發(fā)布時間：2018-02-04 22:55

  本文關(guān)鍵詞： 急性肺損傷 內(nèi)毒素 1-磷酸鞘氨醇 人臍帶間充質(zhì)干細(xì)胞　出處：《安徽醫(yī)科大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：目的:急性肺損傷和急性呼吸窘迫綜合征(acute lung injury/acute respiratory distress syndrome,ALI/ARDS)是臨床危重癥患者高發(fā)病率和死亡率的主要原因,因缺乏有效的治療藥物,所以亟待尋找新的治療手段。近年研究發(fā)現(xiàn)人臍帶間充質(zhì)干細(xì)胞(Human Umbilical Cord-Mesenchymal Stem Cells,h UC-MSCs)可以有效緩解急性肺損傷和急性呼吸窘迫綜合征,而1-磷酸鞘氨醇(sphingosine-1-phosphate,S1P)對增強(qiáng)血管內(nèi)皮細(xì)胞屏障功能具有重要作用,可抵抗肺臟的炎癥反應(yīng),減輕肺損傷。FTY720為其類似物,是修飾后化學(xué)合成物,也是S1P1激動劑,具有免疫抑制作用,有研究結(jié)果表明,小劑量的S1P1激動劑對于小鼠急性肺損傷有保護(hù)作用。因此我們根據(jù)其各自功能及特點(diǎn),推測h UC-MSCs與FTY720之間在治療ALI/ARDS中有協(xié)同性。本實(shí)驗(yàn)研究的目的是探討h UC-MSCs與FTY720治療內(nèi)毒素所致急性肺損傷的協(xié)同作用,以期增加治療效果,并減少各自的治療缺憾,同時對其治療機(jī)理進(jìn)行初步研究。方法:將8-10周齡雌性C57BL/6小鼠隨機(jī)分為5組,分別是正常組、LPS組、h UC-MSCs組、FTY720組、h UC-MSCs與FTY720聯(lián)合給藥組。正常組給予氣管內(nèi)滴注NS,其余各組滴注10 mg/kg內(nèi)毒素制備小鼠急性肺損傷模型,模型建立后24 h,分別給h UC-MSCs組每只小鼠尾靜脈注射2×105人臍帶間充質(zhì)干細(xì)胞,給FTY720組每只小鼠腹腔內(nèi)注射劑量為0.1 mg/kg的FTY720,聯(lián)合給藥組給予尾靜脈注射2×105人臍帶間充質(zhì)干細(xì)胞和腹腔內(nèi)注射劑量為0.1 mg/kg的FTY720。分別于造模后48 h及7 d處死小鼠,觀察各組肺組織病理變化(HE染色)和病理損傷評分、肺組織濕干重比值、肺Micro-CT(活體)、BALF總蛋白和炎癥因子(IL-12p70、IL-10、IL-6、MCP-1、IFN-γ、TNF-α)含量在給藥后48小時和7天分別測得,48小時后分析各組小鼠生存率。提取肺總蛋白,進(jìn)行雙向電泳測定,對獲取的蛋白圖譜進(jìn)行分析,尋找差異表達(dá)的蛋白質(zhì),以觀察用內(nèi)毒素?fù)p傷后肺部蛋白質(zhì)表達(dá)的差異。結(jié)果:1.小鼠肺組織損傷評分和肺CT結(jié)果表明我們成功建立了內(nèi)毒素誘導(dǎo)的小鼠急性肺損傷模型。2.研究發(fā)現(xiàn)h UC-MSCs組、FTY720組、h UC-MSCs與FTY720聯(lián)合給藥組的小鼠給藥后生存率都有所提高、肺病理損傷評分降低、濕干重比值降低、灌洗液中總蛋白含量減少以及灌洗液中炎癥因子IL-6、TNF-α、MCP-1減少。聯(lián)合給藥組與單獨(dú)使用h UC-MSCs或FTY720組相比,聯(lián)合給藥的治療效果最好,但其中生存率和肺濕干重比值的降低并沒有統(tǒng)計(jì)學(xué)意義。3.蛋白質(zhì)雙向電泳結(jié)果表明急性肺損傷小鼠的肺內(nèi)蛋白量是超過正常小鼠的。蛋白質(zhì)點(diǎn)數(shù)為(100±2),其中有21個表達(dá)的蛋白差異點(diǎn),通過蛋白質(zhì)譜鑒定和生物信息檢索結(jié)果顯示蛋白質(zhì)過氧化物酶-6在損傷肺和正常肺中有明顯差異。結(jié)論:氣管內(nèi)滴注內(nèi)毒素建立急性肺損傷小鼠模型較穩(wěn)定;使用h UC-MSCs或FTY720的單獨(dú)給藥組可以減少肺內(nèi)炎癥,h UC-MSCs和FTY720聯(lián)合給藥組與單獨(dú)給藥相比也可以通過彼此之間的相互作用有效緩解急性肺損傷;蛋白質(zhì)雙向電泳結(jié)果表明急性肺損傷小鼠肺組織中蛋白質(zhì)過氧化物酶-6表達(dá)的增加,機(jī)制還待進(jìn)一步研究。h UC-MSCs和FTY720聯(lián)合給藥與分別單獨(dú)給藥相比也許是更優(yōu)的治療策略,其機(jī)制還有待于進(jìn)一步探究。
[Abstract]:Objective: acute lung injury and acute respiratory distress syndrome (acute lung injury/acute respiratory distress syndrome, ALI/ARDS) is a major cause of morbidity and mortality in patients with critical illness, due to lack of effective treatment, so it is urgent to find new treatments. Recent studies have found that human umbilical cord mesenchymal stem cells (Human Umbilical Cord-Mesenchymal Stem Cells, H UC-MSCs) can effectively alleviate the acute lung injury and acute respiratory distress syndrome, and 1- 1-phosphate (sphingosine-1-phosphate, S1P) plays an important role in enhancing vascular endothelial barrier function, inflammation resistance lung, reduce lung injury in.FTY720 for its analogues, is modified by chemical synthesis, is S1P1 agonists have immunosuppressive effects, the results show that small doses of the S1P1 agonist for the mouse acute lung injury protection So we use. According to their respective functions and features, that between H and FTY720 treatment in UC-MSCs ALI/ARDS in collaboration. The purpose of this study is to investigate the synergistic effect of H UC-MSCs and FTY720 in the treatment of acute lung injury induced by endotoxin, in order to increase the treatment effect, and reduce the treatment of their shortcomings, and make a preliminary study on the mechanism of treatment. Methods: 8-10 week old female C57BL/6 mice were randomly divided into 5 groups, namely the normal group, LPS group, H UC-MSCs group, FTY720 group, H UC-MSCs and FTY720 combined group. Normal group received intratracheal instillation of NS, other groups were infusion of 10 mg/kg endotoxin preparation model of acute lung injury of mice model after 24 h, respectively, to the H UC-MSCs group each mouse tail vein injection of 2 x 105 human umbilical cord mesenchymal stem cells, FTY720 group injected dose per mice intraperitoneally for 0.1 mg/kg FTY720, combined treatment groups were given tail vein 2 x 105 intravenous injection of human umbilical cord mesenchymal stem cells and intraperitoneal injection of 0.1 mg/kg FTY720. respectively at 48 h and 7 d mice were sacrificed to observe the pathological changes of lung tissue in each group (HE staining) and the pathological damage score, lung wet dry weight ratio, lung Micro-CT (in vivo), BALF total protein and inflammatory factors (IL-12p70, IL-10, IL-6, MCP-1, IFN- y, TNF- a) content in 48 hours after administration and 7 days were measured, analyzed the survival rate of mice after 48 hours. The total lung protein extraction, two-dimensional electrophoresis determination of protein profiles obtained were analyzed for different expression the protein, in order to observe the differences in protein expression of lung injury after endotoxin. Results: 1. mice lung injury score and lung CT results showed that we successfully established endotoxin induced acute lung injury in mice model of.2. h was found in UC-MSCs group, FTY720 group, H UC-MSCs and FTY720 Combined drug group mice survival rate increased, lung pathology score decreased, wet to dry weight ratio decreased, the total protein content in the lavage fluid and reduce inflammatory factors in the lavage fluid IL-6, TNF- alpha, MCP-1 reduced. The combined dose group and H alone or combined with UC-MSCs FTY720 group compared to drug treatment effect is best, but the lower the survival rate and the lung wet / dry weight ratio was not statistically significant.3. protein two-dimensional electrophoresis results showed that the lung protein in mice with acute lung injury is more than normal mice. The protein points (100 + 2), 21 proteins differentially expressed in the point, through the search the results of protein mass spectrometry and bioinformatics indicated that the protein peroxidase -6 in normal lung and lung injury is significant difference. Conclusion: intratracheal instillation of LPS induced acute lung injury in mice model is stable; the use of H UC-MSCs or FTY720 Single dose group can reduce the inflammation in the lungs, H UC-MSCs and FTY720 combination group and monotherapy compared can effectively alleviate the acute lung injury induced by the interaction between each other; two-dimensional electrophoresis results showed that the increase in protein peroxidase in mouse lung tissue -6 expression in acute lung injury, the mechanism also needs further study of UC-MSCs and.H FTY720 combined with medication and were administered alone may be better than treatment strategies, the mechanism remains to be further explored.

【學(xué)位授予單位】：安徽醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R563.8

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