氟对大鼠睾丸支持细胞雄激素结合蛋白和抑制素B的影响
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摘要
动物实验资料和人群流行病学调查资料证实氟不仅可以直接作用于睾丸和附睾,破坏其结构,造成生殖损伤,还可以通过干扰下丘脑-垂体-性腺轴和睾丸局部微环境,引起生殖毒性。
支持细胞(sertoli cell, SC)是睾丸曲细精管中唯一的体细胞,对维持睾丸内部的生精微环境具有极其重要的作用。首先,支持细胞之间可以形成紧密连接,在一定程度上可以阻止外来化学物对生精细胞的影响。其次,支持细胞还能够分泌各种活性物质,其中研究最多的是雄激素结合蛋白(Androgen Binding Protein, ABP)抑制素B(Inhibin B,INH B)。ABP毙够转运和储存睾酮和二氢睾酮,调节血液和附睾液中雄激素的浓度。INH B能够抑制卵泡刺激素(follicle stimulating hormone, FSH)分泌,不仅在维持下丘脑-垂体-性腺轴的平衡中起着重要作用,而且可直接促进生精细胞的分化和发育。由于位置和功能的特殊性,支持细胞成为许多外源化合物的靶细胞。目的：
以体外培养的大鼠睾丸支持细胞为载体,使其暴露于不同浓度的氟化钠,采用RT-PCR技术和ELISA方法从基因和蛋白质两个水平分析氟化钠对支持细胞合成和分泌ABP和INH B的影响,期望为探讨氟化钠引起雄性内分泌紊乱的机制提供一些线索。方法：1 18～21日龄SD大鼠,采用胰蛋白酶和胶原酶两步法分离纯化支持细胞。分离的支持细胞在37℃、5%CO2条件下进行培养。确定染毒浓度为0、2.5μg/ml、5.0μg/ml、10. 0μg/ml和20.0μg/ml。2体外培养支持细胞按上述确定的浓度染毒24h后,采用RT-PCR技术,以GAPDH为内参,检测支持细胞内ABP和INH B mRNA的相对表达量。3采用ELISA法测定培养液中ABP和INH B的含量。4统计学分析。运用SPSS 12.0统计软件的单因素方差分析(analysis of variance, ANOVA)、Bonfferoni两两比较、Levene方差齐性检验对数据进行统计学分析(检验水准a=0.05)。
结果：
1分离培养的支持细胞存活率达到95%以上,生长良好,且纯度较高。
2①ABP mRNA相对表达量在各浓度组与对照组相比,2.5μg/ml组高于对照,且差异有统计学意义(P<0.05)；5μg/ml组高于对照组,10μg/ml和20μg/ml组低于对照组,但是差异均无统计学意义(P>0.05)。②INH B mRNA相对表达量在各浓度组与对照组比较,2.5μg/ml和5μg/ml组均高于对照组,差异具有统计学意义(P<0.05)；其余两组低于对照组,但是差异均无统计学意义(P>0.05)。
3①各染毒组培养基中的ABP含量均低于对照组,但差异没有统计学意义(P>0.05)。②与对照组相比,2.5μg/ml和5.0μg/ml组培养基中INH B含量高于对照组,但差异没有统计学意义(P>0.05);10μg/ml和20μg/ml组培养基中INHB含量低于对照组,但差异无统计学意义(P>0.05)。
结论：
本研究的染毒浓度范围内,在基因水平,较低浓度的氟(2.5μg/ml)可以影响ABP和INH B mRNA的表达；在蛋白质水平,氟对ABP和INH B没有明显影响。
The findings from animal experiments and epidemiological studies approve that fluorine not only can damage the structure of the testis and the epididymis directly, but also can disturb the function of hypothalamic-pituitary-testicular axis(HPTA) and the microenvironment in the testis, then results in reproductive injury indirectly.
In the testis, there are only two cell types in the seminiferous epithelium, sertoli cells and spermatogenic cells. As the one and only somatic cells in the seminiferous epithelium, sertoli cells are very useful to keep balance of microenvironment in the testis. Firstly, they form the blood-testis barrier by tight junction between adjacent sertoli cells, which can prevent external chemicals from impacting on spermatogenic cells. Secondly, sertoli cells can synthesize and secrete a variety of active substances, of which we pay much attention to are androgen binding protein(ABP) and inibin B(INH B). ABP can bind and transport androgen to maintain high androgen level in the testis and make it available for spermatogenesis. As to INH B, not only keep balance of the HPTA by restraining the secretion of follicle stimulating hormone (FSH)specialy, it can also promote the differentiation and development of spermatogenic directly. As the important locatioan and function of sertoli cells, they become the target cells of many testicular toxicants.
Objective:
The present study was performed in primary culture of rat sertoli cells, after exposuring to sodium fluoride(NaF), the expressions of ABP and INH B mRNA from sertoli cells were measured using RT-PCR technology, the amount of ABP and INH B in medium was tested by ELISA. The object is to provide some clues for the mechanism of the male endocrine disorder resulted by sodium fluoride, in order to offer experimental evidence and related information to the prevention of fluorosis.
Methods:
1 Sertoli cells from testicular tissue of 18～20-day-old Sprague-Dawley rats were sequentially treated with 0.25% pancreatin and 0.1% collagenase, then the seprated sertoli cells were incubated in a humidified atmosphere of 5% CO2 at 37℃. MTT assay was used to evaluate the viability of the cultured cells and determine the sodium fluoride concentrations of exposure,2.5μg/ml,5.0μg/ml, 10.0μg/ml and 20.0μg/ml, serum-free medium as the control group.
2 Cultured sertoli cells exposured to NaF at the concentrations determined by MTT assay for 24h was used for the total RNA extraction, then examined the levels of ABP and INH B mRNA by RT-PCR technology. The level of GAPDH mRNA was used as control normalization. The levels of ABP and INH B expressions were measured by densitometric analysis and standardized by comparison to the GAPDH control using a digital imaging and analysis system.
3 After the cultured sertoli cells exposured to NaF at the concentrations as described above, ELISA methed was employed to test the levels of ABP and INH B in culture medium.
4 Statistical analysis referred to Bonfferoni test for multiple comparisons when significant difference were detected by one-way analysis of variance (ANOVA). A difference at P<0.05 was considered statistically significant.
Results:
1 Sertoli cells isolated from rat testis for primary culture grew well in vitro, the livability exceeded 95%, as well as the purity. According to the growth curve of Sertoli cells, they had the strongest viability and metabolized exuberantly from the third to the seventh day.
2 Compared to the control group, the level of expression of ABP mRNA in 2.5μg/ml was significantly increased(P<0.05). The 5.0μg/ml group was increased, the others were lower than the control group, but there were not statiscally significant (P>0.05).
The levels of expression of INH B mRNA in the 2.5μg/ml and 5μg/ml groups were significantly higher than that of the control group, and the 10μg/ml and 20μg/ml groups were not found to be significantly different(P>0.05), though they were decreased.
3 The levels of ABP in culture medium in the treated groups were not found to be significantly lower than that of the control group(P>0.05).
Compared to the control group, the contents of INH B in culture medium in the 2.5μg/ml and 5.0μg/ml groups were increased, the 10μg/ml and 20μg/ml groups were decreased, but the changes were not statiscally significant (P>0.05).
Conclusions:
Fluoride in the lower concentrations can effect the expression of ABP and INH B and there is no effect on the levels of ABP and INH B in culture medium at the concentrations in present study.
支持细胞(sertoli cell, SC)是睾丸曲细精管中唯一的体细胞,对维持睾丸内部的生精微环境具有极其重要的作用。首先,支持细胞之间可以形成紧密连接,在一定程度上可以阻止外来化学物对生精细胞的影响。其次,支持细胞还能够分泌各种活性物质,其中研究最多的是雄激素结合蛋白(Androgen Binding Protein, ABP)抑制素B(Inhibin B,INH B)。ABP毙够转运和储存睾酮和二氢睾酮,调节血液和附睾液中雄激素的浓度。INH B能够抑制卵泡刺激素(follicle stimulating hormone, FSH)分泌,不仅在维持下丘脑-垂体-性腺轴的平衡中起着重要作用,而且可直接促进生精细胞的分化和发育。由于位置和功能的特殊性,支持细胞成为许多外源化合物的靶细胞。目的：
以体外培养的大鼠睾丸支持细胞为载体,使其暴露于不同浓度的氟化钠,采用RT-PCR技术和ELISA方法从基因和蛋白质两个水平分析氟化钠对支持细胞合成和分泌ABP和INH B的影响,期望为探讨氟化钠引起雄性内分泌紊乱的机制提供一些线索。方法：1 18～21日龄SD大鼠,采用胰蛋白酶和胶原酶两步法分离纯化支持细胞。分离的支持细胞在37℃、5%CO2条件下进行培养。确定染毒浓度为0、2.5μg/ml、5.0μg/ml、10. 0μg/ml和20.0μg/ml。2体外培养支持细胞按上述确定的浓度染毒24h后,采用RT-PCR技术,以GAPDH为内参,检测支持细胞内ABP和INH B mRNA的相对表达量。3采用ELISA法测定培养液中ABP和INH B的含量。4统计学分析。运用SPSS 12.0统计软件的单因素方差分析(analysis of variance, ANOVA)、Bonfferoni两两比较、Levene方差齐性检验对数据进行统计学分析(检验水准a=0.05)。
结果：
1分离培养的支持细胞存活率达到95%以上,生长良好,且纯度较高。
2①ABP mRNA相对表达量在各浓度组与对照组相比,2.5μg/ml组高于对照,且差异有统计学意义(P<0.05)；5μg/ml组高于对照组,10μg/ml和20μg/ml组低于对照组,但是差异均无统计学意义(P>0.05)。②INH B mRNA相对表达量在各浓度组与对照组比较,2.5μg/ml和5μg/ml组均高于对照组,差异具有统计学意义(P<0.05)；其余两组低于对照组,但是差异均无统计学意义(P>0.05)。
3①各染毒组培养基中的ABP含量均低于对照组,但差异没有统计学意义(P>0.05)。②与对照组相比,2.5μg/ml和5.0μg/ml组培养基中INH B含量高于对照组,但差异没有统计学意义(P>0.05);10μg/ml和20μg/ml组培养基中INHB含量低于对照组,但差异无统计学意义(P>0.05)。
结论：
本研究的染毒浓度范围内,在基因水平,较低浓度的氟(2.5μg/ml)可以影响ABP和INH B mRNA的表达；在蛋白质水平,氟对ABP和INH B没有明显影响。
The findings from animal experiments and epidemiological studies approve that fluorine not only can damage the structure of the testis and the epididymis directly, but also can disturb the function of hypothalamic-pituitary-testicular axis(HPTA) and the microenvironment in the testis, then results in reproductive injury indirectly.
In the testis, there are only two cell types in the seminiferous epithelium, sertoli cells and spermatogenic cells. As the one and only somatic cells in the seminiferous epithelium, sertoli cells are very useful to keep balance of microenvironment in the testis. Firstly, they form the blood-testis barrier by tight junction between adjacent sertoli cells, which can prevent external chemicals from impacting on spermatogenic cells. Secondly, sertoli cells can synthesize and secrete a variety of active substances, of which we pay much attention to are androgen binding protein(ABP) and inibin B(INH B). ABP can bind and transport androgen to maintain high androgen level in the testis and make it available for spermatogenesis. As to INH B, not only keep balance of the HPTA by restraining the secretion of follicle stimulating hormone (FSH)specialy, it can also promote the differentiation and development of spermatogenic directly. As the important locatioan and function of sertoli cells, they become the target cells of many testicular toxicants.
Objective:
The present study was performed in primary culture of rat sertoli cells, after exposuring to sodium fluoride(NaF), the expressions of ABP and INH B mRNA from sertoli cells were measured using RT-PCR technology, the amount of ABP and INH B in medium was tested by ELISA. The object is to provide some clues for the mechanism of the male endocrine disorder resulted by sodium fluoride, in order to offer experimental evidence and related information to the prevention of fluorosis.
Methods:
1 Sertoli cells from testicular tissue of 18～20-day-old Sprague-Dawley rats were sequentially treated with 0.25% pancreatin and 0.1% collagenase, then the seprated sertoli cells were incubated in a humidified atmosphere of 5% CO2 at 37℃. MTT assay was used to evaluate the viability of the cultured cells and determine the sodium fluoride concentrations of exposure,2.5μg/ml,5.0μg/ml, 10.0μg/ml and 20.0μg/ml, serum-free medium as the control group.
2 Cultured sertoli cells exposured to NaF at the concentrations determined by MTT assay for 24h was used for the total RNA extraction, then examined the levels of ABP and INH B mRNA by RT-PCR technology. The level of GAPDH mRNA was used as control normalization. The levels of ABP and INH B expressions were measured by densitometric analysis and standardized by comparison to the GAPDH control using a digital imaging and analysis system.
3 After the cultured sertoli cells exposured to NaF at the concentrations as described above, ELISA methed was employed to test the levels of ABP and INH B in culture medium.
4 Statistical analysis referred to Bonfferoni test for multiple comparisons when significant difference were detected by one-way analysis of variance (ANOVA). A difference at P<0.05 was considered statistically significant.
Results:
1 Sertoli cells isolated from rat testis for primary culture grew well in vitro, the livability exceeded 95%, as well as the purity. According to the growth curve of Sertoli cells, they had the strongest viability and metabolized exuberantly from the third to the seventh day.
2 Compared to the control group, the level of expression of ABP mRNA in 2.5μg/ml was significantly increased(P<0.05). The 5.0μg/ml group was increased, the others were lower than the control group, but there were not statiscally significant (P>0.05).
The levels of expression of INH B mRNA in the 2.5μg/ml and 5μg/ml groups were significantly higher than that of the control group, and the 10μg/ml and 20μg/ml groups were not found to be significantly different(P>0.05), though they were decreased.
3 The levels of ABP in culture medium in the treated groups were not found to be significantly lower than that of the control group(P>0.05).
Compared to the control group, the contents of INH B in culture medium in the 2.5μg/ml and 5.0μg/ml groups were increased, the 10μg/ml and 20μg/ml groups were decreased, but the changes were not statiscally significant (P>0.05).
Conclusions:
Fluoride in the lower concentrations can effect the expression of ABP and INH B and there is no effect on the levels of ABP and INH B in culture medium at the concentrations in present study.
引文
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