曲细精管片段培养法和混合细胞共培养法对小鼠生精细胞的增殖分化效应

Proliferation and differentiation effects between seminiferous tubule segments culture and mixed cells coculture on mouse spermatogenic cells in vitro

目的：探讨曲细精管片段培养法和混合细胞共培养法对体外培养生精细胞的增殖、分化效应。方法采用曲细精管片段培养法和生精细胞-支持细胞共培养法对7～8d小鼠生精细胞进行体外培养，通过细胞形态学观察、细胞存活率和精母细胞特异性基因P19、单倍体精子细胞特异性基因TP1检测及染色体倍性分析，比较两种培养法生精细胞的存活、增殖以及分化情况。结果两种培养方法均可见生精细胞增殖，P19／TP1比值呈降低趋势，并可获得少量带鞭毛的精子细胞和长形精子，但共培养法生精细胞增殖速度及存活时间均优于片段法。体外培养各阶段，共培养法所获细胞数和存活率及单倍体精子细胞形成率均显著高于片段培养法（P＜0．05）；P19／TP1比值显著低于组织片段培养法（P＜0．05）。结论生精细胞-支持细胞共培养法的细胞增殖速度、存活率、存活时间及单倍体精子细胞形成率均优于曲细精管片段培养法，是较为理想的小鼠生精细胞体外培养方法。

Objective To investigate the proliferation and differentiation effects for mouse spermatogenic cells between semi -niferous tubule segments culture and mixed cells coculture in vitro .Methods Spermatogenic cells of 7-8 days mouse were separated and cultured by the methods of seminiferous tubule segments culture and mixed cells coculture ,the survival rates ,proliferation and dif-ferentiation rates in two culture methods were evaluated by morphological observation ,viability testing,pachytene-specific phosphopro-tein gene （P19） and haploid sperm cell-specific transition protein gene （TP1） detecting and ploidy analysis of cells .Results In both culture methods ,the rate of P19/TP1 were on a declining curve ,round spermatid and a small number of sperm cells with flagella or e-longating spermatid could be observed .But the proliferation rate and survival time of mixed cells coculture was superior to seminiferous tubule fragments .Compared mixed cells coculture with seminiferous tubule fragments ,cell numbers ,survival rate ,rate of haploid sperm cells was significantly higher （P〈0.05）,the rate of P19/TP1 was significantly lower （P〈0.05）.Conclusion Mixed cells coculture is better method than seminiferous tubules fragment culture with superior proliferation rate ,survival time ,and the rate of haploid sperm cells.