5氮杂胞苷对骨髓间充质干细胞端粒酶逆转录酶及细胞增殖的影响 word格式.docxVIP

胞表团标志物CD44免疫组织化学检测阳性，CD45检测阴性。2、细胞增殖检测:实验组2 (6J!mol/L) OD值24h 时达峰值，显著高于其余各浓度组，P0.05;实验组3(12μmol/L)在12h达峰值;实验组4 (24μmol/L)细胞OD值随培养时间逐渐增高，但始终低于对照组，并里平行关系。3、端粒酶逆转录酶检测:实验组2(6μmollL)TERTmRNA转录、丁ERT蛋白阳性颗粒表达较对照组显著增多(12h、24h) P仪05，其中24小时表达最高，PO.Ol;实验组3(12J!mollL)TERT转录、蛋白阳性颗粒表达在12h时间点高于其余各组P0.05o结论:1、体外培养骨髓间充质干细胞，第3-6 代细胞纯度高、活性好、增殖能力强，是进行相关实验研究的理想细胞模型。2、去甲基化药物氮杂胞背在6μmollL浓度24h时间点是上调BMSCs端粒酶逆转录酶m刚A转录水平、增加蛋白表达、促进细胞增殖的最适浓度和时间点。趴在一定浓度范围一定时间点，5-氮杂胞背发挥去甲基化作用，激活丁ERTmRNA转录，上调TERT蛋白表达，增强端粒酶活性，促进BMSCs增殖，延长BMSCs寿命。关键词端粒酶逆转录闹;氮杂胞背;DNA去甲基化:实时荧光定量;细胞增殖Theeffectof5-Azaonthetelomerasereversetranscriptaseandtheproliferation ofBMSCs【Abstract1 Objective:Toexploretheeffect ofDNAdemethylationwithιAzaon thetelomerasereversetranscriptaseandtheproliferationofBMSC andtounderstandthemechanismofprolonglifespanandmaintainself-renewalbystemcells，soastoprovide seedcellsforclinicalapplication.Method:1、BMSC(Bonemaπowstromalstemcells)cultureinvitro:BMSC仕omthefemurandtibiaofSDratswastakentoculture .丁hecellswereplaced inincubator2(370Cconstanttemperature，5%CO)，culturedthemfor24hours，removedthenon-adherent cells，thenchangedthemedium each2or3day. Whencultured.cellscoveredthebottom70%唰80%areas，wewillSubculture thecells，observethe form ofbone marrow stromal stem cells under inverted microscope andbiological characteristics. 2、Indirectly determine theactivity ofbonemarrowstoromalstemcells :Thefourthgenerationofbonemarrowstromalstemcells，treatedwith弘Azawithdifferentconcentrations(3JlmollL、6JlmolIL、12JlmolIL、24阳nollL)indifferenttime(6h、12h、24h、48h、72h).Thegrowthabilityofcellwas assayed with MTT method. 3.. To determine 出e 回民丁:The fourthgeneration ofbonemaηowstromalstemcells，treatedwith弘Azawithdifferentconcentrations(6μmollL、12μmollL)indifferenttime(6h、12h、24亿48h、72h).Theexpressionof丁ERTatdifferentgroupsweredetectedbyimmunogistochemical staining with SPmethod. The transcription of TERTmRNA at differentgroups were detected by real嗣time polymerase chainreaction (R下PCR) .4、The statistics were prese