CN116083352A - 间充质干细胞体外定向分化为心肌细胞的方法 - Google Patents

间充质干细胞体外定向分化为心肌细胞的方法 Download PDF

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CN116083352A
CN116083352A CN202310300019.7A CN202310300019A CN116083352A CN 116083352 A CN116083352 A CN 116083352A CN 202310300019 A CN202310300019 A CN 202310300019A CN 116083352 A CN116083352 A CN 116083352A
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姚翰博
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Abstract

本发明属于细胞培养领域，具体涉及间充质干细胞体外定向分化为心肌细胞的方法，所述方法包括：(a)采用含有低浓度丁酸钠的DMEM/F12在间歇性低压低氧的状态下诱导培养间充质干细胞；(b)继续培养所述间充质干细胞。试验显示，采用含有低浓度丁酸钠的DMEM/F12在间歇性低压低氧的状态下诱导间充质干细胞24h，细胞Cx43、cTnI及cTnT阳性表达率和蛋白表达量均明显增高。

Description

间充质干细胞体外定向分化为心肌细胞的方法
技术领域
本发明属于干细胞培养技术领域，涉及间充质干细胞体外定向分化为心肌细胞的方法。
背景技术
骨髓间充质干细胞(bone marrow mesenchymal stem cells，BMSCs)在体内特定微环境或体外特定培养条件下，可以分化为心肌样细胞，可用于心肌梗死后的细胞移植治疗，为受损心脏的细胞重建及衰竭心脏的功能恢复提供一种全新的治疗方法。研究表明，通过加入化合物、细胞因子、基因修饰、微环境的条件和物理因素的刺激等都能促进BMSCs向心肌细胞(cardiac myocytes，CMs)分化。
研究表明，间歇性低压低氧可增强心脏对缺血、缺氧的耐受性，减轻缺血/再灌注对心肌的损伤，促进缺血/再灌注后心脏舒缩功能的恢复，具有明显的心脏保护作用。如张翼等人研究发现，给予成年大鼠28天相当于海拔5000米的间歇性低压低氧处理(每天6小时)，动物表现出明显抗缺血/再灌注心律失常、心脏收缩功能的保护作用、心脏组织结构的保护作用和心肌抗氧化能力增强[1]。研究指出，间歇性低压低氧通过对抗缺血/再灌注所致Bcl.2减少、Bax增高，从而增加Bcl.2/Bax比值减少心肌细胞凋亡，对抗缺血/再灌注导致的心肌损伤，发挥心脏保护作用。但有关间歇性低压低氧对骨髓间充质干细胞分化为心肌细胞的影响，未见报道。
参考文献[1]:张翼，钟宁，周兆年.间歇性低氧对大鼠心肌的抗心律失常与抗氧化效应.生理学报,2000,52:89-92.
发明内容
发明人意外发现在某些化学物质的作用下，间歇性低压低氧状态下培养的骨髓间充质干细胞有定向心肌细胞分化的趋势，试验表明，在特定化学物质诱导下的骨髓间充质干细胞更早地出现心肌样细胞的变化。
本发明的目的是提供一种间充质干细胞体外定向分化为心肌细胞的方法，所述方法包括：
(a)在间歇性低压低氧的状态下诱导培养间充质干细胞；
(b)继续培养所述间充质干细胞。
进一步地，所述低压是102～110mmHg。之所以这样限定，是因为在其他范围值的压力下，即使环境中的氧气浓度是合适的，也无法达到令人满意的诱导效果。在氧气浓度限定方面亦是如此，压力的大小和氧气浓度的良好配合是存在一定的范围的。
进一步地，所述低氧是氧气浓度为1～5％。只有在氧气浓度较低的情况下，在低压的条件下才具有一定的诱导效果，这些结论在前期试验中探索得出，本发明中并未列明。
进一步地，步骤(a)诱导培养24～36h。
进一步地，步骤(a)诱导培养期间，持续施加所述低压低氧状态5～60分钟。
进一步地，步骤(a)诱导培养期间，持续施加所述低压低氧状态10～30分钟。虽然前述强调了低压低氧状态对于诱导的有益效果，但是如果整个诱导过程均处于低压低氧状态下对于心肌细胞的增殖和分化反而是不利的。经过多次试验研究发现，将这种低压低氧的状态持续特定时间将会使效果得到明显的加成。
试验结果显示，相比丁酸钠单独诱导组和间歇性低压低氧单独诱导组，联合组细胞Cx43、cTnI及cTnT阳性表达率和蛋白表达量均最高，且均显著高于其余各组，差异具有统计学意义。
进一步地，步骤(a)采用的诱导培养基是含有低浓度丁酸钠的DMEM/F12。令人意外地是，低浓度的丁酸钠与间歇性低压低氧联合诱导表现出强的诱导BM SCs分化为心肌细胞的能力，且丁酸钠浓度越低，这种协同作用越明显。这一点从本发明表3可看出，通过比较不同浓度的丁酸钠与间歇性低压低氧的联合诱导效果，结果显示，采用0.1和0.5nmol/L的丁酸钠与间歇性低压低氧的联合诱导的细胞cTnT阳性表达率最高，且与其余各组差异明显。
进一步地，所述DMEM/F12中丁酸钠的浓度为0.1～0.5nmol/L。
进一步地，所述步骤(b)中继续培养采用的培养基可以是DMEM/F12培养基，只要是能够维持细胞生长的培养基均在本发明范畴内。
本文中所述“DMEM/F12”包含无水氯化钙116.6mg/L、L-亮氨酸59.05mg/L、亚油酸0.042mg/L、五水硫酸铜0.0013mg/L、L-赖氨酸盐酸盐91.25mg/L、硫辛酸0.105mg/L、九水硝酸铁0.05mg/L、L-蛋氨酸17.24mg/L、酚红8.1mg/L、七水硫酸亚铁0.417mg/L、L-苯丙氨酸35.48mg/L、1，4-丁二胺二盐酸盐0.081mg/L、氯化钾311.8mg/L、L-丝氨酸26.25mg/L、丙酮酸钠55mg/L、氯化镁28.64mg/L、L-苏氨酸53.45mg/L、维生素H0.0035mg/L、无水硫酸镁48.84mg/L、L-丙氨酸4.45mg/L、D-泛酸钙2.24mg/L、氯化钠6999.5mg/L、L-天门冬酰胺7.5mg/L、氯化胆碱8.98mg/L、无水磷酸二氢钠54.35mg/L、L-天门冬氨酸6.65mg/L、叶酸2.65mg/L、磷酸氢二钠71.02mg/L、L-半胱氨酸盐酸盐17.56mg/L、i-肌醇12.6mg/L、七水硫酸锌0.432mg/L、L-谷氨酸7.35mg/L、烟酰胺2.02mg/L、L-精氨酸盐酸盐147.5mg/L、L-脯氨酸17.25mg/L、盐酸吡哆醛2mg/L、L-胱氨酸盐酸盐31.29mg/L、L-色氨酸9.02mg/L、盐酸吡哆醇0.031mg/L、L-谷氨酰胺365mg/L、L-酪氨酸38.4mg/L、核黄素0.219mg/L、甘氨酸18.75mg/L、L-缬氨酸52.85mg/L、盐酸硫胺2.17mg/L、L-组氨酸盐酸盐31.48mg/L、D-葡萄糖3151mg/L、胸苷0.365mg/L、L-异亮氨酸54.47mg/L、次黄嘌呤2mg/L、维生素B120.68mg/L。
进一步地，步骤(b)继续培养所述间充质干细胞2～4周。
进一步地，所述间充质干细胞为骨髓间充质干细胞。
附图说明
图1为各组免疫荧光检测结果。
注：标尺示10μm，1A、1B、1C、1D分别为联合组、丁酸钠组、间歇性低压低氧组及对照组cTnT的表达。
具体实施方式
通过下面的实施例可以对本发明进行进一步的描述，然而，本发明的范围并不限于下述实施例。本领域的专业人员能够理解，在不背离本发明的精神和范围的前提下，可以对本发明进行各种变化和修饰。
试验例一
1.材料与方法
1.1BMSCs的诱导及继续培养：取第3代经表面抗原鉴定符合的BMSCs进行诱导培养，分为4组，分别为对照组：采用DMEM/F12培养基诱导；间歇性低压低氧组：采用DMEM/F12培养基诱导培养24h，其中施加间歇性低压低氧的状态30min，氧气浓度为2％，压力为108.8mmHg；丁酸钠组：采用含有0.1nmol/L丁酸钠的DMEM/F12培养基基诱导培养；联合组：采用含有0.1nmol/L丁酸钠的DMEM/F12培养基基诱导培养24h，其中施加间歇性低压低氧的状态30min，氧气浓度为2％，压力为108.8mmHg。各组诱导培养24h后，全部换成DMEM/F12培养基在恒温培养箱中继续培养4周，每3天换液1次，每天镜下观察细胞生长状态。
1.1免疫荧光检测
收集培养4周的细胞，各组细胞采用0.25％胰蛋白酶消化，调整细胞浓度为1×107/L，接种于放入无菌盖玻片的无菌24孔板中，待细胞融合度达到80-90％时，PBS浸洗3次，4％多聚甲醛固定。固定好的细胞爬片弃去多聚甲醛，PBS浸洗后滴加适量的0.05％Triton X-100-PBS，室温通透20min，PBS浸洗滴加正常山羊血清，室温封闭30min；滤纸吸干液体，滴加一抗：Cx43、cTnI及cTnT(1:100比例配制)，4℃湿盒孵育过夜；复温30min，PBST浸洗后滴加羊抗兔IgG/FIFC(1:300比例配制)；37℃恒温箱孵育60min；PBST浸洗后吸干洗液，抗荧光衰减封片剂封片，荧光显微镜下观察，采集图像，分析结果。
1.2Western blotting检测
收集诱导4周后的细胞，弃上清，每1×106个细胞中加入100μL裂解液，震荡，4℃裂解1h；4℃，12000r/min离心10min，将上清吸入另一预冷离心管中，采用酶标仪测定每组蛋白样品中的蛋白浓度。根据各组蛋白样品中的蛋白浓度加入适量的蛋白定量上样缓冲液，混匀，沸水中煮5min使蛋白变性，冷却至室温，将蛋白样品和彩色预染蛋白标准依次加到SDS-PAGE胶上样孔内，电压设置为160V，时间为60min，溴酚蓝跑出凝胶，即可停止电泳，采用美国Bio-rad Trans-Blot Turbo全能型蛋白转印系统，7min完成转膜。封闭液孵育膜120min；孵育一抗，一抗稀释液(β-actin以1:10000的比例稀释，Cx43、cTnI及cTnT以1:1000的比例稀释)孵育膜，摇床慢摇4℃过夜；孵育一抗完成的膜用TBST在摇床上室温快摇10min，3次。配制二抗稀释液，所有二抗比例均为1:10000，摇床室温慢摇2h，孵育二抗完成的膜采用TBST在摇床上室温快摇10min，3次。上机检测，成像仪成像；进行图像分析。
2.结果
2.1免疫荧光检测结果：结果如图1和表1所示，在荧光显微镜下可观察到阳性结果细胞质呈绿色，其中，联合组Cx43、cTnI及cTnT阳性表达率最高，
均显著高于其余各组，差异具有统计学意义(P＜0.01，表1)。
表1四组细胞Cx43、cTnI及cTnT阳性率比较(
Figure BDA0004144812980000041
％)
Figure BDA0004144812980000051
与联合组相比，△P＜0.05，△△P＜0.01。
2.2Western blotting检测结果：由表2可知，以β-actin作为内参蛋白，联合组诱导4周后的细胞中Cx43、cTnI及cTnT的蛋白表达量均最高，且均显著高于其余各组，差异具有统计学意义(P＜0.01，表2)，其次是丁酸钠组。
表2四组细胞Cx43、cTnI及cTnT相对蛋白表达量
Figure BDA0004144812980000052
Figure BDA0004144812980000053
与联合组相比，△P＜0.05，△△P＜0.01。
试验例二
按照试验例一方法考察不同丁酸钠浓度与间歇性低压低氧联合对BMSCs分化为心肌细胞的影响，结果如下表3所示。
表3不同诱导天数cTnT阳性表达率比较(
Figure BDA0004144812980000054
％)
Figure BDA0004144812980000055
与0.1nmol/L同时间段相比，△P＜0.05，△△P＜0.01。
注：间歇性低压低氧参数为：氧气浓度为2％，压力为108.8mmHg，持续时间30min。
由上表可知，低浓度丁酸钠与间歇性低压低氧联合表现出强的诱导BMS Cs分化为心肌细胞的能力，且丁酸钠浓度越低，这种协同作用越明显。
上述实施例为本发明较佳的实施方式，但本发明的实施方式并不受上述实施例的限制，其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化，均应为等效的置换方式，都包含在本发明的保护范围之内。

Claims (10)

1.间充质干细胞体外定向分化为心肌细胞的方法，其特征在于，所述方法包括：
(a)在间歇性低压低氧的状态下诱导培养间充质干细胞；
(b)继续培养所述间充质干细胞。
2.根据权利要求1所述的方法，其特征在于，所述低压是102～110mmHg。
3.根据权利要求2所述的方法，其特征在于，所述低氧是氧气浓度为1～5％。
4.根据权利要求1～3任一所述的方法，其特征在于，步骤(a)诱导培养24～36h。
5.根据权利要求4所述的方法，其特征在于，步骤(a)诱导培养期间，持续施加所述低压低氧状态5～60分钟。
6.根据权利要求5所述的方法，其特征在于，步骤(a)诱导培养期间，持续施加所述低压低氧状态10～30分钟。
7.根据权利要求1～3任一所述的方法，其特征在于，步骤(a)采用的诱导培养基是含有低浓度丁酸钠的DMEM/F12。
8.根据权利要求7所述的方法，其特征在于，所述DMEM/F12中丁酸钠的浓度为0.1～0.5nmol/L。
9.根据权利要求1～3任一所述的方法，其特征在于，步骤(b)继续培养所述间充质干细胞2～4周。
10.根据权利要求1～3任一所述的方法，其特征在于，所述间充质干细胞为骨髓间充质干细胞。
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