新生小鼠心肌干细胞与心肌细胞共培养诱导分化研究
发布时间：2019-06-20 03:19
【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P0.05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。
[Abstract]:Purpose: 1. To understand the presence or absence of myocardial conditioned medium in the differentiation of cardiac muscle cells the effect of CSCs and the contact of the cardiac muscle cells to the CSC s differentiation Methods:1.1-2 day mouse heart tissue was cut into 0.5-1 mm3 fragment, red blood cells were washed with DPBS, and after the tissue mass was slightly digested, the wall was cultured for 11-14 days, and the pancreatic enzymes were used. the differential digestion is carried out and the primary CSCs are collected;2. the primary CSCs are used The primary cells were sorted by beads to obtain c-Kit + CSCs.3. Flow cytometry was used to analyze the surface of unsorted primary cells. CD45,CD34,Sca-1,c-Kit鏍，
本文编号：2502876
[Abstract]:Purpose: 1. To understand the presence or absence of myocardial conditioned medium in the differentiation of cardiac muscle cells the effect of CSCs and the contact of the cardiac muscle cells to the CSC s differentiation Methods:1.1-2 day mouse heart tissue was cut into 0.5-1 mm3 fragment, red blood cells were washed with DPBS, and after the tissue mass was slightly digested, the wall was cultured for 11-14 days, and the pancreatic enzymes were used. the differential digestion is carried out and the primary CSCs are collected;2. the primary CSCs are used The primary cells were sorted by beads to obtain c-Kit + CSCs.3. Flow cytometry was used to analyze the surface of unsorted primary cells. CD45,CD34,Sca-1,c-Kit鏍，
本文编号：2502876
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