神经导向因子Netrin-1对间充质干细胞血管形成能力的作用研究

发布时间：2018-04-22 17:23

本文选题：间充质干细胞 + Netrin-1　；参考：《南京医科大学》2009年博士论文

【摘要】：背景近年研究发现造血干细胞移植可以治疗缺血性血管疾病,促进缺血部位的血管新生和侧枝循环形成,使缺血的组织血供得到改善,这种新的治疗方法成为近年研究的热点之一。间充质干细胞(MSCs)是一类具有自我增殖和分化潜能的多能干细胞,是骨、软骨、血管、肌肉及结缔组织的前体细胞。由于其易于取材和扩增,且移植后在机体保持良好的分化能力,是目前最理想的种子细胞。但是目前MSC移植治疗缺血性疾病也有其缺点,如形成的血管数量有限,移植细胞的存活率低,需移植细胞较多等等,因此,如何提高MSCs的移植效率是当今研究MSCs的焦点。近来的研究发现,神经和血管具有相似性,例如它们循着相同的迁移路线,生存过程中相互依赖等等,因此它们之间可能存在着可以进行分子“交谈(cross talk)”的共同信号,研究发现神经导向因子就扮演如此的角色,它在神经和血管生长过程中均起调节作用。大量研究已证明作为神经导向因子之一的Netrin-1其在神经生长过程中起重要作用,近来还发现其在血管新生方面起着重要作用,它在一定的浓度范围内可以促进血管新生。那么,它和MSC共同移植到机体是否提高MSC的移植效率?即对缺血机体来说其能否促进血管的新生?对缺血组织的功能是否改善?这为缺血性疾病尤其伴有神经功能病变的缺血疾病的治疗提供新的思路。目的分离大鼠骨髓间充质干细胞,并体外培养、扩增,应用Netrin-1蛋白和间充质干细胞联合移植,观察其对局部缺血肢体的血管恢复情况,并对其机制进行探讨,便于进一步应用于临床,给肢体血管受损疾病的治疗带来新的希望。方法在体内研究中,先对24只大鼠给予下肢股动脉结扎造成肢体缺血模型后,随机分为4组(各组6只),对照组、Netrin-1组、MSCs组、MSCs联合Netrin-1组。将分离培养的间充质干细胞和(或)Netrin-1局部注射到缺血组织中。在治疗后7d,14d及28d时观察机体功能变化,同时应用Western blot和ELISA方法检测血或组织中的VEGF水平,最后在治疗28d时,给予数字减影血管造影(DSA)显示侧枝动脉的形成情况、免疫组化检测毛细血管和小动脉的密度。在体外研究中,培养人脐静脉内皮细胞(HUVEC),观察Netrin-1对MSC参与小管形成能力的作用,同时观察Netrin-1对MSC迁移能力的影响,进一步证实Netrin-1对MSC在血管新生中的作用。结果 1、在体内研究过程中,①应用Netrin-1治疗、MSC移植或两者联合治疗时,机体在治疗后7d及14d时局部组织及血VEGF水平明显增高,在移植后28d时VEGF水平有所下降,但仍比治疗前及对照组高,差异具有统计学意义(P0.05);与单纯Netrin-1治疗或MSC治疗组比较,Netrin-1联合MSC治疗组VEGF浓度进一步增加,具有统计学意义(P0.05)。②与空白组比较,Netrin-1治疗、MSC移植治疗或两者联合治疗均能明显改善肢体功能、增加缺血区毛细血管密度及肢体侧枝循环,改善缺血状况,这与机体分泌VEGF水平增多有关;当应用Netrin-1联合MSC治疗时大鼠肢体功能改善更加明显,侧枝循环的建立和缺血区毛细血管密度增加更明显,血管新生更明显,这与VEGF水平进一步增多相关。 2、在体外研究过程中,①应用transwell迁移技术分析体外培养的MSC对Netrin-1(50ng/ml)的应答,结果提示Netrin-1对MSC的细胞迁移数量为69.1士5.63/HPF,VEGF对MSC的细胞迁移数量为(67.5士3.88/HPF),明显高于空白对照组细胞迁移数量(41.0士3.57/HPF),具有统计学意义(P0.05);但Netrin-1加VEGF对MSC的细胞迁移数量为115.5士13.00/HPF,明显高于Netrin-1组、VEGF组及空白对照组,具有显著的统计学意义(P0.05)。Netrin-1加VEGF比两种因子单独应用更易促进MSC的迁移。②定量分析小管形成能力的研究结果提示,Netrin-1组小管形成数量为41.75士2.83/3HPF,VEGF组小管形成数量为48.25士3.42/3HPF,这两组明显高于对照组(11.75士1.35/3HPF),具有统计学意义(P0.05);但是Netrin-1加VEGF组小管形成数量为(73.75士3.42/3HPF),明显高于空白对照组、Netrin-1组及VEGF组,具有统计学意义(P0.05);在培养MSC时加入50 ng/ml Netrin-1与10 ng/ml VEGF比两种因子单独应用更易促进HUVEC的小管形成。结论 MSC移植及Netrin-1局部治疗缺血肢体均可明显改善缺血肢体的功能情况,促进局部的血管新生,改善机体的血流状况;但MSC联合Netrin-1治疗肢体缺血性动物模型,肢体的功能改善更加明显,侧肢循环形成更多,更易促进局部血管新生。
[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.

【学位授予单位】：南京医科大学
【学位级别】：博士
【学位授予年份】：2009
【分类号】：R329

【参考文献】