【摘要】：目的：(1)體外分離培養(yǎng)Balb/c小鼠脂肪間充質(zhì)干細(xì)胞(ADMSCs),對(duì)其表面標(biāo)記分子和多向分化潛能進(jìn)行檢測(cè)。(2)通過(guò)觀察靜脈移植同種異體ADMSCs對(duì)膿毒癥小鼠生存率、體溫、肝腎功能、炎癥因子(TNF-α、IL-6、IL-8)和抗炎癥因子(IL-4、IL-10)的影響,探討ADMSCs對(duì)LPS誘導(dǎo)的膿毒癥小鼠的治療作用及其機(jī)制。 方法：(1)獲取SPF級(jí)Balb/c小鼠腰背部、大網(wǎng)膜處的脂肪組織,采用膠原酶消化法和貼壁法分離獲得Balb/c小鼠ADMSCs,并對(duì)其進(jìn)行體外培養(yǎng)。選擇第3代ADMSCs,采用流式細(xì)胞技術(shù)對(duì)其表面標(biāo)記分子進(jìn)行檢測(cè),通過(guò)化學(xué)誘導(dǎo)法定向誘導(dǎo)細(xì)胞成骨、成脂肪、成軟骨分化,分別予以茜素紅染色,油紅O染色,Alcian blue染色進(jìn)行鑒定。(2)Balb/c小鼠60只,體重22-28g(8-12周齡),隨機(jī)分為空白對(duì)照組20只,膿毒癥對(duì)照組(LPS組)20只,ADMSCs移植組(ADMSCs組)20只。經(jīng)尾靜脈注射LPS(劑量為15mg/kg,濃度1mg/mL)建立膿毒癥模型,空白對(duì)照組注射生理鹽水,15mL/kg。 ADMSCs組于模型建立5min后經(jīng)尾靜脈注射ADMSCs (106,100uL),空白對(duì)照組和LPS組均注射生理鹽水100uL。每4小時(shí)觀察并記錄小鼠生存情況和體溫,直至48h。(3) Balb/c小鼠30只,體重22-28g(8-12周齡)30只小鼠被隨機(jī)分為3組,即空白對(duì)照組10只、LPS組10只和ADMSCs組10只,小鼠經(jīng)尾靜脈注射LPS(劑量為10mg/kg,濃度為1mg/mL)建立膿毒癥模型,其余處理方法同上給藥后6h經(jīng)心臟采血檢測(cè)肝腎功能指標(biāo)SCi、ALT和AST水平,同時(shí)采用酶聯(lián)免疫吸附法檢測(cè)血清炎癥因子和抗炎癥因子TNF-α、IL-4、IL-6、IL-8及IL-10水平。 結(jié)果：(1)成功分離培養(yǎng)出Balb/c小鼠ADMSCs,流式鑒定結(jié)果顯示：CDllb、CD45表達(dá)呈陰性,CD34、CD44、CD106、CD29表達(dá)均呈陽(yáng)性,Balb/c小鼠ADMSCs在特定誘導(dǎo)條件下,具有向骨細(xì)胞、脂肪細(xì)胞和軟骨細(xì)胞分化的潛能。(2)生存率：正常對(duì)照組48h內(nèi)小鼠生存率為100%。LPS組和ADMSCs組小鼠隨時(shí)間延長(zhǎng),生存率逐漸下降。ADMSCs組24h生存率為65%,較同時(shí)間點(diǎn)LPS組(30%)高,差異有統(tǒng)計(jì)學(xué)意義(P0.001)。生存分析結(jié)果顯示ADMSCs組小鼠總體生存率較LPS組高,差異有統(tǒng)計(jì)學(xué)意義(P0.001)。(3)體溫：正常對(duì)照組小鼠體溫在正常范圍內(nèi)波動(dòng),LPS組和ADMSCs組小鼠體溫分別在0-8h、0-12h內(nèi)有下降,而后逐漸回升并保持在正常范圍內(nèi)。LPS組小鼠平均體溫在4h、8h、12h、16h均低于36℃,低于同時(shí)間段正常對(duì)照組,差異有統(tǒng)計(jì)學(xué)意義(P0.001), ADMSCs組小鼠平均體溫在4h、8h、16h均較同時(shí)間段LPS組高,差異有統(tǒng)計(jì)學(xué)意義(P0.001)。(4)肝腎功能：建模后6h, LPS組小鼠血清SCr、ALT及AST水平較正常對(duì)照組高,差異有統(tǒng)計(jì)學(xué)意義(P0.05); ADMSCs組小鼠血清ALT和AST水平較LPS組低,差異有統(tǒng)計(jì)學(xué)意義(P0.05)。(5)炎癥因子和抗炎癥因子水平：建模后6h, LPS組小鼠所有血清炎癥因子水平較正常對(duì)照組升高,差異有統(tǒng)計(jì)學(xué)意義(P0.05); ADMSCs組小鼠血清TNF-α、 IL-4、IL-6、IL-8及IL-10水平均低于LPS組,差異有統(tǒng)計(jì)學(xué)意義(P0.05)。 結(jié)論：(1)本研究所獲得細(xì)胞具有貼壁生長(zhǎng)、干細(xì)胞表型及多向分化能力,符合ADMSCs特征。(2)膿毒癥可引起小鼠低體溫,血清SCr、ALT、AST、TNF-α、IL-4、IL-6、IL-8和IL-10水平升高。(3)靜脈移植同種異體ADMSCs可以降低膿毒癥小鼠血清TNF-α、 IL-4、IL-6、IL-8和IL-10水平,發(fā)揮雙重炎癥調(diào)節(jié)作用,保護(hù)器官功能、改善低體溫、提高生存率。圖6幅,參考文獻(xiàn)114篇
[Abstract]:Objective: (1) To separate and culture the adipose-derived mesenchymal stem cells (ADSCs) in Balb/ c mice, and to test its surface marker and multi-directional differentiation potential. (2) The effects of ADSCs on the survival rate, body temperature, liver and kidney function, inflammation factor (TNF-1, IL-6, IL-8) and anti-inflammatory factors (IL-4, IL-10) in sepsis mice were studied by observing the effects of the administration of the administration of ADSCs on the mice with sepsis induced by LPS. Methods: (1) The adipose tissue at the back of the waist and the large omentum of the SPF Balb/ c mouse was obtained. The ADSCs of Balb/ c mice were obtained by collagenase digestion and the method of attachment. and the surface marker molecules of the third generation ADSCs are selected to be detected by flow cytometry, and the cells are directionally induced into bone, fat and cartilage to be differentiated by a chemical ionization method, and are respectively subjected to fluorescein red dyeing, oil red O dyeing, Alcian blue staining and the like. (2) Balb/ c mice 60, body weight 22-28g (8-12 weeks old) were randomly divided into two groups: control group 20, sepsis control group (LPS group) 20, ADSCs group (ADSCs) 20 The sepsis model was established by the tail vein injection of LPS (dose: 15mg/ kg, concentration: 1mg/ mL), and the blank control group was injected with physiological saline, 15mL/ k. g. The ADSCs (106, 100uL), the blank control group and the LPS group were injected with normal saline 100u after the model was established for 5min. L. The survival and body temperature of the mice were observed and recorded every 4 hours until 48 h. (3) Thirty mice with weight of 22-28g (8-12 weeks old) of Balb/ c mice were randomly divided into three groups, namely, 10 rats in the blank control group, 10 of the LPS group and 10 in the ADSCs group, and the mice were injected with LPS at a dose of 10 mg/ kg and a concentration of 1mg/ mL to establish the sepsis model. The levels of TNF-1, IL-4, IL-6, IL-8 and IL-10 were detected by enzyme-linked immunosorbent assay. Results: (1) The ADSCs of Balb/ c mice were isolated successfully. The results showed that the expression of CD1 lb and CD45 was negative, and the expression of CD34, CD44, CD106 and CD29 was positive. The survival rate of the mice was 100% in the normal control group, and the survival rate of the mice in the LPS group and the ADSCs increased with time. The 24-hour survival rate of the ADSCs group was 65%, and the survival rate was higher than that of the LPS group (30%) at the same time point (P0.05). The survival analysis showed that the overall survival rate of the mice in the ADSCs group was higher than that of the LPS group (P0.05). (3) Body temperature: the body temperature of the normal control group was in the normal range, and the temperature of the mice in the LPS group and the ADSCs group decreased in 0-8h, 0-12h, and then gradually recovered and remained in the normal control group. The mean body temperature of the mice in the LPS group was lower than 36 鈩，

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