新生小鼠心肌干细胞与心肌细胞共培养诱导分化研究
摘要
目的:
1.了解心肌条件培养液有无诱导心肌干细胞(Cardiac stem cells,CSCs)向心肌细胞分化的作用。
2.CSCs与心肌细胞接触对CSCs分化有何影响。
方法:
1.将新生1-2天小鼠心脏组织剪成0.5-1mm3碎片,用DPBS洗涤红细胞,轻微消化组织块后,进行贴壁培养11-14天后,用胰酶进行差速消化并采集原代CSCs。
2.用免疫磁珠分选原代细胞得到c-Kit+ CSCs。
3.流式细胞术分析未分选的原代细胞表面CD45、CD34、Sca-1、c-Kit标志。分选后细胞检测c-Kit标志纯度及细胞周期。
4.结合原位免疫荧光检验c-Kit+的纯度。
5.分选后的c-Kit+ CSCs分3组进行培养。
a.空白对照组,无干预因素,用1%血清培养液培养CSCs 21天。
b.条件液组添加心肌细胞条件培养液诱导CSCs 21天。用1%血清培养液培养心肌细胞48小时后得到心肌条件培养液。方法示意图见附图尾页。
c.混合培养组分别用PKH26和DAPI标记CSCs后与心肌细胞混合共培养8天,期间均以1%血清培养液培养。
PKH26标记的CSCs用于免疫组化鉴定,并与空白组和条件液进行对比; DAPI标记的CSCs,共培养后再进行消化分离并分散传代,改用10%血清培养液,2周内,观察并寻找来源于CSCs,带DAPI标记的单独搏动细胞。
6.观察各组CSCs形态变化,当空白组、条件液组和PKH26标记的混合培养组的细胞完成培养后,用原位免疫组化法鉴定肌钙蛋白T(Troponin-T)和间隙连接蛋白43(connexin43),评估CSCs向心肌细胞分化的程度,每组检测一种蛋白需要6个培养样本,其中1个用于阴性对照。
7.分析试验数据:原位免疫组化每孔随机抽四周+中央五个200倍视野,计数阳性细胞和阴性细胞后进行χ2检验或秩和检验分析,以P<0.05为差异具有显著性。混合培养组中评估在心肌细胞附近的CSCs。
结果:
1.原代未分选的细胞主要表达干细胞标志c-Kit和Sca-1,低表达造血干细胞标志CD34和白细胞标志CD45。
2.分选后得到纯度较高的c-Kit+ CSCs,流式细胞术分析其纯度约96%,原位免疫荧光检验也支持以上结果。
3.分选后的c-Kit+ CSCs中处于S期和G2期的细胞比例约34.6%,说明其染色体复制和细胞分裂较活跃。
4.空白组CSCs在低血清条件下增殖活力较差,部分细胞死亡;条件液组中CSCs增殖活力稍好,细胞平均密度是空白组的2倍。空白组和条件液组均未出现自发搏动的细胞。混合培养组中与心肌细胞接触的CSCs形态饱满,并与心肌细胞同步搏动。分离混合培养的细胞,可见少数单独搏动的带DAPI标记的细胞。
5.原位免疫组化技术:经过21天后,心肌条件培养液不能有效的诱导CSCs分化成熟至表达心肌细胞特异的Troponin-T和connexin43蛋白,条件液组与空白组比较无显著性差异。而CSCs与心肌细胞接触8天后,能表达上述两种蛋白,与空白组比较有显著性差异。
结论
1.心肌条件培养液无诱导CSCs向心肌细胞分化的作用。
2.与心肌细胞接触能有效地诱导CSCs向心肌分化。
Objective:
To investigate whether CSCs can be induced into cardiomyocytes(CM)by Myocardial conditioned medium,whether cellular contact with CM is necessary to CSCs’myocardial differentiation.
Methods:
1. Myocardial tissue isolated from neonatal mice was cut into 0.5-1mm3 fragments,red cells was washed off by PBS buffer.After mild digestion for 5 min with trypsin,the remaining tissue fragments were seeded and cultured for 11-14 days. Small, phase-bright like cells clusters were massive formed,these kind of primary gener- ation of cells were separated and collected by differential pace digestion.
2. The primary cells were sorted with immunobeads to obtain c-Kit+ CSCs.
3. With flow cytometry(FCM),Revealing the cell phenotype of c-Kit、Sca-1、CD34、CD45 expressing of the unsorted primary cells,verifying the purity of the sorted c-Kit+ cells.examining the sorted cells’cell cycle
4. Using In-site immunofluorescence(IFS) to verify the purity of the sorted c-Kit+ cells.
5. There groups were established on the base of sorted c-Kit+ cells.
Blank control group,c-Kit+ cells were cultured solo for 21 days using 1% FBS medium.
Conditioned medium group,c-Kit+ cells were cultured solo for 21 days using Myocardial conditioned medium(1% FBS medium).
Mixed group,c-Kit+ cells labeled with PKH26 or DAPI were co-cultured with CM for 8 days respectively(1% FBS medium).PKH26 labeled cell were undergone Immunohistochemistry in the final,while DAPI labeled cells at the 8th day were separated and pass-generated dispersedly,in the subsequent 2 weeks,Observation were made to spot spontaneous beating events.
6. Performing in-site immunohistochemistry in the blank group Conditioned medium group and PKH26 labelling mixed group to detect Troponin-T and connexin43 expression at the final day to assess the CSCs’myocardial differentiation.
7. Statistical analysis:Five 200x microscope view were randomly taken from every culture tray,one from the centre,four from the peripheral,Positive or negative cell were summed up,and analyzed by statistical method,the accepted level of significance was P<0.05.
Results
1. The primary cells mainly expressed c-Kit(44%) and Sca-1(13%),a small ratio(2-3%) of the cell express CD34、CD45.
2. The Purity of sorted c-Kit+ cells was approximately 96%,which was consistent with the result of IFS
3. The cells of S phase and G2/M phase in the sorted cells were accounted for 34.6%, indicating active proliferation.
4. Cells in the blank group were under growth arrest,partially died,Cells in the Conditioned medium group revealed better growth potential,with 2 times better in mean cell density.CSCs in mixed group which contacted with CM were burly in shape,and beat with CM synchronously.Spontaneous beating cells with DAPI were Observed after separation,although small in number(22 spots).
5. Myocardial conditioned medium had no effect on inducing CSCs’expression of Troponin-T and connexin43,While CSCs in contact with CM expressed Troponin-T and connexin43 in a shorter period of 8 days.
Conclusions
1. Myocardial conditioned medium have no effect on conducting CSCs into myocardial differentiation.
2. Contact with CM is a potent factor in CSCs’myocardial differentiation.
1.了解心肌条件培养液有无诱导心肌干细胞(Cardiac stem cells,CSCs)向心肌细胞分化的作用。
2.CSCs与心肌细胞接触对CSCs分化有何影响。
方法:
1.将新生1-2天小鼠心脏组织剪成0.5-1mm3碎片,用DPBS洗涤红细胞,轻微消化组织块后,进行贴壁培养11-14天后,用胰酶进行差速消化并采集原代CSCs。
2.用免疫磁珠分选原代细胞得到c-Kit+ CSCs。
3.流式细胞术分析未分选的原代细胞表面CD45、CD34、Sca-1、c-Kit标志。分选后细胞检测c-Kit标志纯度及细胞周期。
4.结合原位免疫荧光检验c-Kit+的纯度。
5.分选后的c-Kit+ CSCs分3组进行培养。
a.空白对照组,无干预因素,用1%血清培养液培养CSCs 21天。
b.条件液组添加心肌细胞条件培养液诱导CSCs 21天。用1%血清培养液培养心肌细胞48小时后得到心肌条件培养液。方法示意图见附图尾页。
c.混合培养组分别用PKH26和DAPI标记CSCs后与心肌细胞混合共培养8天,期间均以1%血清培养液培养。
PKH26标记的CSCs用于免疫组化鉴定,并与空白组和条件液进行对比; DAPI标记的CSCs,共培养后再进行消化分离并分散传代,改用10%血清培养液,2周内,观察并寻找来源于CSCs,带DAPI标记的单独搏动细胞。
6.观察各组CSCs形态变化,当空白组、条件液组和PKH26标记的混合培养组的细胞完成培养后,用原位免疫组化法鉴定肌钙蛋白T(Troponin-T)和间隙连接蛋白43(connexin43),评估CSCs向心肌细胞分化的程度,每组检测一种蛋白需要6个培养样本,其中1个用于阴性对照。
7.分析试验数据:原位免疫组化每孔随机抽四周+中央五个200倍视野,计数阳性细胞和阴性细胞后进行χ2检验或秩和检验分析,以P<0.05为差异具有显著性。混合培养组中评估在心肌细胞附近的CSCs。
结果:
1.原代未分选的细胞主要表达干细胞标志c-Kit和Sca-1,低表达造血干细胞标志CD34和白细胞标志CD45。
2.分选后得到纯度较高的c-Kit+ CSCs,流式细胞术分析其纯度约96%,原位免疫荧光检验也支持以上结果。
3.分选后的c-Kit+ CSCs中处于S期和G2期的细胞比例约34.6%,说明其染色体复制和细胞分裂较活跃。
4.空白组CSCs在低血清条件下增殖活力较差,部分细胞死亡;条件液组中CSCs增殖活力稍好,细胞平均密度是空白组的2倍。空白组和条件液组均未出现自发搏动的细胞。混合培养组中与心肌细胞接触的CSCs形态饱满,并与心肌细胞同步搏动。分离混合培养的细胞,可见少数单独搏动的带DAPI标记的细胞。
5.原位免疫组化技术:经过21天后,心肌条件培养液不能有效的诱导CSCs分化成熟至表达心肌细胞特异的Troponin-T和connexin43蛋白,条件液组与空白组比较无显著性差异。而CSCs与心肌细胞接触8天后,能表达上述两种蛋白,与空白组比较有显著性差异。
结论
1.心肌条件培养液无诱导CSCs向心肌细胞分化的作用。
2.与心肌细胞接触能有效地诱导CSCs向心肌分化。
Objective:
To investigate whether CSCs can be induced into cardiomyocytes(CM)by Myocardial conditioned medium,whether cellular contact with CM is necessary to CSCs’myocardial differentiation.
Methods:
1. Myocardial tissue isolated from neonatal mice was cut into 0.5-1mm3 fragments,red cells was washed off by PBS buffer.After mild digestion for 5 min with trypsin,the remaining tissue fragments were seeded and cultured for 11-14 days. Small, phase-bright like cells clusters were massive formed,these kind of primary gener- ation of cells were separated and collected by differential pace digestion.
2. The primary cells were sorted with immunobeads to obtain c-Kit+ CSCs.
3. With flow cytometry(FCM),Revealing the cell phenotype of c-Kit、Sca-1、CD34、CD45 expressing of the unsorted primary cells,verifying the purity of the sorted c-Kit+ cells.examining the sorted cells’cell cycle
4. Using In-site immunofluorescence(IFS) to verify the purity of the sorted c-Kit+ cells.
5. There groups were established on the base of sorted c-Kit+ cells.
Blank control group,c-Kit+ cells were cultured solo for 21 days using 1% FBS medium.
Conditioned medium group,c-Kit+ cells were cultured solo for 21 days using Myocardial conditioned medium(1% FBS medium).
Mixed group,c-Kit+ cells labeled with PKH26 or DAPI were co-cultured with CM for 8 days respectively(1% FBS medium).PKH26 labeled cell were undergone Immunohistochemistry in the final,while DAPI labeled cells at the 8th day were separated and pass-generated dispersedly,in the subsequent 2 weeks,Observation were made to spot spontaneous beating events.
6. Performing in-site immunohistochemistry in the blank group Conditioned medium group and PKH26 labelling mixed group to detect Troponin-T and connexin43 expression at the final day to assess the CSCs’myocardial differentiation.
7. Statistical analysis:Five 200x microscope view were randomly taken from every culture tray,one from the centre,four from the peripheral,Positive or negative cell were summed up,and analyzed by statistical method,the accepted level of significance was P<0.05.
Results
1. The primary cells mainly expressed c-Kit(44%) and Sca-1(13%),a small ratio(2-3%) of the cell express CD34、CD45.
2. The Purity of sorted c-Kit+ cells was approximately 96%,which was consistent with the result of IFS
3. The cells of S phase and G2/M phase in the sorted cells were accounted for 34.6%, indicating active proliferation.
4. Cells in the blank group were under growth arrest,partially died,Cells in the Conditioned medium group revealed better growth potential,with 2 times better in mean cell density.CSCs in mixed group which contacted with CM were burly in shape,and beat with CM synchronously.Spontaneous beating cells with DAPI were Observed after separation,although small in number(22 spots).
5. Myocardial conditioned medium had no effect on inducing CSCs’expression of Troponin-T and connexin43,While CSCs in contact with CM expressed Troponin-T and connexin43 in a shorter period of 8 days.
Conclusions
1. Myocardial conditioned medium have no effect on conducting CSCs into myocardial differentiation.
2. Contact with CM is a potent factor in CSCs’myocardial differentiation.
引文
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[9]Dawn B, Stein AB, Urbanek K, et al. Cardiac stem cells delivered intravascularly traverse the vessel barrier, regenerate infarcted myocardium, and improve cardiac function. Proc Natl Acad Sci U S A. 2005;102:3766–3771.
[10]Bearzi C, Rota M, Hosoda T, et al. Human cardiac stem cells. Proc Natl Acad Sci U S A. 2007;104:14068–14073.
[11]Pouly J, Bruneval P, Mandet C,et al.Cardiac stem cells in the real world. [J] Thorac Cardiovasc Surg. 2008;135:673– 678.
[12]Hajime Kubo, Naser Jaleel, Asangi Kumarapeli,et, al.Increased Cardiac Myocyte Progenitors in Failing Human Hearts.Circulation. 2008;118:649-657
[13]Ming Li, Nawazish Naqvi, Eiji Yahiro,et al.c-kit Is Required for Cardiomyocyte Terminal Differentiation.[J]Circ. Res. 2008;102;677-685 [L2]Palumbo R, Bianchi ME. High mobility group box 1 protein, a cue for stem cell recruitment. Biochem Pharmacol. 2004;68:1165–1170.
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[1]Shinya Fukuhara, Shinji Tomita, Seiji Yamashiro,et al.Direct cell-cell interaction of cardiomyocytes is key for bone marrow stromal cells to go into cardiac lineage in vitro.[J] Thorac Cardiovasc Surg 2003;125:1470-1480
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[3]Martin CM, Meeson AP, Robertson SM,et al. Persistent expression of the ATP-binding cassette transporter, Abcg2, identifies cardiac SP cells in the developing and adult heart. Dev Biol. 2004;265:262–275
[4]Pfister O, Mouquet F, Jain M, et al. CD31- but Not CD31+ cardiac side population cells exhibit functional cardiomyogenic differentiation. Circ Res. 2005;97:52–61
[5]Oyama T, Nagai T, Wada H,et al. Cardiac side population cells have a potential to migrate and differentiate into cardiomyocytes in vitro and in vivo. J Cell Biol. 2007;176:329–341
[6]Martin CM, Ferdous A, Gallardo T,et al. Hypoxia-inducible factor-2alpha transactivates Abcg2 and promotes cytoprotection in cardiac side population cells. Circ Res. 2008;102:1075–1081
[7]Mouquet F, Pfister O, Jain M,et al. Restoration of cardiac progenitor cells after myocardial infarction by self-proliferation and selective homing of bone marrow-derived stem cells. Circ Res. 2005;97:1090–1092
[8] Beltrami AP, Barlucchi L, Torella D et al. Adult cardiac stem cells are multipotent andsupportmyocardial regeneration. Cell, 2003; 114: 763– 776
[9]Dawn B, Stein AB, Urbanek K, et al. Cardiac stem cells delivered intravascularly traverse the vessel barrier, regenerate infarcted myocardium, and improve cardiac function. Proc Natl Acad Sci U S A. 2005;102:3766–3771.
[10]Bearzi C, Rota M, Hosoda T, et al. Human cardiac stem cells. Proc Natl Acad Sci U S A. 2007;104:14068–14073.
[11]Pouly J, Bruneval P, Mandet C,et al.Cardiac stem cells in the real world. [J] Thorac Cardiovasc Surg. 2008;135:673– 678.
[12]Hajime Kubo, Naser Jaleel, Asangi Kumarapeli,et, al.Increased Cardiac Myocyte Progenitors in Failing Human Hearts.Circulation. 2008;118:649-657
[13]Ming Li, Nawazish Naqvi, Eiji Yahiro,et al.c-kit Is Required for Cardiomyocyte Terminal Differentiation.[J]Circ. Res. 2008;102;677-685 [L2]Palumbo R, Bianchi ME. High mobility group box 1 protein, a cue for stem cell recruitment. Biochem Pharmacol. 2004;68:1165–1170.
[15]Taguchi A, Blood DC, del TG,et al. Blockade of RAGE-amphoterin signalling suppresses tumour growth and metastases.Nature. 2000;405:354–360.
[16]Huttunen HJ, Kuja-Panula J, Rauvala H. Receptor for advanced glycation end products signaling induces CREB-dependent chromogranin expression during neuronal differentiation. Biol Chem. 2002;277:38635–38646.
[17]Limana F, Germani A, Zacheo A,et al. Exogenous high-mobility group box 1 protein induces myocardial regeneration after infarction via enhanced cardiac C-kit+ cell proliferation and differentiation. Circ Res.2005;97:e73– e83
[18]Oh H, Bradfute SB, Gallardo TD,et al.Cardiac progenitor cells from adult myocardium: homing, differentiation,and fusion after infarction. Proc Natl Acad Sci U S A. 2003;100:12313–12318.
[19]Matsuura K, Nagai T, Nishigaki N, et, al. Adult cardiac Sca-1-positive cells differentiate into beating cardiomyocytes. J Biol Chem. 2004;279:11384-11391.
[20]Wang X, Hu Q, Nakamura Y, et al. The role of the Sac-1+/CD31- cardiac progenitor cell population in postinfarction left ventricular remodeling. Stem Cells. 2006;24:1779–1788
[21]Messina E, De Angelis L, Frati G, et al. Isolation and expansion of adult cardiac stem cells from human and murine heart. Circ Res. 2004;95:911–921
[22]Smith RR, Barile L, Cho HC,et al. Regenerative potential of cardiosphere-derived cells expanded from percutaneous endomyocardial biopsy specimens. Circulation. 2007;115:896–908.
[23]Quaini F, Urbanek K, Beltrami AP, et al. Chimerism of the transplanted heart. N Engl J Med. 2002;346:5–15
[24]Laugwitz KL, Moretti A, Lam J, Gruber P,et al.Postnatal isl1+ cardioblasts enter fully differentiated cardiomyocyte lineages. Nature. 2005;433:647–653.
[25]Smart N, Risebro CA, Melville AAD, et al. Thymosinβ4 induces adult epicardial progenitor mobilization and neovascularization. Nature. 2007;445:177–182.
[26]Winter E, Gittenberger-de Groot A. Cardiovascular development: towards biomedical applicability. Epicardium-derived cells in cardiogenesis and cardiac regeneration. Cell Mol Life Sci. 2007;64:692–703.
[27]Chen TH, Chang TC, Kang JO,et al.Epicardial induction of fetal cardiomyocyte proliferation via a retinoic acid-inducible trophic factor. Dev Biol.2002;250:198–207.
[28]Munoz-Chapuli R, Gonzalez-Iriarte M, Carmona R,et al. Cellular precursors of the coronary arteries. Tex Heart Inst J. 2002;29:243–249.
[29]Lepilina A, Coon AN, Kikuchi K, et al.A dynamic epicardial injury response supports progenitor cell activity during zebrafish heart regeneration. Cell. 2006;127:607– 619.
[30]Limana F, Zacheo A, Mocini D, et al. Identification of myocardial and vascular precursor cells in human and mouse epicardium. Circ Res.2007;101:1255–1265.
[31]Nian M, Lee P, Khaper N, Liu P. Inflammatory cytokines and postmyocardial infarction remodeling. Circ Res. 2004;94:1543–1553
[32]Jugdutt BI. Ventricular remodeling after infarction and the extracellular collagen matrix: when is enough enough? Circulation. 2003;108: 1395–1403.
[33]Jugdutt BI. Ventricular remodeling after infarction and the extracellular collagen matrix: when is enough enough? Circulation. 2003;108:1395–1403.
[34]Hayakawa K, Takemura G, Kanoh M, Inhibition of granulation tissue cell apoptosis during the subacute stage of myocardial infarction improves cardiac remodeling anddysfunction at the chronic stage. Circulation. 2003;108:104–109.
[35]Susanne Vandervelde, Marja J.A. van Luyn, et al.Stem cell-related cardiac gene expression early after murine myocardial infarction.Cardiovascular Research , 2007;73: 783–793
[36] Vieyra DS, Jackson KA, Goodell MA. Plasticity and tissue regenerative potential of bone marrow-derived cells. Stem Cell Rev. 2005;1:65– 69. [z6]Massimo Cimini, Shafie Fazel, Sun Zhuo,et al.c-Kit Dysfunction Impairs Myocardial Healing After Infarction.Circulation. 2007;116:I-77–I-82
[38]Urbanek K, Rota M, Cascapera S,et al.Cardiac stem cell possesses the growth factor-receptor system that following activation the regenerative the infarcted myocardium improving ventricular function and long term survival. Circ Res.2005;97:663– 673.
[39]Torella D, Rota M, Nurzynska D,et al. Cardiac stem cell and myocyte aging, heart failure, and insulin-like growth factor-1 overexpression. Circ Res. 2004;94:514–524.
[40]Migliaccio E, Giorgio M, Mele S, et al. The p66shc adaptor protein controls oxidative stress response and life span in mammals. Nature. 1999;402:309–313
[41]Napoli C, Martin-Padura I, de Nigris F,et al. Deletion of the p66-Shc longevity gene reduces systemic and tissue oxidative stress, vascular cell apoptosis, and early atherogenesis in mice fed a high-fat diet. Proc Natl Acad Sci U S A. 2003;100:2112–2116.
[42] Marcello Rota, Nicole LeCapitaine, Toru Hosoda, et, al. Diabetes Promotes Cardiac Stem Cell Aging and Heart Failure, Which Are Prevented by Deletion of the p66shc Gene. Circ Res. 2006;99:42-52.