心内膜下移植骨髓单个核细胞和间充质干细胞对猪急性心肌梗死疗效的实验研究

发布时间：2018-08-17 15:07

【摘要】：背景心肌梗死发生后，由于心脏的再生能力极其有限，靠心肌自身的修复机制远远不能弥补因缺血损伤造成的心肌细胞的损失，残存心肌的代偿性肥大以及梗死心肌的纤维化使许多患者最终不可避免的发展成为心力衰竭。药物治疗和冠状动脉血管成形术的飞速发展虽然有效降低了心肌梗死病人的早期死亡率，，改善了患者的生活质量，却不能从根本上解决心肌细胞损失的问题，因此很多病人的预后并不理想。心脏移植作为一种终末治疗手段，也受到免疫排斥、供体不足、高死亡率、费用昂贵等诸多因素的限制。干细胞是一类具有多向分化潜能和自我复制能力的原始的未分化细胞，是形成哺乳类动物各组织器官的原始细胞，它的出现为心肌损伤的治疗带来了新的希望。目前干细胞心肌再生研究已经成为探讨心肌损伤治疗策略的热点。研究表明，干细胞可以通过多种方式发挥修复损伤心肌的治疗作用。对多种干细胞的基础、临床前及临床研究已经证实了干细胞治疗对心脏损伤的安全性和有效性，其中部分已经完成了Ⅲ期临床试验。但干细胞治疗仍然有许多问题未解决，如机制还不十分明确，细胞在体内的存留和植入效率极低，最佳细胞类型、最佳细胞剂量、最佳输注途径及最佳输注时机等仍不清楚，而最直接、首先需要解决的问题则是选择合适的细胞类型，以最佳途径输送到目标区域，由于各个研究的实验设计和操作技术存在很大差别，使不同种类细胞之间的疗效的比较十分困难，因此，我们设计了这个实验，对骨髓单个核细胞(Bone marrow mononuclear cells，BMMNCs)和间充质干细胞(Mesenchymal stemcells，MSCs)对急性心肌梗死的疗效进行头对头的比较。目的 1、探索经皮心内膜下心肌注射技术治疗大动物急性心肌梗死方法； 2、比较心肌内BMMNCs和MSCs的移植，对大动物急性心肌梗死的疗效； 3、进一步观察BMMNCs和MSCs在治疗大动物急性心肌梗死中可能的机制。方法 1、实验用小型猪髂后上嵴穿刺抽取骨髓，密度梯度离心法分离单个核细胞，再体外培养间充质干细胞，应用流式细胞术和多向分化能力对间充质干细胞进行鉴定，应用CM-DiI及GFP分别对骨髓单个核细胞及间充质干细胞进行标记； 2、30只实验用小型猪随机分为3组，每组10只。通过临床心导管技术，应用球囊封堵小型猪左冠状动脉前降支建立小型猪急性心肌梗死模型； 3、制备模型2周后，将标记过的骨髓单个核细胞、间充质干细胞及培养液通过心肌注射器经导管进行心内膜下心肌内注射； 4、通过经胸超声心动图及SPECT对小型猪细胞治疗后的心脏功能及组织灌注进行观察，随访8周； 5、心肌梗死10周后处死动物，取组织荧光显微镜下观察细胞的植入，免疫荧光染色检测植入细胞的分化； 6、 CD31免疫荧光染色观察梗死心肌的新生血管； 7、天狼星红染色并偏振光显微镜下观察心肌纤维化及胶原形成情况； 8、 Western-Blot检测梗死心肌内的MMP表达水平。结果 1、成功从小型猪骨髓中分离培养出了猪骨髓单个核细胞及间充质干细胞，并进行了鉴定及荧光标记； 2、共计30只小型猪接受模型制备，制备过程中死亡8只，其余22只均存活至实验结束，并接受了细胞或培养液注射及超声心动图和SPECT检查；其中MSCs组8只，其余两组每组7只； 3、10周时，经胸超声心动图结果显示，三组之间的左室射血分数（LVEF）、左室收缩末容积(LVESV)、左室舒张末容积(LVEDV)均无明显差异(LVEF：MSCs组51±8.3%，BMMNCs组50±6.2%，CON组48±7.3%，p0.05)，与细胞注射前相比，MSCs组及BMMNCs组的LVEF虽然有所增加，但差异不具有显著性(p0.05)。心梗10周时，MSCs组的室壁运动分数(WMSI)较2周时明显改善(1.96±0.24vs2.35±0.21，p0.05)，而BMMNCs组及CON组则无明显改善。10周时三组之间的WMSI无明显差异(p0.05)。心梗10周时，仅MSCs组的室壁增厚率(WT%)较2周时明显改善(27±4.4%vs18±3.7%，p0.05)，且10周时MSCs组的WT%明显优于BMMNCs组及CON组(27±4.4%vs20±2.1%，18±2.9%，p0.05)； 4、10周时，SPECT结果显示，细胞移植8周即心肌梗死10周后，CON组的灌注缺损没有明显改变，MSCs及BMMNCs治疗均显著减小了梗死心脏的灌注缺损(MSCs：21.7±3.0%vs29.6±5.1%，p0.05；BMMNCs：22.9±3.7%vs29.4±5.6%，p0.05)。且心梗10周时MSCs及BMMNCs治疗组的灌注缺损显著小于CON组（21.7±3.0%，22.9±3.7%vs30.0±5.8%，p0.05)。MSCs组及BMMNCs组治疗后灌注缺损的绝对改变也显著优于CON组（-8.0±2.5%，-6.5±1.9%vs.-2.0±1.3%，p0.05)； 5、组织CD31免疫荧光染色结果显示，10周时， MSCs组及BMMNCs组心脏梗死周边组织的新生血管密度明显高于CON组（16.2±4.1%，14.0±3.4%vs.7.8±2.8%，p0.05）； 6、组织天狼星红染色及偏振光显微镜检查结果显示，MSCs组的缺血区组织内胶原含量明显低于BMMNCs组和CON组（55.4±6.5%vs63.6±7.8%，67.4±7.2%，p0.05）； 7、 Western-Blot检测结果显示，MSCs组梗死周围组织内的MMP-2表达水平明显低于BMMNCs组和CON组(p0.05)，三组的MMP-9表达水平无明显差异。结论 1、本实验建立了经皮心内膜下心肌内注射干细胞，治疗大动物心肌梗死的方法，确认了其可行性； 2、发现BMMNCs和MSCs均可改善心肌灌注，相比BMMNCs，MSCs可提高左室局部收缩功能； 3、 MSCs对心肌损伤的修复作用优于BMMNCs，治疗效果的差异可能与MSC抑制梗死后心肌的纤维化有关。
[Abstract]:background
After myocardial infarction, due to the extremely limited regeneration of the heart, the repair mechanism of the myocardium itself can not compensate for the loss of myocardial cells caused by ischemic injury. The compensatory hypertrophy of the remaining myocardium and the fibrosis of the infarcted myocardium make many patients eventually inevitably develop into heart failure. Although the rapid development of angioplasty can effectively reduce the early mortality and improve the quality of life of patients with myocardial infarction, it can not fundamentally solve the problem of myocardial cell loss, so the prognosis of many patients is not ideal. High mortality, high cost, and many other factors.
Stem cells are a kind of primitive undifferentiated cells with multi-directional differentiation potential and self-replication ability. They are the primitive cells that form various tissues and organs of mammals. Their appearance brings new hope for the treatment of myocardial injury. Stem cells can be used to repair damaged myocardium in many ways. Pre-clinical and clinical studies have confirmed the safety and effectiveness of stem cell therapy for heart injury, and some of them have completed phase III clinical trials.
However, there are still many unsolved problems in stem cell therapy, such as the mechanism is not very clear, the cell survival and implantation efficiency in vivo is very low, the best cell type, the best cell dose, the best infusion route and the best infusion timing are still unclear, and the most direct, the first problem to be solved is to select the appropriate cell type, in order to optimize. It is difficult to compare the efficacy of different types of cells because of the great differences in experimental design and operational techniques. Therefore, we designed this experiment for bone marrow mononuclear cells (BMMNCs) and mesenchymal stem cells (MSCs). Head to head comparison was performed on the efficacy of acute myocardial infarction.
objective
1, explore the method of percutaneous endocardial injection in the treatment of acute myocardial infarction in large animals.
2, compare the effects of BMMNCs and MSCs transplantation on acute myocardial infarction in large animals.
3, we further observed the possible mechanisms of BMMNCs and MSCs in the treatment of acute myocardial infarction in large animals.
Method
1 * the bone marrow was extracted from the posterior superior iliac crest of a miniature pig. The mononuclear cells were isolated by density gradient centrifugation. Then mesenchymal stem cells were cultured in vitro. The mesenchymal stem cells were identified by flow cytometry and multidirectional differentiation, and bone marrow mononuclear cells and mesenchymal stem cells were labeled by CM-DiI and GFP respectively.
2,30 * miniature pigs were randomly divided into 3 groups, 10 in each group. A mini pig model of acute myocardial infarction was established by means of * * catheterization.
3. Two weeks after the establishment of the model, the labeled bone marrow mononuclear cells, mesenchymal stem cells and culture medium were injected into the subendocardial myocardium via a catheter through a myocardial syringe.
4, cardiac function and tissue perfusion were observed after transthoracic echocardiography and SPECT * for 8 weeks.
5. The animals were sacrificed 10 weeks after myocardial infarction. The implanted cells were observed under tissue fluorescence microscope and the differentiation of implanted cells was detected by immunofluorescence staining.
6, CD31 immunofluorescence staining was used to observe the neovascularization of infarcted myocardium.
7, Sirius red staining and polarized light microscopy were used to observe myocardial fibrosis and collagen formation.
8, Western-Blot detected MMP expression in infarcted myocardium.
Result
1, * * the pig bone marrow mononuclear cells and mesenchymal stem cells were successfully isolated and identified from the bone marrow of small pigs.
2 * a total of 30 miniature pigs were prepared by model preparation, and 8 died during the preparation process. The remaining 22 survived to the end of the experiment, and received cell or culture injection and echocardiography and SPECT examination. Among them, 8 were in group MSCs and 7 in the other two groups.
At 3 and 10 weeks, transthoracic echocardiography showed no significant difference in LVEF, LVESV and LVEDV among the three groups (LVEF: MSCs group 51 65507 There was no significant difference (p0.05). At 10 weeks after MI, the wall motion fraction (WMSI) of MSCs group was significantly improved compared with that of 2 weeks (1.96.24 vs 2.35.21, p0.05), while there was no significant difference between BMMNCs group and CON group at 10 weeks (p0.05). At 10 weeks after MI, the wall thickening rate (WT%) of MSCs group was significantly improved compared with that of 2 weeks (27. WT% in MSCs group was significantly higher than that in BMMNCs group and CON group at 10 weeks (27 + 4.4% vs 20 + 2.1%, 18 + 2.9%, p0.05).
At 4 and 10 weeks, SPECT results showed that there was no significant change in perfusion defect in CON group after 8 weeks of cell transplantation or 10 weeks of myocardial infarction. MSCs and BMMNCs treatment significantly reduced perfusion defect in infarcted heart (MSCs: 21.7 +3.0% vs 29.6 +5.1%, p0.05; BMMNCs: 22.9 +3.7% vs 29.4 +5.6%, p0.05). The defect was significantly smaller than that in CON group (21.7 (+ 3.0%), 22.9 (+ 3.7%) vs. 30.0 (+ 5.8%) and BMMNCs group (p0.05). The absolute change of perfusion defect in MSCs group and BMMNCs group was also significantly better than that in CON group (- 8.0 (+ 2.5%), - 6.5 (+ 1.9%) vs. - 2.0 (+ 1.3%), p0.05).
5. CD31 immunofluorescence staining showed that the density of neovascularization in the periphery of myocardial infarction in MSCs group and BMMNCs group was significantly higher than that in CON group at 10 weeks (16.2 + 4.1%, 14.0 + 3.4% vs. 7.8 + 2.8%, p0.05).
6. The results of Sirius red staining and polarized light microscopy showed that the content of collagen in ischemic tissue of MSCs group was significantly lower than that of BMMNCs group and CON group (55.4 (+ 6.5%) vs 63.6 (+ 7.8%), 67.4 (+ 7.2%), P 0.05).
7. Western-Blot test results showed that the expression of MMP-2 in the periinfarct tissues of MSCs group was significantly lower than that of BMMNCs group and CON group (p0.05). There was no significant difference in the expression of MMP-9 among the three groups.
conclusion
1. The method of percutaneous subendocardial injection of stem cells for the treatment of myocardial infarction in large animals was established, and its feasibility was confirmed.
2, it was found that both BMMNCs and MSCs could improve myocardial perfusion. Compared with BMMNCs, MSCs could improve left ventricular regional systolic function.
3. MSCs are superior to BMMNCs in repairing myocardial injury. The difference of therapeutic effect may be related to the inhibition of myocardial fibrosis by MSCs after infarction.
【学位授予单位】：第四军医大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R542.22

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