本文選題：UCMSCS + ECFCs　； 參考：《中南大學(xué)》2014年碩士論文

【摘要】：目的：研究探討臍帶間充質(zhì)干細(xì)胞(umbilical cord mesenchymal stem cells, UCMSCs)、內(nèi)皮克隆形成細(xì)胞(endothelial colony-forming cells,ECFCs)、細(xì)胞培養(yǎng)基質(zhì)及兩種細(xì)胞聯(lián)合治療糖尿病小鼠皮膚創(chuàng)面的有效性,觀察單種細(xì)胞、細(xì)胞自分泌因子、聯(lián)合細(xì)胞治療糖尿病創(chuàng)面修復(fù)的作用,并探討相關(guān)的作用機(jī)制。 方法：1.建立糖尿病小鼠皮膚創(chuàng)傷模型：將36只7周齡db/db小鼠適應(yīng)性飼養(yǎng)1周后稱重、測血糖大于16.7mmol/L。麻醉后用直徑為6mmm的打孔器在小鼠背部制備圓形創(chuàng)面。2.動(dòng)物分組：A組：內(nèi)皮克隆形成細(xì)胞(ECFCs)組(n=6)：B組：ECFC-CM (conditioned medium)組(n=6)；C組：臍帶來源的間充質(zhì)干細(xì)胞(MSC)組(n=6)；D組：MSC-CM (conditioned medium)組(n=6)：E組：ECFCs及UCMSCs組,即混合細(xì)胞組(n=6)；F組：PBS對照組(n=6)。3.細(xì)胞移植：模型建立成功后,在A組、B組分別沿創(chuàng)面旁開2-3mm左右4個(gè)位點(diǎn)進(jìn)行多點(diǎn)皮下注射CM-Dil標(biāo)記的ECFCs和UCMSCs(細(xì)胞注射總量為1*10^6個(gè)/只,共100u1)；C組、D組分別采用同上的方法皮下注射用M199培養(yǎng)的同等細(xì)胞數(shù)(1*10^6個(gè)) ECFCs和UCMSCs培養(yǎng)基的上清液(100ul/只),即ECFC-CM、UCMSC-CM; E組皮下注射ECFCs及UCMSCs(即0.5*10^6個(gè)ECFCs及0.5*10^6個(gè)UCMSCs混合細(xì)胞組)；F組為對照組,注射PBS(100ul/只)。移植后,應(yīng)用水膠體敷料覆蓋創(chuàng)面。4.取材觀察指標(biāo)：用照相機(jī)每隔一日對所有小鼠創(chuàng)面進(jìn)行拍照,記錄創(chuàng)面愈合情況,同時(shí)記錄血糖及體重。分別在細(xì)胞及其培養(yǎng)基移植后第18天取材ECFCs及UCMSCs組小鼠的全層皮膚組織,并制作成冰凍切片,在熒光顯微鏡下觀察CM-DiI標(biāo)記后的細(xì)胞在移植后的具體定位；采用H-E染色法及vWF免疫組化法觀察皮膚組織的新生毛細(xì)血管密度變化；采用Western Blot方法檢測促血管生成因子及信號通路蛋白的表達(dá)。 結(jié)果：細(xì)胞及培養(yǎng)基質(zhì)移植治療后,每2天監(jiān)測小鼠隨機(jī)體重及血糖,各組小鼠體重隨著時(shí)間的延長逐漸增加,組間體重?zé)o明顯差異(P0.05)；血糖均大于16.7mmol/L,組間血糖無明顯差異(P0.05)；創(chuàng)緣可見鮮紅色、顆粒狀、柔軟濕潤的肉芽組織逐漸形成并填補(bǔ)創(chuàng)口組織缺損。移植細(xì)胞組及CM組較對照組(PBS組)傷口愈合時(shí)間明顯縮短,其中混合細(xì)胞組傷口愈合最快,其愈合速率較其他各組有統(tǒng)計(jì)學(xué)差異(P0.05),ECFC、ECFC-CM、UCMSCs、MSC-CM愈合速率無明顯差異(P0.05),但比PBS對照組傷口愈合時(shí)間短。移植細(xì)胞及其自分泌因子第18天后,用Western Blot檢測創(chuàng)緣皮膚組織中VEGF、AKT、pAKT、ERK1/2的蛋白表達(dá)量,結(jié)果顯示：ECFCs、 ECFC-CM、UCMSCs、UCMSC-CM及聯(lián)合移植組的VEGF蛋白含量明顯高于PBSs組,差異有統(tǒng)計(jì)學(xué)意義(單獨(dú)移植組P0.05,聯(lián)合移植組P0.01),細(xì)胞移植及自分泌因子組可使VEGF分泌增加。聯(lián)合移植組VEGF蛋白表達(dá)量高于單獨(dú)移植組,差異有統(tǒng)計(jì)學(xué)意義(P0.01),提示聯(lián)合細(xì)胞移植后VEGF蛋白表達(dá)量最高。單獨(dú)細(xì)胞及自分泌因子移植組VEGF蛋白表達(dá)無明顯變化,組間比較無差異(P0.05)。而信號通路蛋白AKT、pAKT、ERK1/2蛋白測定結(jié)果提示,ECFCs、 ECFC-CM、UCMSCs、UCMSC-CM及聯(lián)合移植組的表達(dá)量明顯高于PBSs組,差異有統(tǒng)計(jì)學(xué)意義(單獨(dú)移植組與PBS組相比,P0.05；聯(lián)合移植組與PBS組相比,P0.01)；聯(lián)合移植組蛋白表達(dá)量最高,與單獨(dú)移植及自分泌因子組相比,差異有統(tǒng)計(jì)學(xué)意義(P0.01)；單獨(dú)移植組及自分泌組組間比較無差異(P0.05)。提示ECFCs及UCMSCs在促進(jìn)創(chuàng)面血管新生、促進(jìn)組織修復(fù)中可能存在自分泌VEGF發(fā)揮作用,而聯(lián)合細(xì)胞移植可能更有利于VEGF自分泌而促進(jìn)血管新生及組織修復(fù)。 結(jié)論：1. UCMSCs、ECFCs及其自分泌因子移植能有效促進(jìn)新生血管形成,加速組織修復(fù)。2. UCMSCs和ECFCs兩種細(xì)胞聯(lián)合移植具有協(xié)同作用,比單獨(dú)移植一種細(xì)胞或自分泌因子促血管新生及組織修復(fù)作用更顯著。3. ECFCs及其自分泌因子對糖尿病小鼠皮膚創(chuàng)面的修復(fù)作用可達(dá)到與UCMSCs及其自分泌因子相同的效果,為臨床細(xì)胞移植治療組織修復(fù)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.
【學(xué)位授予單位】：中南大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R587.1

【共引文獻(xiàn)】

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