静脉移植同种异体ADMSCs对LPS诱导的脓毒症小鼠的治疗作用
发布时间：2019-01-01 12:45
【摘要】：目的：(1)体外分离培养Balb/c小鼠脂肪间充质干细胞(ADMSCs),对其表面标记分子和多向分化潜能进行检测。(2)通过观察静脉移植同种异体ADMSCs对脓毒症小鼠生存率、体温、肝肾功能、炎症因子(TNF-α、IL-6、IL-8)和抗炎症因子(IL-4、IL-10)的影响,探讨ADMSCs对LPS诱导的脓毒症小鼠的治疗作用及其机制。 方法：(1)获取SPF级Balb/c小鼠腰背部、大网膜处的脂肪组织,采用胶原酶消化法和贴壁法分离获得Balb/c小鼠ADMSCs,并对其进行体外培养。选择第3代ADMSCs,采用流式细胞技术对其表面标记分子进行检测,通过化学诱导法定向诱导细胞成骨、成脂肪、成软骨分化,分别予以茜素红染色,油红O染色,Alcian blue染色进行鉴定。(2)Balb/c小鼠60只,体重22-28g(8-12周龄),随机分为空白对照组20只,脓毒症对照组(LPS组)20只,ADMSCs移植组(ADMSCs组)20只。经尾静脉注射LPS(剂量为15mg/kg,浓度1mg/mL)建立脓毒症模型,空白对照组注射生理盐水,15mL/kg。 ADMSCs组于模型建立5min后经尾静脉注射ADMSCs (106,100uL),空白对照组和LPS组均注射生理盐水100uL。每4小时观察并记录小鼠生存情况和体温,直至48h。(3) Balb/c小鼠30只,体重22-28g(8-12周龄)30只小鼠被随机分为3组,即空白对照组10只、LPS组10只和ADMSCs组10只,小鼠经尾静脉注射LPS(剂量为10mg/kg,浓度为1mg/mL)建立脓毒症模型,其余处理方法同上给药后6h经心脏采血检测肝肾功能指标SCi、ALT和AST水平,同时采用酶联免疫吸附法检测血清炎症因子和抗炎症因子TNF-α、IL-4、IL-6、IL-8及IL-10水平。 结果：(1)成功分离培养出Balb/c小鼠ADMSCs,流式鉴定结果显示：CDllb、CD45表达呈阴性,CD34、CD44、CD106、CD29表达均呈阳性,Balb/c小鼠ADMSCs在特定诱导条件下,具有向骨细胞、脂肪细胞和软骨细胞分化的潜能。(2)生存率：正常对照组48h内小鼠生存率为100%。LPS组和ADMSCs组小鼠随时间延长,生存率逐渐下降。ADMSCs组24h生存率为65%,较同时间点LPS组(30%)高,差异有统计学意义(P0.001)。生存分析结果显示ADMSCs组小鼠总体生存率较LPS组高,差异有统计学意义(P0.001)。(3)体温：正常对照组小鼠体温在正常范围内波动,LPS组和ADMSCs组小鼠体温分别在0-8h、0-12h内有下降,而后逐渐回升并保持在正常范围内。LPS组小鼠平均体温在4h、8h、12h、16h均低于36℃,低于同时间段正常对照组,差异有统计学意义(P0.001), ADMSCs组小鼠平均体温在4h、8h、16h均较同时间段LPS组高,差异有统计学意义(P0.001)。(4)肝肾功能：建模后6h, LPS组小鼠血清SCr、ALT及AST水平较正常对照组高,差异有统计学意义(P0.05); ADMSCs组小鼠血清ALT和AST水平较LPS组低,差异有统计学意义(P0.05)。(5)炎症因子和抗炎症因子水平：建模后6h, LPS组小鼠所有血清炎症因子水平较正常对照组升高,差异有统计学意义(P0.05); ADMSCs组小鼠血清TNF-α、 IL-4、IL-6、IL-8及IL-10水平均低于LPS组,差异有统计学意义(P0.05)。 结论：(1)本研究所获得细胞具有贴壁生长、干细胞表型及多向分化能力,符合ADMSCs特征。(2)脓毒症可引起小鼠低体温,血清SCr、ALT、AST、TNF-α、IL-4、IL-6、IL-8和IL-10水平升高。(3)静脉移植同种异体ADMSCs可以降低脓毒症小鼠血清TNF-α、 IL-4、IL-6、IL-8和IL-10水平,发挥双重炎症调节作用,保护器官功能、改善低体温、提高生存率。图6幅,参考文献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，
本文编号：2397588
[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，
本文编号：2397588
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