人肝细胞生长因子基因修饰的骨髓间充质干细胞对大鼠急性肺损伤的作用研究

发布时间：2018-04-23 18:26

本文选题：肝细胞生长因子 + 骨髓间充质干细胞　；参考：《福建医科大学》2016年硕士论文

【摘要】：目的:观察人肝细胞生长因子基因修饰的骨髓间充质干细胞对脂多糖诱导的大鼠急性肺损伤的影响。方法:7周龄近交系雄性F344大鼠60只,体重200~250 g,将其随机分为3组(n=20),对照组(Control组)、空载体慢病毒转导的骨髓间充质干细胞(EGFP-MSCs)治疗组和人肝细胞生长因子基因转导的骨髓间充质干细胞(h HGF-MSCs)治疗组。所有大鼠均采用脂多糖(Lipopolysaccharide,LPS)7 mg/kg气管滴注建立急性肺损伤模型。Control组大鼠经气管给予LPS后立即经尾静脉注射低糖培养基(DMEM)1ml,EGFP-MSCs治疗组大鼠经气管给予LPS后立即经尾静脉注射含5×105个EGFP-MSCs的DMEM 1ml,h HGF-MSCs治疗组大鼠经气管给予LPS后立即经尾静脉注射含5×105个h HGF-MSCs的DMEM 1ml。细胞注射后7天,激光共聚焦显微镜下观察DAPI标记的MSCs肺组织分布情况。LPS注射后1天,3天,7天,每时点随机抽取6只大鼠,留取标本,测定肺湿干比(wet-to-dry ratio,W/D),肺泡灌洗液(bronchoalveolar lavage fluid,BALF)中总细胞计数和中性粒细胞计数及肺组织病理损伤严重度评分;化学比色法测定肺组织髓过氧化物酶(myeloperoxidase,MPO)活性;BCA法测定大鼠BALF中的总蛋白浓度;酶联免疫吸附测定法(ELISA)检测大鼠血清中人HGF和鼠HGF蛋白水平以及肺组织匀浆和肺泡灌洗液中肿瘤坏死因子α(TNF-α)、白介素10(IL-10)、白介素6(IL-6)蛋白含量;实时荧光定量聚合酶链式反应(RT-q PCR)测定大鼠肺组织匀浆中TNF-αm RNA、IL-10 m RNA、IL-6 m RNA的表达;DNA断裂的原位末端标记法(TUNEL)测定肺泡上皮细胞的凋亡指数(apoptotic index,AI)。另取F334大鼠36只,随机分为三组(n=12),分组及建模情况同上,LPS注射后90天内进行生存分析。结果:(1)EGFP-MSCs治疗组及h HGF-MSCs治疗组大鼠肺间质和肺泡腔可见绿色荧光蛋白阳性细胞分布。(2)h HGF-MSCs治疗组大鼠血清中表达h HGF蛋白,Control组和EGFP-MSCs治疗组未表达h HGF蛋白。与Control组相比,EGFP-MSCs治疗组大鼠血清中r HGF在第3天明显增高(P0.05),h HGF-MSCs治疗组大鼠血清中r HGF在第1天和第3天明显增高(P0.05,P0.01)。与EGFP-MSCs治疗组相比,h HGF-MSCs治疗组大鼠血清中r HGF在第3天明显增高(P0.05)。(3)与Control组相比,EGFP-MSCs治疗组及h HGF-MSCs治疗组大鼠肺部病理损伤评分在各时点均降低(P0.05,P0.01),差异均具有统计学意义。与EGFP-MSCs治疗组相比,h HGF-MSCs治疗组大鼠肺部病理损伤评分在第3天显著降低(P0.05)。(4)与Control组相比,EGFP-MSCs治疗组及h HGF-MSCs治疗组大鼠肺组织W/D在各时间点均明显降低(P0.01)。(5)与Control组相比,EGFP-MSCs治疗组及h HGF-MSCs治疗组大鼠肺泡灌洗液总蛋白含量在第7天明显降低(P0.05)。(6)与Control组相比,h HGF-MSCs治疗组大鼠肺泡灌洗液总细胞计数和中性粒细胞计数在第3天和第7天均明显降低(P0.05,P0.01)。与Control组相比,EGFP-MSCs治疗组大鼠肺泡灌洗液总细胞计数和中性粒细胞计数在第7天明显降低(P0.01)。与EGFP-MSCs治疗组相比,h HGF-MSCs治疗组肺泡灌洗液总细胞计数和中性粒细胞计数在第1天均降低(P0.01)。(7)h HGF-MSCs治疗组MPO活性值在第7天小于Control组(P0.05)。Control组和EGFP-MSCs治疗组在各时间点MPO活性值没有显著差别。(8)与Control组相比EGFP-MSCs治疗组和h HGF-MSCs治疗组大鼠BALF中TNF-α和IL-6含量在第1天和第7天均降低(P0.05,P0.01)。与Control组相比,EGFP-MSCs治疗组大鼠BALF中IL-10含量在第1天和第7天增高(P0.01)。h HGF-MSCs治疗组的BALF中IL-10与Control组相比,在各时点均增高(P0.05)。与EGFP-MSCs治疗组相比,h HGF-MSCs治疗组BALF中IL-6在第7天降低(P0.01),IL-10在第3天和第7天增高(P0.05,P0.01)。(9)与Control组相比,h HGF-MSCs治疗组大鼠肺组织中TNF-α、IL-6含量在各时点均降低(P0.01),IL-10含量在各时点均增高(P0.01)。与Control组相比,EGFP-MSCs治疗组大鼠肺组织中TNF-α、IL-6含量在各时点均降低(P0.01),IL-10在第1天增高(P0.05)。与EGFP-MSCs治疗组相比h HGF-MSCs治疗组TNF-α值在第一天显著低于MSC治疗组(P0.01),IL-6在第3天降低(P0.01),IL-10在第3天和第7天增高(P0.05,P0.01)。(10)与Control组相比,h HGF-MSCs治疗组大鼠肺组织中TNF-α和IL-6m RNA含量在各时点均降低(P0.01),IL-10 m RNA在各时点均增高(P0.01)。与Control组相比,TNF-α和IL-6 m RNA含量在各时点均降低(P0.05,P0.01),IL-10 m RNA含量在第3天和第7天增高(P0.05)。与EGFP-MSCs治疗组相比,h HGF-MSCs治疗组大鼠肺组织中TNF-αm RNA含量在第一天降低(P0.05),IL-10 m RNA含量在各时点均增高(P0.05,P0.01)。(11)与Control组相比,h HGF-MSCs治疗组大鼠肺泡上皮细胞AI在第3天和第7天降低(P0.05)。EGFP-MSCs治疗组大鼠肺泡上皮细胞AI与Control组无显著差别。(12)与Control组相比,EGFP-MSCs治疗组和h HGF-MSCs治疗组大鼠生存率明显增加(P0.05)。结论:h HGF-MSCs和EGFP-MSCs均可显著减轻LPS诱发的大鼠急性肺损伤的炎症反应,提高生存率及生存时间,并且h HGF-MSCs的治疗效果优于EGFP-MSCs的治疗效果。
[Abstract]:Objective: To observe the effect of human hepatocyte growth factor gene modified bone marrow mesenchymal stem cells (MSCs) on lipopolysaccharide induced acute lung injury in rats. Methods: 60 male F344 rats of 7 weeks old inbred line, weighing 200~250 g, were randomly divided into 3 groups (n=20), the control group (Control group), the bone marrow mesenchymal stem cells (EGFP-MS) transduced by no-load lentivirus (EGFP-MS) Cs) treatment group and human hepatocyte growth factor gene transduced bone marrow mesenchymal stem cells (H HGF-MSCs) treatment group. All rats were treated with lipopolysaccharide (Lipopolysaccharide, LPS) 7 mg/kg endotracheal drip to establish acute lung injury model.Control group,.Control group rats were given the low sugar medium (DMEM) 1ml, EGFP-MSCs treatment by intravenous injection of the tail vein. The rats in the treatment group were injected into the trachea for LPS immediately after the injection of DMEM 1ml containing 5 * 105 EGFP-MSCs. The rats in the H HGF-MSCs treatment group were injected with 5 * 105 h HGF-MSCs DMEM 1ml. cells after the administration of the trachea via the trachea and injected into the trachea for 7 days. The lung tissue distribution of the DAPI markers was observed under the laser confocal microscope and 1 after the injection. Days, 3 days, 7 days, 6 rats were randomly selected at every point, and specimens were collected to determine the total cell count of the lung wet dry ratio (wet-to-dry ratio, W/D), the bronchoalveolar lavage fluid (BALF), the neutrophils count and the severity score of the lung tissue, and the lung tissue myeloperoxidase (myeloperoxidase, MP) was measured by chemical colorimetry. O) activity; BCA assay was used to determine the total protein concentration in rat BALF; enzyme linked immunosorbent assay (ELISA) was used to detect the level of human HGF and rat HGF protein in the rat serum, and the tumor necrosis factor alpha (TNF- alpha) in lung homogenate and alveolar lavage fluid, interleukin 10 (IL-10), and interleukin 6 (IL-6) protein content; real time fluorescence quantitative polymerase chain reaction (RT-q). PCR) the expression of TNF- alpha m RNA, IL-10 m RNA and IL-6 m RNA in the lung tissue homogenate of rats; the apoptotic index of alveolar epithelial cells was measured by the in-situ end labeling method of DNA fracture (TUNEL), and 36 rats were randomly divided into three groups, divided into groups and modeling conditions, and the survival analysis was carried out within 90 days after injection. The results were: (1) the distribution of green fluorescent protein positive cells in the interstitial and alveolar cavities of the EGFP-MSCs treatment group and the H HGF-MSCs treatment group. (2) the expression of H HGF protein in the serum of the H HGF-MSCs group and the H HGF protein in the Control group and the EGFP-MSCs treatment group were not expressed in the third days. Higher (P0.05), the serum R HGF in the H HGF-MSCs group was significantly higher in first days and third days (P0.05, P0.01). Compared with the EGFP-MSCs treatment group, the R HGF in the serum of the H HGF-MSCs treatment group was significantly higher than that in the third day. (3) the lung pathological damage score of the rats in the treatment group and the treatment group was at the time points. Both decreased (P0.05, P0.01), the difference was statistically significant. Compared with the EGFP-MSCs treatment group, the lung pathological damage score of the H HGF-MSCs group was significantly lower (P0.05). (4) the lung tissue of the EGFP-MSCs treatment group and the H HGF-MSCs treatment group was significantly lower (P0.01) at all time points compared with the Control group (5) and the group phase. The total protein content of alveolar lavage fluid in the EGFP-MSCs treatment group and the H HGF-MSCs group decreased significantly (P0.05) in seventh days. (6) the total cell count and neutrophils count of the alveolar lavage fluid in the H HGF-MSCs group were significantly decreased (P0.05, P0.01) compared with the Control group (P0.05, P0.01). Compared with the Control group, the EGFP-MSCs treatment group was compared with the Control group. The total cell count and neutrophils count of alveolar lavage fluid in rats decreased significantly on seventh days (P0.01). Compared with the EGFP-MSCs treatment group, the total cell count and neutrophils count of alveolar lavage solution in the H HGF-MSCs group decreased (P0.01) at first days. (7) the MPO activity of the H HGF-MSCs treatment group was less than the Control group (P0.05).Control group at seventh days. There was no significant difference in MPO activity at all time points in the EGFP-MSCs treatment group. (8) the content of TNF- A and IL-6 in BALF in the EGFP-MSCs treatment group and the H HGF-MSCs group decreased (P0.05, P0.01) in the BALF group and the H HGF-MSCs treatment group (P0.05, P0.01). Compared with the Control group, IL-10 in the HGF-MSCs treatment group increased at all time points (P0.05). Compared with the EGFP-MSCs treatment group, the IL-6 in the H HGF-MSCs treatment group decreased (P0.01) at seventh days, and the IL-10 was increased on the third and seventh days. (9) the concentrations of the IL-6 in the lung tissue of the rats were compared with those in the group. Both decreased (P0.01) and IL-10 content increased at all time points (P0.01). Compared with group Control, TNF- alpha in lung tissue of EGFP-MSCs treatment group decreased (P0.01) at all time points (P0.01) and IL-10 increased at first days (P0.05). Decreased days (P0.01), IL-10 increased at third and seventh days (P0.05, P0.01). (10) the levels of TNF- A and IL-6m RNA in the lung tissue of the H HGF-MSCs group were lower than those in the Control group (P0.01). The content of 10 m RNA increased at third days and seventh days (P0.05). Compared with the EGFP-MSCs treatment group, the TNF- alpha m RNA content in the lung tissue of the H HGF-MSCs group decreased (P0.05) in the first day (P0.05), and the IL-10 m was increased at all time points. (11) the alveolar epithelial cells of the rats were compared with the third and seventh days. There was no significant difference between the AI and the Control groups in the alveolar epithelial cells in the.EGFP-MSCs treatment group (P0.05). (12) compared with the Control group, the survival rate of the EGFP-MSCs treatment group and the H HGF-MSCs treatment group increased significantly (P0.05). Conclusion: H HGF-MSCs and EGFP-MSCs can significantly reduce the inflammatory response to acute lung injury induced by rats and improve the survival rate. And survival time, and the therapeutic effect of H HGF-MSCs is better than that of EGFP-MSCs.

【学位授予单位】：福建医科大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R614

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