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目的:建立一种长时程的有效获得高度纯化成年大鼠背根节(dorsal root ganglions,DRG)神经元的方法。方法:将SD大鼠的DRG剥膜、消化制成单细胞悬液,牛血清白蛋白(bovine serum albumin,BSA)分层离心去除大部分非神经元细胞后接种于多聚赖氨酸处理的盖玻片上;培养1 d后,胰酶消化再次制成细胞悬液,BSA二次分层离心,再次接种于多聚赖氨酸处理的盖玻片上。BSA二次分层离心后的神经元为实验一(T1组)、单次离心的神经元为实验组二(T2组)、未经离心处理的神经元为对照组(C组)。各组除离心次数外,其余各方法相同。相差显微镜下观察上述各组培养神经元,结合βtubulinⅢ免疫荧光组化染色及MTT分析检测神经元纯化效果及细胞活力。结果:培养3 d后,T1组神经元比例达88.43±6.13%,较T2组、C组显著增高,差别具有统计学意义(P<0.05);MTT的结果显示与T2组、C组比较,T1组神经元的相对活力略微下降,但无统计学差异(P>0.05)。结论:二次纯化法是一种简单有效的成年大鼠DRG神经元纯化方法。
OBJECTIVE: To establish a long-term effective method for obtaining highly purified adult rat dorsal root ganglions (DRG) neurons. Methods: The DRG of SD rats was stripped of the membrane, digested to form a single cell suspension, and bovine serum albumin (BSA) was layered and centrifuged to remove most of the non-neuronal cells and inoculated into polylysine-treated Cover slides; cultured for 1 d, trypsinized again made into cell suspension, BSA secondary stratification, re-seeded in polylysine treated cover glass. The neurons after BSA secondary delamination were experimental group one (group T1), single centrifugation neurons group two (group T2), and those without centrifugation as control group (group C). In addition to the number of centrifuges in each group, the rest of the same method. The neurons were cultured under the phase contrast microscope. The neuronal purification and cell viability were detected by immunofluorescence staining and MTT assay. Results: After 3 days of culture, the proportion of neurons in T1 group was 88.43 ± 6.13%, which was significantly higher than that in T2 and C groups (P <0.05). The results of MTT showed that compared with T2 and C groups, The relative activity of neurons in T1 group decreased slightly, but there was no significant difference (P> 0.05). Conclusion: The secondary purification method is a simple and effective purification method of adult rat DRG neurons.