本文選題：間充質(zhì)干細(xì)胞 + Netrin-1　； 參考：《南京醫(yī)科大學(xué)》2009年博士論文

【摘要】：背景 近年研究發(fā)現(xiàn)造血干細(xì)胞移植可以治療缺血性血管疾病,促進(jìn)缺血部位的血管新生和側(cè)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(diǎn)之一。 間充質(zhì)干細(xì)胞(MSCs)是一類具有自我增殖和分化潛能的多能干細(xì)胞,是骨、軟骨、血管、肌肉及結(jié)締組織的前體細(xì)胞。由于其易于取材和擴(kuò)增,且移植后在機(jī)體保持良好的分化能力,是目前最理想的種子細(xì)胞。但是目前MSC移植治療缺血性疾病也有其缺點(diǎn),如形成的血管數(shù)量有限,移植細(xì)胞的存活率低,需移植細(xì)胞較多等等,因此,如何提高M(jìn)SCs的移植效率是當(dāng)今研究MSCs的焦點(diǎn)。 近來的研究發(fā)現(xiàn),神經(jīng)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(jìn)行分子“交談(cross talk)”的共同信號(hào),研究發(fā)現(xiàn)神經(jīng)導(dǎo)向因子就扮演如此的角色,它在神經(jīng)和血管生長(zhǎng)過程中均起調(diào)節(jié)作用。 大量研究已證明作為神經(jīng)導(dǎo)向因子之一的Netrin-1其在神經(jīng)生長(zhǎng)過程中起重要作用,近來還發(fā)現(xiàn)其在血管新生方面起著重要作用,它在一定的濃度范圍內(nèi)可以促進(jìn)血管新生。那么,它和MSC共同移植到機(jī)體是否提高M(jìn)SC的移植效率?即對(duì)缺血機(jī)體來說其能否促進(jìn)血管的新生?對(duì)缺血組織的功能是否改善?這為缺血性疾病尤其伴有神經(jīng)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(zhì)干細(xì)胞,并體外培養(yǎng)、擴(kuò)增,應(yīng)用Netrin-1蛋白和間充質(zhì)干細(xì)胞聯(lián)合移植,觀察其對(duì)局部缺血肢體的血管恢復(fù)情況,并對(duì)其機(jī)制進(jìn)行探討,便于進(jìn)一步應(yīng)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(nèi)研究中,先對(duì)24只大鼠給予下肢股動(dòng)脈結(jié)扎造成肢體缺血模型后,隨機(jī)分為4組(各組6只),對(duì)照組、Netrin-1組、MSCs組、MSCs聯(lián)合Netrin-1組。將分離培養(yǎng)的間充質(zhì)干細(xì)胞和(或)Netrin-1局部注射到缺血組織中。在治療后7d,14d及28d時(shí)觀察機(jī)體功能變化,同時(shí)應(yīng)用Western blot和ELISA方法檢測(cè)血或組織中的VEGF水平,最后在治療28d時(shí),給予數(shù)字減影血管造影(DSA)顯示側(cè)枝動(dòng)脈的形成情況、免疫組化檢測(cè)毛細(xì)血管和小動(dòng)脈的密度。在體外研究中,培養(yǎng)人臍靜脈內(nèi)皮細(xì)胞(HUVEC),觀察Netrin-1對(duì)MSC參與小管形成能力的作用,同時(shí)觀察Netrin-1對(duì)MSC遷移能力的影響,進(jìn)一步證實(shí)Netrin-1對(duì)MSC在血管新生中的作用。 結(jié)果 1、在體內(nèi)研究過程中,①應(yīng)用Netrin-1治療、MSC移植或兩者聯(lián)合治療時(shí),機(jī)體在治療后7d及14d時(shí)局部組織及血VEGF水平明顯增高,在移植后28d時(shí)VEGF水平有所下降,但仍比治療前及對(duì)照組高,差異具有統(tǒng)計(jì)學(xué)意義(P0.05);與單純Netrin-1治療或MSC治療組比較,Netrin-1聯(lián)合MSC治療組VEGF濃度進(jìn)一步增加,具有統(tǒng)計(jì)學(xué)意義(P0.05)。②與空白組比較,Netrin-1治療、MSC移植治療或兩者聯(lián)合治療均能明顯改善肢體功能、增加缺血區(qū)毛細(xì)血管密度及肢體側(cè)枝循環(huán),改善缺血狀況,這與機(jī)體分泌VEGF水平增多有關(guān);當(dāng)應(yīng)用Netrin-1聯(lián)合MSC治療時(shí)大鼠肢體功能改善更加明顯,側(cè)枝循環(huán)的建立和缺血區(qū)毛細(xì)血管密度增加更明顯,血管新生更明顯,這與VEGF水平進(jìn)一步增多相關(guān)。 2、在體外研究過程中,①應(yīng)用transwell遷移技術(shù)分析體外培養(yǎng)的MSC對(duì)Netrin-1(50ng/ml)的應(yīng)答,結(jié)果提示Netrin-1對(duì)MSC的細(xì)胞遷移數(shù)量為69.1士5.63/HPF,VEGF對(duì)MSC的細(xì)胞遷移數(shù)量為(67.5士3.88/HPF),明顯高于空白對(duì)照組細(xì)胞遷移數(shù)量(41.0士3.57/HPF),具有統(tǒng)計(jì)學(xué)意義(P0.05);但Netrin-1加VEGF對(duì)MSC的細(xì)胞遷移數(shù)量為115.5士13.00/HPF,明顯高于Netrin-1組、VEGF組及空白對(duì)照組,具有顯著的統(tǒng)計(jì)學(xué)意義(P0.05)。Netrin-1加VEGF比兩種因子單獨(dú)應(yīng)用更易促進(jìn)MSC的遷移。②定量分析小管形成能力的研究結(jié)果提示,Netrin-1組小管形成數(shù)量為41.75士2.83/3HPF,VEGF組小管形成數(shù)量為48.25士3.42/3HPF,這兩組明顯高于對(duì)照組(11.75士1.35/3HPF),具有統(tǒng)計(jì)學(xué)意義(P0.05);但是Netrin-1加VEGF組小管形成數(shù)量為(73.75士3.42/3HPF),明顯高于空白對(duì)照組、Netrin-1組及VEGF組,具有統(tǒng)計(jì)學(xué)意義(P0.05);在培養(yǎng)MSC時(shí)加入50 ng/ml Netrin-1與10 ng/ml VEGF比兩種因子單獨(dú)應(yīng)用更易促進(jìn)HUVEC的小管形成。 結(jié)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(jìn)局部的血管新生,改善機(jī)體的血流狀況;但MSC聯(lián)合Netrin-1治療肢體缺血性動(dòng)物模型,肢體的功能改善更加明顯,側(cè)肢循環(huán)形成更多,更易促進(jìn)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(xué)位授予單位】：南京醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2009
【分類號(hào)】：R329

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