论文部分内容阅读
BACKGROUND: Schwann cells (SCs) are neuroglial cells of peripheral nerve and play a key role in repairing peripheral nerve injury; therefore, it provides an important evidence for transplantation of SCs which are characterized by active proliferation and adult high-purity in vitro after nerve injury in clinic, and also develops a new therapeutic way for nerve injury. OBJECTIVE: To investigate an effective technique for isolating adult activated Schwann cells. DESIGN: Controlled observational study. SETTING: Mudanjiang Medical College. MATERIALS: The experiment was completed at the Department of Medical Genetics of Harbin Medical University from March 2003 to April 2005. Health female Wistar rats, aged 2 months, weighting 150-160 g, were randomly divided into 3 groups with 5 in each group. METHODS: The right sciatic nerves from 15 Wistar rats were exposed and transected at the mid thigh under pentobarbital anesthesia (4 mg/kg, i.p). Seven days later, the distal segments of the predegenerated nerves were removed and used to produce adult Schwann cell cultures. The distal segment of the predegenerated nerve, 20 mm in length, was resected. The nerve was cut into pieces 1 mm in length and incubated for 3 hours under CO2 at 37 ℃ with an enzyme mixture of 0.05% collagenase/dispase. Rats were divided into 3 groups: ① Group 1: The nerve fragments were explanted in poly-L-lysine and laminin-coated dishes with BS medium from the 1st to the 6th day. On the 6th day, the fragments were removed into a new poly-L-lysine-laminin-coated dish and the BS medium was changed to BS with 10% FBS. The nerve fragments were replaced repeatedly in the same way in new dishes on the 12th and the 18th days. ② Group 2: For the first 3 days, the nerve fragments were fed with BS with 10% FBS. This medium was changed to BS medium on the third day. The nerve fragments were removed to another dish on day 6 and BS medium was changed to BS with 25 mL/L FBS. Hereafter the culture method was the same as for group 1. ③ Group 3: For the first 6 days, nerve fragments were incubated in a dish not coated with poly-L-lysine and laminin, in BS medium supplemented with 8×107 U/L of penicillin-streptomycin. On the 6th day, the nerve fragments were removed to a poly-L-lysine-laminin-coated dish and cultured in BS with 25 mL/L FBS. On the 12th day, the nerve fragments were explanted a second dish and fed with BS containing 100 mL/L FBS. On the 18th day, they were explanted to a third poly-L-lysine-laminin-coated dish. SCs were obtained from all 3 dishes on the 21st day. Finally, purity and density of SCs were identified and proliferation index was calculated at the same time. MAIN OUTCOME MEASURES: Purity and density of SCs cultured with various methods in the three groups for 21 days. RESULTS: ① Isolation and proliferation of SCs: In the group 1, they increased in number after 4 days and both purity and density of cultured SCs were significantly higher than those from group 2. In the group 2, there were few fibroblasts. In the group 3, both purity and density of cultured SCs were remarkably higher than in those from groups 1 or 2. Then optimal proliferation was soon seen and the rapid expansion of SC populations suppressed the development of contaminating fibroblasts. On the 21st day, SCs proliferated to achieve maximal density and were too crowded to be counted. With Chi-square test, the data of the purity and the density were analyzed from groups 1 to 3, the result indicated χ2=430.47, P < 0.05. ② Characterization and proliferation rate of SCs: Immunostaining for S100 protein was evident in the cell soma and the processes of all three groups in cultures of SCs. SCs in vitro demonstrated typical bi- or tri-polar morphology, had oval nuclei, and stained brightly for S100. The proliferation rate of SCs was assessed with double fluorescence staining for BrdU and S100 on the 21st day of all three groups in cultures. About 40%-50% of the total SCs in the each group showed BrdU incorporation. CONCLUSION: The method is to use predegeneration in vivo, differential speed culture supplemented with the penicillin-streptomycin in low concentration, and changing of the concentration of FBS in the BS medium from 0 to 100 mL/L. This method allows remarkable suppression of fibroblast growth and attainment of SC proliferation and purity, in a short time, from adult nerves.
BACKGROUND: Schwann cells (SCs) are neuroglial cells of peripheral nerve and play a key role in repairing peripheral nerve injury; therefore, it provide an important evidence for transplantation of SCs which are characterized by active proliferation and adult high-purity in vitro after nerve injury in clinic, and also develops a new therapeutic way for nerve injury. OBJECTIVE: To investigate an effective technique for isolating adult activated Schwann cells. DESIGN: Controlled observational study. SETTING: Mudanjiang Medical College. MATERIALS: The experiment was completed at the Department of Medical Genetics of Harbin Medical University from March 2003 to April 2005. Methods: Female right Wistar rats, aged 2 months, weighting 150-160 g, were randomly divided into 3 groups with 5 in each group. METHODS: The right sciatic nerves from 15 Wistar rats were exposed and transected at the mid thigh under pentobarbital anesthesia (4 mg / kg, ip). Seven days later, the distal segments of the predegene rated nerves were removed and used to produce adult Schwann cell cultures. The distal segment of the predegenerated nerve, 20 mm in length, was resected. The nerve was cut into pieces 1 mm in length and incubated for 3 hours under CO2 at 37 ° C with Group 1: The nerve fragments were explanted in poly-L-lysine and laminin-coated dishes with BS medium from the 1st to the 6th day. On the enzyme mixture of 0.05% collagenase / dispase. 6th day, the fragments were removed into a new poly-L-lysine-laminin-coated dish and the BS medium was changed to BS with 10% FBS. The nerve fragments were replaced repeatedly in the same way in new dishes on the 12th and the 18th days. ② Group 2: For the first 3 days, the nerve fragments were fed with BS with 10% FBS. This medium was changed to BS medium on the third day. The nerve fragments were removed to another dish on day 6 and BS medium was changed to BS with 25 mL / L FBS. Hereafter the culture method was the same as for group 1. ③ Group 3: For the first 6 days, the nerve fragments were incubated in a dish not coated with poly-L-lysine and laminin, in BS medium supplemented with 8 × 107 U / L of penicillin-streptomycin. 6th day, the nerve fragments were removed to a poly-L-lysine-laminin-coated dish and cultured in BS with 25 mL / L FBS. On the 12th day, the nerve fragments were explanted a second dish and fed with BS containing 100 mL / L FBS. On the 18th day, they were explanted to a third poly-L-lysine-laminin-coated dish. SCs were obtained from all 3 dishes on the 21st day. Finally, purity and density of SCs were identified and proliferation MAIN OUTCOME MEASURES: Purity and density of SCs cultured with various methods in the three groups for 21 days. RESULTS: ① Isolation and proliferation of SCs: In the group 1, they increased in number after 4 days and both both purity and density of cultured SCs were significantly higher than those from group 2. In the group 2 , there were few fibroblasts. In the group 3, both purity and density of cultured SCs were remarkably higher than those from groups 1 or 2. Then optimal proliferation was soon seen and the rapid expansion of SC populations suppressed the development of contaminating fibroblasts. With Chi-square test, the data of the purity and the density were analyzed from groups 1 to 3, the result shows χ2 = 430.47, P < 0.05. ② Characterization and proliferation rate of SCs: Immunostaining for S100 protein was evident in the cell soma and the processes of all three groups in cultures of SCs. SCs in vitro demonstrated typical bi- or tri-polar morphology, had oval nuclei, and stained brightly for S100. The proliferation rate of SCs was assessed with double fluorescence staining for BrdU and S100 on the 21st day of all three groups in cultures. About 40% -50% of the total SCs in the each group showed BrdU incor method. CONCLUSION: The method is to use predegeneration in vivo, differential speed culture supplemented with the penicillin-streptomycin in low concentration, and changing the concentration of FBS in the BS medium from 0 to 100 mL / L. This method permits remarkable suppression of fibroblast growth and attainment of SC proliferation and purity, in a short time, from adult nerves.