血小板衍生生长因子和表皮生长因子对山羊SSCs凋亡相关基因的影响
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摘要
为了研究表皮生长因子和血小板衍生生长因子-BB对山羊SSCs中凋亡相关基因的影响,本研究将60~75日龄的山羊睾丸(n=6)剪碎后,利用两步酶消化法得到睾丸单细胞悬液,然后通过两次差速贴壁法分离纯化山羊SSCs,并采用免疫荧光染色法对山羊SSCs进行鉴定。随后,单独或联合添加EGF和PDGF-BB,体外培养山羊SSCs 6d,运用real time PCR技术检测不同处理组中BAD、BCL2和BAX基因的相对表达量。结果表明,睾丸单细胞悬液经两次差速贴壁纯化后,能够获得纯度较高的SSCs。染色结果显示,纯化后的细胞大量表达SSCs的标记分子THY1和PLZF。与对照组相比,3个试验组的BCL2基因表达量均上调。其中,联合添加20ng/mL PDGF-BB+25ng/mL EGF处理组的中细胞的抗凋亡能力显著高于其他组(P<0.05),25ng/mL EGF组的BCL2表达水平显著高于对照组(P<0.05),然而20ng/mL PDGF-BB组中的抗凋亡基因BCL2表达水平与对照组相比差异不显著(P>0.05);与其他所有试验组相比,对照组的促凋亡基因BAX和BAD的表达水平显著升高(P<0.05),而试验组之间差异均不显著(P>0.05)。
To investigate the effects of epidermal growth factor(EGF)and platelet-derived growth factor-BB(PDGF-BB)on apoptosis-related genes of goat spermatogonial stem cells(SSCs),6 testis obtared from 60-to 75-day-old goats were minced and digested using a modified two-step enzymatic digestion process.Then,goat SSCs were purified twice by differential plating,and were identified by indirect immunofluorescence cell analysis.Subsequently,EGF and PDGF-BB were added into Dulbecco's modified eagle medium(DMEM),and the relative expression of BAD,BCL2 and BAX genes in different treatment groups was detected by real time PCR after goat SSCs were cultured for 6 days.The results showed that high purity SSCs were obtained from testicular single-cell suspension after twice differential plating.The result of immunofluorescence staining showed that the purified cells expressed the SSCs markers(THY1 and PLZF).Compared with the control group,the expression of BCL2 was upregulated in the three experimental groups.And the BCL2 expression of the 20 ng/mL PDGF-BB plus 25 ng/mL EGF group was significantly higher than that in other groups(P<0.05).The BCL2 level in 25 ng/mL EGF group was significantly higher than that of the control(P<0.05),however,the expression level of anti-apoptotic gene BCL2 in 20 ng/mL PDGF-BB group was not significant compared to the control group(P>0.05).Comapred with all other experimental groups,the expression levels of apoptosis genes BAX and BAD in the control group were significantly increased(P<0.05),while the differences between the experimental groups were not significant(P>0.05).
To investigate the effects of epidermal growth factor(EGF)and platelet-derived growth factor-BB(PDGF-BB)on apoptosis-related genes of goat spermatogonial stem cells(SSCs),6 testis obtared from 60-to 75-day-old goats were minced and digested using a modified two-step enzymatic digestion process.Then,goat SSCs were purified twice by differential plating,and were identified by indirect immunofluorescence cell analysis.Subsequently,EGF and PDGF-BB were added into Dulbecco's modified eagle medium(DMEM),and the relative expression of BAD,BCL2 and BAX genes in different treatment groups was detected by real time PCR after goat SSCs were cultured for 6 days.The results showed that high purity SSCs were obtained from testicular single-cell suspension after twice differential plating.The result of immunofluorescence staining showed that the purified cells expressed the SSCs markers(THY1 and PLZF).Compared with the control group,the expression of BCL2 was upregulated in the three experimental groups.And the BCL2 expression of the 20 ng/mL PDGF-BB plus 25 ng/mL EGF group was significantly higher than that in other groups(P<0.05).The BCL2 level in 25 ng/mL EGF group was significantly higher than that of the control(P<0.05),however,the expression level of anti-apoptotic gene BCL2 in 20 ng/mL PDGF-BB group was not significant compared to the control group(P>0.05).Comapred with all other experimental groups,the expression levels of apoptosis genes BAX and BAD in the control group were significantly increased(P<0.05),while the differences between the experimental groups were not significant(P>0.05).
引文
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[16]卢璐璐.绵羊BAD基因影响常年发情性状的功能研究[D].北京:北京畜牧兽医研究所,2015.
[17]WANG J,CAO H,XUE X,et al.Effect of vitamin C on growth of caprine spermatogonial stem cells in vitro[J].Theriogenology,2014,81(4):545-555.
[18]YAN J,XIANG J,LIN Y,et al.Inactivation of BAD by IKK inhibits TNFα-induced apoptosis independently of NF-κB activation[J].Cell,2013,152(1-2):304-315.
[19]SCHIMMER A D,HEDLEY D W,PHAM N A,et al.BAD induces apoptosis in cells over-expressing Bcl-2 or Bcl-xL without loss of mitochondrial membrane potential[J].Leukemia&Lymphoma,2001,42(3):429-443.
[20]FERNANDO R,FOSTER J S,BIBLE A,et al.Breast cancer cell proliferation is inhibited by BAD:regulation of cyclin D1[J].Journal of Biological Chemistry,2007,282(39):28 864-28 873.
[21]葛晨霞,王龙涛,李向军,等.Bad基因在黄体期绵羊生殖器官中表达的比较研究[J].中国兽医学报,2011,31(3):428-432.
[22]CZABOTAR P E,LESSENE G,STRASSER A,et al.Control of apoptosis by the BCL-2protein family:implications for physiology and therapy[J].Nature Reviews Molecular Cell Biology,2014,15(1):49-63.
[23]LIU Y,SUN S Y,OWONIKOKO T K,et al.Rapamycin induces Bad phosphorylation in association with its resistance to human lung cancer cells[J].Molecular Cancer Therapeutics,2012,11(1):45-56.
[2]GERHARDT K.Spermatogonial stem cells at the helm of gametogenesis[J].Biology of Reproduction,2016,95(5):94.doi:10.1095/biolreprod.116.144766.
[3]ZHANG P,CHEN X,ZHENG Y,et al.Long-term propagation of porcine undifferentiated spermatogonia[J].Stem Cells and Development,2017,26(15):1 121-1 131.
[4]MU H,LI N,WU J,et al.PLZF-induced upregulation of CXCR4promotes dairy goat male germline stem cell proliferation by targeting Mir146a[J].Journal of Cellular Biochemistry,2015,117(4):844-852.
[5]AIZAWA Y,LEIPZIG N,ZAHIR T,et al.The effect of immobilized platelet derived growth factor AA on neural stem/progenitor cell differentiation on cell-adhesive hydrogels[J].Biomaterials,2008,29(35):4 676-4 683.
[6]LEVEN P,PEKNY M,GEBRE-MEDHIN S,et al.Mice deficient for PDGF B show renal,cardiovascular,and hematological abnormalities[J].Genes&Development,1994,8(16):1 875-1 887.
[7]ARTEMENKO Y,GAGNON A,AUBIN D,et al.Anti-adipogenic effect of PDGF is reversed by PKC inhibition[J].Journal of Cellular Physiology,2005,204(2):646-653.
[8]KASSAB M,ABD-ELMAKSOUD A,ALI M A.Localization of the epidermal growth factor(EGF)and epidermal growth factor receptor(EGFR)in the bovine testis[J].Journal of Molecular Histology,2007,38(3):207-214.
[9]BLOTTNER S,SCHN J,JEWGENOW K.Seasonally activated spermatogenesis is correlated with increased testicular production of testosterone and epidermal growth factor in mink(Mustela vison)[J].Theriogenology,2006,66(6-7):1 593-1 598.
[10]BEUMER T L,ROEPERS-GAJADIEN H L,GADEMAN I S,et al.Apotposis regulation in the testis:involvement of Bcl-2family members[J].Molecular Reproduction and Development,2000,56(3):353-359.
[11]TAN Y,DEMETER M R,RUAN H,et al.BAD Ser-155Phosphorylation Regulates BAD/Bcl-XL Interaction and Cell Survival[J].Journal of Biological Chemistry,2000,275(33):25 865-25 869.
[12]KELEKAR A,CHANG B S,HARLAN J E,et al.BAD is a BH-3domain-containing protein that forms an inactivating dimer with Bcl-xL[J].Molecular and Cellular Biology,1997,17(12):7 040-7 046.
[13]王菊花.山羊精原干细胞体外增殖及其分化的调控[D].合肥:安徽农业大学,2014.
[14]SHINOHARA T,INOUE K,OGONUKI N,et al.Birth of offspring following transplantation of cryopreserved immature pieces and in-vitro microinsemination[J].Human Reproduction,2002,17(12):3 039-3 045.
[15]HEIDARI B,GIFANI M,SHIRAZI A,et al.Enrichment of undifferentiated type A spermatogonia from goat testis using discontinuous percoll density gradient and differential plating[J].Avicenna Journal of Medical Biotechnology,2014,6(2):94-103.
[16]卢璐璐.绵羊BAD基因影响常年发情性状的功能研究[D].北京:北京畜牧兽医研究所,2015.
[17]WANG J,CAO H,XUE X,et al.Effect of vitamin C on growth of caprine spermatogonial stem cells in vitro[J].Theriogenology,2014,81(4):545-555.
[18]YAN J,XIANG J,LIN Y,et al.Inactivation of BAD by IKK inhibits TNFα-induced apoptosis independently of NF-κB activation[J].Cell,2013,152(1-2):304-315.
[19]SCHIMMER A D,HEDLEY D W,PHAM N A,et al.BAD induces apoptosis in cells over-expressing Bcl-2 or Bcl-xL without loss of mitochondrial membrane potential[J].Leukemia&Lymphoma,2001,42(3):429-443.
[20]FERNANDO R,FOSTER J S,BIBLE A,et al.Breast cancer cell proliferation is inhibited by BAD:regulation of cyclin D1[J].Journal of Biological Chemistry,2007,282(39):28 864-28 873.
[21]葛晨霞,王龙涛,李向军,等.Bad基因在黄体期绵羊生殖器官中表达的比较研究[J].中国兽医学报,2011,31(3):428-432.
[22]CZABOTAR P E,LESSENE G,STRASSER A,et al.Control of apoptosis by the BCL-2protein family:implications for physiology and therapy[J].Nature Reviews Molecular Cell Biology,2014,15(1):49-63.
[23]LIU Y,SUN S Y,OWONIKOKO T K,et al.Rapamycin induces Bad phosphorylation in association with its resistance to human lung cancer cells[J].Molecular Cancer Therapeutics,2012,11(1):45-56.
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