植块法与酶消化法结合培养原代大鼠雪旺细胞

Study of culturing primary Schwann cells of rats by explant and enzyme digestion technique

目的:探讨利用植块法与酶消化法相结合培养大鼠原代雪旺细胞的可行性,并研究其增殖规律。方法:取10只新生2～3d的SD大鼠双侧坐骨神经,剥除神经外膜,剪成约1mm3大小的碎块,用0.03%胶原酶和0.25%胰蛋白酶按1∶2对神经碎块消化12min,加入少量含10%胎牛血清的DMED培养基小心吹打细胞及组织块,再植于培养皿中培养。用S-100免疫组化染色鉴定雪旺细胞,结合Hoechst3342染色计算其纯度,在显微镜下确定细胞数量。结果:在植块培养24h后即有大量细胞迁出,2～6d细胞生长迅速,10d以后生长较为缓慢,其倍增时间为2.3d。经S-100染色证实所培养细胞为雪旺细胞,结合Hoechst3342染色可得其培养8d时纯度为95.1%,传代后纯度可达96.3%。10只新生鼠培养12d后可得细胞数约为4.8×106个。结论:利用植块法与酶消化法相结合的方法可以迅速获得大量高纯度的雪旺细胞,其增殖速度在前6d最快,倍增时间为2.3d。

Objective：To culture primary Schwann cells（SCs） using explant and enzyme digestion technique. Method：The bilateral sciatic nerve of the 2-3 days newbom rats was harvested,the epineurium was separated and the nerve was dissected into discrete fascicles which were 1-3mm in size. 0.03% collagenase and 0.25% trypsin in ratio of 1：2 were used to dissociate the nerve segments,then explants were planted in culture dishes incubated at 37℃ in 5% CO2.The SCs were identified by S-100 immunochemistry staining.The purity of the cultured SCs was determined by comparing the number of Hoechst-labelled nuclei with the number of S-100 immunoreactive cells under a microscope.Cell population was counted under microscope.Result：A great number of SCs emigrated from the explants 24 hours later,the SCs grew fast witnin 2-6 days,but 10 days later the cell growing slowed down.The doubling generation time was 2.3 days.The purity of SCs reached 95.1% for the primary cells and 96.3% for the second generation.With this modified protocol 4.8×10^6 cells could be obtained from ten rats by 12 days culture.Conclusion：A great quantity of highly purified SCs can be obtained with explant and enzyme digestion technique.In the first 6 days the proliferation of SCs is fastest. The doubling generation time is 2.3 days.