【摘要】：背景 心肌梗死發(fā)生后，由于心臟的再生能力極其有限，靠心肌自身的修復機制遠遠不能彌補因缺血損傷造成的心肌細胞的損失，殘存心肌的代償性肥大以及梗死心肌的纖維化使許多患者最終不可避免的發(fā)展成為心力衰竭。藥物治療和冠狀動脈血管成形術的飛速發(fā)展雖然有效降低了心肌梗死病人的早期死亡率，，改善了患者的生活質量，卻不能從根本上解決心肌細胞損失的問題，因此很多病人的預后并不理想。心臟移植作為一種終末治療手段，也受到免疫排斥、供體不足、高死亡率、費用昂貴等諸多因素的限制。 干細胞是一類具有多向分化潛能和自我復制能力的原始的未分化細胞，是形成哺乳類動物各組織器官的原始細胞，它的出現為心肌損傷的治療帶來了新的希望。目前干細胞心肌再生研究已經成為探討心肌損傷治療策略的熱點。研究表明，干細胞可以通過多種方式發(fā)揮修復損傷心肌的治療作用。對多種干細胞的基礎、臨床前及臨床研究已經證實了干細胞治療對心臟損傷的安全性和有效性，其中部分已經完成了Ⅲ期臨床試驗。 但干細胞治療仍然有許多問題未解決，如機制還不十分明確，細胞在體內的存留和植入效率極低，最佳細胞類型、最佳細胞劑量、最佳輸注途徑及最佳輸注時機等仍不清楚，而最直接、首先需要解決的問題則是選擇合適的細胞類型，以最佳途徑輸送到目標區(qū)域，由于各個研究的實驗設計和操作技術存在很大差別，使不同種類細胞之間的療效的比較十分困難，因此，我們設計了這個實驗，對骨髓單個核細胞(Bone marrow mononuclear cells，BMMNCs)和間充質干細胞(Mesenchymal stemcells，MSCs)對急性心肌梗死的療效進行頭對頭的比較。 目的 1、探索經皮心內膜下心肌注射技術治療大動物急性心肌梗死方法； 2、比較心肌內BMMNCs和MSCs的移植，對大動物急性心肌梗死的療效； 3、進一步觀察BMMNCs和MSCs在治療大動物急性心肌梗死中可能的機制。 方法 1、實驗用小型豬髂后上嵴穿刺抽取骨髓，密度梯度離心法分離單個核細胞，再體外培養(yǎng)間充質干細胞，應用流式細胞術和多向分化能力對間充質干細胞進行鑒定，應用CM-DiI及GFP分別對骨髓單個核細胞及間充質干細胞進行標記； 2、30只實驗用小型豬隨機分為3組，每組10只。通過臨床心導管技術，應用球囊封堵小型豬左冠狀動脈前降支建立小型豬急性心肌梗死模型； 3、制備模型2周后，將標記過的骨髓單個核細胞、間充質干細胞及培養(yǎng)液通過心肌注射器經導管進行心內膜下心肌內注射； 4、通過經胸超聲心動圖及SPECT對小型豬細胞治療后的心臟功能及組織灌注進行觀察，隨訪8周； 5、心肌梗死10周后處死動物，取組織熒光顯微鏡下觀察細胞的植入，免疫熒光染色檢測植入細胞的分化； 6、 CD31免疫熒光染色觀察梗死心肌的新生血管； 7、天狼星紅染色并偏振光顯微鏡下觀察心肌纖維化及膠原形成情況； 8、 Western-Blot檢測梗死心肌內的MMP表達水平。 結果 1、成功從小型豬骨髓中分離培養(yǎng)出了豬骨髓單個核細胞及間充質干細胞，并進行了鑒定及熒光標記； 2、共計30只小型豬接受模型制備，制備過程中死亡8只，其余22只均存活至實驗結束，并接受了細胞或培養(yǎng)液注射及超聲心動圖和SPECT檢查；其中MSCs組8只，其余兩組每組7只； 3、10周時，經胸超聲心動圖結果顯示，三組之間的左室射血分數（LVEF）、左室收縮末容積(LVESV)、左室舒張末容積(LVEDV)均無明顯差異(LVEF：MSCs組51±8.3%，BMMNCs組50±6.2%，CON組48±7.3%，p0.05)，與細胞注射前相比，MSCs組及BMMNCs組的LVEF雖然有所增加，但差異不具有顯著性(p0.05)。心梗10周時，MSCs組的室壁運動分數(WMSI)較2周時明顯改善(1.96±0.24vs2.35±0.21，p0.05)，而BMMNCs組及CON組則無明顯改善。10周時三組之間的WMSI無明顯差異(p0.05)。心梗10周時，僅MSCs組的室壁增厚率(WT%)較2周時明顯改善(27±4.4%vs18±3.7%，p0.05)，且10周時MSCs組的WT%明顯優(yōu)于BMMNCs組及CON組(27±4.4%vs20±2.1%，18±2.9%，p0.05)； 4、10周時，SPECT結果顯示，細胞移植8周即心肌梗死10周后，CON組的灌注缺損沒有明顯改變，MSCs及BMMNCs治療均顯著減小了梗死心臟的灌注缺損(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周時MSCs及BMMNCs治療組的灌注缺損顯著小于CON組（21.7±3.0%，22.9±3.7%vs30.0±5.8%，p0.05)。MSCs組及BMMNCs組治療后灌注缺損的絕對改變也顯著優(yōu)于CON組（-8.0±2.5%，-6.5±1.9%vs.-2.0±1.3%，p0.05)； 5、組織CD31免疫熒光染色結果顯示，10周時， MSCs組及BMMNCs組心臟梗死周邊組織的新生血管密度明顯高于CON組（16.2±4.1%，14.0±3.4%vs.7.8±2.8%，p0.05）； 6、組織天狼星紅染色及偏振光顯微鏡檢查結果顯示，MSCs組的缺血區(qū)組織內膠原含量明顯低于BMMNCs組和CON組（55.4±6.5%vs63.6±7.8%，67.4±7.2%，p0.05）； 7、 Western-Blot檢測結果顯示，MSCs組梗死周圍組織內的MMP-2表達水平明顯低于BMMNCs組和CON組(p0.05)，三組的MMP-9表達水平無明顯差異。 結論 1、本實驗建立了經皮心內膜下心肌內注射干細胞，治療大動物心肌梗死的方法，確認了其可行性； 2、發(fā)現BMMNCs和MSCs均可改善心肌灌注，相比BMMNCs，MSCs可提高左室局部收縮功能； 3、 MSCs對心肌損傷的修復作用優(yōu)于BMMNCs，治療效果的差異可能與MSC抑制梗死后心肌的纖維化有關。
[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.
【學位授予單位】：第四軍醫(yī)大學
【學位級別】：博士
【學位授予年份】：2013
【分類號】：R542.22

【共引文獻】

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