气相色谱/质谱法检测壬基酚在大鼠睾丸支持细胞中的分布

Distribution of nonylphenol in rat testes sertoli cells by chromatography-mass spectrometry

壬基酚(NP)是一种环境内分泌干扰物,具有类雌激素作用,它的高稳定性和脂溶性的特点使其能够在有机体,特别是脂肪组织内聚集,对人类的生殖系统产生毒害作用,因其分子结构与细胞膜的磷脂分子很相似,故与磷脂分子有很高的亲和力.为了探讨NP对生殖细胞的毒理机制,了解其在细胞内的分布情况,建立大鼠睾丸支持细胞NP含量的高效气相色谱及质谱(即GC/MS)检测方法,观察NP在细胞中的分布.实验采用原代培养的大鼠睾丸支持细胞,通过带有CY3标记的抗体对其质膜上特异表达的卵泡刺激素受体(FSHR)进行免疫荧光染色,鉴定支持细胞.然后用10μmol/L和20μmol/L的NP分别作用细胞3 h6、h1、2 h2、4 h后,超声破碎,并对10μmol/L NP处理的样品用改进的三步离心法进行细胞质膜分离,GC/MS方法检测NP在细胞中的总含量,以及细胞膜中NP含量占细胞中的总含量的比例.结果显示培养的细胞均呈现绿色荧光,都表达FSHR.两步离心的质膜分离方法,膜纯度为33.22%,改进的三步离心法膜纯度为78.60%.另外,分别在10μmol/L和20μmol/L NP浓度下,随着作用时间的延长,NP在细胞中的总含量逐渐增加至12 h,在24 h时突然下降至3 h的水平.细胞膜中NP含量占细胞中的总含量的比例,随着作用时间的延长而降低.由此得出结论是:FSHR可以作为大鼠睾丸支持细胞的鉴定指标;三步离心的质膜分离方法得到的膜纯度远远高于两步离心法;虽然NP与磷脂分子有很高的亲和力,但随着作用时间的延长,NP并没有停留在细胞膜里,而是能够穿过细胞膜进入到细胞内,并主要分布在细胞内.

Nonylphenol （NP）is a kind of xenoestrogens. NP possesses high stability and lipid solubility and can accumulate in organisms, especially in the fatty tissue. NP was demonstrated to exhibit toxicity effect on the reproductive systems of human. Based on the structural comparability and high affinity between NP and membrane phospholipid moleculars, we intended to find out the toxicity mechanism and the distribution of NP in the reproductive cells. We used gas chromatographwmass spectrometry（GC/MS）to determine the concentration of NP in the different part of the rat testes sertoli cells. In this study, we used the rat testes sertoli cells in primary culture and identified it by testing its special receptor-follicle stimulating hormone receptor（FSHR）. We applied immunofluorescence technology to identify sertoli cells by using FSHR antibody with fluorecein isothiocyante（FITC）. Then we treated sertoli cells with NP in 10 gmol/L and 20 ~mol/L for 3 h,6 h,12 h,24 h,respectively. All samples were homogenized by ultrasonic treatment. Additionally, the half samples treated inl0 /,mol/L NP was separated the membrane from the plasma through amending three step centrifugal technique. The total concentration of NP in the whole cells was measured by using GC/MS, as well as the concentration of NP in the cell membranes. Then we calculated the proportion of NP concentration in cell membranes compared with that in the whole cells. The results showed that all the cells displayed the green fluorescence in primary culture, this meant all of them can express FSHR, they were Sertoli cells. Themembrane purity of two-step centrifugal technique was 33.22%, whereas the purity of three-step centrifugal technique was 78.60%. The concentration of NP in Sertoli cells from 10 μmol/L, and 20 %mol/L groups gradually increased with prolonged exposure time to 12 h. However, after 24 h exposure, the concentration of NP in both groups dramatically decreased to the level at 3 h. The proportion of NP concentration in the cell membranes compared with that in the whole cells was gradually decreased with prolonged exposure time. From these results, we drew a conclusion that FSttR can be regarded as an index for identifying sertoli cells. The membrane purity taken from three-step centrifugal technique was much higher than that taken from two-step centrifugal technique. Though NP holds high affinity with cell membrane phospholipid moleculars, with the extension o,r exposure times treated to sertoli cells, it didn＇t settle in the cell membrane and could enter sertoli cells while it drilling through the membrane and distributed mainly in the inside of sertoli cells.