别接种至培养皿中,盖玻片分别以左旋多聚赖氨酸(PLL组)、丝素蛋白纳米纤维1000 nm组(TSF组)包被,复合培养不同时间点行倒置相差显微镜观察细胞形态、生长及与材料黏附情况,抗S-100免疫荧光染色鉴定两组SCs并计算其纯度,MTT法测定2组SCs的增殖情况并行材料毒性评价,ELISA法检测2组细胞分泌BDNF浓度。
结果:1.每只大鼠可切取背根神经节36～43个。消化成单个细胞后计数,实验组可提取(7.5±0.6)×10~6个SCs,高于对照组(3.5±0.4)×10~6个SCs,差异有统计学意义(P<0.05)。SCs第3 d开始进入对数增殖期,随培养时间延长,两组细胞数量及增殖吸光度(A)值均呈上升趋势;培养3、4、5、6 d,实验组细胞数及A值明显高于对照组,差异有统计学意义(P<0.05)。经S-100免疫细胞化学法检测,实验组SCs纯度为92.08%±3.45%,对照组为77.50%±3.57%,差异有统计学意义(P<0.05)。ELISA法检测示实验组培养3 d和5 d的BDNF浓度均高于对照组,差异有统计学意义(P<0.05)。2.倒置相差显微镜示丝素蛋白纳米纤维上培养的SCs与对照组相比,细胞沿丝素纤维分布,细胞突起方向与丝素蛋白走向较一致,形态特征无显著差异。培养3 d时,S-100免疫荧光染色示PLL组SCs纯度为91.25%±2.57%,TSF组为91.51%±1.34%(P>0.05);SCs在丝素蛋白纳米纤维上仍保留其特有的表现型,TSF组轴突长度及复杂度均高于PLL组。MTT检测示培养1、3、5、7、9 d两组吸光度值比较差异无统计学意义(P>0.05)。培养1、3、5、7、9 d,细胞相对增殖率均大于90%,根据GB/T 16886标准,细胞毒性为1级,无毒。ELISA法检测示培养3 d和5 d两组SCs分泌的BDNF浓度水平比较,差异无统计学意义(P>0.05)。
结论:取材于背根神经节能获得数量更多、纯度和活性更高的SCs,能满足周围神经修复研究的需要。柞蚕丝素蛋白纳米三维多孔支架生物相容性良好,安全无毒,有望成为修复周围神经损伤的组织工程支架材料。
Objective:To establish the methods to get high activated, purified and adequate Schwann cells (schwann cells, SCs),and provide sufficient seed cells for peripheral nerve repairment. To find a material which support SCs adhesion and proliferation and to provide a three diamensions bioscaffold,which has ideal biocompatibility and can guide the growth of SCs.
Methods:1. Six Sprague‐Dawley rats(5 days old)were divided into sciatic nerve group (control group) and DRG (dorsal root ganglia, DRG) group (experime‐ ntal group,then the sciatic nerves and DRGs were harvested respectively. They were digested by co‐enzyme and dispersed by medium containing serum. Freshly isolated SCs from rats were cultured, purified and subcultured. 1st generation of SCs were choosed. Growth curve of SCs were drawed by the counting method and the proliferation of SCs was detected by MTT assay in 8 days in culture. Immunocyto‐ chemistry of Anti‐S100 was used to detected the purity of SCs. ELISA (enzyme‐linked immunosorbent assay, ELISA) was used to detected BDNF(brain‐derived neurotrophic factor, BDNF) concentration. 2. Silk fibrion nanofibers, which diameter is 1000nm, were prepared by using electrospinning techniques. 3. Freshly isolated SCs from 5 days SD rats’s DRG were purified by the modifided differential adherent velocity method and cultured. 1st generation of SCs were seeded on the poly‐l‐ lysine(PLL group) and tussah silk fibrioin nanofibers(TSF group).The coverslips were coated in Petri dish. At different time points, the morphological features, growth and adhesion of cells were observed using phase contrast inverted microscopy. Immunocytochemistry of Anti‐S100 was used to identitied the purity of SCs in two group. The proliferation of SCs was determined by MTT assay. The cytoxicity of TSF was evaluated. ELISA was used to detected BDNF concentration.
Results: 1. Each rat could get 36～43 DRGs. Single cells were counted after digestion. A total of(7.5±0.6)×10~6 SCs could be obtained in experimental group higher than (3.5±0.4)×10~6 SCs in control group .There was significant difference between two groups (P< 0.05). SCs reach logarithm proliferation phase in 3 days. The cells’s quantity and A value of two groups all show upward trend along of time proceed. The number and A value of experimental group is higher than control group. There were significant difference between two groups(P<0.05). Immunocytochemist‐ ry of Anti‐S‐100 detects SCs. The purity of experimental group (92.08%±3.45% ) is higher than control group (77.50%±3.57%) . The difference is significant (P<0.05). Elisa results showed that the density of BDNF at 3d and 5d in experimental group was higher than control group. The difference is significant (P<0.05). 2. The morphology of SCs on the TSF group was better than PLL group. The cells distributed along the fibers, and the cell protrusions were in the same as that of the fibers. Immunofluorescence showed that the purity was 91.25%±2.57% in the PLL group and 91.51%±1.34% in the TSF group 3 days after subculture(P>0.05). SCs still maintained the characteristic phenotype in TSF nanofibers. The axons’s length and diversity of SCs in TSF group was higher than PLL group. The A value at1,3,5,7,9 days was detected by MTT assay and didn’t have significant differences by contrast in two growps(P>0.05). The relative growth rates were above 90% at different time points. The cytotoxicity of the material was grade 1 and nonevenomous according to GB/T 16886 standard. Elisa results showed that the density of BDNF at 3 days and 5 days in SF group was similar to PLL group. No significant difference (P>0.05).
Conclusion: The sufficient amount, higher purity and viability of SCs which drawing from DRG can meet the needs of studies on peripheral nerve repairment. Three diamensions scaffold which was made of TSF nanofibers have good compatibility and nonvenomous. It’s a promising tissue engineered scaffold for the repair of peripheral nerve injury.
方法:1.选用6只5 d龄SD大鼠,切取背根神经节(实验组)和坐骨神经(对照组)。采用联合酶消化加机械吹打法分离培养SCs,并进行纯化及传代培养。取第1代SCs,用计数法绘制培养8 d内SCs生长曲线,MTT法检测8 d内SCs增殖情况,抗S-100免疫细胞化学法检测SCs纯度,酶联免疫吸附试验(enzyme-linked immunosorbent assay,ELISA)检测脑源性神经营养因子(brain-derived neurotrophic factor,BDNF)浓度。2.采用静电纺丝技术制备1000 nm直径的丝素蛋白纳米纤维。3.将从新生SD大鼠背根神经节分离的SCs经改良差速贴壁纯化培养后,取第1代细胞分别接种至培养皿中,盖玻片分别以左旋多聚赖氨酸(PLL组)、丝素蛋白纳米纤维1000 nm组(TSF组)包被,复合培养不同时间点行倒置相差显微镜观察细胞形态、生长及与材料黏附情况,抗S-100免疫荧光染色鉴定两组SCs并计算其纯度,MTT法测定2组SCs的增殖情况并行材料毒性评价,ELISA法检测2组细胞分泌BDNF浓度。
结果:1.每只大鼠可切取背根神经节36～43个。消化成单个细胞后计数,实验组可提取(7.5±0.6)×10~6个SCs,高于对照组(3.5±0.4)×10~6个SCs,差异有统计学意义(P<0.05)。SCs第3 d开始进入对数增殖期,随培养时间延长,两组细胞数量及增殖吸光度(A)值均呈上升趋势;培养3、4、5、6 d,实验组细胞数及A值明显高于对照组,差异有统计学意义(P<0.05)。经S-100免疫细胞化学法检测,实验组SCs纯度为92.08%±3.45%,对照组为77.50%±3.57%,差异有统计学意义(P<0.05)。ELISA法检测示实验组培养3 d和5 d的BDNF浓度均高于对照组,差异有统计学意义(P<0.05)。2.倒置相差显微镜示丝素蛋白纳米纤维上培养的SCs与对照组相比,细胞沿丝素纤维分布,细胞突起方向与丝素蛋白走向较一致,形态特征无显著差异。培养3 d时,S-100免疫荧光染色示PLL组SCs纯度为91.25%±2.57%,TSF组为91.51%±1.34%(P>0.05);SCs在丝素蛋白纳米纤维上仍保留其特有的表现型,TSF组轴突长度及复杂度均高于PLL组。MTT检测示培养1、3、5、7、9 d两组吸光度值比较差异无统计学意义(P>0.05)。培养1、3、5、7、9 d,细胞相对增殖率均大于90%,根据GB/T 16886标准,细胞毒性为1级,无毒。ELISA法检测示培养3 d和5 d两组SCs分泌的BDNF浓度水平比较,差异无统计学意义(P>0.05)。
结论:取材于背根神经节能获得数量更多、纯度和活性更高的SCs,能满足周围神经修复研究的需要。柞蚕丝素蛋白纳米三维多孔支架生物相容性良好,安全无毒,有望成为修复周围神经损伤的组织工程支架材料。
Objective:To establish the methods to get high activated, purified and adequate Schwann cells (schwann cells, SCs),and provide sufficient seed cells for peripheral nerve repairment. To find a material which support SCs adhesion and proliferation and to provide a three diamensions bioscaffold,which has ideal biocompatibility and can guide the growth of SCs.
Methods:1. Six Sprague‐Dawley rats(5 days old)were divided into sciatic nerve group (control group) and DRG (dorsal root ganglia, DRG) group (experime‐ ntal group,then the sciatic nerves and DRGs were harvested respectively. They were digested by co‐enzyme and dispersed by medium containing serum. Freshly isolated SCs from rats were cultured, purified and subcultured. 1st generation of SCs were choosed. Growth curve of SCs were drawed by the counting method and the proliferation of SCs was detected by MTT assay in 8 days in culture. Immunocyto‐ chemistry of Anti‐S100 was used to detected the purity of SCs. ELISA (enzyme‐linked immunosorbent assay, ELISA) was used to detected BDNF(brain‐derived neurotrophic factor, BDNF) concentration. 2. Silk fibrion nanofibers, which diameter is 1000nm, were prepared by using electrospinning techniques. 3. Freshly isolated SCs from 5 days SD rats’s DRG were purified by the modifided differential adherent velocity method and cultured. 1st generation of SCs were seeded on the poly‐l‐ lysine(PLL group) and tussah silk fibrioin nanofibers(TSF group).The coverslips were coated in Petri dish. At different time points, the morphological features, growth and adhesion of cells were observed using phase contrast inverted microscopy. Immunocytochemistry of Anti‐S100 was used to identitied the purity of SCs in two group. The proliferation of SCs was determined by MTT assay. The cytoxicity of TSF was evaluated. ELISA was used to detected BDNF concentration.
Results: 1. Each rat could get 36～43 DRGs. Single cells were counted after digestion. A total of(7.5±0.6)×10~6 SCs could be obtained in experimental group higher than (3.5±0.4)×10~6 SCs in control group .There was significant difference between two groups (P< 0.05). SCs reach logarithm proliferation phase in 3 days. The cells’s quantity and A value of two groups all show upward trend along of time proceed. The number and A value of experimental group is higher than control group. There were significant difference between two groups(P<0.05). Immunocytochemist‐ ry of Anti‐S‐100 detects SCs. The purity of experimental group (92.08%±3.45% ) is higher than control group (77.50%±3.57%) . The difference is significant (P<0.05). Elisa results showed that the density of BDNF at 3d and 5d in experimental group was higher than control group. The difference is significant (P<0.05). 2. The morphology of SCs on the TSF group was better than PLL group. The cells distributed along the fibers, and the cell protrusions were in the same as that of the fibers. Immunofluorescence showed that the purity was 91.25%±2.57% in the PLL group and 91.51%±1.34% in the TSF group 3 days after subculture(P>0.05). SCs still maintained the characteristic phenotype in TSF nanofibers. The axons’s length and diversity of SCs in TSF group was higher than PLL group. The A value at1,3,5,7,9 days was detected by MTT assay and didn’t have significant differences by contrast in two growps(P>0.05). The relative growth rates were above 90% at different time points. The cytotoxicity of the material was grade 1 and nonevenomous according to GB/T 16886 standard. Elisa results showed that the density of BDNF at 3 days and 5 days in SF group was similar to PLL group. No significant difference (P>0.05).
Conclusion: The sufficient amount, higher purity and viability of SCs which drawing from DRG can meet the needs of studies on peripheral nerve repairment. Three diamensions scaffold which was made of TSF nanofibers have good compatibility and nonvenomous. It’s a promising tissue engineered scaffold for the repair of peripheral nerve injury.
引文
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2. Fu SY, Gordon T. The cellular and molecular basis of peripheral nerve regeneration. Mol Neurobiol, 1997,14(1-2):67-116.
3. Goodrum JF, Bouldin TW. The cell biology of myelin degeneration and regeneration in the peripheral nervous system. J Neuropathol Exp Neurol, 1996,55(9):943-953.
4. Wei Y, Zhou J, Zheng Z, et al. An improved method for isolating Schwann cells from postnatal rat sciatic nerves. Cell Tissue Res, 2009,337(3):361-369.
5. Pannunzio ME, Jou IM, Long A, et al. A new method of selecting Schwann cells from adult mouse sciatic nerve. J Neurosci Methods, 2005,149(1):74-81.
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