本文選題：肝細胞生長因子 + 骨髓間充質(zhì)干細胞��； 參考：《福建醫(yī)科大學》2016年碩士論文

【摘要】：目的:觀察人肝細胞生長因子基因修飾的骨髓間充質(zhì)干細胞對脂多糖誘導的大鼠急性肺損傷的影響。方法:7周齡近交系雄性F344大鼠60只,體重200~250 g,將其隨機分為3組(n=20),對照組(Control組)、空載體慢病毒轉(zhuǎn)導的骨髓間充質(zhì)干細胞(EGFP-MSCs)治療組和人肝細胞生長因子基因轉(zhuǎn)導的骨髓間充質(zhì)干細胞(h HGF-MSCs)治療組。所有大鼠均采用脂多糖(Lipopolysaccharide,LPS)7 mg/kg氣管滴注建立急性肺損傷模型。Control組大鼠經(jīng)氣管給予LPS后立即經(jīng)尾靜脈注射低糖培養(yǎng)基(DMEM)1ml,EGFP-MSCs治療組大鼠經(jīng)氣管給予LPS后立即經(jīng)尾靜脈注射含5×105個EGFP-MSCs的DMEM 1ml,h HGF-MSCs治療組大鼠經(jīng)氣管給予LPS后立即經(jīng)尾靜脈注射含5×105個h HGF-MSCs的DMEM 1ml。細胞注射后7天,激光共聚焦顯微鏡下觀察DAPI標記的MSCs肺組織分布情況。LPS注射后1天,3天,7天,每時點隨機抽取6只大鼠,留取標本,測定肺濕干比(wet-to-dry ratio,W/D),肺泡灌洗液(bronchoalveolar lavage fluid,BALF)中總細胞計數(shù)和中性粒細胞計數(shù)及肺組織病理損傷嚴重度評分;化學比色法測定肺組織髓過氧化物酶(myeloperoxidase,MPO)活性;BCA法測定大鼠BALF中的總蛋白濃度;酶聯(lián)免疫吸附測定法(ELISA)檢測大鼠血清中人HGF和鼠HGF蛋白水平以及肺組織勻漿和肺泡灌洗液中腫瘤壞死因子α(TNF-α)、白介素10(IL-10)、白介素6(IL-6)蛋白含量;實時熒光定量聚合酶鏈式反應(RT-q PCR)測定大鼠肺組織勻漿中TNF-αm RNA、IL-10 m RNA、IL-6 m RNA的表達;DNA斷裂的原位末端標記法(TUNEL)測定肺泡上皮細胞的凋亡指數(shù)(apoptotic index,AI)。另取F334大鼠36只,隨機分為三組(n=12),分組及建模情況同上,LPS注射后90天內(nèi)進行生存分析。結果:(1)EGFP-MSCs治療組及h HGF-MSCs治療組大鼠肺間質(zhì)和肺泡腔可見綠色熒光蛋白陽性細胞分布。(2)h HGF-MSCs治療組大鼠血清中表達h HGF蛋白,Control組和EGFP-MSCs治療組未表達h HGF蛋白。與Control組相比,EGFP-MSCs治療組大鼠血清中r HGF在第3天明顯增高(P0.05),h HGF-MSCs治療組大鼠血清中r HGF在第1天和第3天明顯增高(P0.05,P0.01)。與EGFP-MSCs治療組相比,h HGF-MSCs治療組大鼠血清中r HGF在第3天明顯增高(P0.05)。(3)與Control組相比,EGFP-MSCs治療組及h HGF-MSCs治療組大鼠肺部病理損傷評分在各時點均降低(P0.05,P0.01),差異均具有統(tǒng)計學意義。與EGFP-MSCs治療組相比,h HGF-MSCs治療組大鼠肺部病理損傷評分在第3天顯著降低(P0.05)。(4)與Control組相比,EGFP-MSCs治療組及h HGF-MSCs治療組大鼠肺組織W/D在各時間點均明顯降低(P0.01)。(5)與Control組相比,EGFP-MSCs治療組及h HGF-MSCs治療組大鼠肺泡灌洗液總蛋白含量在第7天明顯降低(P0.05)。(6)與Control組相比,h HGF-MSCs治療組大鼠肺泡灌洗液總細胞計數(shù)和中性粒細胞計數(shù)在第3天和第7天均明顯降低(P0.05,P0.01)。與Control組相比,EGFP-MSCs治療組大鼠肺泡灌洗液總細胞計數(shù)和中性粒細胞計數(shù)在第7天明顯降低(P0.01)。與EGFP-MSCs治療組相比,h HGF-MSCs治療組肺泡灌洗液總細胞計數(shù)和中性粒細胞計數(shù)在第1天均降低(P0.01)。(7)h HGF-MSCs治療組MPO活性值在第7天小于Control組(P0.05)。Control組和EGFP-MSCs治療組在各時間點MPO活性值沒有顯著差別。(8)與Control組相比EGFP-MSCs治療組和h HGF-MSCs治療組大鼠BALF中TNF-α和IL-6含量在第1天和第7天均降低(P0.05,P0.01)。與Control組相比,EGFP-MSCs治療組大鼠BALF中IL-10含量在第1天和第7天增高(P0.01)。h HGF-MSCs治療組的BALF中IL-10與Control組相比,在各時點均增高(P0.05)。與EGFP-MSCs治療組相比,h HGF-MSCs治療組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)與Control組相比,h HGF-MSCs治療組大鼠肺組織中TNF-α、IL-6含量在各時點均降低(P0.01),IL-10含量在各時點均增高(P0.01)。與Control組相比,EGFP-MSCs治療組大鼠肺組織中TNF-α、IL-6含量在各時點均降低(P0.01),IL-10在第1天增高(P0.05)。與EGFP-MSCs治療組相比h HGF-MSCs治療組TNF-α值在第一天顯著低于MSC治療組(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)與Control組相比,h HGF-MSCs治療組大鼠肺組織中TNF-α和IL-6m RNA含量在各時點均降低(P0.01),IL-10 m RNA在各時點均增高(P0.01)。與Control組相比,TNF-α和IL-6 m RNA含量在各時點均降低(P0.05,P0.01),IL-10 m RNA含量在第3天和第7天增高(P0.05)。與EGFP-MSCs治療組相比,h HGF-MSCs治療組大鼠肺組織中TNF-αm RNA含量在第一天降低(P0.05),IL-10 m RNA含量在各時點均增高(P0.05,P0.01)。(11)與Control組相比,h HGF-MSCs治療組大鼠肺泡上皮細胞AI在第3天和第7天降低(P0.05)。EGFP-MSCs治療組大鼠肺泡上皮細胞AI與Control組無顯著差別。(12)與Control組相比,EGFP-MSCs治療組和h HGF-MSCs治療組大鼠生存率明顯增加(P0.05)。結論:h HGF-MSCs和EGFP-MSCs均可顯著減輕LPS誘發(fā)的大鼠急性肺損傷的炎癥反應,提高生存率及生存時間,并且h HGF-MSCs的治療效果優(yōu)于EGFP-MSCs的治療效果。
[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.

【學位授予單位】：福建醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2016
【分類號】：R614

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