發(fā)布時(shí)間：2018-07-28 07:21

【摘要】：目的建立小鼠背部皮膚全層缺損模型,應(yīng)用AMD3100進(jìn)行骨髓動(dòng)員,研究在骨髓動(dòng)員情況下單核巨噬細(xì)胞的亞型變化及其對(duì)創(chuàng)面愈合的影響。 方法將小鼠分為實(shí)驗(yàn)組和對(duì)照組,單劑量實(shí)驗(yàn)組造模前40分鐘給于腹腔注射5mg/kg AMD3100,對(duì)照組注射等量PBS。同時(shí)于小鼠背部造出5mm的創(chuàng)面。確定6小時(shí)、12小時(shí)、1天、3天、7天、14天為取材的時(shí)間點(diǎn),通過(guò)大體觀察、組織切片、免疫組化、流式細(xì)胞術(shù)等手段來(lái)觀察愈合情況及單核細(xì)胞動(dòng)力學(xué)變化。另外,采用每隔24小時(shí)腹腔重復(fù)注射AMD31005mg/Kg,對(duì)照組則注射等劑量PBS。術(shù)后1天、2天、3天觀察傷后愈合情況。 結(jié)果除1只單次注射實(shí)驗(yàn)組小鼠術(shù)后約3小時(shí)死亡外,其他的小鼠均存活良好。實(shí)驗(yàn)組創(chuàng)面較對(duì)照組愈合明顯,創(chuàng)傷后7天時(shí),單次注射組和對(duì)照組創(chuàng)面面積分別為1.28±0.95mm2、3.14±2.84mm2,p0.05.連續(xù)注射組實(shí)驗(yàn)組創(chuàng)面愈合較對(duì)照組較慢,創(chuàng)傷后3天時(shí),連續(xù)注射組和對(duì)照組創(chuàng)面面積分別是17.65±2.60mm2、15.12±2.74mm2,p0.05.組織切片HE染色顯示創(chuàng)傷后早期(6小時(shí)-24小時(shí))單次注射實(shí)驗(yàn)組中性粒細(xì)胞、單核細(xì)胞等炎癥浸潤(rùn)較對(duì)照組提前出現(xiàn),以血管周圍明顯,伴毛細(xì)血管擴(kuò)張、水腫等。創(chuàng)傷后3天,單次注射實(shí)驗(yàn)組微血管生成較對(duì)照組多,細(xì)胞數(shù)量增多,以核大深染的類上皮細(xì)胞為多。創(chuàng)傷后7-14天,單次注射實(shí)驗(yàn)組纖維母細(xì)胞增多,微血管增生。內(nèi)皮細(xì)胞及成纖維母細(xì)胞增多。免疫組化顯示單次注射實(shí)驗(yàn)組的α-SMA陽(yáng)性增高,微血管密度增加。單次注射實(shí)驗(yàn)組CD68表達(dá)較對(duì)照組高。流式細(xì)胞術(shù)顯示單次注射實(shí)驗(yàn)組Gr-1highCD11b+細(xì)胞群于創(chuàng)傷后6小時(shí)、12小時(shí)、1天、3天、7天占門內(nèi)細(xì)胞總數(shù)分別為46.78±5.70%,19.09±9.25%,14.53±4.19%,4.15±1.81%,11.51±8.78%。對(duì)照組分別為23.58±5.85%,27.63±1.42%,15.72±4.97%,9.90±0.60%,9.33±0.91%。在6小時(shí)、3天時(shí)二者有顯著性差異。在皮膚組織中,Gr-1highCD11b+細(xì)胞群在6小時(shí)、1天和3天時(shí)占比例分別為：1.35±0.06%,3.72±1.82%,2.71±1.68%,而對(duì)照組為0.86±0.18%,3.00±0.59%和1.66±0.15%。在6小時(shí)二者有顯著性差異。 結(jié)論通過(guò)單劑量AMD3100阻斷SDF-1/CXCR4軸,能夠從骨髓中動(dòng)員出大量的單核細(xì)胞等造血干細(xì)胞,以Gr-1highCD11b+單核細(xì)胞群較多,而Gr-1lowCD11b+細(xì)胞群較少。單核細(xì)胞在傷后早期大量迅速招募到皮膚受損組織,清除傷口內(nèi)的組織細(xì)胞碎片異物等,以盡快結(jié)束炎性反應(yīng)期盡早啟動(dòng)抗炎修復(fù)期。在炎癥中后期,組織中單核巨噬細(xì)胞增多,通過(guò)發(fā)揮巨噬細(xì)胞啟動(dòng)組織修復(fù)再生能力等,從而促進(jìn)創(chuàng)面的愈合。
[Abstract]:Objective to establish a full-thickness skin defect model in mice and to investigate the changes of mononuclear macrophage subtypes and their effects on wound healing by bone marrow mobilization (AMD3100). Methods mice were divided into experimental group and control group. The mice were injected intraperitoneally with 5mg/kg AMD3100 40 minutes before the model was made in the single dose group, and the control group was injected with the same amount of 5mg/kg. At the same time, the wounds of 5mm were made on the back of mice. The time points of 6 hours, 12 hours, 1 day, 3 days, 7 days and 14 days were determined to observe the healing and the dynamic changes of monocyte by gross observation, tissue section, immunohistochemistry, flow cytometry and so on. In addition, AMD31005 mg / kg was injected intraperitoneally every 24 hours, while the control group was injected with PBSat the same dose. Wound healing was observed 1 day 2 days and 3 days after operation. Results except for one single injection, the mice in the experimental group died about 3 hours after operation, the other mice survived well. The wound healing in the experimental group was more obvious than that in the control group, and the wound area in the single injection group and the control group was 1.28 鹵0.95mm2and 3.14 鹵2.84mm2p0.05at 7 days after trauma, respectively. Wound healing in the continuous injection group was slower than that in the control group, and the wound area in the continuous injection group and the control group was 17.65 鹵2.60 mm ~ (2) and 15.12 鹵2.74 mm ~ (2) (p 0.05) respectively at 3 days after trauma. The HE staining showed that neutrophils were injected into the experimental group at the early stage (6 hours to 24 hours) after trauma. The inflammatory infiltration of monocytes appeared earlier than that of the control group, especially around the blood vessels, accompanied by capillary dilatation, edema and so on. On the 3rd day after trauma, the microvascular formation and the number of cells in the experimental group were more than those in the control group, especially in the large and deep stained epithelioid cells. At 7-14 days after trauma, fibroblasts and microvascular hyperplasia were increased in the experimental group. Endothelial cells and fibroblasts increased. Immunohistochemical staining showed that 偽 -SMA positive and microvessel density increased in single injection group. The expression of CD68 in the experimental group was higher than that in the control group. Flow cytometry showed that the total number of Gr-1highCD11b cells in the door was 46.78 鹵5.70 鹵19.09 鹵9.25 鹵4.19 鹵4.15 鹵1.81 鹵11.51 鹵8.78, respectively. The control group (23.58 鹵5.85) was 27.63 鹵1.42 (15.72 鹵4.97) and 9.90 鹵0.60 (9.33 鹵0.91). There was a significant difference between them at 6 hours and 3 days. The percentage of Gr-1high CD11b cells in skin tissue at 6 hours and 3 days was 1: 1.35 鹵0.06 and 3.72 鹵1.82 and 2.71 鹵1.68, respectively, compared with 0.86 鹵0.18 鹵0.59% and 1.66 鹵0.15 in the control group. There was a significant difference between the two groups at 6 hours. Conclusion A large number of hematopoietic stem cells, such as monocytes, can be mobilized from bone marrow by blocking SDF-1/CXCR4 axis by single dose AMD3100. The monocytes of Gr-1highCD11b are more than those of Gr-1lowCD11b. Mononuclear cells were rapidly recruited into damaged skin tissue in the early stage of injury and removed tissue cell fragments from the wound so as to start the anti-inflammatory repair phase as soon as possible after the end of the inflammatory reaction period. In the middle and late stage of inflammation, the mononuclear macrophages in the tissues increased, and the ability of tissue repair and regeneration was activated by the macrophages, so as to promote the wound healing.
【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2013
【分類號(hào)】：R641

【共引文獻(xiàn)】

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