去分化法提高骨髓间充质干细胞心脏修复潜能的研究

Ischemic heart disease complicating myocardial infarction is a common clinical condition for which effective treatment is still lacking. Recent studies showed that bone marrow mesenchymal stem cells (MSCs) have a role in cardiac repair but are limited to improve cardiac function. Our previous study showed that basic fibroblast growth factor (bFGF) can promote homing and differentiation of MSCs when delivered by coronary venous retroperfusion, but the abilities of survival and differentiation of the transplanted cells remained poor. In previous studies, some cells from humans and other mammals have dedifferentiation potential, which can revert from terminally differentiated cells back to an earlier, more primitive phenotype, and in turn exert the capacity of tissue repair and regeneration. Recent study showed that MSCs-derived neurons are able to revert back to dedifferentiated MSCs (De-MSCs), which have enhanced survival and dedifferentiation potential. Thus, we hypothesized that De-MSCs would have improved survival and higher differentiation capacity by inducing dedifferentiation of MSCs-derived cardiomyocyte-like cells. In this project, we first plan to explore and evaluate different conditions to establish an ideal dedifferentiation system of MSCs-derived cardiomyocyte-like cells. The survival ability and cardiomyocyte differentiation potential of De-MSCs will be evaluated. In addition, the survival and differentiation capacity of De-MSCs as well as its effect on angiogenesis, cell apoptosis, cardiac function will also be investigated. It is expected that the dedifferentiation method might provide a more effective and convenient cell source for cardiac repair.

缺血性心脏病所致的心肌梗死仍缺乏有效治疗方法。将骨髓间充质干细胞（MSCs）导入受损心肌有一定的心脏修复作用，但心脏功能改善不明显。我们前期经冠状静脉逆行灌注bFGF能促进MSCs归巢与分化，但移植细胞的存活与分化率仍较低。研究发现，人和哺乳动物的部分细胞存在去分化潜能，即从高度分化状态逆转为早期的原始状态，并参与组织修复和再生。新近发现，采用神经细胞诱导剂使MSCs转化为更原始的去分化MSCs（De-MSCs），能提高其存活能力与定向分化潜能。据此我们推测，通过特定条件将MSCs诱导为能向心肌细胞分化的De-MSCs后，能提高其存活和心肌分化能力。我们拟摸索并评价不同诱导条件，建立MSCs去分化诱导体系。在体外评价De-MSCs的存活能力与心肌细胞分化潜能；观察心肌内注射De-MSCs的存活与分化能力以及对血管再生、细胞凋亡和心脏功能的影响。有望为心脏细胞治疗寻找更为高效便捷的细胞来源。

研究背景.骨髓间充质干细胞（bone marrow mesenchymal stem cells, BMMSCs）作为心肌细胞再生治疗研究的重要细胞来源，但目前由BMMSCs来源的心肌细胞诱导效率低下，并不能满足目前科研与临床工作的需要。.研究内容.本研究采用密度梯度离心法获得BMMSCs，进行培养传代，并加入诱导因子，采用T3处理由BMMSCs诱导的心肌样细胞。应用采用免疫荧光和流式细胞法，选择心肌细胞标志基因蛋白cTnI、GATA4、Cx-43和α-actinin作为检测目标，观察BMMSCs向心肌细胞分化的效率和成熟度。采用实时定量逆转录聚合酶链式反应（realtime RT-PCR）检测和Western blot 检测cTnI、GATA4、Cx-43、α-actinin的mRNA和蛋白表达水平。 .应用大鼠心肌梗死模型，通过超声心动图监测心功能，应用Lambeth Conventions评价方法进行心律失常评分，心肌组织HE染色、Masson染色及心肌标志物免疫荧光染色等方法，观察BMMSCs对心肌修复潜能的影响。.结果.细胞免疫荧光：cTnI、GATA4、Cx-43及α-actinin在对照组表达均为阴性，在标准诱导组表达阳性，在T3诱导组表达进一步升高。流式细胞术：cTnI 、Cx-43阳性细胞在B组和C组与A组相比较明显升高；C组表达阳性率与B组相比较明显升高（p<0.05）。cTnI和Cx-43蛋白在A组中呈阳性表达，B组与A组相比较表达明显升高（P<0.01），C组与B组相比较表达进一步升高（P<0.01）。Masson染色显示，移植28天后心肌梗死面积在D组明显小于A、B、C组，C组明显小于B组，B组与A组无明显差别。B组、C组、D组LVEF较A组明显上升，C组和D组优于B组，D组优于C组。心律失常评分：B组和C组与A组相比较，未见明显差异；D组与A组相比较，心律失常评分明显降低（p<0.05）。HE染色： D组心肌梗死边缘区聚集的炎症细胞明显减少。心肌组织免疫荧光：对各组心肌组织进行共显影显示，可见部分细胞呈EGFP与TnI、α-actin、VIII因子和Cx-43共显影，而D组的共显影细胞率较A、B和C组明显增多。.结论.BMMSCs向心肌样细胞分化率增加、成熟度更高；经过处理的BMMSCs来源的心肌细胞保留一定增殖潜能，为实验研究及临床应用