小鼠精原干细胞体外分化及相关基因表达的研究
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摘要
目的：体外培养小鼠精原干细胞(mouse spermatogonial stem cells,mSSCs),并通过全反式视黄酸(ATRA, all-trans retinoic acid)进行诱导分化,观察小鼠精原干细胞在诱导中的变化情况,检测Stra8、mina53、Oct-4、GCNF在mSSCs体外分化中的表达,有助于研究精原干细胞体外分化的最佳条件和分子生物学特性,从精原干细胞体外分化的角度理解和认识此过程基因表达调控机制
方法：本研究体外分离培养6-8天BABL/C小鼠睾丸的SSCs,进行形态学鉴定,并通过含10% FBS、ATRA浓度为1.0×10-5mol/L的L-DMEM培养基对精原干细胞进行体外诱导分化,观察mSSCs加入诱导剂后的形态学变化,用间接免疫荧光技术从c-kit、Oct-4、GCNF三个标志物表达情况观察诱导分化情况,通过RT-PCR检测mSSCs诱导分化前后(0h,2h,12h,3d,5d) Stra8、mina53、Oct-4、GCNF的mRNA的水平,初步了解Stra8、mina53、Oct-4、GCNF的表达状况,以及与mSSCs体外分化的相关情况。
结果：1、形态学变化显示ATRA使mSSCs向精母细胞方向发生了分化,并通过间接免疫荧光反应进行鉴定；2、Stra8在mSSCs诱导分化2 h、12 h、3 d、5 d的水平高于成年小鼠和未诱导分化的水平,其中12h的表达水平最高,3d、5d时水平低于2 h,各对照组间差异具有统计学显著性；mina53的表达在诱导后增加,其中诱导2 h、12h时水平高于3d、5d及成年小鼠和未诱导mSSCs的水平,但诱导3d、5d组与成年小鼠组差异不具统计学显著性。3、Oct-4在1nSSCs诱导前呈表达水平最高,诱导2h、12h后下降,3d组和5d组均为阴性。4、GCNF的表达在诱导2h后表达开始增强,3d时表达水平最高,而5d时表达为阴性,在诱导组与成年小鼠睾丸之间表达差异具有显著统计学意义。
结论：(1)ATRA在体外诱导mSSCs分化成功。(2)Stra8基因在ATRA诱导mSSCs体外分化中可能发挥了启动减数分裂的作用,但Stra8、mina53基因在SSCs分化中的表达是否具有相关性是不能确定的。(3)在ATRA诱导mSSCs体外分化过程中,GCNF和Oct-4的表达呈负相关。
Objective:To study the expression of Stra8、mina53、Oct-4 and GCNF in orientation-induced differentiation in mSSCs in vitro, which wouid be useful for learning the optimal condition of culturing mSSCs and the biological characteristics of mSSCs and recognizing the genetic mechanisim of the differentiation in mSSCs.
Methods:1、Separated and cultured spermatogonial stem cells of testis of mice aged 6-8 days in vitro. Induced mSSCs differentiate into spermatocyte orientation in vitro by ATRA.2、Markers(c-kit、Oct-4、GCNF) were detected by indirect immunofluorescene in mSSCs before and after induction..3、Spermatogonial stem cells were induced by ATRA for 2h、12h、72h.Meanwhile the levels of Stra8、mina53、Oct-4 and GCNF mRNA expression were detected by RT-PCR method.
Results:1、The morphology change of mSSCs indirect immunoinfluorescence showed cultured mSSCs had been differetioned by the induction of ATRA.2、The levels of stra8 mRNA of induced mSSCs for 2h、12h、3d were higher than those of adult mouse testis and uninduced mSSCs,however,the level that mSSCs had been induced for 12 h was the highest of all,and those of 3d and5d were below to which induced for 12 h.The differences between all groups were statistical (p<0.05).3、The expression of mina53 had been increased after mSSCs were induced.The differnces between the 3d-group and the 5d-group weren't statistical(p>0.05). (4) The expression of Oct-4 in undifferentionated mSSCs was the strongest,it had been degenerated since the mSSCs was induced for 2h,it was nagative when mSSCs was induced for 3d and 5d. (5) The expression of GCNF was strengthened since the mSSCs was induced for 12h by ATRA,but it was far lower than the expression in the adult testis of mice, and it was negative in mSSCs induced for 5d.
Conclusion:(1)ATRA might have the capability that induces mSSCs differentiate into spermatocyte orientation in vitro.(23)The gene Stra8 maybe initiate the meiosis of mSSCs in its differentiateion by ATRA,but it is yet uncertain that there could be the correlation beween gene Stra8 and gene mina53 in the differentiateion of mSSCs in vitro while induced by ATRA. (3) The expressions of GCNF and Oct-4 are in inverse correlations in the orientation-induced differentiation in mSSCs in vitro by ATRA.
方法：本研究体外分离培养6-8天BABL/C小鼠睾丸的SSCs,进行形态学鉴定,并通过含10% FBS、ATRA浓度为1.0×10-5mol/L的L-DMEM培养基对精原干细胞进行体外诱导分化,观察mSSCs加入诱导剂后的形态学变化,用间接免疫荧光技术从c-kit、Oct-4、GCNF三个标志物表达情况观察诱导分化情况,通过RT-PCR检测mSSCs诱导分化前后(0h,2h,12h,3d,5d) Stra8、mina53、Oct-4、GCNF的mRNA的水平,初步了解Stra8、mina53、Oct-4、GCNF的表达状况,以及与mSSCs体外分化的相关情况。
结果：1、形态学变化显示ATRA使mSSCs向精母细胞方向发生了分化,并通过间接免疫荧光反应进行鉴定；2、Stra8在mSSCs诱导分化2 h、12 h、3 d、5 d的水平高于成年小鼠和未诱导分化的水平,其中12h的表达水平最高,3d、5d时水平低于2 h,各对照组间差异具有统计学显著性；mina53的表达在诱导后增加,其中诱导2 h、12h时水平高于3d、5d及成年小鼠和未诱导mSSCs的水平,但诱导3d、5d组与成年小鼠组差异不具统计学显著性。3、Oct-4在1nSSCs诱导前呈表达水平最高,诱导2h、12h后下降,3d组和5d组均为阴性。4、GCNF的表达在诱导2h后表达开始增强,3d时表达水平最高,而5d时表达为阴性,在诱导组与成年小鼠睾丸之间表达差异具有显著统计学意义。
结论：(1)ATRA在体外诱导mSSCs分化成功。(2)Stra8基因在ATRA诱导mSSCs体外分化中可能发挥了启动减数分裂的作用,但Stra8、mina53基因在SSCs分化中的表达是否具有相关性是不能确定的。(3)在ATRA诱导mSSCs体外分化过程中,GCNF和Oct-4的表达呈负相关。
Objective:To study the expression of Stra8、mina53、Oct-4 and GCNF in orientation-induced differentiation in mSSCs in vitro, which wouid be useful for learning the optimal condition of culturing mSSCs and the biological characteristics of mSSCs and recognizing the genetic mechanisim of the differentiation in mSSCs.
Methods:1、Separated and cultured spermatogonial stem cells of testis of mice aged 6-8 days in vitro. Induced mSSCs differentiate into spermatocyte orientation in vitro by ATRA.2、Markers(c-kit、Oct-4、GCNF) were detected by indirect immunofluorescene in mSSCs before and after induction..3、Spermatogonial stem cells were induced by ATRA for 2h、12h、72h.Meanwhile the levels of Stra8、mina53、Oct-4 and GCNF mRNA expression were detected by RT-PCR method.
Results:1、The morphology change of mSSCs indirect immunoinfluorescence showed cultured mSSCs had been differetioned by the induction of ATRA.2、The levels of stra8 mRNA of induced mSSCs for 2h、12h、3d were higher than those of adult mouse testis and uninduced mSSCs,however,the level that mSSCs had been induced for 12 h was the highest of all,and those of 3d and5d were below to which induced for 12 h.The differences between all groups were statistical (p<0.05).3、The expression of mina53 had been increased after mSSCs were induced.The differnces between the 3d-group and the 5d-group weren't statistical(p>0.05). (4) The expression of Oct-4 in undifferentionated mSSCs was the strongest,it had been degenerated since the mSSCs was induced for 2h,it was nagative when mSSCs was induced for 3d and 5d. (5) The expression of GCNF was strengthened since the mSSCs was induced for 12h by ATRA,but it was far lower than the expression in the adult testis of mice, and it was negative in mSSCs induced for 5d.
Conclusion:(1)ATRA might have the capability that induces mSSCs differentiate into spermatocyte orientation in vitro.(23)The gene Stra8 maybe initiate the meiosis of mSSCs in its differentiateion by ATRA,but it is yet uncertain that there could be the correlation beween gene Stra8 and gene mina53 in the differentiateion of mSSCs in vitro while induced by ATRA. (3) The expressions of GCNF and Oct-4 are in inverse correlations in the orientation-induced differentiation in mSSCs in vitro by ATRA.
引文
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18、唐红,石国庆,精原干细胞体外培养研究进展[J],动物医学进展。2008,29(6)：87—89
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2、Rooij DG. Proliferation and differentiation of spermatogonial stem cells[J], Reproduction.2001 Mar;121(3):347-54.
3、Feng LX, Chen YL, Dettin L, Reijo Pera RA, Herr JC, Goldberg E, Dyml M. Generation and in Vitro Differentiation of a Spermatogonial Cell Line[J],Science,2002,19 (297):392-395
4、Geijsen N, Horoschak M, Kim K, Gribnau J, Eggan K, Daley GQ. Derivation of embryonic germ cells and male gametes from embryonic stem sells[J], Nature 2004,427:148-154.
5、McLean DJ, Friel PJ, Johnston DS, Griswold MD. Characterization of Spermatogonial Stem Cell Maturation and Differentiation in Neonatal Mice[J],Biol Reprod.2003 Sep 3
6、韩济南,候艳秋,吴绍华(审校),精原干细胞分化及自我更新调控机制研究进展[J],四川解剖学杂志,2007,15(4)：94-95
7、李彦锋(综述),李小红,郭应禄(审校)雄性生殖细胞体外分化研究现状[J],中华男科学,2004,10(2)；521-921
8、Sousa M, Cremades N, Alves C, et al. Development potential of human spermatogenic cells co-culture with Sertoli cells[J] Hum Reprod.2002,17 (1):1612172.
9.刘勇军贺斌(综述),王介东(审校)精原干细胞移植的研究进展[J],Iot Reprod Health/Fam Plan,2008,27(2):121-124
10, Parks J E, Lee DR, Huang S, et al. Prospects for spermatogenesis in vitro[J],.Therio Genology 2003,59(1):73-86.
11、Jean-Pierre Dadoune, New insights into male gametogenesis:what about the spermatogonial stem cell niche?[J],FOLIA HISTOCHEMICA ET CYTOBIOLOGICA.2007,45(3):141-147
12、李巍,窦中英,华进联,王华岩,Stra8基因的激活与原干细胞特异分化研究[J],生物工程学报,2007,23(7)：639644
13、Ericka L. Anderson, Andrew E. Baltus, Hermien L. Roepers-Gajadien et al.Stra8 and its inducer, retinoic acid, regulate meiotic initiation in both spermatogenesis and oogenesis in mice[J],PNAS,2008,105(39):14976-14980
14、Tsuneoka, M., Koda, Y., Soejima, M., Teye K, Kimura.A novel Myc target gene, mina53, that is involved in cell proliferation[J],Journal of Biological Chemistry,2002,277,35450-35459.
15、Teye K, Tsuneoka M, Arima N, Koda Y, Nakamura Y,Ueta Y, Shirouzu, K. Kimura. Increased expression of a Myc target gene mina53 in human colon cancer[J], American Journal of Pathology,2004,164,205-216.
16、Makoto Tsuneoka, Yoshitake Nishimune,Keisuke Ohta et al,Expression of Mina53, a product of a Myc target gene in mouse testis[J],international journal of indrology,2006,29,323-330
17、李莲军,李德雪,小鼠精原干细胞体外培养的研究[李莲军博士学位论文],北京,军需大学,2002.
18、唐红,石国庆,精原干细胞体外培养研究进展[J],动物医学进展。2008,29(6)：87—89
19、徐富翠,吴泽才,韩艺,等.以骨髓基质细胞为饲养层培养小鼠精原干细胞[J].中国组织工程研究与临床康复,2007.15：2822-2825.
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