雌二醇对人羊膜间充质干细胞增殖、凋亡的影响
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摘要
目的:研究不同浓度雌二醇(estradiol,E2)对人羊膜间充质干细胞(human amniotic mesenchymal stromal cells,HAMSCs)体外培养中增殖、凋亡的影响。方法:采用改良酶消化法提取HAMSCs,流式细胞学方法和免疫荧光法鉴定提取细胞。HAMSCs培养方法分为对照组和E2组(分别加入浓度为10-5、10-6、10-7、10-8 mol/L E2)。CCK-8检测各组HAMSCs增殖情况;q RT-PCR检测干细胞特异基因Oct-4和凋亡相关基因Bcl-2、Bax的表达;Western blot检测凋亡相关蛋白Bcl-2、Bax的表达。结果:CCK-8检测显示各浓度E2组和对照组的HAMSCs增殖活性在1~6 d都随时间的变化而变化(F时间=2 418.132,P时间=0.000);各浓度E2组在1~4 d的增殖率与对照组无明显差异;4 d后,各浓度E2组的增殖率明显高于对照组(F组别=246.601,P组别=0.000),各浓度组间无明显差异。q RT-PCR检测显示10-6 mol/L E2组(1.00±0.10)的Oct-4 m RNA表达水平较对照组(0.58±0.16)和其他E2浓度组(1.31±0.22、0.81±0.04、0.78±0.07)上调(P=0.000,P=0.020,P=0.001,P=0.000);除10-6 mol/L E2组(0.83±0.47,P=0.084)外,其他E2浓度组(0.49±0.25、0.38±0.19、0.32±0.10)抗凋亡基因Bcl-2基因表达量均较对照组(1.06±0.13)下调(P=0.038,P=0.015,P=0.010)。10-5 mol/L E2组(2.20±0.58)的Bax基因较对照组、其他E2浓度组(1.00±0.05、1.52±0.38、1.38±0.17、1.17±0.19)上调(P=0.001,P=0.028,P=0.012,P=0.003)。Western blot检测提示10-5 mol/L E2组(0.514±0.014)的促凋亡蛋白表达Bax较对照组(0.201±0.017)明显上调(P=0.000),而抗凋亡蛋白Bcl-2(0.366±0.024)与对照组(0.215±0.080)之间差异无统计学意义(P=0.055)。而10-6 mol/L E2组(0.378±0.016)的促凋亡蛋白Bax较其他浓度E2组(0.514±0.014、0.547±0.036、0.577±0.028)表达降低(P=0.000)。结论:E2促进HAMSCs的增殖呈时间依赖性,高浓度E2(10-5 mol/L)促进HAMSCs凋亡,较高浓度E2(10-6 mol/L)维持HAMSCs活性并保护其分化潜能。
Objective:To explore the effects of different concentrations of estradiol(E2)on proliferation and apoptosis in human amniotic mesenchymal stromal cells(HAMSCs). Methods:The improved enzyme digestion method was used to isolated culture HAMSCs.Flow cytometry analysis and immunofluorescence staining were used to identify the cultured cell. HAMSCs were divided into control group and E2 group(E2 with concentration of 10-5,10-6,10-7,10-8 mol/L were added respectively). The proliferative ability of HAMSCs in each group was measured by cell counting Kit-8 assay(CCK-8). Real-time PCR method was applied to measure the expression of the m RNA associated with pluripotency(Oct-4) and the expression of apoptosis-related genes Bcl-2 and Bax. The expression of apoptosis-related protein Bcl-2 and Bax was measured by Western blot. Results:CCK-8 showed that the proliferation activity of HAMSCs in each concentration E2 group and control group changed with time(Ftime=2418.132,Ptime=0.000). There was no significant difference between the the proliferation rate of the E2 group of each concentration in the 1 to 4 days and the control group,while the proliferation rate of each concentration E2 group was significantly higher than that of the control group after 4 days(Fgroup=246.601,Pgroup=0.000),and there was no significant difference among the concentration groups. Real-time PCR analysis showed that m RNA expression of Oct-4 was significant higher in10-6 mol/L E2 group(1.00±0.10)than in control group(0.58±0.16)and other E2 concentration group(1.31±0.22,0.81±0.04,0.78±0.07)(P=0.000,P=0.020,P=0.001,P=0.001). In addition to 10-6 mol/L E2 group(0.83±0.47)(P=0.084),the expression of Bcl-2 m RNA was significant lower in other E2 concentration group(0.49 ±0.25,0.38 ±0.19,0.32 ±0.10)than in control group(1.06 ±0.13)(P=0.038,P=0.015,P=0.010),and m RNA expression of Bcl-2 was significant higher in 10-5 mol/L E2 group(2.20±0.58)than in control group(1.00±0.05)and other E2 concentration group(1.52±0.38,1.38±0.17,1.17±0.19)(P=0.001,P=0.028,P=0.012,P=0.003). Western blot showed that protein expression of Bax was significant higher in 10-5 mol/L E2(0.514±0.014)group than control group(0.201±0.017)(P=0.000). Nevertheless,protein expression of Bcl-2 was no significant higher in 10-5 mol/L E2 group(0.366±0.024)than in control group(0.215±0.080)(P=0.055). Protein expression of Bax was significant lower in 10-6 mol/L E2 group(0.378±0.016)than in other E2 concentration group(0.514±0.014,0.547±0.036,0.577±0.028)(P=0.000). Conclusions:The present study demonstrates E2 can promote the proliferation of HAMSCs,proliferative effect was time dependency. And high concentration of E2 group(10-5 mol/L)can promote apoptosis,however higher concentration of E2 group(10-6 mol/L)can maintain activity of HAMSCs and protect pluripotency.
Objective:To explore the effects of different concentrations of estradiol(E2)on proliferation and apoptosis in human amniotic mesenchymal stromal cells(HAMSCs). Methods:The improved enzyme digestion method was used to isolated culture HAMSCs.Flow cytometry analysis and immunofluorescence staining were used to identify the cultured cell. HAMSCs were divided into control group and E2 group(E2 with concentration of 10-5,10-6,10-7,10-8 mol/L were added respectively). The proliferative ability of HAMSCs in each group was measured by cell counting Kit-8 assay(CCK-8). Real-time PCR method was applied to measure the expression of the m RNA associated with pluripotency(Oct-4) and the expression of apoptosis-related genes Bcl-2 and Bax. The expression of apoptosis-related protein Bcl-2 and Bax was measured by Western blot. Results:CCK-8 showed that the proliferation activity of HAMSCs in each concentration E2 group and control group changed with time(Ftime=2418.132,Ptime=0.000). There was no significant difference between the the proliferation rate of the E2 group of each concentration in the 1 to 4 days and the control group,while the proliferation rate of each concentration E2 group was significantly higher than that of the control group after 4 days(Fgroup=246.601,Pgroup=0.000),and there was no significant difference among the concentration groups. Real-time PCR analysis showed that m RNA expression of Oct-4 was significant higher in10-6 mol/L E2 group(1.00±0.10)than in control group(0.58±0.16)and other E2 concentration group(1.31±0.22,0.81±0.04,0.78±0.07)(P=0.000,P=0.020,P=0.001,P=0.001). In addition to 10-6 mol/L E2 group(0.83±0.47)(P=0.084),the expression of Bcl-2 m RNA was significant lower in other E2 concentration group(0.49 ±0.25,0.38 ±0.19,0.32 ±0.10)than in control group(1.06 ±0.13)(P=0.038,P=0.015,P=0.010),and m RNA expression of Bcl-2 was significant higher in 10-5 mol/L E2 group(2.20±0.58)than in control group(1.00±0.05)and other E2 concentration group(1.52±0.38,1.38±0.17,1.17±0.19)(P=0.001,P=0.028,P=0.012,P=0.003). Western blot showed that protein expression of Bax was significant higher in 10-5 mol/L E2(0.514±0.014)group than control group(0.201±0.017)(P=0.000). Nevertheless,protein expression of Bcl-2 was no significant higher in 10-5 mol/L E2 group(0.366±0.024)than in control group(0.215±0.080)(P=0.055). Protein expression of Bax was significant lower in 10-6 mol/L E2 group(0.378±0.016)than in other E2 concentration group(0.514±0.014,0.547±0.036,0.577±0.028)(P=0.000). Conclusions:The present study demonstrates E2 can promote the proliferation of HAMSCs,proliferative effect was time dependency. And high concentration of E2 group(10-5 mol/L)can promote apoptosis,however higher concentration of E2 group(10-6 mol/L)can maintain activity of HAMSCs and protect pluripotency.
引文
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[2]Díaz-Prado S,Mui觡os-López E,Hermida-Gómez T,et al.Human amniotic membrane as an alternative source of stem cells for regenerative medicine[J].Differentiation,2011,81(3):162-167.
[3]Kim SW,Zhang HZ,Kim CE,et al.Amniotic mesenchymal stem cells have robust angiogenic properties and are effective in treating hindlimb ischaemia[J].Cardiovasc Res,2012,93(3):525-534.
[4]Timmers L,Lim SK,Hoefer IE,et al.Human mesenchymal stem cell-conditioned medium improves cardiac function following myocardial infarction[J].Stem Cell Res,2011,6(3):206-214.
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[6]Gan L,Duan H,Xu Q,et al.Human amniotic mesenchymal stromal cell transplantation improves endometrial regeneration in rodent models of intrauterine adhesions[J].Cytotherapy,2017,19(5):603-616.
[7]方宁,张路,宋秀军,等.人羊膜间充质干细胞的分离、培养及鉴定[J].遵义医学院学报,2009,32(3):234-236.
[8]Celojevic D,Petersen A,Karlsson JO,et al.Effects of 17β-estradiol on proliferation,cell viability and intracellular redox status in native human lens epithelial cells[J].Mol Vis,2011,17:1987-1996.
[9]Xu J,Hu FF,Cui YG,et al.Effect of estradiol on proliferation and differentiation of side population stem/progenitor cells from murine endometrium[J].Reprod Biol Endocrinol,2011,9(1):103.
[10]谢幸,苟文丽.妇产科学[M].第8版.北京:人民卫生出版社,2013:32-36.
[11]蔡艾杞,吴本媛,王佳.新鲜羊膜移植对中、重度宫腔粘连月经模式的影响[J].重庆医科大学学报,2017,42(4):24-428.
[12]张惠娟,丛姗,梁美萍,等.人羊膜间充质干细胞分离培养:胰蛋白酶及胶原酶消化时间及浓度的选择[J].中国组织工程研究,2014,18(6):944-949.
[13]朴正福,丁淑芹,张海燕,等.人羊膜间充质干细胞的分离及分化潜能的研究[J].生物医学工程与临床,2010,14(1):15-19.
[14]Parolini O,Alviano F,Bagnara GP,et al.Concise review:isolation and characterization of cells from human term placenta:outcome of the first international Workshop on Placenta Derived Stem Cells[J].Stem Cells,2008,26(2):300-311.
[15]Yun SP,Lee MY,Ryu JM,et al.Role of HIF-1alpha and VEGF in human mesenchymal stem cell proliferation by 17beta-estradiol:involvement of PKC,PI3K/Akt,and MAPKs[J].Am J Physiol Cell Physiol,2009,296(2):C317-326.
[16]刘德宝,张复文,任振华.雌激素受体α介导小鼠骨髓间充质干细胞成骨分化[J].中国骨质疏松杂志,2014,20(3):228-233.
[17]赵叶琳,赵晓寅,孙凌云,等.雌激素对人脐带来源间充质细胞骨架和超微结构的影响[J].中国医药生物技术,2011,6(3):198-203.
[18]Hendrick V,Altshuler LL,Suri R.Hormonal changes in the postpartum and implications for postpartum depression[J].Psychosomatics,1998,39(2):93-101.
[19]Cha Y,Kwon SJ,Seol W,et al.Estrogen receptor-alpha mediates the effects of estradiol on telomerase activity in human mesenchymal stem cells[J].Mol Cells,2008,26(5):454-458.
[20]Ayaloglu-Butun F,Terzioglu-Kara E,Tokcaer-Keskin Z,et al.The effect of estrogen on bone marrow-derived rat mesenchymal stem cell maintenance:inhibiting apoptosis through the expression of Bcl-x L and Bcl-2[J].Stem Cell Rev,2012,8(2):393-401.
[21]Ding J,Xu H,Faiola F,et al.Oct4 links multiple epigenetic pathways to the pluripotency network[J].Cell Res,2012,22(1):155-167.
[2]Díaz-Prado S,Mui觡os-López E,Hermida-Gómez T,et al.Human amniotic membrane as an alternative source of stem cells for regenerative medicine[J].Differentiation,2011,81(3):162-167.
[3]Kim SW,Zhang HZ,Kim CE,et al.Amniotic mesenchymal stem cells have robust angiogenic properties and are effective in treating hindlimb ischaemia[J].Cardiovasc Res,2012,93(3):525-534.
[4]Timmers L,Lim SK,Hoefer IE,et al.Human mesenchymal stem cell-conditioned medium improves cardiac function following myocardial infarction[J].Stem Cell Res,2011,6(3):206-214.
[5]Liu X,Wang Z,Wang R,et al.Direct comparison of the potency of human mesenchymal stem cells derived,from amnion tissue,bone marrow and adipose tissue at inducing dermal fibroblast,responses to cutaneous wounds[J].Int J Mol Med,2013,31(2):407-415.
[6]Gan L,Duan H,Xu Q,et al.Human amniotic mesenchymal stromal cell transplantation improves endometrial regeneration in rodent models of intrauterine adhesions[J].Cytotherapy,2017,19(5):603-616.
[7]方宁,张路,宋秀军,等.人羊膜间充质干细胞的分离、培养及鉴定[J].遵义医学院学报,2009,32(3):234-236.
[8]Celojevic D,Petersen A,Karlsson JO,et al.Effects of 17β-estradiol on proliferation,cell viability and intracellular redox status in native human lens epithelial cells[J].Mol Vis,2011,17:1987-1996.
[9]Xu J,Hu FF,Cui YG,et al.Effect of estradiol on proliferation and differentiation of side population stem/progenitor cells from murine endometrium[J].Reprod Biol Endocrinol,2011,9(1):103.
[10]谢幸,苟文丽.妇产科学[M].第8版.北京:人民卫生出版社,2013:32-36.
[11]蔡艾杞,吴本媛,王佳.新鲜羊膜移植对中、重度宫腔粘连月经模式的影响[J].重庆医科大学学报,2017,42(4):24-428.
[12]张惠娟,丛姗,梁美萍,等.人羊膜间充质干细胞分离培养:胰蛋白酶及胶原酶消化时间及浓度的选择[J].中国组织工程研究,2014,18(6):944-949.
[13]朴正福,丁淑芹,张海燕,等.人羊膜间充质干细胞的分离及分化潜能的研究[J].生物医学工程与临床,2010,14(1):15-19.
[14]Parolini O,Alviano F,Bagnara GP,et al.Concise review:isolation and characterization of cells from human term placenta:outcome of the first international Workshop on Placenta Derived Stem Cells[J].Stem Cells,2008,26(2):300-311.
[15]Yun SP,Lee MY,Ryu JM,et al.Role of HIF-1alpha and VEGF in human mesenchymal stem cell proliferation by 17beta-estradiol:involvement of PKC,PI3K/Akt,and MAPKs[J].Am J Physiol Cell Physiol,2009,296(2):C317-326.
[16]刘德宝,张复文,任振华.雌激素受体α介导小鼠骨髓间充质干细胞成骨分化[J].中国骨质疏松杂志,2014,20(3):228-233.
[17]赵叶琳,赵晓寅,孙凌云,等.雌激素对人脐带来源间充质细胞骨架和超微结构的影响[J].中国医药生物技术,2011,6(3):198-203.
[18]Hendrick V,Altshuler LL,Suri R.Hormonal changes in the postpartum and implications for postpartum depression[J].Psychosomatics,1998,39(2):93-101.
[19]Cha Y,Kwon SJ,Seol W,et al.Estrogen receptor-alpha mediates the effects of estradiol on telomerase activity in human mesenchymal stem cells[J].Mol Cells,2008,26(5):454-458.
[20]Ayaloglu-Butun F,Terzioglu-Kara E,Tokcaer-Keskin Z,et al.The effect of estrogen on bone marrow-derived rat mesenchymal stem cell maintenance:inhibiting apoptosis through the expression of Bcl-x L and Bcl-2[J].Stem Cell Rev,2012,8(2):393-401.
[21]Ding J,Xu H,Faiola F,et al.Oct4 links multiple epigenetic pathways to the pluripotency network[J].Cell Res,2012,22(1):155-167.
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