从细胞凋亡途径研究骨髓间充质干细胞抗急性肺损伤作用机制
发布时间：2018-08-30 08:37
【摘要】：[研究背景和目的] 急性肺损伤(acute lung injury,ALI)是临床上常见的危重病,起病急剧,发展迅速,病情凶险,预后恶劣,死亡率高,发病机制复杂,目前临床上缺乏有效的治疗措施。因此,寻找新的治疗策略是目前临床上治疗ALI亟待解决的重要问题。 肺泡上皮细胞丧失和过度凋亡是ALI发展过程中的重要事件,决定ALI发生、发展和预后,在ALI发生、发展过程中起重要作用。因此,抑制肺泡上皮细胞凋亡和促进肺泡上皮修复被认为是目前ALI治疗的重要目标和作用的细胞靶点。 骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMMSC)是一种具有自我更新和多向分化潜能的多功能干细胞,目前被认为是一种较为理想的治疗性细胞,在细胞替代治疗、器官组织的修复、再生和重构以及基因治疗等方面具有广阔临床应用前景。新近研究表明,输入外源性或迁移的内源性干细胞(stem cells)能定植在损伤的肺组织内,参与受损的肺组织的修复和重构,能够治疗ALI/ARDS.肺纤维化、慢性阻塞性肺疾病(chrocnic obstructive pulmonary disease, COPD),这为急、慢性肺部疾病治疗提供了新的策略和新思路。然而,干细胞治疗急、慢性肺部疾病作用和机制尚不清楚。 综上所述,我们认为BMMSC可以作为ALI的治疗性细胞,推测其治疗作用和机制可能与抗炎症、抗肺水肿、抗凋亡及其旁分泌效应有关。为了证明这-假说,本研究在LPS诱导的小鼠ALI模型上研究BMMSC抗炎症、抗肺水肿和抗凋亡作用,在肺泡Ⅱ细胞与BMMSC共培养体系上研究BMMSC抗肺泡Ⅱ上皮细胞凋亡的旁分泌效应机制,为呼吸性疾病提供新的治疗策略和作用的靶点。 [研究方法] 1.BMMSC体外生长生物学特性研究方法 (1)采用全骨髓贴壁法体外分离培养BMMSC; (2)光学显微镜下计数细胞生长数量、观测细胞培养形态和定向诱导分化的细胞形态； (3)流式细胞仪分析BMMSC表面标志物和细胞周期。 2.在整体水平上,研究BMMSC的抗炎、抗水肿和抗凋亡作用的评价方法： (1)气管内滴注LPS建立急性肺损伤小鼠模型,实验随机分为3组(每组n=18只)：①对照组：经气管滴注PBS,30min后尾静脉注射100μl的PBS；②损伤组：经气管滴注LPS,30min后尾静脉注射100μ1的PBS；③治疗组：经气管滴注LPS,30min后尾静脉注入100μl BMMSC的PBS悬液; (2)Gustavo Matute-Bello病理学评分方法光镜下观察肺组织损伤程度； (3)血气分析仪检测动脉血氧分压(PaO2)； (4)荧光显微镜下观察BMMSC在肺内的定植； (5)常规方法测定肺湿干(W/D)比值； (6)ELISA测定肺组织髓过氧化物酶水平和肺泡灌洗液(BALF)中IL-6、TNF-a、IL-10含量； (7)考马斯亮蓝(CBB)染料结合法测定BALF中蛋白含量; (8)血细胞计数仪检测BALF中中性粒细胞数量； (9)TUNEL原位检测肺组织细胞凋亡率； (10)免疫组化染色检测肺组织肺泡上皮细胞caspase3、bcl-2和bax表达水平; (11)透射电镜观察肺泡Ⅱ性上皮细胞凋亡。 3.在细胞水平上,研究BMMSC抗肺泡Ⅱ型上皮细胞凋亡作用机制的评价方法 (1)选用人肺泡上皮细胞株(A549细胞株)与人骨髓间充质干细胞株(第3代hBMMSC株),建立Transwell非接触分层共培养体系。实验分4组(每组6孔,5×106细胞/孔)：①空白对照组,PBS+A549;②LPS损伤组,LPS+A549;③BMMSC对照组,PBS+A549+BMMSC④BMMSC干预组,LPS+A549+BMMSC。 (2)Annexin V/PI双染色流式细胞仪检测A549细胞凋亡率； (3)Western blotting检测A549细胞caspase3、bcl-2和bax蛋白表达水平; (4)透射电镜观察A549细胞凋亡特征性超微结构； (5)ELISA测定共培养液中的KGF、HGF含量； (6)荧光定量PCR检测BMMSC KGF和HGF mRNA水平。 [研究结果] 1.BMMSC体外生长生物学特性研究结果 (1)4-6周龄小鼠BMMSC在5%～10%胎牛血清浓度和5～8×107/L接种密度时生长活力强,生长速度最快,收获细胞数量最多,被设定为获得高浓度、高纯度、高活性的BMMSC的最佳条件； (2)在设定的最佳条件下,体外培养小鼠骨髓BMMSC呈集落性生长,大部分细胞为G0/G1期未分化细胞。细胞形态主要呈梭形,类似成纤维细胞,其表达CD14-、CD34-、CD45和CD90+、CD105+、CD106+,具有贴壁生长特性和多向分化能力,符合间充质干细胞的体外生长的主要生物学特性。 2.BMMSC在急性肺损伤中的抗炎、抗水肿和抗凋亡作用研究结果 (1)BMMS能够定植于LPS致肺损伤小鼠的肺组织内,减轻LPS致ALI小鼠的肺组织结构损伤程度,提高动脉血Pa02水平； (2)小鼠BMMSC能降低LPS致ALI小鼠的肺组织MPO含量和PMN数量、IL-6和TNF-α水平,提高IL-10含量; (3)小鼠BMMSC能降低LPS致ALI小鼠肺W/D比值和肺泡内蛋白质含量； (4)小鼠BMMSC能够减少LPS致ALI小鼠肺组织肺泡上皮细胞凋亡及促凋亡蛋白caspase3和bax的表达,增加抗凋亡蛋白bcl-2表达。 3.BMMSC体外抗肺泡Ⅱ型上皮细胞凋亡作用机制研究结果 (1)LPS能体外诱导体外培养的A549细胞凋亡增加,最适的浓度和最佳时间为10μg/ml LPS作用于A549细胞24h; (2)LPS能促进A549细胞促凋亡蛋白caspase-3、bax表达,降低抗凋亡蛋白bcl-2表达； (3)BMMSC能促进A549细胞抗凋亡蛋白bcl-2表达,抑制促凋亡蛋白caspase-3、bax蛋白表达； (4)与LPS诱导的A549细胞共培养,BMMSC能够旁分泌生长因子KGF和HGF,增加KGF和HGF mRNA表达。 [结论] 1.在设定的4-6周龄小鼠,5%-10%胎牛血清浓度和5-8×107/L接种密度的最佳条件下,小鼠BMMSC表达CD14-.CD34-.CD45和CD90+.CD105+.CD106+,具有贴壁生长特性和多向分化能力,符合间充质干细胞体外生长的主要生物学特性。 2.小鼠BMMSC可定植在LPS致ALI小鼠的肺组织内,能减轻其肺组织结构损伤程度和改善肺功能,具有抗炎症反应和抗肺水肿和抗肺泡上皮细胞凋亡的作用。 3.与LPS诱导的A549细胞共培养,BMMSC能促进A549细胞表达抗凋亡蛋白bcl-2和抑制促凋亡蛋白caspase-3和bax的表达,增加旁分泌生长因子KGF、HGF和KGF.HGF mRNA表达水平,具有抗细胞凋亡和旁分泌作用。 4BMMSC旁分泌生长因子KGF和HGF可能是其抗细胞凋亡作用的机制之一
[Abstract]:[background and purpose]
Acute lung injury (ALI) is a common clinical critical disease with a rapid onset, rapid development, dangerous condition, poor prognosis, high mortality and complex pathogenesis. At present, there is no effective treatment in clinic. Therefore, it is an important problem to find a new treatment strategy to treat ALI.
Loss of alveolar epithelial cells and excessive apoptosis are important events in the development of ALI, which determine the occurrence, development and prognosis of ALI and play an important role in the occurrence and development of ALI.
Bone marrow mesenchymal stem cells (BMMSC) are a kind of multifunctional stem cells with self-renewal and multi-differentiation potential. At present, BMMSC is considered to be an ideal therapeutic cell. It has broad clinical applications in cell replacement therapy, organ tissue repair, regeneration and reconstruction, and gene therapy. Recent studies have shown that exogenous or migrating stem cells can be implanted in damaged lung tissue, participate in the repair and reconstruction of damaged lung tissue, and can treat ALI/ARDS. Pulmonary fibrosis, chronic obstructive pulmonary disease (COPD), which is the treatment of acute and chronic lung diseases. Therapy offers new strategies and new ideas. However, the role and mechanism of stem cell therapy for acute and chronic lung diseases are still unclear.
In conclusion, we suggest that BMMSC can be used as a therapeutic cell for ALI, and its therapeutic effect and mechanism may be related to anti-inflammation, anti-pulmonary edema, anti-apoptosis and paracrine effect. To study the paracrine effect of BMMSC on apoptosis of alveolar II epithelial cells in co-culture system with BMMSC, and to provide a new therapeutic strategy and target for respiratory diseases.
[research methods]
Research methods of biological characteristics of 1.BMMSC in vitro growth
(1) BMMSC was isolated and cultured in vitro by whole bone marrow adherent method.
(2) The number of cell growth was counted under optical microscope, and the morphology of cell culture and differentiation were observed.
(3) flow cytometry was used to analyze the surface markers and cell cycle of BMMSC.
2. to evaluate the anti-inflammatory, anti edema and anti apoptotic effects of BMMSC on the whole.
(1) Acute lung injury model was established by intratracheal LPS infusion in mice, and the experiment was randomly divided into three groups (n = 18 mice in each group): control group: PBS was dripped through trachea, 100 ml PBS was injected into tail vein after 30 minutes; injury group: LPS was dripped through trachea, 100 mu 1 PBS was injected into tail vein after 30 minutes; treatment group: LPS was dripped through trachea, and 100 mu 1 PBS was injected into tail vein after 30 minutes. PBS suspension of the L BMMSC.
(2) Gustavo Matute-Bello pathological grading method was used to observe the degree of lung tissue injury under light microscope.
(3) arterial blood oxygen partial pressure (PaO2) was detected by blood gas analyzer.
(4) the colonization of BMMSC in lung was observed under fluorescence microscope.
(5) the ratio of lung wet dry (W/D) was measured by routine method.
(6) ELISA determined the level of myeloperoxidase in lung tissue and IL-6, TNF-a and IL-10 in bronchoalveolar lavage fluid (BALF).
(7) Coomassie brilliant blue (CBB) dye binding assay was used to determine the protein content in BALF.
(8) the number of neutrophils in BALF was detected by blood cell counter.
(9) the apoptosis rate of lung tissue was detected by TUNEL in situ.
(10) immunohistochemical staining was used to detect the expression levels of Caspase3, Bcl-2 and Bax in alveolar epithelial cells of lung tissue.
(11) apoptosis of alveolar epithelial cells was observed by transmission electron microscope.
3. to evaluate the mechanism of BMMSC on apoptosis of alveolar type II epithelial cells at cellular level.
(1) Transwell non-contact stratified co-culture system was established by using human alveolar epithelial cell line (A549 cell line) and human bone marrow mesenchymal stem cell line (hBMMSC line of the third generation). The experiment was divided into four groups (6 holes in each group, 5 *106 cells/hole): blank control group, PBS + A549; LPS + A549; LPS + A549; BMMSC control group, PBS + A549 + MSC intervention group, LPS + A + A549 + MSC; BMMSC intervention group. 549+BMMSC.
(2) Annexin V/PI double staining flow cytometry was used to detect the apoptosis rate of A549 cells.
(3) Western blotting was used to detect the expression level of Caspase3, Bcl-2 and Bax protein in A549 cells.
(4) transmission electron microscopy was used to observe the characteristic ultrastructure of apoptosis in A549 cells.
(5) ELISA was used to determine the content of KGF and HGF in co culture medium.
(6) fluorescence quantitative PCR was used to detect BMMSC KGF and HGF mRNA levels.
[results]
Biological characteristics of 1.BMMSC in vitro growth
(1) BMMSC of 4-6 week-old mice grew vigorously at the concentration of 5%-10% fetal bovine serum and the density of 5-8 *107/L. The growth rate was the fastest and the number of harvested cells was the largest. BMMSC with high concentration, purity and activity was set as the best condition.
(2) Under the optimal conditions, BMMSC in vitro grew in colonies, most of which were undifferentiated cells in G0/G1 phase. The cells were spindle-shaped, similar to fibroblasts, and expressed CD14-, CD34-, CD45 and CD90+, CD105+, CD106+, which had the characteristics of adherent growth and multi-directional differentiation, and accorded with mesenchymal stem cells in vitro. The main biological characteristics of growth.
Anti inflammatory, anti edema and anti apoptotic effects of 2.BMMSC in acute lung injury
(1) BMMS can be implanted into the lung tissue of LPS-induced lung injury mice, reduce the degree of lung tissue damage in LPS-induced ALI mice, and increase the level of Pa02 in arterial blood.
(2) BMMSC could decrease the content of MPO, the quantity of PMN, the levels of IL-6 and TNF-alpha, and increase the content of IL-10 in lung tissue of ALI mice induced by LPS.
(3) mouse BMMSC can reduce the W/D ratio and protein content in alveolar of LPS induced ALI mice.
(4) BMMSC could decrease the apoptosis of alveolar epithelial cells and the expression of pro-apoptotic proteins caspase-3 and bax, and increase the expression of anti-apoptotic protein Bcl-2 in ALI mice induced by LPS.
Effect of 3.BMMSC on apoptosis of alveolar type II epithelial cells in vitro
(1) LPS could induce apoptosis of A549 cells in vitro. The optimal concentration and time of LPS treatment were 10 ug/ml for 24 hours.
(2) LPS can promote the expression of Pro apoptotic protein caspase-3 and Bax in A549 cells, and decrease the expression of anti apoptotic protein bcl-2.
(3) BMMSC could promote the expression of anti-apoptotic protein Bcl-2 and inhibit the expression of pro-apoptotic protein caspase-3 and Bax in A549 cells.
(4) BMMSC can paracrine growth factor KGF and HGF and increase the expression of KGF and HGF mRNA in A549 cells co-cultured with LPS.
[Conclusion]
1. BMMSC expressed CD14-. CD34-. CD45 and CD90+. CD105+. CD106+ under the optimal conditions of 4-6 weeks old mice, 5% - 10% fetal bovine serum concentration and 5-8 *107/L inoculation density. BMMSC had the characteristics of adherent growth and multidirectional differentiation, which accorded with the main biological characteristics of mesenchymal stem cell growth in vitro.
2. BMMSC can be implanted in the lung tissue of ALI mice induced by LPS. It can alleviate the degree of lung tissue damage and improve lung function. It has the effects of anti-inflammatory reaction, anti-pulmonary edema and anti-alveolar epithelial cell apoptosis.
3. BMMSC can promote the expression of anti-apoptotic protein Bcl-2 and inhibit the expression of pro-apoptotic protein caspase-3 and bax, increase the expression of paracrine growth factor KGF, HGF and KGF.
4BMMSC paracrine growth factor KGF and HGF may be one of the mechanisms of its anti apoptotic effect.
【学位授予单位】：昆明医科大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R363
本文编号：2212552
[Abstract]:[background and purpose]
Acute lung injury (ALI) is a common clinical critical disease with a rapid onset, rapid development, dangerous condition, poor prognosis, high mortality and complex pathogenesis. At present, there is no effective treatment in clinic. Therefore, it is an important problem to find a new treatment strategy to treat ALI.
Loss of alveolar epithelial cells and excessive apoptosis are important events in the development of ALI, which determine the occurrence, development and prognosis of ALI and play an important role in the occurrence and development of ALI.
Bone marrow mesenchymal stem cells (BMMSC) are a kind of multifunctional stem cells with self-renewal and multi-differentiation potential. At present, BMMSC is considered to be an ideal therapeutic cell. It has broad clinical applications in cell replacement therapy, organ tissue repair, regeneration and reconstruction, and gene therapy. Recent studies have shown that exogenous or migrating stem cells can be implanted in damaged lung tissue, participate in the repair and reconstruction of damaged lung tissue, and can treat ALI/ARDS. Pulmonary fibrosis, chronic obstructive pulmonary disease (COPD), which is the treatment of acute and chronic lung diseases. Therapy offers new strategies and new ideas. However, the role and mechanism of stem cell therapy for acute and chronic lung diseases are still unclear.
In conclusion, we suggest that BMMSC can be used as a therapeutic cell for ALI, and its therapeutic effect and mechanism may be related to anti-inflammation, anti-pulmonary edema, anti-apoptosis and paracrine effect. To study the paracrine effect of BMMSC on apoptosis of alveolar II epithelial cells in co-culture system with BMMSC, and to provide a new therapeutic strategy and target for respiratory diseases.
[research methods]
Research methods of biological characteristics of 1.BMMSC in vitro growth
(1) BMMSC was isolated and cultured in vitro by whole bone marrow adherent method.
(2) The number of cell growth was counted under optical microscope, and the morphology of cell culture and differentiation were observed.
(3) flow cytometry was used to analyze the surface markers and cell cycle of BMMSC.
2. to evaluate the anti-inflammatory, anti edema and anti apoptotic effects of BMMSC on the whole.
(1) Acute lung injury model was established by intratracheal LPS infusion in mice, and the experiment was randomly divided into three groups (n = 18 mice in each group): control group: PBS was dripped through trachea, 100 ml PBS was injected into tail vein after 30 minutes; injury group: LPS was dripped through trachea, 100 mu 1 PBS was injected into tail vein after 30 minutes; treatment group: LPS was dripped through trachea, and 100 mu 1 PBS was injected into tail vein after 30 minutes. PBS suspension of the L BMMSC.
(2) Gustavo Matute-Bello pathological grading method was used to observe the degree of lung tissue injury under light microscope.
(3) arterial blood oxygen partial pressure (PaO2) was detected by blood gas analyzer.
(4) the colonization of BMMSC in lung was observed under fluorescence microscope.
(5) the ratio of lung wet dry (W/D) was measured by routine method.
(6) ELISA determined the level of myeloperoxidase in lung tissue and IL-6, TNF-a and IL-10 in bronchoalveolar lavage fluid (BALF).
(7) Coomassie brilliant blue (CBB) dye binding assay was used to determine the protein content in BALF.
(8) the number of neutrophils in BALF was detected by blood cell counter.
(9) the apoptosis rate of lung tissue was detected by TUNEL in situ.
(10) immunohistochemical staining was used to detect the expression levels of Caspase3, Bcl-2 and Bax in alveolar epithelial cells of lung tissue.
(11) apoptosis of alveolar epithelial cells was observed by transmission electron microscope.
3. to evaluate the mechanism of BMMSC on apoptosis of alveolar type II epithelial cells at cellular level.
(1) Transwell non-contact stratified co-culture system was established by using human alveolar epithelial cell line (A549 cell line) and human bone marrow mesenchymal stem cell line (hBMMSC line of the third generation). The experiment was divided into four groups (6 holes in each group, 5 *106 cells/hole): blank control group, PBS + A549; LPS + A549; LPS + A549; BMMSC control group, PBS + A549 + MSC intervention group, LPS + A + A549 + MSC; BMMSC intervention group. 549+BMMSC.
(2) Annexin V/PI double staining flow cytometry was used to detect the apoptosis rate of A549 cells.
(3) Western blotting was used to detect the expression level of Caspase3, Bcl-2 and Bax protein in A549 cells.
(4) transmission electron microscopy was used to observe the characteristic ultrastructure of apoptosis in A549 cells.
(5) ELISA was used to determine the content of KGF and HGF in co culture medium.
(6) fluorescence quantitative PCR was used to detect BMMSC KGF and HGF mRNA levels.
[results]
Biological characteristics of 1.BMMSC in vitro growth
(1) BMMSC of 4-6 week-old mice grew vigorously at the concentration of 5%-10% fetal bovine serum and the density of 5-8 *107/L. The growth rate was the fastest and the number of harvested cells was the largest. BMMSC with high concentration, purity and activity was set as the best condition.
(2) Under the optimal conditions, BMMSC in vitro grew in colonies, most of which were undifferentiated cells in G0/G1 phase. The cells were spindle-shaped, similar to fibroblasts, and expressed CD14-, CD34-, CD45 and CD90+, CD105+, CD106+, which had the characteristics of adherent growth and multi-directional differentiation, and accorded with mesenchymal stem cells in vitro. The main biological characteristics of growth.
Anti inflammatory, anti edema and anti apoptotic effects of 2.BMMSC in acute lung injury
(1) BMMS can be implanted into the lung tissue of LPS-induced lung injury mice, reduce the degree of lung tissue damage in LPS-induced ALI mice, and increase the level of Pa02 in arterial blood.
(2) BMMSC could decrease the content of MPO, the quantity of PMN, the levels of IL-6 and TNF-alpha, and increase the content of IL-10 in lung tissue of ALI mice induced by LPS.
(3) mouse BMMSC can reduce the W/D ratio and protein content in alveolar of LPS induced ALI mice.
(4) BMMSC could decrease the apoptosis of alveolar epithelial cells and the expression of pro-apoptotic proteins caspase-3 and bax, and increase the expression of anti-apoptotic protein Bcl-2 in ALI mice induced by LPS.
Effect of 3.BMMSC on apoptosis of alveolar type II epithelial cells in vitro
(1) LPS could induce apoptosis of A549 cells in vitro. The optimal concentration and time of LPS treatment were 10 ug/ml for 24 hours.
(2) LPS can promote the expression of Pro apoptotic protein caspase-3 and Bax in A549 cells, and decrease the expression of anti apoptotic protein bcl-2.
(3) BMMSC could promote the expression of anti-apoptotic protein Bcl-2 and inhibit the expression of pro-apoptotic protein caspase-3 and Bax in A549 cells.
(4) BMMSC can paracrine growth factor KGF and HGF and increase the expression of KGF and HGF mRNA in A549 cells co-cultured with LPS.
[Conclusion]
1. BMMSC expressed CD14-. CD34-. CD45 and CD90+. CD105+. CD106+ under the optimal conditions of 4-6 weeks old mice, 5% - 10% fetal bovine serum concentration and 5-8 *107/L inoculation density. BMMSC had the characteristics of adherent growth and multidirectional differentiation, which accorded with the main biological characteristics of mesenchymal stem cell growth in vitro.
2. BMMSC can be implanted in the lung tissue of ALI mice induced by LPS. It can alleviate the degree of lung tissue damage and improve lung function. It has the effects of anti-inflammatory reaction, anti-pulmonary edema and anti-alveolar epithelial cell apoptosis.
3. BMMSC can promote the expression of anti-apoptotic protein Bcl-2 and inhibit the expression of pro-apoptotic protein caspase-3 and bax, increase the expression of paracrine growth factor KGF, HGF and KGF.
4BMMSC paracrine growth factor KGF and HGF may be one of the mechanisms of its anti apoptotic effect.
【学位授予单位】：昆明医科大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R363
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相关期刊论文 前4条
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