雷公藤甲素致小鼠睾丸精母细胞损伤的敏感靶细胞群研究

Investigation of the Sensitive Target Cell of Spermatocytes Injury Induced by Triptolide in Mice Testes

目的研究雷公藤甲素(TL)致小鼠精母细胞损伤的敏感靶细胞群。方法7~8周龄健康雄性C57BL/6小鼠,每天经口灌胃给予0.125、0.25和0.5 mg·kg^-1的TL,分别于给药第3、7、11和15天解剖,摘取睾丸用甲醛固定,进行石蜡切片和苏木素-伊红(HE)染色,用于确定TL致精母细胞损伤敏感靶细胞群的最适时间点和剂量。并通过免疫组化染色,依据γ-H2AX在精母细胞的特征性分布,明确区分和计数不同分化阶段的初级精母细胞,依据不同阶段精母细胞下降百分比,确定精母细胞损伤的敏感靶细胞群。结果HE染色提示,TL给药第11天睾丸生精细胞损伤具有最佳的剂量-效应关系(病变程度从0.125 mg·kg^-1组的轻微损伤,到0.25 mg·kg^-1组的轻度至中度损伤,最后到0.5 mg·kg^-1组的重度损伤),0.125和0.25 mg·kg^-1组损伤程度适中,均适用于敏感靶细胞群的区分。γ-H2AX免疫组化染色显示,在精母细胞不同分化阶段,γ-H2AX的免疫组化染色呈现不同的细胞核内分布特征:细线期在整个核内呈斑点状分布至覆盖整个细胞核,偶线期集中于核染色质,粗线期在核边缘呈单点状分布,双线期则在核内部呈单点状着色。计数结果显示,0.125 mg·kg^-1组各分化阶段初级精母细胞绝对数量轻微下降(P>0.05),0.25 mg·kg^-1组各分化阶段初级精母细胞绝对数量显著下降(P<0.01或0.001);在粗线期及以前各分化阶段初级精母细胞中细线期精母细胞下降百分比明显更高,且在0.25 mg·kg^-1组下降百分比与粗线期初级精母细胞相比具有统计学意义(P<0.05)。结论γ-H2AX免疫组化染色可以明确区分不同阶段初级精母细胞;TL给药后,细线期初级精母细胞最可能是TL致睾丸精母细胞损伤的敏感靶细胞。

OBJECTIVE To investigate the sensitive target cell population of spermatocyte injury induced by TL in mice.METHODS Seven to eight-week-old healthy male C57BL/6 mice were orally administered triptolid(TL)of 0.125,0.25 and 0.5 mg·kg^-1 per day,and dissected on days 3,7,11 and 15 respectively.The extracted testes were fixed with formaldehyde.Paraffin sections and hematoxylin-eosin(HE)staining were performed to determine the optimal time point and dose level to be applied for sensitive target cell population analysis of spermatocyte injury induced by TL.The primary spermatocytes in different stages were clearly distinguished and counted based on the characteristic distribution profile ofγ-H2AX in spermatocytes under immunohistochemical staining.The sensitive target cell populations of spermatocyte injury were determined according to the decreased percentage of spermatocytes in different stages.RESULTS HE staining showed that the best dose-effect relationship of spermatogenic cell injury in the testes was present on day 11 after TL administration(The severity of the lesion ranged from a minimal degree in the 0.125 mg·kg^-1 group to a mild to moderate injury in the 0.25 mg·kg^-1 group,and finally to a marked injury in the 0.5 mg·kg^-1 group).The degree of injury in the 0.125 and 0.25 mg·kg^-1 groups was appropriate and suitable for determination of sensitive target cell populations.γ-H2AX immunohistochemical staining indicated that theγ-H2AX showed different distribution characteristics in nucleus in different stages of spermatocyte differentiation:scattered throughout the nucleus in a few discrete foci to fill the whole nuclear in leptotene;assembled in the chromatin regions in zygotene;located on the edge of the nucleus in a single foci in the pachytene;located in the nucleus in a single foci in the diplotene.The counting results showed that the absolute number of primary spermatocytes in all differentiating stages decreased slightly without statistical significance(P>0.05)in the 0.125 mg·kg^-1 dose group;the absolute number of primary spermatocytes decreased significantly with statistical significance(P<0.01 or 0.001)in the 0.25 mg·kg^-1 dose group.There was higher decreased percentage of the leptotene primary spermatocyte among the differentiating stages before pachytene stage and with statistical significance(P<0.05)at 0.25 mg·kg^-1 when compared with the pachytene primary spermatocyte.CONCLUSIONγ-H2AX immunohistochemical staining can clearly distinguish primary spermatocytes at different stages.The leptotene primary spermatocytes are the most likely sensitive target cells in the testicular spermatocyte-injury induced by TL administration.