携带缺氧诱导有丝分裂因子的骨髓间充质干细胞对急性肺损伤的作用研究
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摘要
实验目的
急性肺损伤(acute lung injury,ALI)存在发病率和病死率高的特点,目前临床各种治疗手段尚未能使其病死率明显下降,本研究以脂多糖(lipopolysaccharide,LPS)诱导炎症性肺损伤为研究对象,探讨炎症性肺损伤新的治疗措施及缺氧诱导有丝分裂因子(hypoxia-induced mitogenic factor,HIMF)这一肺损伤过程中新近发现的炎症因子的作用。由于HIMF旁分泌和自分泌作用于局部肺组织,本课题以骨髓间充质干细胞(mesenchymal stem cell,MSC)作为载体携带HIMF进入肺损伤局部,在除外HIMF旁分泌和自分泌作用的影响下观察单纯MSC和MSC联合HIMF在炎症损伤的不同阶段对疾病发展的影响和可能的作用。
实验方法
采用经典的全骨髓分离法从Balb/c小鼠中分离MSC进行原代培养、传代及性质鉴定。在确定分离的MSC为目的细胞后,与小鼠肺上皮细胞(MLE12)在炎症微环境中体外共培养,观察对MSC功能的影响;另一方面包装含目的基因HIMF的慢病毒过表达载体,转导MSC确定转导效率、转导最佳MOI值、转导时间等。在LPS诱导小鼠肺炎症性损伤的动物实验中,分为三组包括单纯生理盐水组、MSC组、MSC-HIMF组进行研究。观察不同时间点不同干预组中反映炎症损伤各指标(W/D、MPO、肺组织和BALF中炎症因子TNF-α和抗炎因子IL-10表达、BALF总蛋白水平)的变化和肺泡上皮细胞凋亡水平的变化,探讨MSC和HIMF在炎症性肺损伤中的作用。
实验结果
1.全骨髓贴壁法可以成功获得高纯度的MSC,并经流式细胞仪检测、多胚层分化实验得以验证和确认。
2.体外共培养炎症条件下MSC表达抗炎因子明显增加,具有免疫调节作用。
3.炎症条件下MSC细胞周期并没有发生显著变化,仍以G0/G1期细胞为主。
4.Lentivirus系统可以成功转导目的基因HIMF至MSC,并稳定表达HIMF。实验确定最佳MOI值为5(poly=5ug/ml),最佳体内植入时间为转导后第5天。GFP作为体内示踪剂操作简便且易于观察。
5.肺组织病理评分:MSCs治疗组的肺损伤程度较对照组明显减轻,差异有统计学意义;MSC-HIMF治疗组急性期肺损伤程度并没有减轻,并且较对照组相比,炎症持续存在,有慢性化趋势,部分肺组织存在胶原沉积增加。
6.肺炎症损伤指标:较对照组相比,MSC干预组表现为W/D降低、MPO活性下降、肺内和肺泡灌洗液中炎症因子含量的降低和抗炎因子表达的明显增加,提示MSC干预可明显减轻肺内的急性炎症反应;而MSC-HIMF组在治疗过程中W/D比值增加、MPO活性增强、肺内和肺泡灌洗液中炎症因子含量升高,并且维持到d14天,肺泡灌洗液中总蛋白含量明显增加。提示HIMF可促进肺内炎症反应,且炎症损伤持续存在。
7.MSC减轻肺炎症损伤的机制:MSC治疗组肺组织和BALF中均出现明显的炎症因子含量的降低和抗炎因子表达的明显增加,推测MSC在炎症性肺损伤中发挥保护作用的可能原因之一是其调节了肺组织局部的免疫状态,当然在急性期MSC可以与肺部受损细胞融合或转化发挥治疗作用。
结论
1.MSC可成功体外分离纯化、维持细胞功能,慢病毒转导MSC可作为一种可行的携带外源基因的方法。
2.MSC干预LPS诱导的炎症损伤可明显减轻肺内的急性炎症反应,可能机制之一为调节肺组织局部的免疫状态。
3.HIMF可促进肺内的炎症反应,且炎症反应呈现慢性化趋势。
Objective
Acute lung injury(ALI),with its high incidence rate and mortality,lacks specific treatment strategies currently.Lipopolysaccharide(LPS) plays important role in the pathogenesis of ALI.The research explored intervention roles of mesenchymal stem cells(MSC) in LPS-induced inflammatory lung injury and role of hypoxia-induced mitogenic factor(HIMF).As HIMF was an autocrine and paracrine cytokine,MSCs were designed as a vehicle carrying HIMF into the locus of lung injury to explore the roles of MSC and HIMF in the different period of acute lung injury.
Methods
MSCs were successfully isolated from Balb/c mice,proliferated,subcultured and identified its phenotype and capacity to differentiate.After coculture with mouse lung epithelium 12(MLE12) under LPS exposure,the function of MSCs was observed.On the other hand,Lentiviral vector carrying HIMF was constructed and identified its efficiency in MSC transduction.In the animal model of ALI which LPS was inhaled,mice were administered with saline,MSC or MSC-HIMF.To evaluate its difference in ALI,several aspects were valued including W/D ratio,MPO activities, proinflammation and anti-inflammation cytokines,apoptosis index etc.
Results
1.MSCs were successfully isolated with high purity and were analyzed by FACS and multiple differentiation.
2.Under inflammatory environment,MSCs played immunomodulation role with increased anti-inflammation cytokines expression.
3.Inflammatory environment had no influence on MSC cycle,which was G0/G1 dominated.
4.Lentivirus vector succeeded in transducing HIMF into MSC with steady HIMF expression.
5.Pathological scores:MSC treatment can significantly alleviate lung damage compared with control,MSC-HIMF administration could not attenuate acute inflammatory reation,meanwhile it promoted chronic inflammation.
6.Lung injury factors:compared with control,MSC treatment showed marked reduction in W/D ratio,MPO activities,proinflammation cytokines level and elevation in anti-inflammation cytokines;MSC-HIMF administration increased W/D ratio,MPO activities,upregulated proinflammation cytokines level until 14 days with high protein content in BALF.
7.We speculated that MSC reduced lung injury by immunomodution of local immune state when observing reduced proinflammation cytokines level and increased anti-inflammation cytokines level in MSC treatment,and MSC can also exert its role by merging with injured lung cells or differentiating into lung cells.
Conclusions
1.MSC can be isolated and cultured for expansion in vitro;Lentivirus system is a proper way to carry exogenous gene.
2.MSC intervention can significantly attenuate acute lung injury by immunomodution and reduce fibrosis process in certain extent.
3.HIMF has proinflammatory effects in acute lung injury and promotes chronic inflammation.
急性肺损伤(acute lung injury,ALI)存在发病率和病死率高的特点,目前临床各种治疗手段尚未能使其病死率明显下降,本研究以脂多糖(lipopolysaccharide,LPS)诱导炎症性肺损伤为研究对象,探讨炎症性肺损伤新的治疗措施及缺氧诱导有丝分裂因子(hypoxia-induced mitogenic factor,HIMF)这一肺损伤过程中新近发现的炎症因子的作用。由于HIMF旁分泌和自分泌作用于局部肺组织,本课题以骨髓间充质干细胞(mesenchymal stem cell,MSC)作为载体携带HIMF进入肺损伤局部,在除外HIMF旁分泌和自分泌作用的影响下观察单纯MSC和MSC联合HIMF在炎症损伤的不同阶段对疾病发展的影响和可能的作用。
实验方法
采用经典的全骨髓分离法从Balb/c小鼠中分离MSC进行原代培养、传代及性质鉴定。在确定分离的MSC为目的细胞后,与小鼠肺上皮细胞(MLE12)在炎症微环境中体外共培养,观察对MSC功能的影响;另一方面包装含目的基因HIMF的慢病毒过表达载体,转导MSC确定转导效率、转导最佳MOI值、转导时间等。在LPS诱导小鼠肺炎症性损伤的动物实验中,分为三组包括单纯生理盐水组、MSC组、MSC-HIMF组进行研究。观察不同时间点不同干预组中反映炎症损伤各指标(W/D、MPO、肺组织和BALF中炎症因子TNF-α和抗炎因子IL-10表达、BALF总蛋白水平)的变化和肺泡上皮细胞凋亡水平的变化,探讨MSC和HIMF在炎症性肺损伤中的作用。
实验结果
1.全骨髓贴壁法可以成功获得高纯度的MSC,并经流式细胞仪检测、多胚层分化实验得以验证和确认。
2.体外共培养炎症条件下MSC表达抗炎因子明显增加,具有免疫调节作用。
3.炎症条件下MSC细胞周期并没有发生显著变化,仍以G0/G1期细胞为主。
4.Lentivirus系统可以成功转导目的基因HIMF至MSC,并稳定表达HIMF。实验确定最佳MOI值为5(poly=5ug/ml),最佳体内植入时间为转导后第5天。GFP作为体内示踪剂操作简便且易于观察。
5.肺组织病理评分:MSCs治疗组的肺损伤程度较对照组明显减轻,差异有统计学意义;MSC-HIMF治疗组急性期肺损伤程度并没有减轻,并且较对照组相比,炎症持续存在,有慢性化趋势,部分肺组织存在胶原沉积增加。
6.肺炎症损伤指标:较对照组相比,MSC干预组表现为W/D降低、MPO活性下降、肺内和肺泡灌洗液中炎症因子含量的降低和抗炎因子表达的明显增加,提示MSC干预可明显减轻肺内的急性炎症反应;而MSC-HIMF组在治疗过程中W/D比值增加、MPO活性增强、肺内和肺泡灌洗液中炎症因子含量升高,并且维持到d14天,肺泡灌洗液中总蛋白含量明显增加。提示HIMF可促进肺内炎症反应,且炎症损伤持续存在。
7.MSC减轻肺炎症损伤的机制:MSC治疗组肺组织和BALF中均出现明显的炎症因子含量的降低和抗炎因子表达的明显增加,推测MSC在炎症性肺损伤中发挥保护作用的可能原因之一是其调节了肺组织局部的免疫状态,当然在急性期MSC可以与肺部受损细胞融合或转化发挥治疗作用。
结论
1.MSC可成功体外分离纯化、维持细胞功能,慢病毒转导MSC可作为一种可行的携带外源基因的方法。
2.MSC干预LPS诱导的炎症损伤可明显减轻肺内的急性炎症反应,可能机制之一为调节肺组织局部的免疫状态。
3.HIMF可促进肺内的炎症反应,且炎症反应呈现慢性化趋势。
Objective
Acute lung injury(ALI),with its high incidence rate and mortality,lacks specific treatment strategies currently.Lipopolysaccharide(LPS) plays important role in the pathogenesis of ALI.The research explored intervention roles of mesenchymal stem cells(MSC) in LPS-induced inflammatory lung injury and role of hypoxia-induced mitogenic factor(HIMF).As HIMF was an autocrine and paracrine cytokine,MSCs were designed as a vehicle carrying HIMF into the locus of lung injury to explore the roles of MSC and HIMF in the different period of acute lung injury.
Methods
MSCs were successfully isolated from Balb/c mice,proliferated,subcultured and identified its phenotype and capacity to differentiate.After coculture with mouse lung epithelium 12(MLE12) under LPS exposure,the function of MSCs was observed.On the other hand,Lentiviral vector carrying HIMF was constructed and identified its efficiency in MSC transduction.In the animal model of ALI which LPS was inhaled,mice were administered with saline,MSC or MSC-HIMF.To evaluate its difference in ALI,several aspects were valued including W/D ratio,MPO activities, proinflammation and anti-inflammation cytokines,apoptosis index etc.
Results
1.MSCs were successfully isolated with high purity and were analyzed by FACS and multiple differentiation.
2.Under inflammatory environment,MSCs played immunomodulation role with increased anti-inflammation cytokines expression.
3.Inflammatory environment had no influence on MSC cycle,which was G0/G1 dominated.
4.Lentivirus vector succeeded in transducing HIMF into MSC with steady HIMF expression.
5.Pathological scores:MSC treatment can significantly alleviate lung damage compared with control,MSC-HIMF administration could not attenuate acute inflammatory reation,meanwhile it promoted chronic inflammation.
6.Lung injury factors:compared with control,MSC treatment showed marked reduction in W/D ratio,MPO activities,proinflammation cytokines level and elevation in anti-inflammation cytokines;MSC-HIMF administration increased W/D ratio,MPO activities,upregulated proinflammation cytokines level until 14 days with high protein content in BALF.
7.We speculated that MSC reduced lung injury by immunomodution of local immune state when observing reduced proinflammation cytokines level and increased anti-inflammation cytokines level in MSC treatment,and MSC can also exert its role by merging with injured lung cells or differentiating into lung cells.
Conclusions
1.MSC can be isolated and cultured for expansion in vitro;Lentivirus system is a proper way to carry exogenous gene.
2.MSC intervention can significantly attenuate acute lung injury by immunomodution and reduce fibrosis process in certain extent.
3.HIMF has proinflammatory effects in acute lung injury and promotes chronic inflammation.
引文
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1.Qiangsong Tong,Liduan Zheng,Li Lin,et al.VEGF is upregulated by hypoxia-induced mitogenic factor via the PI-3K/Akt-NF-κB signaling pathway.Respir Res.2006,7:37-51
2.Qiangsong Tong,Liduan Zheng,Qiaohua Kang,et al.Upregulation of hypoxia-induced mitogenic factor in bacterial lipopolysaccharide-induced acute lung injury.FEBS Lett.2006,580(9):2207-2215
3.Aparna Reniguntaa,Christiane Hilda,Frank Rosea,et al.Human relmb is a mitogenic factor in lung cells and induced in hypoxia.FEBS Lett.2006,580(3):900-903
4.Xingwu Teng,Dechun Li,Hunter C.FIZZ1/RELMa,a Novel Hypoxia-lnduced Mitogenic Factor in Lung With Vasoconstdctive and Angiogenic Properties.Circ Res.2003,92(10):1065-1067
5. Qiangsong Tong, Liduan Zheng, Jeffrey Dodd-o,et al.Hypoxia-Induced Mitogenic Factor Modulates Surfactant Protein B and C Expression in Mouse Lung. Am J Respir Cell Mol Biol.2006, 34(1): 28-38
6. Tianju Liu, Hong Jin, Matthew Ullenbruch,et al.Regulation of Found in Inflammatory Zone 1 Expression in Bleomycin-Induced Lung Fibrosis: Role of IL-4/IL-13 and Mediation via STAT-6.J Immunol. 2004, 173(5): 3425-3431
7. Kazuyo Yamaji-Kegan, Qingning Su, Daniel J,et al. Hypoxia-induced mitogenic factor has proangiogenic and proinflammatory effects in the lung via VEGF and VEGF receptor-2. Am J Physiol Lung Cell Mol Physiol. 2006, 291(6): 1159-1168
2. M Dominici1, K Le Blanc2,I Mueller3,et al.Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy.2006, 8(4): 315-317
3. Aparna Reniguntaa, Christiane Hilda, Frank Rosea,et al.Human relmb is a mitogenic factor in lung cells and induced in hypoxia. FEBS Lett. 2006, 580(3): 900-903
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1.Qiangsong Tong,Liduan Zheng,Li Lin,et al.VEGF is upregulated by hypoxia-induced mitogenic factor via the PI-3K/Akt-NF-κB signaling pathway.Respir Res.2006,7:37-51
2.Qiangsong Tong,Liduan Zheng,Qiaohua Kang,et al.Upregulation of hypoxia-induced mitogenic factor in bacterial lipopolysaccharide-induced acute lung injury.FEBS Lett.2006,580(9):2207-2215
3.Aparna Reniguntaa,Christiane Hilda,Frank Rosea,et al.Human relmb is a mitogenic factor in lung cells and induced in hypoxia.FEBS Lett.2006,580(3):900-903
4.Xingwu Teng,Dechun Li,Hunter C.FIZZ1/RELMa,a Novel Hypoxia-lnduced Mitogenic Factor in Lung With Vasoconstdctive and Angiogenic Properties.Circ Res.2003,92(10):1065-1067
5. Qiangsong Tong, Liduan Zheng, Jeffrey Dodd-o,et al.Hypoxia-Induced Mitogenic Factor Modulates Surfactant Protein B and C Expression in Mouse Lung. Am J Respir Cell Mol Biol.2006, 34(1): 28-38
6. Tianju Liu, Hong Jin, Matthew Ullenbruch,et al.Regulation of Found in Inflammatory Zone 1 Expression in Bleomycin-Induced Lung Fibrosis: Role of IL-4/IL-13 and Mediation via STAT-6.J Immunol. 2004, 173(5): 3425-3431
7. Kazuyo Yamaji-Kegan, Qingning Su, Daniel J,et al. Hypoxia-induced mitogenic factor has proangiogenic and proinflammatory effects in the lung via VEGF and VEGF receptor-2. Am J Physiol Lung Cell Mol Physiol. 2006, 291(6): 1159-1168