外源性bFGF和CNTF对大鼠MDSCs增殖的影响
摘要
目前,MDSCs的研究存在许多热点问题,体外细胞培养中的纯化、扩增、定向分化等难题尚在探索中。人们在探讨适宜的干细胞培养条件的同时,特别关注生物活性因子对干细胞增殖的影响。bFGF和CNTF作为生物活性因子的成员,因其生物活性的多效性而备受基础与临床研究的重视。
本文进行的有关研究,旨在进一步探讨成体干细胞的培养条件,为有关研究与应用奠定实验基础。本实验通过酶消化法分离大鼠MDSCs,然后用密度梯度离心法和差速贴壁法纯化获取MDSCs,进行体外原代和传代培养。并通过免疫细胞化学法(desmin、Sca-1)对所获得的细胞进行鉴定。通过MTT比色法检测不同浓度(6.25、12.50、25.00、50.00、100.00ng/ml)的bFGF和CNTF单独应用96h以及二者(100.0ng/ml)联合作用24h、48h、72h和96h对MDSCs增殖的影响。MTT的OD值显示:
1.与阴性对照组比较,除6.25(ng/ml)组外,12.50ng/ml至100.00ng/ml实验组的MDSCs均有明显的增殖(P<0.05)。在6.25ng/ml至50.00ng/ml期间,MDSCs的增殖随bFGF和CNTF浓度升高而显著增强(P<0.05)。
2.在6.25ng/ml至50.00ng/ml期间,bFGF与CNTF对MDSCs的促增殖作用没有明显差异(P>0.05)。随其浓度升高,在50.00ng/ml时,bFGF对MDSCs的促增殖作用明显低于CNTF组,并存在显著性差异(P<0.05)。但50.00ng/ml组促增殖作用接近顶峰,与100.00ng/ml组比较,差异无统计学意义(P>0.05)。
3.bFGF和CNTF的促增殖效应随培养时间的延长而逐渐明显,但促增殖作用的显效时间有明显差异。与阴性对照组比较,出现显著促增殖效应的时间(P<0.05)分别为:bFGF单独作用96 h、CNTF单独作用72h、二者联合应用48h。实验组在24h内尽管MDSCs数量增多,但没有显著性差异(P>0.05)。
4.在48h-96h期间,bFGF和CNTF联合组促进增殖的作用最强,并与其它实验组间存在显著差异(P<0.05)。
本实验提示:bFGF和CNTF均可促进体外培养的大鼠MDSCs增殖,而且具有协同增效作用。但是,bFGF和CNTF促进MDSCs增殖作用具有显效时间的差异性,而且在12.5-50ng/ml期间,促MDSCs增殖作用还呈浓度依赖性。
At present, there are plenty hot spots about the MDSCs research. The purifying, amplifying, and directional differentiation of vitro cell culture is still under exploration. Special attention usually has been paid to the effects of the biological activity factor on the stem cell proliferation while the appropriate cell culture conditions are mentioned. bFGF and CNTF, as a member of biological activity factors, are of great importance both for its biological activity and effectiveness in the basic and clinical researches.
In this thesis, researches and experiments have been designed to explore the adult stem cell culture conditions, which may lay the basic experimental foundation for the future study and application. MDSCs of rat has been isolated through the enzymatic digestion, then purified by density gradient centrifugation and differential adhesion and vitro and cultured. The MTT colorimetric has been employed to assay the effects of bFGF with different concentrations (6.25, 12.50, 25.00, 50.00, 100.00ng/ml), CNTF using alone 96h, and the role of the above two combined with 100.0ng/ml, in 24h, 48h, 72h, and 96h on the proliferation of MDSCs. The obtained cells have been identified with the immunocytochemical method (desmin, Sca-1). The OD of the MTT shows:
1. Compared with the negative control group, there is a significant proliferation of MDSCs in all the other experimental groups (P<0.05), except the 6.25 (ng/ml). Among the 6.25ng/ml to 50.00ng/ml, MDSCs proliferation significantly enhances with the increased concentrations of bFGF and CNTF (P<0.05).
2. There is no significant difference in the MDSCs proliferation between bFGF and CNTF among the 6.25ng/ml to 50.00ng/ml (P>0.05). With its concentration increased to 50.00ng/ml, the bFGF group is significantly lower than CNTF group in the MDSCs proliferation, and the significant differences exists (P<0.05). However, the proliferation effects of the 50.00ng/m group is close to capacity, and there is no significant statistical difference when compared with the 100.00ng/ml group (P>0.05).
3. The proliferation effects of the bFGF and CNTF becomes clear with the extension of the culture time, while there is significant difference in the proliferation of effectual time. Compared with the negative control group, the significant proliferative effect of time presents at bFGF alone 96 h, CNTF alone 72h, combined 48h, respectively (P <0.05). At 24h in the experimental groups, even though the number of MDSCs increases, but there is no significant difference (P >0.05).
4. At 48h-96h period, the proliferation effects is of the strongest in bFGF and CNTF combined group, and there is significant difference exists when compared with the other experimental groups (P <0.05).
Note: Both of the bFGF and CNTF can promote the vitro cultured rat MDSCs proliferation, and there is a synergy in the proliferation. However, bFGF and CNTF to promote the proliferation of MDSCs is with appearent different time, and at 12.5-50ng/ml period, the role of promoting the proliferation of MDSCs also appears a dose-dependent manner.
本文进行的有关研究,旨在进一步探讨成体干细胞的培养条件,为有关研究与应用奠定实验基础。本实验通过酶消化法分离大鼠MDSCs,然后用密度梯度离心法和差速贴壁法纯化获取MDSCs,进行体外原代和传代培养。并通过免疫细胞化学法(desmin、Sca-1)对所获得的细胞进行鉴定。通过MTT比色法检测不同浓度(6.25、12.50、25.00、50.00、100.00ng/ml)的bFGF和CNTF单独应用96h以及二者(100.0ng/ml)联合作用24h、48h、72h和96h对MDSCs增殖的影响。MTT的OD值显示:
1.与阴性对照组比较,除6.25(ng/ml)组外,12.50ng/ml至100.00ng/ml实验组的MDSCs均有明显的增殖(P<0.05)。在6.25ng/ml至50.00ng/ml期间,MDSCs的增殖随bFGF和CNTF浓度升高而显著增强(P<0.05)。
2.在6.25ng/ml至50.00ng/ml期间,bFGF与CNTF对MDSCs的促增殖作用没有明显差异(P>0.05)。随其浓度升高,在50.00ng/ml时,bFGF对MDSCs的促增殖作用明显低于CNTF组,并存在显著性差异(P<0.05)。但50.00ng/ml组促增殖作用接近顶峰,与100.00ng/ml组比较,差异无统计学意义(P>0.05)。
3.bFGF和CNTF的促增殖效应随培养时间的延长而逐渐明显,但促增殖作用的显效时间有明显差异。与阴性对照组比较,出现显著促增殖效应的时间(P<0.05)分别为:bFGF单独作用96 h、CNTF单独作用72h、二者联合应用48h。实验组在24h内尽管MDSCs数量增多,但没有显著性差异(P>0.05)。
4.在48h-96h期间,bFGF和CNTF联合组促进增殖的作用最强,并与其它实验组间存在显著差异(P<0.05)。
本实验提示:bFGF和CNTF均可促进体外培养的大鼠MDSCs增殖,而且具有协同增效作用。但是,bFGF和CNTF促进MDSCs增殖作用具有显效时间的差异性,而且在12.5-50ng/ml期间,促MDSCs增殖作用还呈浓度依赖性。
At present, there are plenty hot spots about the MDSCs research. The purifying, amplifying, and directional differentiation of vitro cell culture is still under exploration. Special attention usually has been paid to the effects of the biological activity factor on the stem cell proliferation while the appropriate cell culture conditions are mentioned. bFGF and CNTF, as a member of biological activity factors, are of great importance both for its biological activity and effectiveness in the basic and clinical researches.
In this thesis, researches and experiments have been designed to explore the adult stem cell culture conditions, which may lay the basic experimental foundation for the future study and application. MDSCs of rat has been isolated through the enzymatic digestion, then purified by density gradient centrifugation and differential adhesion and vitro and cultured. The MTT colorimetric has been employed to assay the effects of bFGF with different concentrations (6.25, 12.50, 25.00, 50.00, 100.00ng/ml), CNTF using alone 96h, and the role of the above two combined with 100.0ng/ml, in 24h, 48h, 72h, and 96h on the proliferation of MDSCs. The obtained cells have been identified with the immunocytochemical method (desmin, Sca-1). The OD of the MTT shows:
1. Compared with the negative control group, there is a significant proliferation of MDSCs in all the other experimental groups (P<0.05), except the 6.25 (ng/ml). Among the 6.25ng/ml to 50.00ng/ml, MDSCs proliferation significantly enhances with the increased concentrations of bFGF and CNTF (P<0.05).
2. There is no significant difference in the MDSCs proliferation between bFGF and CNTF among the 6.25ng/ml to 50.00ng/ml (P>0.05). With its concentration increased to 50.00ng/ml, the bFGF group is significantly lower than CNTF group in the MDSCs proliferation, and the significant differences exists (P<0.05). However, the proliferation effects of the 50.00ng/m group is close to capacity, and there is no significant statistical difference when compared with the 100.00ng/ml group (P>0.05).
3. The proliferation effects of the bFGF and CNTF becomes clear with the extension of the culture time, while there is significant difference in the proliferation of effectual time. Compared with the negative control group, the significant proliferative effect of time presents at bFGF alone 96 h, CNTF alone 72h, combined 48h, respectively (P <0.05). At 24h in the experimental groups, even though the number of MDSCs increases, but there is no significant difference (P >0.05).
4. At 48h-96h period, the proliferation effects is of the strongest in bFGF and CNTF combined group, and there is significant difference exists when compared with the other experimental groups (P <0.05).
Note: Both of the bFGF and CNTF can promote the vitro cultured rat MDSCs proliferation, and there is a synergy in the proliferation. However, bFGF and CNTF to promote the proliferation of MDSCs is with appearent different time, and at 12.5-50ng/ml period, the role of promoting the proliferation of MDSCs also appears a dose-dependent manner.
引文
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[2]何涛,黄红军,等.肌源性干细胞的研究进展[J].中华实验外科杂志, 2006, 23(3):383-384.
[3] Torrente Y , Camirand G., et al. Identification of a putative pathway for the muscle homing of stem cells in a muscular dystrophy model[J]. J Cell Biol, 2003, 162(3):511-520.
[4] Qu-Petersen Z, Deasy B, et al. Identification of a novel population of muscle stem cells in mice: potential for muscle regeneration[J]. J Cell Biol, 2002, 157(5):851-864.
[5] Asakura A, Seale P, et al. Myogenic specification of side population cells in skeletal muscle[J]. J Cell Biol, 2002, 159(1):123-134.
[6] Chen J C, Goldhamer D J. Skeletal muscle stem cells[J]. Reprod Biol Endocrinol, 2003, 1:101.
[7] McLoon L K, Wirtschafter J. Activated Satellite Cells in Extraocular Muscles of Normal Adult Monkeys and Humans[J]. Invest Ophthalmol Vis Sci, 2003, 44(5):1927-1932.
[8] McCroskery S, Thomas M, et al. Myostatin negatively regulates satellite cell activation and self-renewal[J]. J Cell Biol, 2003, 162(6):1135-1147.
[9] Adachi N, Sato K, et al. Muscle derived, cell based ex vivo gene therapy for treatment of full thickness articular cartilage defects[J]. J Rheumatol, 2002, 29(9):1920-1930.
[10] Schienda J, Engleka K A, et al. Somitic origin of limb muscle satellite and side population cells[J]. Proc Natl Acad Sci U S A, 2006, 103(4):945-950.
[11] Goodell M A, Brose K, et al. Isolation and functional properties of murine hematopoietic stem cells that are replicating in vivo[J]. J Exp Med, 1996, 183(4):1797-1806.
[12] Goodell M A. Introduction: Focus on hematology. CD34(+) or CD34(-): does it
[13] Gussoni E, Soneoka Y, et al. Dystrophin expression in the mdx mouse restored by stem cell transplantation[J]. Nature, 1999, 401(6751):390-394.
[14]陈振兵,洪光祥,等.差速贴壁法体外培养失神经骨骼肌肌源性干细胞的生物学特性[J].中国临床康复, 2006, 10(33):7-9.
[15] Qu Z, Balkir L, et al. Development of Approaches to Improve Cell Survival in Myoblast Transfer Therapy[J]. J Cell Biol, 1998, 142(5):1257-1267.
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