【作者】 宋晓平；

【导师】 窦忠英；

【作者基本信息】 西北农林科技大学 ， 临床兽医学， 2003， 博士

【摘要】 胚胎干细胞(ES细胞)，是存在于早期胚胎内细胞团(ICM)的一种多潜能细胞。在体外抑制分化培养时，ES细胞可无限增殖并保持正常的二倍体核型，一旦撤除分化抑制因素，或添加适当诱导剂，ES细胞可发生谱系分化或定向分化。本研究在回顾ES细胞研究文献的基础上，进行了小鼠ES细胞的分离与克隆、ES-D3细胞系的扩增和分化能力检测，以及应用成骨细胞条件培养基、维生素C、β-磷酸甘油和地塞米松进行了定向诱导ES-D3细胞系向成骨细胞的分化。 1．MEF的分离与生长曲线 小鼠胎儿成纤维细胞(MEF)在24孔培养板内以密度5.0×10~3/mL，每孔接种1mL，间隔2d或4d换液1次，细胞增殖缓慢，倍增时间(Td)分别为47.13和47.96h，细胞活力较差。以密度5.0×10~4／mL，每孔接种1mL，每间隔2d换液1次，MEF增殖较快，Td为53.12h，第2～3d时需及时传代，否则细胞易出现老化卷层，间隔4d换液时细胞增殖速度减慢，Td为57.80h。以密度2.0×10~4／mL，每孔接种1mL，间隔4d换液1次，MEF增殖速度快，Td为35.38h，第3～5d时传代较适宜，间隔2d换液时MEF增殖减慢，Td延长至41.56h。 2．丝裂霉素C对MEF的影响 MEF以密度3×10~4／mL接种24孔培养板，在24h后分别经10μg／mL丝裂霉素C处理30、90和180min。在培养的第3，5，7，9，11d时，MEF增殖抑制与未经丝裂霉素C处理比较，均存在极显著差异(P＜0.01)，但三种不同的处理时间组间均无显著性差异(P＞0.05)。这说明，丝裂霉素C处理30min即可极显著抑制MEF的增殖，并可至少维持11d。 3．小鼠ES细胞的分离培养 应用MEF饲养层，以DMEM培养基添加15％FBS、10ng／mL rmLIF、0.1mmol／L2-Me、80U／mL青霉素、100U／mL链霉素，3.5dpc昆明系小鼠囊胚24h后脱去透明带，但极少有囊胚贴壁，48h时囊胚贴壁率为39％，72h贴壁率达74％，ES细胞集落形成率为8％，体外最高传5代。 4．ES-D3细胞系的扩增 以MEF为饲养层，DMEM培养基(含0.1mmol／L 2-Me+80U／mL青霉素+100U／mL链霉素)中添加15％KSR或15％FBS，，ES-D3细胞扩增培养48h时具有典型的集落形态，ES细胞增殖速度快，每天换液1次，间隔48h传代1次；添加15％NBS，维持ES细胞的有效增殖和存活效果较差，传第3代时消失。以STO细胞作饲养层，添加15％FBS，在倒置显微镜下观察，ES-D3细胞不易与周围的STO饲养层细胞区别，形成的集落较平坦，生长状态不如MEF饲养层好。 5.ES一D3细胞系的自主分化 ES一D3细胞悬浮培养，24h后可聚集成小的细胞团，第2~3d时形成大量的类胚体(EBs)。继续悬浮培养第6~8d时，EBs外围细胞开始脱落，延长悬浮培养时间至第10d时EBs大量死亡。悬浮培养的ES细胞及EBs易贴壁生长，特别是培养的第1~Zd，贴壁细胞较多，用吸头轻轻吹打可使贴壁的细胞和EBs重新悬浮。悬浮培养过程中未观察到囊状胚体的形成，但当EBs贴壁培养至第6d后出现囊状类胚体。ES一D3源的EBs在贴壁培养第9一1 Od时出现神经样细胞、成纤维样细胞，第14d时，EBs生长晕周围重新出现ES细胞集落，这些集落可存在较长时间。第23d后出现典型的长扁平状血管内皮细胞，并可排列成索状且形成血管样结构，随后血管内皮细胞逐渐消失。第3ld时，HE染色还显示存在一些多角形细胞。 6.小鼠成骨细胞条件培养基的制备 小鼠头顶骨经0.25%胰蛋白酶和0.04%EDI’A充分除去骨片周围及骨缝处的结缔组织，再按常规植块法培养，骨片周围出现典型的成骨细胞的时间较早，原代培养24h后即出现梭状、三角形或多边形细胞，BCIP加BT液体底物系统染色显淡蓝紫色。随培养时间的延长，细胞数量逐渐增多，8一IOd后相邻骨片间细胞汇合，密度较大处呈现重叠生长现象。传代培养后细胞生长速度明显加快，3~4d即可铺满皿底，差速豁附传代培养可获得较纯的成骨细胞。收集传代培养48h的成骨细胞培养液，4000r/m离心20min，0.22娜滤膜过滤，可制得成骨细胞条件培养基。 7.Es一D3细胞系的成骨细胞诱导分化 在EBs单细胞贴壁培养的第14~2 ld，在DMEM培养基(15%FBS+0.1~of几2一Me+80U/mL青霉素+looU/mL链霉素)中，添加60%成骨细胞条件培养基，第22d时1%茜素红染色钙结节呈鲜红色，成骨细胞诱导形成率为 10.04%，与对照组比较，差异极显著(P<0.01)。首次证明，条件培养液具有诱导ES细胞向成骨细胞分化的作用。 添加50林创mL维生素C+50~。比p一磷酸甘油，诱导分化的细胞分泌物形成网状结构，茜素红染色阳性细胞分布在这些网状结构中，成骨细胞诱导形成率为7.43%，与对照组比较差异显著(P<0.05);添加50林留mL维生素C+50~ol几日-磷酸甘油+1林mol/L地塞米松，茜素红染色阳性细胞分散而均匀，但未出现网状结构，成骨细胞诱导形成率提高至27.57%，与对照组比较差异极显著(P<0.01)，与添加50林创mL_维生素C+50~ol几p一磷酸甘油比较，差异极显著(P<0.01)。在国内，首次证明维生素C和p一磷酸甘油具有诱导ES细胞向成骨细胞分化的作用，地塞米松能促进维生素C和p一磷酸甘油的这种诱导作用。更多还原

【Abstract】 Embryonic stem (ES) cells are pluripotent cells derived from the inner cell mass (ICM) of early blastrocyts, which possess the ability of unlimitted proliferation and maintain normal dipoid keptypes under differentiation-inhibited culture in vitro, they will differentiate into multiple lineage cell types or occur commentent and differentiation without differentiation-inhibited factors or hi presence of certain induced-factors.. Based on the review of previous researches of ES cells, we conducted to isolate mouse ES cells from the ICM of early blasteocyts, to proliferate ES cell D3 line and to examine their ability of spontaneous differentiation in vitro, as well as to induce the ES cell D3 line into osteoblasts by conditioned-medium, ascorbic acid, β -glycerophosphate and/or dexame-thasone.1. Isolation and growth curve of MEFThe mouse embryonic fibroblasts (MEF) proliferated slowly that the time of doubling (Td) was 47.13h or 47.96h, respectively, and their growth state is poor when seeded into each well of 24-well plates at 5.0±103 cells with replacing fresh medium at 2- or 4-day intervals, whereas MEF flourished at 2.0±104 cells per well with replacing fresh medium at 4-day intervals (Td-35.38h) and it was appropriate to passage after 3 to 5 days of culture, and there was no benefits to the growth of the cells as replacing fresh medium at 2-day intervals (Td=41.56). The cell proliferation become rapidly when plated at 5.0±104 cells per well with replacing fresh medium at 2-day intervals (Td=53.12h), and needed to be subcultured after the culture of 2 to 3 days, if not, the cultured cells were easily expire, and on the contrary, the speed of the cell proliferation decreased as replacing fresh medium at 4-day intervals (Td=57.80h).2. Effects of mitomycin C on MEFThe MEF were inactivated by mitomycin C for 30, 90, 180min, respectively, at 24h after seeded into each well of 24-well culture plate at 3±104 cells. The low number of proliferating cells in all experimetial groups was significantly different from that of the control group (P<0.01) at the 3th, 5th, 7th, 9th, llth day of culture, respectively. Incontrast, there was not significantly different among the three treated groups (P>0.05). The results indicated that the MEF inactivated by 10μg/mL mitomycin C for 30 min were inhibited efficiently and maintained not to proliferate for 11 days at least.3. Isolation and culture of ES cells from mouse blastocytsWhen intact blastocyts from 3.5 dpc mouse cultured on the mitomycin C-inactivated MEF in Doulbecco’s modified minimal essential medium (DMEM) supplemented with 15% fetal bovine serum (FBS), 10ng/mL rmLIF,0.1mmol/L 2-mercaptoethanol, 80U/mL penicillin and 100U/mL streptomycin, the zona pellucida disengaged and removed from their blastocyts within 24h, but few blastocyts without zona pellucida attached to culture dish. The ratio of attached blastocyts was 39% and 74% at 48 h and 72 h of culture, respectively. The cloning ratio of ES cells was only 8 percent of growing ICM, and the ES clonies disappeared after going 5 passage at best.4. Proliferation of ES cell D3 lineES cell D3 line maintained the flourish and undifferentiated state with typical clonies at 48 h on the mitomycin C-inhibited MEF in DMEM supplemented with 15% Knockout Serum Replacement (KSR) or 15% fetal bovine serum (FBS). They propagated rapidly and needed to replace fresh medium every day and to subculture at 2-day intervals, whereas did not maintain the efficient proliferation and survive supplemented wth 15% newborn calf serum (NBS). The clonies of ES cells were flatter on the mitomycin C-inhibited STO fibroblasts, not easily differentiated from the feeder layer cells surround them, than that on the MEF in DMEM supplemented 15%FBS.5. Spontaneous differentiation of ES cell D3 line in vitroES cell D3 line via suspension culture can congregate small mass within 24 h, and large numbers of aggregates called embryoid bodies (EBs) gradually formed after 2 to 3 days of culture. During the 6th to the 8th days o更多还原