目的 探讨不同浓度川芎嗪(TMP)的玻璃化保存液对大鼠施万细胞凋亡及调控基因表达的影响,以便获得川芎嗪的最佳浓度.方法 20只SD大鼠,切取双侧坐骨神经(共40条),将其随机分成A、B、C、D、E组,每组8条,分别用含不同浓度川芎嗪的玻璃化保存液在-20 ℃保存3周(A组:0 mg/L、B组:80 mg/L、C组:160 mg/L、D组:320 mg/L、E组:640 mg/L).3周后取神经进行苏木素-伊红(HE)染色光镜检查;免疫组化法检测Bcl-2、Bax;末端标记法(TUNEL)检测细胞凋亡.结果 D组神经施万细胞凋亡数量明显少于其余各组施万细胞凋亡数量,A组神经施万细胞凋亡数量明显多于其余各组施万细胞凋亡数量;D组Bcl-2的阳性表达率最高,且明显高于其余各组,A组Bcl-2的阳性表达率最低,且明显低于其余各组;D组Bax阳性表达率明显低于其余各组,A组Bax阳性表达率明显高于其余各组.结论 川芎嗪能通过调节Bcl-2和Bax的表达而有效抑制施万细胞的凋亡,在-20 ℃含川芎嗪的玻璃化液保存大鼠坐骨神经,川芎嗪的浓度以320 mg/L最佳.%Objective To investigate the effects of vitreous cryopreservation with different concentration of tetramethylpyrazine on Schwann s cell apoptosis and the expression of regulating genes, in order to get the optimum concentration of tetramethylpyrazine. Methods 40 sciatic nerves obtained from 20 SD rats were randomly divided into 5 groups (n=8) : A, B, C, D, and E groups, and preserved in vitreous cryopreservation solution with the concentration of tetramethylpyrazine: 0mg/L, 80mg/L, 160mg/L, 320mg/L,and 640 mg/L, respectively. All the groups were cryopreserved at-20℃ for 3 weeks. After 3 weeks, all the nerves were obtained for tests; HE staining for light-microscope test, Schwann s cell apoptosis was measured quantitatively by the terminal transferase UTP nick end labeling (TUNEL), and the expressions of Bcl-2 and Bax were determined by immunohisto-chemisty. Results The number of Schwann S cell apoptosis in D group was significantly smaller than that in other groups, and the number of Schwann S cell apoptosis in A group was significantly more than that in other groups (P<0. 05). The expression of Bcl-2 in D group was significantly higher than that in other groups, and the expression of Bcl-2 in A group was significantly lower than that in other groups (P<0. 05). The expression of Bax in D group was significantly lower than that in other groups, and the expression of Bax in A group was significantly higher than that in other groups (P<0. 05). Conclusion These findings suggest that at -20℃ , vitreous cryopreservation with tetramethylpyrazine can depress Schwann S cell apoptosis by regulating the expression of Bcl-2 and Bax, and the optimum concentration of tetramethylpyrazine in vitreous cryopreservation is 320 mg/L.
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