本文選題：間充質干細胞 + Netrin-1��； 參考：《南京醫(yī)科大學》2009年博士論文

【摘要】：背景 近年研究發(fā)現造血干細胞移植可以治療缺血性血管疾病,促進缺血部位的血管新生和側枝循環(huán)形成,使缺血的組織血供得到改善,這種新的治療方法成為近年研究的熱點之一。 間充質干細胞(MSCs)是一類具有自我增殖和分化潛能的多能干細胞,是骨、軟骨、血管、肌肉及結締組織的前體細胞。由于其易于取材和擴增,且移植后在機體保持良好的分化能力,是目前最理想的種子細胞。但是目前MSC移植治療缺血性疾病也有其缺點,如形成的血管數量有限,移植細胞的存活率低,需移植細胞較多等等,因此,如何提高MSCs的移植效率是當今研究MSCs的焦點。 近來的研究發(fā)現,神經和血管具有相似性,例如它們循著相同的遷移路線,生存過程中相互依賴等等,因此它們之間可能存在著可以進行分子“交談(cross talk)”的共同信號,研究發(fā)現神經導向因子就扮演如此的角色,它在神經和血管生長過程中均起調節(jié)作用。 大量研究已證明作為神經導向因子之一的Netrin-1其在神經生長過程中起重要作用,近來還發(fā)現其在血管新生方面起著重要作用,它在一定的濃度范圍內可以促進血管新生。那么,它和MSC共同移植到機體是否提高MSC的移植效率?即對缺血機體來說其能否促進血管的新生?對缺血組織的功能是否改善?這為缺血性疾病尤其伴有神經功能病變的缺血疾病的治療提供新的思路。 目的 分離大鼠骨髓間充質干細胞,并體外培養(yǎng)、擴增,應用Netrin-1蛋白和間充質干細胞聯合移植,觀察其對局部缺血肢體的血管恢復情況,并對其機制進行探討,便于進一步應用于臨床,給肢體血管受損疾病的治療帶來新的希望。 方法 在體內研究中,先對24只大鼠給予下肢股動脈結扎造成肢體缺血模型后,隨機分為4組(各組6只),對照組、Netrin-1組、MSCs組、MSCs聯合Netrin-1組。將分離培養(yǎng)的間充質干細胞和(或)Netrin-1局部注射到缺血組織中。在治療后7d,14d及28d時觀察機體功能變化,同時應用Western blot和ELISA方法檢測血或組織中的VEGF水平,最后在治療28d時,給予數字減影血管造影(DSA)顯示側枝動脈的形成情況、免疫組化檢測毛細血管和小動脈的密度。在體外研究中,培養(yǎng)人臍靜脈內皮細胞(HUVEC),觀察Netrin-1對MSC參與小管形成能力的作用,同時觀察Netrin-1對MSC遷移能力的影響,進一步證實Netrin-1對MSC在血管新生中的作用。 結果 1、在體內研究過程中,①應用Netrin-1治療、MSC移植或兩者聯合治療時,機體在治療后7d及14d時局部組織及血VEGF水平明顯增高,在移植后28d時VEGF水平有所下降,但仍比治療前及對照組高,差異具有統(tǒng)計學意義(P0.05);與單純Netrin-1治療或MSC治療組比較,Netrin-1聯合MSC治療組VEGF濃度進一步增加,具有統(tǒng)計學意義(P0.05)。②與空白組比較,Netrin-1治療、MSC移植治療或兩者聯合治療均能明顯改善肢體功能、增加缺血區(qū)毛細血管密度及肢體側枝循環(huán),改善缺血狀況,這與機體分泌VEGF水平增多有關;當應用Netrin-1聯合MSC治療時大鼠肢體功能改善更加明顯,側枝循環(huán)的建立和缺血區(qū)毛細血管密度增加更明顯,血管新生更明顯,這與VEGF水平進一步增多相關。 2、在體外研究過程中,①應用transwell遷移技術分析體外培養(yǎng)的MSC對Netrin-1(50ng/ml)的應答,結果提示Netrin-1對MSC的細胞遷移數量為69.1士5.63/HPF,VEGF對MSC的細胞遷移數量為(67.5士3.88/HPF),明顯高于空白對照組細胞遷移數量(41.0士3.57/HPF),具有統(tǒng)計學意義(P0.05);但Netrin-1加VEGF對MSC的細胞遷移數量為115.5士13.00/HPF,明顯高于Netrin-1組、VEGF組及空白對照組,具有顯著的統(tǒng)計學意義(P0.05)。Netrin-1加VEGF比兩種因子單獨應用更易促進MSC的遷移。②定量分析小管形成能力的研究結果提示,Netrin-1組小管形成數量為41.75士2.83/3HPF,VEGF組小管形成數量為48.25士3.42/3HPF,這兩組明顯高于對照組(11.75士1.35/3HPF),具有統(tǒng)計學意義(P0.05);但是Netrin-1加VEGF組小管形成數量為(73.75士3.42/3HPF),明顯高于空白對照組、Netrin-1組及VEGF組,具有統(tǒng)計學意義(P0.05);在培養(yǎng)MSC時加入50 ng/ml Netrin-1與10 ng/ml VEGF比兩種因子單獨應用更易促進HUVEC的小管形成。 結論 MSC移植及Netrin-1局部治療缺血肢體均可明顯改善缺血肢體的功能情況,促進局部的血管新生,改善機體的血流狀況;但MSC聯合Netrin-1治療肢體缺血性動物模型,肢體的功能改善更加明顯,側肢循環(huán)形成更多,更易促進局部血管新生。
[Abstract]:background
In recent years, it has been found that hematopoietic stem cell transplantation can treat ischemic vascular disease, promote angiogenesis and collateral circulation in ischemic parts, and improve the blood supply of ischemic tissue. This new treatment has become one of the hot topics in recent years.
Mesenchymal stem cells (MSCs) are a kind of pluripotent stem cells with the potential of self proliferation and differentiation. It is the precursor cells of bone, cartilage, blood vessels, muscles and connective tissue. It is the most likely seed cell because of its easy selection and expansion and good differentiation ability after transplantation. But MSC transplantation is currently used to treat ischemic disease. It also has its shortcomings, such as the limited number of blood vessels, low survival rate of transplanted cells, more transplant cells, and so on. Therefore, how to improve the efficiency of MSCs transplantation is the focus of the study of MSCs.
Recent studies have found that nerves and blood vessels have similarities, such as they follow the same route of migration, interdependence in the process of survival, and so on, so there may be a common signal that can carry on a molecular "cross talk". In the long process, all of them play the role of regulation.
A large number of studies have shown that Netrin-1, one of the nerve leading factors, plays an important role in the process of nerve growth, and recently it has been found to play an important role in angiogenesis. It can promote angiogenesis in a certain concentration range. Then, it and MSC are transplanted to the body to improve the efficiency of MSC transplantation? That is, ischemia. Can the body promote angiogenesis and improve the function of ischemic tissue? This provides a new way of thinking for the treatment of ischemic diseases, especially with neuropathy.
objective
The rat bone marrow mesenchymal stem cells were isolated and cultured in vitro. The Netrin-1 protein and mesenchymal stem cells were transplanted together to observe the vascular recovery of the local ischemic limbs, and the mechanism was discussed so as to be further applied to the clinic and bring new hope for the treatment of limb vascular damaged diseases.
Method
In the study in vivo, 24 rats were divided into 4 groups (6 rats in each group) after ligation of the femoral artery in the lower extremities. The control group, group Netrin-1, group MSCs, MSCs combined with Netrin-1 group. The isolated mesenchymal stem cells and / or Netrin-1 were injected into the ischemic tissue locally. The body work was observed at 7d, 14d and 28d after treatment. At the same time, the Western blot and ELISA methods were used to detect the VEGF level in the blood or tissue. At the end of the treatment of 28d, digital subtraction angiography (DSA) was given to show the formation of the collateral artery and the density of the capillary and the small arteries by immunohistochemistry. In the study, human umbilical vein endothelial cells (HUVEC) were cultured, and Netrin-1 was observed. The role of MSC in the formation of tubules was observed, and the effect of Netrin-1 on MSC migration ability was also observed. The role of Netrin-1 in the angiogenesis of MSC was further confirmed.
Result
1, during the study in vivo, the level of local tissue and blood VEGF increased significantly at 7d and 14d after treatment with Netrin-1 therapy, MSC transplantation or combined treatment. The level of VEGF decreased at 28d after transplantation, but still higher than before and in the control group. The difference was statistically significant (P0.05), with the simple Netrin-1 treatment or MSC treatment. Compared with the treatment group, the VEGF concentration in the Netrin-1 combined with the MSC treatment group was further increased and had statistical significance (P0.05). (2) compared with the blank group, Netrin-1 treatment, MSC transplantation therapy or both combined treatment could obviously improve the limb function, increase the capillary density and limb collateral circulation in the ischemic area, improve the ischemic condition, and secrete the VEGF water with the body. The improvement of limb function in rats with Netrin-1 combined with MSC was more obvious, the establishment of the collateral circulation and the increase of capillary density in the ischemic area were more obvious, and the angiogenesis was more obvious, which was related to the further increase of VEGF level.
2, in the process of in vitro study, (1) the Transwell migration technique was used to analyze the response of MSC to Netrin-1 (50ng/ml) in vitro. The results suggested that the number of Netrin-1 cells migrated to MSC, and the number of VEGF to MSC was (67.5 M. 3.88/HPF), which was significantly higher than the number of cell migration (41 3.57/HPF) in the blank control group. Statistical significance (P0.05), but the number of cells migrated to MSC by Netrin-1 plus VEGF was 115.5 m 13.00/HPF, obviously higher than that in group Netrin-1, VEGF group and blank control group, with significant statistical significance (P0.05).Netrin-1 plus VEGF compared with two factors more easily promoted MSC migration. 2. Quantitative analysis of tubule formation ability The number of tubules in group Netrin-1 was 41.75 2.83/3HPF, and the number of VEGF tubules was 48.25 3.42/3HPF. The two groups were significantly higher than those of the control group (11.75 1.35/3HPF), with statistical significance (P0.05), but the number of tubules in Netrin-1 plus VEGF group was (73.75 3.42/ 3HPF), obviously higher than that in the blank control group, Netrin-1 group and VEGF group. P0.05, MSC 50 VEGF Netrin-1 and 10 ng/ml VEGF than two factors alone promoted HUVEC formation.
conclusion
MSC transplantation and Netrin-1 local treatment of ischemic limbs can obviously improve the function of ischemic limbs, promote the local angiogenesis and improve the blood flow status of the body, but MSC combined with Netrin-1 in the treatment of limb ischemic animal model, the function of limb improvement is more obvious, the side limb ring formation is more, and it is easier to promote local angiogenesis.

【學位授予單位】：南京醫(yī)科大學
【學位級別】：博士
【學位授予年份】：2009
【分類號】：R329

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