UCMSCs和ECFCs联合移植及培养基质促进糖尿病小鼠创面修复的作用

发布时间：2018-05-24 02:27

本文选题：UCMSCS + ECFCs　；参考：《中南大学》2014年硕士论文

【摘要】：目的：研究探讨脐带间充质干细胞(umbilical cord mesenchymal stem cells, UCMSCs)、内皮克隆形成细胞(endothelial colony-forming cells,ECFCs)、细胞培养基质及两种细胞联合治疗糖尿病小鼠皮肤创面的有效性,观察单种细胞、细胞自分泌因子、联合细胞治疗糖尿病创面修复的作用,并探讨相关的作用机制。方法：1.建立糖尿病小鼠皮肤创伤模型：将36只7周龄db/db小鼠适应性饲养1周后称重、测血糖大于16.7mmol/L。麻醉后用直径为6mmm的打孔器在小鼠背部制备圆形创面。2.动物分组：A组：内皮克隆形成细胞(ECFCs)组(n=6)：B组：ECFC-CM (conditioned medium)组(n=6)；C组：脐带来源的间充质干细胞(MSC)组(n=6)；D组：MSC-CM (conditioned medium)组(n=6)：E组：ECFCs及UCMSCs组,即混合细胞组(n=6)；F组：PBS对照组(n=6)。3.细胞移植：模型建立成功后,在A组、B组分别沿创面旁开2-3mm左右4个位点进行多点皮下注射CM-Dil标记的ECFCs和UCMSCs(细胞注射总量为1*10^6个/只,共100u1)；C组、D组分别采用同上的方法皮下注射用M199培养的同等细胞数(1*10^6个) ECFCs和UCMSCs培养基的上清液(100ul/只),即ECFC-CM、UCMSC-CM; E组皮下注射ECFCs及UCMSCs(即0.5*10^6个ECFCs及0.5*10^6个UCMSCs混合细胞组)；F组为对照组,注射PBS(100ul/只)。移植后,应用水胶体敷料覆盖创面。4.取材观察指标：用照相机每隔一日对所有小鼠创面进行拍照,记录创面愈合情况,同时记录血糖及体重。分别在细胞及其培养基移植后第18天取材ECFCs及UCMSCs组小鼠的全层皮肤组织,并制作成冰冻切片,在荧光显微镜下观察CM-DiI标记后的细胞在移植后的具体定位；采用H-E染色法及vWF免疫组化法观察皮肤组织的新生毛细血管密度变化；采用Western Blot方法检测促血管生成因子及信号通路蛋白的表达。结果：细胞及培养基质移植治疗后,每2天监测小鼠随机体重及血糖,各组小鼠体重随着时间的延长逐渐增加,组间体重无明显差异(P0.05)；血糖均大于16.7mmol/L,组间血糖无明显差异(P0.05)；创缘可见鲜红色、颗粒状、柔软湿润的肉芽组织逐渐形成并填补创口组织缺损。移植细胞组及CM组较对照组(PBS组)伤口愈合时间明显缩短,其中混合细胞组伤口愈合最快,其愈合速率较其他各组有统计学差异(P0.05),ECFC、ECFC-CM、UCMSCs、MSC-CM愈合速率无明显差异(P0.05),但比PBS对照组伤口愈合时间短。移植细胞及其自分泌因子第18天后,用Western Blot检测创缘皮肤组织中VEGF、AKT、pAKT、ERK1/2的蛋白表达量,结果显示：ECFCs、 ECFC-CM、UCMSCs、UCMSC-CM及联合移植组的VEGF蛋白含量明显高于PBSs组,差异有统计学意义(单独移植组P0.05,联合移植组P0.01),细胞移植及自分泌因子组可使VEGF分泌增加。联合移植组VEGF蛋白表达量高于单独移植组,差异有统计学意义(P0.01),提示联合细胞移植后VEGF蛋白表达量最高。单独细胞及自分泌因子移植组VEGF蛋白表达无明显变化,组间比较无差异(P0.05)。而信号通路蛋白AKT、pAKT、ERK1/2蛋白测定结果提示,ECFCs、 ECFC-CM、UCMSCs、UCMSC-CM及联合移植组的表达量明显高于PBSs组,差异有统计学意义(单独移植组与PBS组相比,P0.05；联合移植组与PBS组相比,P0.01)；联合移植组蛋白表达量最高,与单独移植及自分泌因子组相比,差异有统计学意义(P0.01)；单独移植组及自分泌组组间比较无差异(P0.05)。提示ECFCs及UCMSCs在促进创面血管新生、促进组织修复中可能存在自分泌VEGF发挥作用,而联合细胞移植可能更有利于VEGF自分泌而促进血管新生及组织修复。结论：1. UCMSCs、ECFCs及其自分泌因子移植能有效促进新生血管形成,加速组织修复。2. UCMSCs和ECFCs两种细胞联合移植具有协同作用,比单独移植一种细胞或自分泌因子促血管新生及组织修复作用更显著。3. ECFCs及其自分泌因子对糖尿病小鼠皮肤创面的修复作用可达到与UCMSCs及其自分泌因子相同的效果,为临床细胞移植治疗组织修复提供了新的思路。
[Abstract]:Objective: To investigate the effectiveness of umbilical cord mesenchymal stem cells (umbilical cord mesenchymal stem cells (UCMSCs), endothelial colony-forming cells, ECFCs), cell culture matrix and two kinds of cells in the treatment of skin wounds in diabetic mice, and observe single cell, cell autocrine factor, combined cell treatment. Objective to investigate the role of diabetic wound repair and explore the related mechanisms.
Methods: 1. the model of skin trauma in diabetic mice was established: 36 7 weeks old db/db mice were reared for 1 weeks and weighed for 1 weeks, and the blood glucose was larger than that of 16.7mmol/L.. The circular wound surface of the mouse with the diameter of 6mmm was used to prepare the circular wound.2. animals in the mouse back: A group: ECFCs group (n=6):B group: ECFC-CM (conditioned). Medium group (n=6); group C: group of umbilical cord derived mesenchymal stem cells (MSC) group (n=6); D group: MSC-CM (conditioned medium) group (n=6):E group: ECFCs and mixed cell group (mixed cell group). CM-Dil labeled ECFCs and UCMSCs were injected subcutaneously (the total amount of cell injection was 1*10^6 / only 100u1), and in group C, the same cell number (1*10^6) ECFCs and UCMSCs culture medium (100ul/ only) in group D was subcutaneously injected by M199. Cs and 0.5*10^6 UCMSCs mixed cell group); F group was the control group, PBS (100ul/ only) was injected. After transplantation, the application of hydrocolloid dressing to cover the surface of the wound surface was observed. All the mice were photographed every other day with a camera, the wound healing was recorded, blood sugar and weight were recorded at the same time. After the transplantation of the cells and the culture medium respectively. The whole layer skin tissue of ECFCs and UCMSCs mice was obtained on the 18 day and made into frozen section. Under the fluorescence microscope, the specific location of the cells after the CM-DiI labeling was observed. The density change of the newborn capillaries in skin tissues was observed by H-E staining and vWF immunohistochemical method, and the Western Blot method was used to detect the angiogenesis. Expression of adult factor and signal pathway protein.
Results: after the cell and culture matrix transplantation, the random weight and blood sugar were monitored every 2 days. The weight of the mice in each group increased gradually with time, and there was no significant difference in weight between the groups (P0.05). The blood sugar was greater than 16.7mmol/L, and there was no significant difference between the groups (P0.05). The wound healing time of the transplant cell group and the CM group was significantly shorter than the control group (group PBS), and the wound healing was the fastest in the mixed cell group, and the healing rate was significantly different from the other groups (P0.05), ECFC, ECFC-CM, UCMSCs, MSC-CM healing rate had no significant difference (P0.05), but the wound healing was better than that of the PBS control group. Western Blot was used to detect the protein expression of VEGF, AKT, pAKT, ERK1/2 in the skin tissue of the invasive skin tissue after eighteenth days. The results showed that the content of VEGF protein in ECFCs, ECFC-CM, UCMSCs, UCMSC-CM and combined transplantation group was significantly higher than that in the PBSs group. The difference was statistically significant (a single transplant group, combined transplantation) Group P0.01), cell transplantation and autocrine factor group could increase the secretion of VEGF. The expression of VEGF protein in the combined transplantation group was higher than that of the single transplant group. The difference was statistically significant (P0.01), suggesting that the expression of VEGF protein was the highest after the combined cell transplantation. There was no significant change in the expression of VEGF protein in the transplantation group of the individual cell and autocrine factor. There was no difference between the groups. (P0.05). The results of signal pathway protein AKT, pAKT, and ERK1/2 protein showed that the expression of ECFCs, ECFC-CM, UCMSCs, UCMSC-CM and combined transplantation group was significantly higher than that of the PBSs group. The difference was statistically significant (compared with the PBS group, P0.05; the combined transplantation group was compared with the PBS group); the protein expression in the combined transplantation group was the highest, and the individual transplantation group was the highest. The difference between the transplantation and the autocrine factor group was statistically significant (P0.01), and there was no difference between the individual transplantation group and the autocrine group (P0.05). It suggested that ECFCs and UCMSCs could promote the angiogenesis of the wound and promote the autocrine VEGF in the tissue repair, and the combined cell transplantation may be more beneficial to the autocrine and promote the blood of the VEGF. Tube regeneration and tissue repair.
Conclusion: 1. UCMSCs, ECFCs and its autocrine transplantation can effectively promote the formation of neovascularization, accelerate tissue repair of the combined transplantation of two cells,.2. UCMSCs and ECFCs, and more significant than the individual transplantation of a cell or autocrine factor to promote angiogenesis and tissue repair, which is more significant to.3. ECFCs and its autocrine factor to diabetes. The repair effect of mouse skin wound can achieve the same effect as UCMSCs and its autocrine factor, which provides a new way for clinical cell transplantation for tissue repair.
【学位授予单位】：中南大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R587.1

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