三种不同来源的围产期间充质干细胞条件培养基的抗癌能力比较
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摘要
目的:探究羊水、脐带和胎盘三种不同来源间充质干细胞中,抑癌效果最好的间充质条件培养基。方法:应用羊水、脐带和胎盘三种不同来源间充质干细胞的不同浓度的条件培养基分别培养HELA、SKOV-3、MCF-7三种癌细胞,CCK-8法检测培养72 h后不同癌细胞的细胞活性;应用羊水、脐带和胎盘三种不同来源间充质干细胞条件培养基培养HELA细胞24、48、72、96 h后,利用CCK-8法检测癌细胞的细胞活性;采用Annexin V-FITC/PI通过流式细胞术检测条件培养基培养HELA细胞72 h后的癌细胞凋亡情况;qPCR鉴定条件培养基培养HELA细胞72 h后的癌症相关基因LOXL2、LIF、CatB、FOXQ1的表达,同时对不同间充质干细胞条件培养基培养后迁移的HELA细胞数量进行计算。结果:三种不同浓度的条件培养基均对癌细胞有抑制作用,其中浓度为100%条件培养基对癌细胞的抑制作用最好;三种不同来源间充质干细胞条件培养基均对癌细胞有抑制作用,且羊水间充质干细胞条件培养基培养HELA细胞72 h对癌细胞的抑制作用最强;流式细胞术与qPCR结果表明间充质干细胞条件培养基抑制HELA细胞生长的作用机制是通过促进其凋亡进行的;羊水间充质干细胞条件培养基培养后HELA细胞迁移数量最少。结论:羊水间充质干细胞是最适合用于抑制癌细胞生长的细胞,可以促进癌细胞凋亡,浓度为100%条件培养基培养癌细胞72 h对癌细胞抑制效果最好。
Objective:To explore the best anti-tumor cells conditioned medium in three different kinds of source of mesenchymal stem cells:Amniotic fluid,umbilical cord,and placenta.Methods:Amniotic fluid-derived mesenchymal stem cells,umbilical cord-derived mesenchymal stem cells,placenta-derived mesenchymal stem cells were cultured for 72 h,then got the conditioned medium.The HELA,SKOV-3 and MCF-7 were cultured for 72 h with conditioned medium of three concentrations respectively,then the cell viability was detected by CCK-8.The HELA cells cultured in three conditioned medium for 24 h,48 h,72 h,96 h,then got the cells activity with CCK-8.The apoptosis of HELA cells cultured in conditioned medium for 72 h were detected by Flow cytometry.The qPCR was used to identify the expression of cancer-related genes LOXL2,LIF,CatB and FOXQ1 in HELA cells after 72 h of conditioned medium culture.The number of migrated HELA cells was calculated after different conditioned medium culture.Results:Three different concentrations of conditioned medium all inhibited the growth of the cancer cells,of which the concentration of 100%conditioned medium had the best inhibitory effect on cancer cells.Three different sources of conditioned medium all had inhibitory effects on cancer cells,of which the cells cutured with conditioned medium for 72 h had the best inhibitory effect on cancer cells.Besides,the results of flow cytometry and qPCR indicated that the mechanism of mesenchymal stem cell conditioned medium inhibiting the growth of HELA cells was through the promotion of apoptosis.The conditioned medium from amniotic fluid mesenchymal stem cells had the least number of migration of HELA cells.Conclusion:The results showed that the conditioned medium from amniotic fluid-derived mesenchymal stem cells was the best anti-tumor cells.It can promote the apoptosis of cancer cells.The concentration of 100% conditioned medium cutured for 72 h has the best inhibitory effect on cancer cells.
Objective:To explore the best anti-tumor cells conditioned medium in three different kinds of source of mesenchymal stem cells:Amniotic fluid,umbilical cord,and placenta.Methods:Amniotic fluid-derived mesenchymal stem cells,umbilical cord-derived mesenchymal stem cells,placenta-derived mesenchymal stem cells were cultured for 72 h,then got the conditioned medium.The HELA,SKOV-3 and MCF-7 were cultured for 72 h with conditioned medium of three concentrations respectively,then the cell viability was detected by CCK-8.The HELA cells cultured in three conditioned medium for 24 h,48 h,72 h,96 h,then got the cells activity with CCK-8.The apoptosis of HELA cells cultured in conditioned medium for 72 h were detected by Flow cytometry.The qPCR was used to identify the expression of cancer-related genes LOXL2,LIF,CatB and FOXQ1 in HELA cells after 72 h of conditioned medium culture.The number of migrated HELA cells was calculated after different conditioned medium culture.Results:Three different concentrations of conditioned medium all inhibited the growth of the cancer cells,of which the concentration of 100%conditioned medium had the best inhibitory effect on cancer cells.Three different sources of conditioned medium all had inhibitory effects on cancer cells,of which the cells cutured with conditioned medium for 72 h had the best inhibitory effect on cancer cells.Besides,the results of flow cytometry and qPCR indicated that the mechanism of mesenchymal stem cell conditioned medium inhibiting the growth of HELA cells was through the promotion of apoptosis.The conditioned medium from amniotic fluid mesenchymal stem cells had the least number of migration of HELA cells.Conclusion:The results showed that the conditioned medium from amniotic fluid-derived mesenchymal stem cells was the best anti-tumor cells.It can promote the apoptosis of cancer cells.The concentration of 100% conditioned medium cutured for 72 h has the best inhibitory effect on cancer cells.
引文
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[2] Li X,Bai J,Ji X,et al.Comprehensive characterization of four different populations of human mesenchymal stem cells as regards their immune properties,proliferation and differentiation[J].Int J Mol Med,2014,34(3):695-704.
[3] Collins E,Gu F,Qi M,et al.Differential efficacy of human mesenchymal stem cells based on source of origin[J].J Immunol,2014,193(9):4381-4390.
[4] Martinelli D,Pereira RC,Mogni M,et al.A humanized system to expand in vitro amniotic fluid-derived stem cells intended for clinical application[J].Cytotherapy,2016,18(3):438-451.
[5] Von Bahr L,Sundberg B,Lonnies L,et al.Long-term complications,immunologic effects,and role of passage for outcome in mesenchymal stromal cell therapy[J].Biol Blood Marrow Transplant,2012,18(4):557-564.
[6] Anasiz Y,Ozgul RK,Uckan-Cetinkaya D.A new chapter for mesenchymal stem cells:Decellularized extracellular matrices[J].Stem Cell Rev,2017,13(5):587-597.
[7] Ghafarzadeh M,Eatemadi A,Fakhravar Z.Human amniotic fluid derived mesenchymal stem cells cause an anti-cancer effect on breast cancer cell line in vitro[J].Cell Mol Biol(Noisy-le-grand),2016,62(6):102-106.
[8] Ramdasi S,Sarang S,Viswanathan C.Potential of mesenchymal stem cell based application in cancer[J].Int J Hematol Oncol Stem Cell Res,2015,9(2):95-103.
[9] Janowski M,Lyczek A,Engels C,et al.Cell size and velocity of injection are major determinants of the safety of intracarotid stem cell transplantation[J].J Cereb Blood Flow Metab,2013,33(6):921-927.
[10] Bhere D,Shah K.Stem cell-based therapies for cancer[J].Adv Cancer Res,2015,127:159-189.
[11] Mareschi K,Castiglia S,Sanavio F,et al.Immunoregulatory effects on T lymphocytes by human mesenchymal stromal cells isolated from bone marrow,amniotic fluid,and placenta[J].Experimental Hematology,2016,44(2):138-150.
[12] Macrin D,Joseph JP,Pillai AA,et al.Eminent sources of adult mesenchymal stem cells and their therapeutic imminence[J].Stem Cell Rev,2017,13(6):741-756.
[13] Marquez-Curtis LA,Janowska-Wieczorek A,Mcgann LE,et al.Mesenchymal stromal cells derived from various tissues:Biological,clinical and cryopreservation aspects[J].Cryobiology,2015,71(2):181-197.
[14] Danisovic L,Bohac M,Zamborsky R,et al.Comparative analysis of mesenchymal stromal cells from different tissue sources in respect to articular cartilage tissue engineering[J].Gen Physiol Biophys,2016,35(2):207-214.
[15] El OR,Beroud J,Stoltz JF,et al.Umbilical cord mesenchymal stem cells:The new gold standard for mesenchymal stem cell-based therapies[J]?Tissue Eng Part B Rev,2014,20(5):523-544.
[16] Motaln H,Schichor C,Lah TT.Human mesenchymal stem cells and their use in cell-based therapies[J].Cancer,2010,116(11):2519-2530.
[17] Kamerkar S,Lebleu VS,Sugimoto H,et al.Exosomes facilitate therapeutic targeting of oncogenic KRAS in pancreatic cancer[J].Nature,2017,546(7659):498-503.
[18] Kordelas L,Rebmann V,Ludwig AK,et al.MSC-derived exosomes:A novel tool to treat therapy-refractory graft-versus-host disease[J].Leukemia,2014,28(4):970-973.
[19] Reza-Zaldivar EE,Hernandez-Sapiens MA,Minjarez B,et al.Potential effects of MSC-derived exosomes in neuroplasticity in alzheimer's disease[J].Front Cell Neurosci,2018,12:317.
[20] Cheng X,Zhang G,Zhang L,et al.Mesenchymal stem cells deliver exogenous miR-21 via exosomes to inhibit nucleus pulposus cell apoptosis and reduce intervertebral disc degeneration[J].J Cell Mol Med,2018,22(1):261-276.
[21] Hanahan D,Weinberg RA.Hallmarks of cancer:The next generation[J].Cell,2011,144(5):646-674.
[22] Stotz M,Szkandera J,Stojakovic T,et al.The lymphocyte to monocyte ratio in peripheral blood represents a novel prognostic marker in patients with pancreatic cancer[J].Clin Chem Lab Med,2015,53(3):499-506.
[23] Maj M,Bajek A,Nalejska E,et al.Influence of mesenchymal stem cells conditioned medium on proliferation of urinary tract cancer cell lines and their sensitivity to ciprofloxacin[J].J Cell Biochem,2017,118(6):1361-1368.
[24] Eltoukhy HS,Sinha G,Moore CA,et al.Immune modulation by a cellular network of mesenchymal stem cells and breast cancer cell subsets:Implication for cancer therapy[J].Cell Immunol,2017,326:33-41.