WO2016107563A1

WO2016107563A1 - Fatty mesenchymal progenitor cell complex for treating upper respiratory tract illnesses

Info

Publication number: WO2016107563A1
Application number: PCT/CN2015/099568
Authority: WO
Inventors: 刘佳; 曹卫; 戴成祥; 蔡松柏; 郑成小; 张丽; 王立群; 汪文; 王飞; 孙中伟
Original assignee: 西比曼生物科技（上海）有限公司; 西比曼生物科技（无锡）有限公司
Priority date: 2014-12-29
Filing date: 2015-12-29
Publication date: 2016-07-07

Abstract

Provided in the present invention is a use of fatty mesenchymal progenitor cells in the prevention or treatment of upper respiratory tract illnesses (e.g., chronic obstructive pulmonary diseases or asthma). The fatty mesenchymal progenitor cells can be used for preparing a pharmaceutical composition for treating upper respiratory tract illnesses. The pharmaceutical composition is administrated to the subject in need, and can improve the indexes thereof (pulmonary function, airway inflammation, quality of life score, airway hyperreactivity, etc.), thereby promoting the repair of pathological changes in the upper respiratory tract.

Description

一种用于治疗上呼吸道疾病的脂肪间充质祖细胞复合物Adipose mesenchymal progenitor cell complex for treating upper respiratory tract diseases

技术领域Technical field

本发明涉及脂肪干细胞应用领域，具体地，本发明提供了一种脂肪间充质祖细胞用于治疗慢性阻塞性肺疾病的用途。The present invention relates to the field of application of adipose stem cells, and in particular to the use of adipose mesenchymal progenitor cells for the treatment of chronic obstructive pulmonary diseases.

背景技术Background technique

慢性阻塞性肺疾病(chronic obstructive pulmonary disease,COPD)是一种严重危害人类健康的常见病、多发病，严重影响患者的生命质量，病死率高，并给患者及其家庭以及社会带来沉重的经济负担。世界银行和世界卫生组织的资料表明，至2020年，慢阻肺将位居世界疾病经济的第5位。Chronic obstructive pulmonary disease (COPD) is a common and frequently-occurring disease that seriously endangers human health, seriously affects the quality of life of patients, has a high mortality rate, and brings heavy burdens to patients, their families and society. economic burden. According to the World Bank and the World Health Organization, by 2020, COPD will rank fifth in the world's disease economy.

COPD的发病机制尚未完全明了，吸入有害颗粒或气体可引起肺内氧化应激、蛋白酶和抗蛋白酶失衡及肺部炎症反应。慢阻肺目前尚不能根治，其稳定期与急性加重期处理方法不同。COPD稳定期的处理目标是减轻当前症状，降低未来风险。处于COPD稳定期的患者可进行腹式呼吸及缩唇呼吸锻炼并服用药物，支气管舒张剂(β2受体激动剂、抗胆碱药、茶碱类药物)是控制慢阻肺症状的主要治疗措施。短期按需服用可缓解症状，长期规则应用可预防和减轻症状，增加运动耐力，但不能使所有患者的FEV1得到改善。现有的手段大多属于对症治疗，根据COPD诊治指南，现有的可行性治疗方法包括药物治疗、氧疗、康复治疗及外科治疗，但这些治疗手段仅仅可以减轻症状，阻止病情发展，改善生活质量，尚缺乏可以从根本上逆转疾病的方法。因此，患者与医师均期待一种新型、疗效可靠、不良反应不明显及经济的治疗方法。The pathogenesis of COPD is not fully understood. Inhalation of harmful particles or gases can cause oxidative stress in the lungs, protease and anti-protease imbalance, and pulmonary inflammation. Chronic obstructive pulmonary disease is currently not cured, and its stabilization period is different from that in acute exacerbation. The goal of COPD stabilization is to reduce current symptoms and reduce future risks. Patients in the stable phase of COPD can perform abdominal breathing and lip-reducing exercises and take drugs. Bronchodilators (β2 receptor agonists, anticholinergic drugs, theophylline drugs) are the main treatment measures for controlling chronic obstructive pulmonary disease. Short-term on-demand medications can alleviate symptoms, and long-term regular use can prevent and alleviate symptoms and increase exercise tolerance, but not all patients with improved FEV1. Most of the existing methods belong to symptomatic treatment. According to the guidelines for diagnosis and treatment of COPD, the existing feasible treatment methods include drug treatment, oxygen therapy, rehabilitation therapy and surgical treatment, but these treatments can only alleviate symptoms, prevent the development of the disease and improve the quality of life. There is still a lack of ways to fundamentally reverse the disease. Therefore, both patients and physicians expect a new type of treatment with reliable efficacy, unreasonable adverse reactions and economics.

支气管哮喘(简称哮喘)是常见的慢性呼吸道疾病之一，近年来其患病率在全球范围内有逐年增加的趋势。哮喘目前尚不能根治，以抑制炎症为主的规范治疗仅能够控制哮喘临床症状。Bronchial asthma (referred to as asthma) is one of the common chronic respiratory diseases. In recent years, its prevalence has increased year by year in the world. Asthma is currently not cured, and standardized treatments that inhibit inflammation can only control the clinical symptoms of asthma.

目前治疗哮喘的药物可以分为控制药物和缓解药物。大部分支气管哮喘(简称哮喘)患者可获得有效的控制，但其中约有5％～8％的患者尽管经过规范化的治疗，症状仍难以控制，这些患者属于难治性哮喘。其临床表现类型分为：(1)激素依赖性/抵抗性哮喘，这类患者对激素治疗反应差，表现出不同程度的激素抵抗，需要长期依赖大剂量吸入激素，甚至是口服激素。(2)脆性哮喘，分为两型。Ⅰ型的特点是尽管大剂量吸入激素治疗，峰值呼吸仍然大幅度波动和反复哮喘发作；Ⅱ型的特点是哮喘控制良好的情况下，突然发作致死性的哮喘。(3)致死性哮喘，尽管这类患者在使用“适当”的治疗方法，但仍会发生致命或濒死的哮喘发作。目前，难治性哮喘仍无切实有效的治疗方法。The drugs currently used to treat asthma can be divided into control drugs and relief drugs. Most patients with bronchial asthma (abbreviated as asthma) have effective control, but about 5% to 8% of them are difficult to control despite standardized treatment, and these patients are refractory asthma. Its clinical manifestations are divided into: (1) hormone-dependent/resistance asthma, which responds poorly to hormone therapy and exhibits varying degrees of hormonal resistance, requiring long-term dependence on large doses of inhaled hormones, or even oral hormones. (2) Brittle asthma, divided into two types. Type I is characterized by a large fluctuation in peak breathing and repeated asthma attacks despite high-dose inhaled steroid therapy; type II is characterized by a sudden onset of lethal asthma in the case of well-controlled asthma. (3) Lethal asthma, despite Such patients are using "appropriate" treatments, but still have fatal or sudden deaths. At present, there is still no effective treatment for refractory asthma.

另外，长期高剂量吸入激素可能会导致患者出现一些全身不良反应，包括皮肤瘀斑、肾上腺功能抑制、骨密度降低、白内障、青光眼等。其他一些治疗也存在疗效差，不良反应大的缺点。因此，患者与医师均期待一种新型、疗效可靠、不良反应不明显及经济的治疗方法。In addition, long-term high-dose inhaled hormone may cause some systemic adverse reactions in patients, including skin ecchymosis, adrenal function inhibition, bone density reduction, cataract, glaucoma and so on. Other treatments also have the disadvantages of poor efficacy and large adverse reactions. Therefore, both patients and physicians expect a new type of treatment with reliable efficacy, unreasonable adverse reactions and economics.

干细胞是一类具有自我更新和分化潜能的细胞，它在一定的环境下能分化为多种功能细胞。一直以来，骨髓都是用作造血祖细胞组织来源的金标准，但随着研究与应用越来越多，骨髓来源的干细胞在临床使用的不足之处亦越发突显，如从采集到临床使用的过程耗时过长，采集骨髓对供体本身的伤害，培养成本较高，较低的HLA配型成功率等，制约了其在临床治疗中的广泛应用。Stem cells are a class of cells with self-renewal and differentiation potential that can differentiate into multiple functional cells under certain circumstances. Bone marrow has long been used as the gold standard for the origin of hematopoietic progenitor cells, but with the increasing number of research and application, the insufficiency of bone marrow-derived stem cells in clinical use has become more prominent, such as from collection to clinical use. The process takes too long, collecting bone marrow damage to the donor itself, higher culture cost, lower HLA matching success rate, etc., which restricts its wide application in clinical treatment.

综上所述，本领域迫切需要一种能够有效治疗上呼吸道疾病，特别是慢性阻塞性肺疾病和哮喘的方法。In summary, there is an urgent need in the art for a method for effectively treating upper respiratory tract diseases, particularly chronic obstructive pulmonary disease and asthma.

发明内容Summary of the invention

本发明的目的是提供一种能够有效治疗慢性阻塞性肺疾病和哮喘等上呼吸道疾病的方法。It is an object of the present invention to provide a method for effectively treating upper respiratory tract diseases such as chronic obstructive pulmonary disease and asthma.

本发明的第一方面，提供了一种脂肪间充质祖细胞的用途，所述脂肪间充质祖细胞用于制备治疗慢性阻塞性肺疾病和哮喘的药物组合物。In a first aspect of the invention, there is provided the use of a fat mesenchymal progenitor cell for the preparation of a pharmaceutical composition for the treatment of chronic obstructive pulmonary disease and asthma.

在另一优选例中，在所述药物组合物中，所述的脂肪间充质祖细胞的浓度为脂肪间充质祖细胞的浓度为0.1～10×10⁶个/ml，较佳地为0.2～5×10⁶个/ml，更佳地为0.5～2×10⁶个/ml。In another preferred embodiment, the concentration of the adipose mesenchymal progenitor cells in the pharmaceutical composition is a concentration of adipose mesenchymal progenitor cells of 0.1 to 10 × 10 ⁶ /ml, preferably 0.2 to 5 × 10 ⁶ /ml, more preferably 0.5 to 2 × 10 ⁶ /ml.

在另一优选例中，所述的脂肪间充质祖细胞是传代培养的脂肪间充质祖细胞。In another preferred embodiment, the adipose mesenchymal progenitor cells are subcultured adipose mesenchymal progenitor cells.

在另一优选例中，所述的组合物中，所述脂肪间充质祖细胞的含量为1×10⁶/ml-1×10⁸/ml。In another preferred embodiment, the adipose mesenchymal progenitor cells are present in the composition in an amount of from 1 × 10 ⁶ /ml to 1 × 10 ⁸ /ml.

在另一优选例中，所述的脂肪间充质祖细胞是通过基质血管组分培养3-10代纯化扩增获得。In another preferred embodiment, the adipose mesenchymal progenitor cells are obtained by purification and expansion of the stromal vascular fraction for 3-10 passages.

在另一优选例中，在所述组合物中，主要活性成分仅为脂肪间充质祖细胞。In another preferred embodiment, in the composition, the main active ingredient is only adipose mesenchymal progenitor cells.

在另一优选例中，所述的治疗指慢性阻塞性肺和哮喘等上呼吸道疾病的病理症状改善。 In another preferred embodiment, the treatment refers to an improvement in pathological symptoms of upper respiratory tract diseases such as chronic obstructive pulmonary disease and asthma.

在另一优选例中，所述的治疗指减轻或逆转慢性阻塞性肺引起的肺病理改变。In another preferred embodiment, the treatment refers to alleviating or reversing pulmonary pathological changes caused by chronic obstructive pulmonary disease.

在另一优选例中，所述的脂肪间充质祖细胞是通过SVF培养P3-P10代纯化扩增获得的细胞。In another preferred embodiment, the adipose mesenchymal progenitor cells are cells obtained by purification and purification of P3-P10 passages by SVF culture.

在另一优选例中，所述的脂肪间充质祖细胞具有分化为骨，软骨和脂肪的能力。In another preferred embodiment, the adipose mesenchymal progenitor cells have the ability to differentiate into bone, cartilage and fat.

在另一优选例中，所述的脂肪间充质祖细胞为表达选自下组的表面标记的脂肪间充质祖细胞：CD29、CD90、CD73、CD49d、CD44、CD105、SCA-1。In another preferred embodiment, the adipose mesenchymal progenitor cells are adipose mesenchymal progenitor cells expressing a surface marker selected from the group consisting of CD29, CD90, CD73, CD49d, CD44, CD105, SCA-1.

在另一优选例中，所述的脂肪间充质祖细胞为不表达选自下组的表面标记的脂肪间充质祖细胞：CD34、CD45、CD14、Actin、HLA-DR、CD106、CD80、CD86、CD31。In another preferred embodiment, the adipose mesenchymal progenitor cells are adipose mesenchymal progenitor cells that do not express a surface marker selected from the group consisting of CD34, CD45, CD14, Actin, HLA-DR, CD106, CD80, CD86, CD31.

在另一优选例中，所述的药物组合物包括：脂肪间充质祖细胞，和药学上可接受的载体。In another preferred embodiment, the pharmaceutical composition comprises: adipose mesenchymal progenitor cells, and a pharmaceutically acceptable carrier.

在另一优选例中，所述的药学上可接受的载体为输液剂载体和/或注射剂载体，优选地选自下组：生理盐水、葡萄糖盐水，或其组合。In another preferred embodiment, the pharmaceutically acceptable carrier is an infusion vehicle carrier and/or an injection vehicle carrier, preferably selected from the group consisting of physiological saline, dextrose saline, or a combination thereof.

在另一优选例中，所述的治疗包括选自下组的一项或多项指标改善：肺功能、气道炎症、生活质量评分。In another preferred embodiment, the treatment comprises one or more indicator improvements selected from the group consisting of lung function, airway inflammation, and quality of life score.

在另一优选例中，所述的脂肪间充质祖细胞具有选自下组的任一或多种特征：In another preferred embodiment, the adipose mesenchymal progenitor cells have any one or more of the characteristics selected from the group consisting of:

(i)90％以上的细胞具有表面抗原CD29；(i) more than 90% of the cells have the surface antigen CD29;

(ii)90％以上的细胞具有表面抗原CD90；(ii) more than 90% of the cells have the surface antigen CD90;

(iii)90％以上的细胞具有表面抗原CD73；(iii) more than 90% of the cells have the surface antigen CD73;

(iv)90％以上的细胞具有表面抗原CD49d；(iv) more than 90% of the cells have the surface antigen CD49d;

(v)90％以上的细胞具有表面抗原CD44；(v) more than 90% of the cells have the surface antigen CD44;

(vi)90％以上的细胞具有表面抗原CD105；(vi) more than 90% of the cells have the surface antigen CD105;

(vii)90％以上的细胞具有表面抗原SCA-1。(vii) More than 90% of the cells have the surface antigen SCA-1.

在另一优选例中，95％以上的细胞具有表面抗原CD29。In another preferred embodiment, more than 95% of the cells have the surface antigen CD29.

在另一优选例中，95％以上的细胞具有表面抗原CD90。In another preferred embodiment, more than 95% of the cells have the surface antigen CD90.

在另一优选例中，95％以上的细胞具有表面抗原CD73。In another preferred embodiment, more than 95% of the cells have the surface antigen CD73.

在另一优选例中，95％以上的细胞具有表面抗原CD49d。In another preferred embodiment, more than 95% of the cells have the surface antigen CD49d.

在另一优选例中，95％以上的细胞具有表面抗原CD44。In another preferred embodiment, more than 95% of the cells have the surface antigen CD44.

在另一优选例中，95％以上的细胞具有表面抗原CD105。 In another preferred embodiment, more than 95% of the cells have the surface antigen CD105.

在另一优选例中，95％以上的细胞具有表面抗原SCA-1。In another preferred embodiment, more than 95% of the cells have the surface antigen SCA-1.

在另一优选例中，95-100％(优选98-100％，更优选99-100％)的细胞具有表面抗原CD29。In another preferred embodiment, 95-100% (preferably 98-100%, more preferably 99-100%) of the cells have the surface antigen CD29.

在另一优选例中，95-100％(优选98-100％，更优选99-100％)的细胞具有表面抗原CD90。In another preferred embodiment, 95-100% (preferably 98-100%, more preferably 99-100%) of the cells have the surface antigen CD90.

在另一优选例中，95-100％(优选98-100％，更优选99-100％)的细胞具有表面抗原CD73。In another preferred embodiment, 95-100% (preferably 98-100%, more preferably 99-100%) of the cells have the surface antigen CD73.

在另一优选例中，95-100％(优选98-100％，更优选99-100％)的细胞具有表面抗原CD49d。In another preferred embodiment, 95-100% (preferably 98-100%, more preferably 99-100%) of the cells have the surface antigen CD49d.

在另一优选例中，95-100％(优选98-100％，更优选99-100％)的细胞具有表面抗原CD44。In another preferred embodiment, 95-100% (preferably 98-100%, more preferably 99-100%) of the cells have the surface antigen CD44.

在另一优选例中，95-100％(优选98-100％，更优选99-100％)的细胞具有表面抗原CD105。In another preferred embodiment, 95-100% (preferably 98-100%, more preferably 99-100%) of the cells have the surface antigen CD105.

在另一优选例中，95-100％(优选98-100％，更优选99-100％)的细胞具有表面抗原SCA-1。In another preferred embodiment, 95-100% (preferably 98-100%, more preferably 99-100%) of the cells have the surface antigen SCA-1.

(viii)在细胞群中，5％以下的细胞具有表面抗原CD34；(viii) in the cell population, less than 5% of the cells have the surface antigen CD34;

(ix)在细胞群中，5％以下的细胞具有表面抗原CD45；(ix) in the cell population, less than 5% of the cells have the surface antigen CD45;

(x)在细胞群中，5％以下的细胞具有表面抗原Actin；(x) in the cell population, less than 5% of the cells have a surface antigen Actin;

(xi)在细胞群中，5％以下的细胞具有表面抗原HLA-DR；(xi) in the cell population, less than 5% of the cells have the surface antigen HLA-DR;

(xii)在细胞群中，5％以下的细胞具有表面抗原CD14。(xii) In the cell population, less than 5% of the cells have the surface antigen CD14.

(xiv)在细胞群中，5％以下的细胞具有表面抗原CD106(xiv) In the cell population, less than 5% of cells have the surface antigen CD106

(xv)在细胞群中，5％以下的细胞具有表面抗原CD80(xv) In the cell population, less than 5% of cells have the surface antigen CD80

(xvi)在细胞群中，5％以下的细胞具有表面抗原CD86(xvi) In the cell population, less than 5% of cells have the surface antigen CD86

(xvii)在细胞群中，5％以下的细胞具有表面抗原CD31。(xvii) In the cell population, 5% or less of cells have the surface antigen CD31.

在另一优选例中，2％以下的细胞具有表面抗原CD34。In another preferred embodiment, less than 2% of the cells have the surface antigen CD34.

在另一优选例中，2％以下的细胞具有表面抗原CD45。In another preferred embodiment, less than 2% of the cells have the surface antigen CD45.

在另一优选例中，2％以下的细胞具有表面抗原CD106。In another preferred embodiment, less than 2% of the cells have the surface antigen CD106.

在另一优选例中，2％以下的细胞具有表面抗原CD80。 In another preferred embodiment, less than 2% of the cells have the surface antigen CD80.

在另一优选例中，1％以下的细胞具有表面抗原CD34。In another preferred embodiment, less than 1% of the cells have the surface antigen CD34.

在另一优选例中，1％以下的细胞具有表面抗原CD45。In another preferred embodiment, less than 1% of the cells have the surface antigen CD45.

在另一优选例中，1％以下的细胞具有表面抗原CD106。In another preferred embodiment, less than 1% of the cells have the surface antigen CD106.

在另一优选例中，1％以下的细胞具有表面抗原CD80。In another preferred embodiment, less than 1% of the cells have the surface antigen CD80.

在另一优选例中，2％以下的细胞具有表面抗原Actin。In another preferred embodiment, less than 2% of the cells have the surface antigen Actin.

在另一优选例中，2％以下的细胞具有表面抗原HLA-DR。In another preferred embodiment, less than 2% of the cells have the surface antigen HLA-DR.

在另一优选例中，2％以下的细胞具有表面抗原CD14。In another preferred embodiment, less than 2% of the cells have the surface antigen CD14.

在另一优选例中，2％以下的细胞具有表面抗原CD86。In another preferred embodiment, less than 2% of the cells have the surface antigen CD86.

在另一优选例中，2％以下的细胞具有表面抗原CD31。In another preferred embodiment, less than 2% of the cells have the surface antigen CD31.

在另一优选例中，1％以下的细胞具有表面抗原Actin。In another preferred embodiment, less than 1% of the cells have the surface antigen Actin.

在另一优选例中，1％以下的细胞具有表面抗原HLA-DR。In another preferred embodiment, less than 1% of the cells have the surface antigen HLA-DR.

在另一优选例中，1％以下的细胞具有表面抗原CD14。In another preferred embodiment, less than 1% of the cells have the surface antigen CD14.

在另一优选例中，1％以下的细胞具有表面抗原CD86。In another preferred embodiment, less than 1% of the cells have the surface antigen CD86.

在另一优选例中，1％以下的细胞具有表面抗原CD31。In another preferred embodiment, less than 1% of the cells have the surface antigen CD31.

在另一优选例中，0.01-5％(优选0.1-4％，更优选1-3％)的细胞具有表面抗原CD34。In another preferred embodiment, 0.01 to 5% (preferably 0.1 to 4%, more preferably 1-3%) of the cells have the surface antigen CD34.

在另一优选例中，0.01-1％(优选0.05-0.5％，更优选0.08-0.2％)的细胞具有表面抗原CD45。In another preferred embodiment, 0.01-1% (preferably 0.05-0.5%, more preferably 0.08-0.2%) of the cells have the surface antigen CD45.

在另一优选例中，0.01-3％(优选0.05-2％，更优选0.1-1％)的细胞具有表面抗原CD14。In another preferred embodiment, 0.01 to 3% (preferably 0.05 to 2%, more preferably 0.1 to 1%) of the cells have the surface antigen CD14.

在另一优选例中，0.01-1％(优选0.05-0.5％，更优选0.08-0.2％)的细胞具有表面抗原HLA-DR。In another preferred embodiment, 0.01-1% (preferably 0.05-0.5%, more preferably 0.08-0.2%) of the cells have the surface antigen HLA-DR.

在另一优选例中，0.01-3％(优选0.05-2％，更优选0.1-1％)的细胞具有表面抗原Actin。In another preferred embodiment, 0.01 to 3% (preferably 0.05 to 2%, more preferably 0.1 to 1%) of the cells have the surface antigen Actin.

在另一优选例中，0.01-1％(优选0.05-0.5％，更优选0.08-0.2％)的细胞具有表面抗原CD80。In another preferred embodiment, 0.01-1% (preferably 0.05-0.5%, more preferably 0.08-0.2%) of the cells have the surface antigen CD80.

在另一优选例中，0.01-3％(优选0.05-2％，更优选0.1-1％)的细胞具有表面抗原CD86。In another preferred embodiment, 0.01 to 3% (preferably 0.05 to 2%, more preferably 0.1 to 1%) of the cells have the surface antigen CD86.

在另一优选例中，0.01-1％(优选0.05-0.5％，更优选0.08-0.2％)的细胞具有表面抗原CD31。In another preferred embodiment, 0.01-1% (preferably 0.05-0.5%, more preferably 0.08-0.2%) of the cells have a table Surface antigen CD31.

本发明的第二方面，提供了一种用于预防或治疗慢性阻塞性肺疾病和哮喘等上呼吸道疾病的药物组合物，所述的药物组合物包括：有效量的脂肪间充质祖细胞，以及药学上可接受的载体。According to a second aspect of the present invention, there is provided a pharmaceutical composition for preventing or treating an upper respiratory tract disease such as chronic obstructive pulmonary disease and asthma, the pharmaceutical composition comprising: an effective amount of adipose mesenchymal progenitor cells, And a pharmaceutically acceptable carrier.

在另一优选例中，所述的载体是输液剂载体和/或注射剂载体。In another preferred embodiment, the carrier is an infusion carrier and/or an injection carrier.

在另一优选例中，所述的药学上可接受的载体包括(但并不限于)：盐水、缓冲液、葡萄糖、水、甘油、乙醇、生理盐水、葡萄糖盐水，及其组合。In another preferred embodiment, the pharmaceutically acceptable carrier includes, but is not limited to, saline, buffer, dextrose, water, glycerol, ethanol, physiological saline, dextrose saline, and combinations thereof.

在另一优选例中，所述的药物组合物中，脂肪间充质祖细胞的浓度为0.1～10×10⁶个/ml，较佳地为0.2～5×10⁶个/ml，更佳地为0.5～2×10⁶个/ml。In another preferred embodiment, the concentration of the adipose mesenchymal progenitor cells in the pharmaceutical composition is 0.1 to 10 × 10 ⁶ /ml, preferably 0.2 to 5 × 10 ⁶ /ml, more preferably The ground is 0.5 to 2 x 10 ⁶ /ml.

在另一优选例中，所述的注射试剂的体积为10-1000mL，较佳地为100mL。In another preferred embodiment, the volume of the injection reagent is from 10 to 1000 mL, preferably 100 mL.

(v)90％以上的细胞具有表面抗原CD44。(v) More than 90% of the cells have the surface antigen CD44.

(vi)90％以上的细胞具有表面抗原CD105。(vi) More than 90% of the cells have the surface antigen CD105.

在另一优选例中，95％以上的细胞具有表面抗原CD105。In another preferred embodiment, more than 95% of the cells have the surface antigen CD105.

(x)在细胞群中，5％以下的细胞具有表面抗原Actin； (x) in the cell population, less than 5% of the cells have a surface antigen Actin;

(xiii)在细胞群中，5％以下的细胞具有表面抗原CD106。(xiii) In the cell population, 5% or less of cells have the surface antigen CD106.

(xiv)在细胞群中，5％以下的细胞具有表面抗原CD80。(xiv) In the cell population, less than 5% of the cells have the surface antigen CD80.

(xv)在细胞群中，5％以下的细胞具有表面抗原CD86。(xv) In the cell population, cells of 5% or less have the surface antigen CD86.

(xvi)在细胞群中，5％以下的细胞具有表面抗原CD31。(xvi) In the cell population, cells of 5% or less have the surface antigen CD31.

在另一优选例中，2％以下的细胞具有表面抗原CD80。In another preferred embodiment, less than 2% of the cells have the surface antigen CD80.

在另一优选例中，所述的脂肪间充质祖细胞是用含血清培养基培养纯化扩增的细胞，或所述的脂肪间充质祖细胞是用无血清培养基培养纯化扩增的细胞。 In another preferred embodiment, the adipose mesenchymal progenitor cells are purified and cultured in a serum-containing medium, or the adipose mesenchymal progenitor cells are purified and cultured in a serum-free medium. cell.

在另一优选例中，所述的脂肪间充质祖细胞还表达细胞因子，且所述的细胞因子选自下组：TGF-β1，HGF，VEGF，IL6、IL8、IL10，或其组合。In another preferred embodiment, the adipose mesenchymal progenitor cells further express a cytokine, and the cytokine is selected from the group consisting of TGF-β1, HGF, VEGF, IL6, IL8, IL10, or a combination thereof.

在另一优选例中，所述的脂肪间充质祖细胞表达细胞因子TGF-β1的量为1000-1300pg/ml/10⁶细胞。In another preferred embodiment, the adipose mesenchymal progenitor cells express the cytokine TGF-β1 in an amount of 1000-1300 pg/ml/10 ⁶ cells.

在另一优选例中，所述的脂肪间充质祖细胞表达细胞因子HGF的量为9000-10000pg/ml/10⁶细胞。In another preferred embodiment, the adipose mesenchymal progenitor cells express cytokine HGF in an amount of 9000-10000 pg/ml/10 ⁶ cells.

在另一优选例中，所述的脂肪间充质祖细胞表达细胞因子VEGF的量为700–800pg/ml/10⁶细胞。In another preferred embodiment, the adipose mesenchymal progenitor cells express the cytokine VEGF in an amount of 700 - 800 pg / ml / 10 ⁶ cells.

在另一优选例中，所述的脂肪间充质祖细胞表达细胞因子IL6的量为5000–8000pg/ml/10⁶细胞。In another preferred embodiment, the adipose mesenchymal progenitor cells express the cytokine IL6 in an amount of 5000 - 8000 pg / ml / 10 ⁶ cells.

在另一优选例中，所述的脂肪间充质祖细胞表达细胞因子IL8的量为6000–10000pg/ml/10⁶细胞。In another preferred embodiment, the adipose mesenchymal progenitor cells express the cytokine IL8 in an amount of 6000 - 10000 pg / ml / 10 ⁶ cells.

在另一优选例中，所述的脂肪间充质祖细胞表达细胞因子IL10的量为500～1000pg/ml/10⁶细胞。In another preferred embodiment, the adipose mesenchymal progenitor cells express the cytokine IL10 in an amount of 500 to 1000 pg/ml/10 ⁶ cells.

在另一优选例中，所述的间充质祖细胞具有成软骨、成骨及成脂分化能力。In another preferred embodiment, the mesenchymal progenitor cells have cartilage, osteogenic and adipogenic differentiation capabilities.

在另一优选例中，所述的药物组合物为注射制剂，较佳地为静脉注射制剂。In another preferred embodiment, the pharmaceutical composition is an injectable preparation, preferably an intravenous preparation.

在另一优选例中，所述的药物组合物中，所述的脂肪间充质祖细胞具有分化为骨，软骨和/或脂肪的能力。In another preferred embodiment, the adipose-derived mesenchymal progenitor cells have the ability to differentiate into bone, cartilage and/or fat.

本发明的第三方面，提供了一种预防或治疗慢性阻塞性肺疾病的方法，所述方法包括步骤：给需要的对象施用脂肪间充质祖细胞，或如本发明第二方面所述的药物组合物。According to a third aspect of the invention, a method for preventing or treating chronic obstructive pulmonary disease, the method comprising the steps of: administering adipose mesenchymal progenitor cells to a subject in need thereof, or according to the second aspect of the invention Pharmaceutical composition.

在另一优选例中，所述的对象为人或非人哺乳动物。In another preferred embodiment, the subject is a human or non-human mammal.

在另一优选例中，所述的非人哺乳动物为鼠、兔、牛、羊、猪等。In another preferred embodiment, the non-human mammal is a mouse, a rabbit, a cow, a sheep, a pig, or the like.

应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。It is to be understood that within the scope of the present invention, the various technical features of the present invention and the various technical features specifically described hereinafter (as in the embodiments) may be combined with each other to constitute a new or preferred technical solution. Due to space limitations, we will not repeat them here.

附图说明DRAWINGS

图1脂肪间充质祖细胞分化的油红O染色、阿辛蓝染色和茜素红染色实验图； Fig. 1 Experimental diagram of oil red O staining, acin blue staining and alizarin red staining for differentiation of adipose mesenchymal progenitor cells;

图2油红O染色对照组(阴性)；Figure 2 Oil red O staining control group (negative);

图3haMPC流式检测试验图谱；Figure 3haMPC flow detection test map;

图4实施例2中尾静脉注射组的气道高反应性测试结果图；Figure 4 is a graph showing the results of airway hyperresponsiveness test in the tail vein injection group in Example 2;

图5实施例2中尾静脉注射组的肺组织病理测试结果图；Figure 5 is a diagram showing the results of pathological test of lung tissue in the tail vein injection group in Example 2;

图6实施例2中气管滴注组的气道高反应性测试结果图；Figure 6 is a diagram showing the results of airway hyperresponsiveness test of the tracheal instillation group in Example 2;

图7实施例2中气管滴注组的肺组织病理测试结果图；Figure 7 is a diagram showing the results of pathological examination of lung tissue in the tracheal instillation group in Example 2;

图8实施例3中尾静脉注射组的肺功能检测测试结果图；Figure 8 is a graph showing the results of lung function test in the tail vein injection group in Example 3;

图9实施例3中尾静脉注射组的肺组织病理检测测试结果图；Figure 9 is a diagram showing the results of pathological examination of lung tissue in the tail vein injection group in Example 3;

图10实施例3中尾静脉注射组的肺组织病理检测测试结果图；Figure 10 is a diagram showing the results of pathological examination of lung tissue in the tail vein injection group in Example 3;

图11实施例3中气管内滴注组的肺功能检测测试结果图；Figure 11 is a graph showing the results of lung function test of the intratracheal instillation group in Example 3;

图12实施例3中气管内滴注组的肺组织病理检测测试结果图；Figure 12 is a graph showing the results of pathological examination of lung tissue in the intratracheal instillation group in Example 3;

图13实施例3中气管内滴注组的肺组织病理检测测试结果图；Figure 13 is a graph showing the results of pathological examination of lung tissue in the intratracheal instillation group in Example 3;

图14实施例4中FISH检测测试结果图；Figure 14 is a diagram showing the results of the FISH test in Example 4;

图15实施例1中脂肪间充质祖细胞的免疫调节作用结果图。Figure 15 is a graph showing the results of immunomodulatory effects of adipose mesenchymal progenitor cells in Example 1.

具体实施方式detailed description

本发明人经过长期而深入的研究，意外地发现，脂肪间充质祖细胞具有极其优异的预防或治疗慢性阻塞性肺疾病和哮喘等上呼吸道疾病的作用。具体地，给需要的对象施用本发明的含有脂肪间充质祖细胞的药物组合物，对于上呼吸道疾病疾病有显著的预防或治疗作用。在此基础上，发明人完成了本发明。After long-term and intensive research, the present inventors have unexpectedly found that adipose mesenchymal progenitor cells have an extremely excellent effect of preventing or treating chronic obstructive pulmonary diseases and upper respiratory diseases such as asthma. Specifically, the pharmaceutical composition containing the adipose-derived mesenchymal progenitor cells of the present invention is administered to a subject in need thereof, and has a significant preventive or therapeutic effect on diseases of the upper respiratory tract disease. On this basis, the inventors completed the present invention.

术语the term

如本文所用，术语“以上”和“以下”包括本数，例如“95％以上”指≥95％，“0.2％以下”指≤0.2％。As used herein, the terms "above" and "below" include the number, such as "95% or more" means ≥ 95%, and "0.2% or less" means ≤ 0.2%.

如本文所用，术语“脂肪基质血管成分”和“脂肪来源的基质血管成分”可互换使用。As used herein, the terms "fatty stromal vascular component" and "fat derived stromal vascular component" are used interchangeably.

如本文所用，术语“脂肪间充质祖细胞”、“脂肪间充质干细胞”和“脂肪来源的间充质祖细胞”，“脂肪来源的间充质干细胞”可互换使用。As used herein, the terms "adipose mesenchymal progenitor cells", "adipose mesenchymal stem cells" and "fat-derived mesenchymal progenitor cells", "fat-derived mesenchymal stem cells" are used interchangeably.

特别地，在本发明中，术语“95％以上”包括96％以上，97％以上，98％以上，99％以上，或99.5％以上。In particular, in the present invention, the term "95% or more" includes 96% or more, 97% or more, 98% or more, 99% or more, or 99.5% or more.

脂肪 Fat

自体脂肪是整形和抗衰老治疗的优良来源，脂肪组织材料可以来源于腰部、臀部、腹部、大腿、上臂等部位。本领域技术人员可采用通用的技术方法获得自体脂肪组织，包括(但不限于)抽吸、手术分离等方法。Autologous fat is an excellent source of plastic and anti-aging treatments. Adipose tissue materials can be derived from the waist, hips, abdomen, thighs, upper arms and other parts. Those skilled in the art can obtain autologous adipose tissue using general technical methods including, but not limited to, methods of aspiration, surgical separation, and the like.

在本发明中，脂肪组织或脂肪原料没有特别限制，可以是来源于动物或人的任何部位的脂肪组织，优选人的脂肪组织。较佳地，脂肪组织可以是腰部、臀部、腹部、大腿、上臂等部位的组织。In the present invention, the adipose tissue or the fat raw material is not particularly limited, and may be an adipose tissue derived from any part of an animal or a human, preferably a human adipose tissue. Preferably, the adipose tissue may be a tissue of a part such as a waist, a buttocks, an abdomen, a thigh, an upper arm or the like.

脂肪间充质祖细胞(Human adipose derived mesenchymal progenitor cells)Human adipose derived mesenchymal progenitor cells

间充质祖细胞可以分泌广谱的具有免疫调节和/或再生活性的生物因子。在本发明中，优选地采用脂肪来源的间充质祖细胞(Human adipose derived msenchymal progenitor cells,haMPCs)。其中，一种最为优选的脂肪来源的间充质祖细胞为不含CD34+的细胞，通常通过SVF培养P3-P10代纯化扩增获得。Mesenchymal progenitor cells secrete a broad spectrum of biological factors with immunomodulatory and/or regenerative activity. In the present invention, human adipose derived msenchymal progenitor cells (haMPCs) are preferably used. Among them, one of the most preferred adipose-derived mesenchymal progenitor cells is a CD34+-free cell, which is usually obtained by SVF culture P3-P10 generation purification and amplification.

在本发明中，脂肪间充质祖细胞的制备方法可以包括步骤：洗涤脂肪组织，然后用胶原酶消化，离心分离基质血管成分，除去油脂和胶原酶，培养原代细胞，得到传代后的脂肪间充质祖细胞。In the present invention, the method for preparing adipose mesenchymal progenitor cells may include the steps of: washing adipose tissue, then digesting with collagenase, centrifuging the stromal vascular component, removing oil and collagenase, culturing the primary cell, and obtaining the fat after passage. Mesenchymal progenitor cells.

脂肪间充质祖细胞的抗原检测Antigen detection of adipose mesenchymal progenitor cells

用本发明使用的脂肪间充质祖细胞具有很高的纯度，基本上不含有其他类型的细胞或干细胞。这可通过细胞表面抗原的检测加以验证。The adipose mesenchymal progenitor cells used in the present invention are of high purity and substantially free of other types of cells or stem cells. This can be verified by detection of cell surface antigens.

脂肪间充质祖细胞具有多种特异性抗原和受体，主要有CD3、CD13、D29、CD34、CD45、CD49e、CD59、CD73、CD90、CD105、HLA-ABC等。Adipose mesenchymal progenitor cells have a variety of specific antigens and receptors, mainly CD3, CD13, D29, CD34, CD45, CD49e, CD59, CD73, CD90, CD105, HLA-ABC and so on.

CD34抗原是一种高度糖基化Ⅰ型跨膜蛋白，它选择性的表达于人类造血干细胞(HSC)，祖细胞(PC)和血管内皮细胞(EC)表面，带有CD34的脂肪间充质祖细胞在总干细胞的比例优选为≤0.2％，更佳地，≤0.1％。CD34 antigen is a highly glycosylated type I transmembrane protein that is selectively expressed on human hematopoietic stem (HSC), progenitor (PC) and vascular endothelial (EC) surfaces with adipose mesenchyme of CD34. The proportion of progenitor cells in total stem cells is preferably ≤ 0.2%, more preferably ≤ 0.1%.

CD45存在于所有造血细胞的表面，包括造血干细胞和破骨细胞。带有CD45的脂肪间充质祖细胞在总干细胞的比例优选为≤0.1％。CD45 is present on the surface of all hematopoietic cells, including hematopoietic stem cells and osteoclasts. The proportion of adipose mesenchymal progenitor cells bearing CD45 in total stem cells is preferably ≤ 0.1%.

CD29、CD44、CD105、CD90、CD106等主要存在于脂肪间充质祖细胞表面。CD29, CD44, CD105, CD90, CD106, etc. are mainly present on the surface of adipose mesenchymal progenitor cells.

带有CD29的脂肪间充质祖细胞在总干细胞的比例优选为≥90％，更佳地≥95％。 The proportion of adipose mesenchymal progenitor cells bearing CD29 in total stem cells is preferably ≥ 90%, more preferably ≥ 95%.

带有CD73的脂肪间充质祖细胞在总干细胞的比例优选为≥80％，更佳地≥85％。The proportion of adipose mesenchymal progenitor cells bearing CD73 in total stem cells is preferably ≥ 80%, more preferably ≥ 85%.

带有CD49d的脂肪间充质祖细胞在总干细胞的比例优选为≥80％，更佳地≥85％。The proportion of adipose mesenchymal progenitor cells bearing CD49d in total stem cells is preferably ≥ 80%, more preferably ≥ 85%.

带有CD105的脂肪间充质祖细胞在总干细胞的比例优选为≥70％，更佳地≥75％。The proportion of adipose mesenchymal progenitor cells bearing CD105 in total stem cells is preferably ≥ 70%, more preferably ≥ 75%.

带有CD90的脂肪间充质祖细胞在总干细胞的比例优选为≥70％，更佳地≥75％。The proportion of adipose mesenchymal progenitor cells bearing CD90 in total stem cells is preferably ≥ 70%, more preferably ≥ 75%.

带有SCA-1的脂肪间充质祖细胞在总干细胞的比例优选为≥70％，更佳地≥75％。The proportion of adipose mesenchymal progenitor cells bearing SCA-1 in total stem cells is preferably ≥ 70%, more preferably ≥ 75%.

本领域内技术人员可以使用通用的方法检测脂肪间充质祖细胞的纯度和分化程度，如流式细胞仪法。检测时，加入不同的与有针对性的特异抗体，抗体可以是完整的单克隆或多克隆抗体，也可以是具有免疫活性的抗体片段，如Fab’或(Fab)2片段；抗体重链；抗体轻链；遗传工程改造的单链Fv分子(Ladner等人，美国专利No.4,946,778)；或嵌合抗体，如具有鼠抗体结合特异性但仍保留来自人的抗体部分的抗体。加入抗体与细胞表面的抗原结合一定时间，用流式细胞仪对细胞进行自动分析和分选。One skilled in the art can use a general method to detect the purity and degree of differentiation of adipose mesenchymal progenitor cells, such as flow cytometry. At the time of detection, different specific and targeted specific antibodies are added, and the antibody may be a complete monoclonal or polyclonal antibody, or may be an immunologically active antibody fragment, such as a Fab' or (Fab) 2 fragment; an antibody heavy chain; An antibody light chain; a genetically engineered single chain Fv molecule (Ladner et al., U.S. Patent No. 4,946,778); or a chimeric antibody, such as an antibody having murine antibody binding specificity but still retaining antibody portions from humans. The antibody is added to the antigen on the cell surface for a certain period of time, and the cells are automatically analyzed and sorted by flow cytometry.

药物组合物Pharmaceutical composition

本发明还提供了一种药物组合物，它含有有效量的脂肪间充质祖细胞，以及药学上可接受的载体。The invention also provides a pharmaceutical composition comprising an effective amount of adipose mesenchymal progenitor cells, and a pharmaceutically acceptable carrier.

通常，可将脂肪间充质祖细胞和脂肪基质血管成分配制于无毒的、惰性的和药学上可接受的水性载体介质中，如生理盐水中，其中pH通常约为5-8，较佳地，pH约为7-8。In general, the adipose mesenchymal progenitor cells and the adipose matrix vascular component can be formulated in a non-toxic, inert, and pharmaceutically acceptable aqueous carrier medium, such as physiological saline, wherein the pH is usually about 5-8, preferably. Ground, pH is about 7-8.

如本文所用，术语“有效量”或“有效剂量”是指可对人和/或动物产生功能或活性的且可被人和/或动物所接受的量。在本发明的优选实施例中，所述的有效量为：10±5×10⁶个细胞。优选地，所述的有效量细胞一次性注射完毕。As used herein, the term "effective amount" or "effective amount" refers to an amount that can produce a function or activity on a human and/or animal and that can be accepted by a human and/or animal. In a preferred embodiment of the invention, the effective amount is: 10 ± 5 x 10 ⁶ cells. Preferably, the effective amount of cells is injected in one shot.

如本文所用，“药学上可接受的”的成分是适用于人和/或哺乳动物而无过度不良副反应(如毒性、刺激和***反应)的，即具有合理的效益/风险比的物质。术语“药学上可接受的载体”指用于治疗剂给药的载体，包括。在本发明中，可以使用的药学上可接受的载体并没有特别的限制，可以是一种或多种相容性固体或液体填料或凝胶物质，它们适合于人使用，而且必须有足够的纯度和足够低的毒性。“相容性”在此指的是组合物中各组份能和本发明的脂肪间充质祖细胞相互掺和，而不明显降低其治疗效果。本发明药学上可以接受的载体部分例子有生理上可接受的无菌含水或无水溶液、分散液、悬浮液或乳液，和用于重新溶解成无菌的可注射溶液或分散液的无菌粉末，适宜的含水和非水载体、稀释剂、溶剂或赋形剂包括水、乙醇、多元醇及其适宜的混合物。除了上述的常规载体外，也可以根据脂肪间充质祖细胞的性质设计优化的载体。所述的载体优选为输液剂载体和/或注射剂载体。As used herein, a "pharmaceutically acceptable" ingredient is one that is suitable for use in humans and/or mammals without excessive adverse side effects (eg, toxicity, irritation, and allergies), ie, having a reasonable benefit/risk ratio. Operation The phrase "pharmaceutically acceptable carrier" refers to a carrier for the administration of a therapeutic agent, including. In the present invention, the pharmaceutically acceptable carrier which can be used is not particularly limited and may be one or more compatible solid or liquid fillers or gel substances which are suitable for human use and must have sufficient Purity and low enough toxicity. By "compatibility" it is meant herein that the components of the composition are capable of intermingling with the adipose mesenchymal progenitor cells of the invention without significantly reducing the therapeutic effect thereof. Examples of pharmaceutically acceptable carrier parts of the invention are physiologically acceptable sterile aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Suitable aqueous and nonaqueous vehicles, diluents, solvents or vehicles include water, ethanol, polyols and suitable mixtures thereof. In addition to the conventional vectors described above, optimized vectors can also be designed based on the nature of adipose mesenchymal progenitor cells. The carrier is preferably an infusion carrier and/or an injection carrier.

本发明的药物组合物含有安全有效量的脂肪间充质祖细胞，脂肪基质血管成分以及药学上可接受的载体。这类载体包括(但并不限于)：盐水、缓冲液、葡萄糖、水、甘油、乙醇、及其组合。通常药物制剂应与给药方式相匹配，本发明的药物组合物可以被制成针剂形式，例如用生理盐水或含有葡萄糖和其他辅剂的水溶液通过常规方法进行制备。所述的药物组合物宜在无菌条件下制造。活性成分的给药量是治疗有效量。本发明的药物制剂还可制成缓释制剂。The pharmaceutical compositions of the present invention comprise a safe and effective amount of adipose mesenchymal progenitor cells, a fatty matrix vascular component, and a pharmaceutically acceptable carrier. Such carriers include, but are not limited to, saline, buffer, dextrose, water, glycerol, ethanol, and combinations thereof. Usually, the pharmaceutical preparation should be matched to the mode of administration, and the pharmaceutical composition of the present invention can be prepared into an injection form, for example, by a conventional method using physiological saline or an aqueous solution containing glucose and other adjuvants. The pharmaceutical composition is preferably manufactured under sterile conditions. The amount of active ingredient administered is a therapeutically effective amount. The pharmaceutical preparation of the present invention can also be formulated into a sustained release preparation.

本发明所述脂肪间充质祖细胞和脂肪来源基质血管成分的有效量可随给药的模式和待治疗的疾病的严重程度等而变化。优选的有效量的选择可以由本领域普通技术人员根据各种因素来确定(例如通过临床试验)。所述的因素包括但不限于：所述药代动力学参数例如生物利用率、代谢、半衰期等；患者所要治疗的疾病的严重程度、患者的体重、患者的免疫状况、给药的途径等。The effective amount of the adipose mesenchymal progenitor cells and the adipose-derived matrix vascular component of the present invention may vary depending on the mode of administration and the severity of the disease to be treated and the like. The selection of a preferred effective amount can be determined by one of ordinary skill in the art based on various factors (e.g., by clinical trials). Such factors include, but are not limited to, the pharmacokinetic parameters such as bioavailability, metabolism, half-life, etc.; the severity of the condition to be treated by the patient, the weight of the patient, the immune status of the patient, the route of administration, and the like.

本发明的药物组合物优选为皮下或静脉注射试剂。在另一优选例中，所述的皮下或静脉注射试剂中，脂肪间充质祖细胞的浓度为0.1～10×10⁶个/ml，较佳地为0.2～5×10⁶个/ml，更佳地为0.5～2×10⁶个/ml。所述的脂肪间充质祖细胞优选地与细胞悬液配成悬浮液体使用，在另一优选例中，所述的细胞悬液为生理盐水。The pharmaceutical composition of the invention is preferably a subcutaneous or intravenous injection. In another preferred embodiment, the concentration of the adipose mesenchymal progenitor cells in the subcutaneous or intravenous injection is 0.1 to 10 × 10 ⁶ /ml, preferably 0.2 to 5 × 10 ⁶ /ml. More preferably, it is 0.5 to 2 x 10 ⁶ /ml. The adipose mesenchymal progenitor cells are preferably used in combination with a cell suspension to form a suspension liquid. In another preferred embodiment, the cell suspension is physiological saline.

所述的药物组合物的注射方式没有特别限制，可以是单次注射制剂，也可以是多次注射的制剂组合。在本发明的一种优选实施例中，所述的药物组合物为单次注射剂。The injection method of the pharmaceutical composition is not particularly limited, and may be a single injection preparation or a combination of preparations for multiple injections. In a preferred embodiment of the invention, the pharmaceutical composition is a single injection.

在本发明中，所述的药物组合物优选为静脉注射制剂或者气管滴注制剂。 In the present invention, the pharmaceutical composition is preferably an intravenous preparation or a tracheal instillation preparation.

本发明的主要优点：The main advantages of the invention:

(1)提供了一种用于治疗上呼吸道疾病的脂肪间充质祖细胞复合物，所述的脂肪间充质祖细胞组合物施用后可以对上呼吸道疾病起到一定的治疗或逆转效果。(1) A fat mesenchymal progenitor cell complex for treating an upper respiratory tract disease, which can exert a certain therapeutic or reversal effect on an upper respiratory tract disease after administration.

(2)脂肪间充质祖细胞来源丰富、取材方便、免疫原性低，在临床应用具有广阔前景。(2) Adipose mesenchymal progenitor cells are rich in source, convenient in obtaining materials and low in immunogenicity, and have broad prospects in clinical application.

下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数按重量计算。The invention is further illustrated below in conjunction with specific embodiments. It is to be understood that the examples are not intended to limit the scope of the invention. The experimental methods in the following examples which do not specify the specific conditions are usually in accordance with conventional conditions or according to the conditions recommended by the manufacturer. Percentages and parts are by weight unless otherwise stated.

实施例1脂肪间充质祖细胞的培养Example 1 Culture of adipose mesenchymal progenitor cells

一、试剂和耗材First, reagents and supplies

1.无菌手术器械及耗材1. Sterile surgical instruments and consumables

(1)无菌长柄手术镊子5把(1) 5 sterile sterile handles

(2)无菌100目滤网(2) Sterile 100 mesh filter

(3)灭菌40目滤网(3) Sterilization 40 mesh filter

(4)50ml离心管(4) 50ml centrifuge tube

2.无菌试剂：2. Sterile reagents:

(1)DMEM(基础培养基)、(1) DMEM (basic medium),

Iscove’s Modification of DMEM(Corning，基础培养基)；

Stem Cell Factor(SCF)(Corning,补充因子)Iscove's Modification of DMEM (Corning, basal medium);

Stem Cell Factor (SCF) (Corning, supplement factor)

(2)I型胶原酶(现配现用)：0.1％胶原酶I配制方法：称取0.1g胶原酶I粉末溶解于100ml未加任何因子的培养基中，用之前37℃进行预热。(2) Type I collagenase (currently used): 0.1% collagenase I preparation method: 0.1 g of collagenase I powder was weighed and dissolved in 100 ml of medium without any factor, and preheated at 37 ° C before.

(3)氯化钠注射液(3) Sodium chloride injection

二.实施方案2. Implementation plan

1.洗涤脂肪组织，除去血细胞。向离心管中加入20ml氯化钠注射液，拧紧盖子，剧烈晃动3分钟以充分洗涤脂肪组织，接着静止3-5分钟，使不同相分离，吸去下层水相；重复以上操作三次，直到下层液变为清澈。1. Wash the adipose tissue and remove the blood cells. Add 20 ml of sodium chloride injection to the centrifuge tube, tighten the lid, shake vigorously for 3 minutes to fully wash the adipose tissue, and then stand still for 3-5 minutes to separate the different phases. Aspirate the lower aqueous phase; repeat the above three times until the lower layer becomes clear.

2.分离基质血管组分(SVF)：加入等量新配制的预热的胶原酶Ⅱ溶液，37℃，200rpm，消化40～60分钟，将消化后的组织用无菌100目滤网过滤，室温1500rpm离心5分钟，得到的沉淀即为SVF。2. Separation of stromal vascular fraction (SVF): Add an equal amount of freshly prepared pre-warmed collagenase II solution, digest for 40-60 minutes at 37 ° C, 200 rpm, and filter the digested tissue with a sterile 100 mesh filter. After centrifugation at 1500 rpm for 5 minutes at room temperature, the resulting precipitate was SVF.

3.细胞培养：离心后，SVF沉积于离心管底部，用移液管自上而下小心除去上层油脂和下层的胶原酶溶液。适量DMEM重悬细胞，细胞在无血清，无抗生素的培养基中，在低氧(氧气的体积百分比为1-20％(v/v))进行培养，细胞融合达80％时传代细胞。培养至3-10代收获脂肪间充质祖细胞。3. Cell culture: After centrifugation, SVF was deposited on the bottom of the centrifuge tube, and the upper layer of the oil and the lower layer of the collagenase solution were carefully removed from the top with a pipette. The cells were resuspended in an appropriate amount of DMEM, and the cells were cultured in a serum-free, antibiotic-free medium under hypoxia (1-20% by volume of oxygen (v/v)), and passaged cells at a cell fusion of 80%. Cultured to 3-10 passages to harvest adipose mesenchymal progenitor cells.

4.细胞回输：将收获的细胞注入生理盐水，制备成细胞悬液，以备回输使用。4. Cell reinfusion: The harvested cells are injected into physiological saline to prepare a cell suspension for use in returning.

三.结论:III. Conclusion:

1.细胞分离培养实验结果1. Cell separation and culture experiment results

经上述方法分离的细胞得率:5×10⁵～1×10⁶cells/ml脂肪Cell yield isolated by the above method: 5 × 10 ⁵ to 1 × 10 ⁶ cells / ml of fat

细胞培养后，收获P3-P7代细胞，细胞量大于5×10⁹。After the cells were cultured, P3-P7 cells were harvested, and the amount of cells was greater than 5 × 10 ⁹ .

2.脂肪干细胞分化的免疫染色分析2. Immunostaining analysis of adipose stem cell differentiation

成脂诱导对照及油红O染色实验Adipogenic induction control and oil red O staining experiment

以实施例1所培养的细胞作为本发明组，进行成软骨、成骨、成脂分化能力测试。体外向软骨方向分化培养3-4周后阿辛蓝染色表明以实施例1所培养的细胞在体外具有向软骨分化的能力(图1B)；体外向骨方向分化培养3-4周后茜素红染色表明以实施例1所培养的细胞在体外具有向骨分化的能力(图1C)；体外向脂肪方向分化培养3-4周后油红O染色表明以实施例1所培养的细胞在体外具有向脂肪分化的能力(图1A)。The cells cultured in Example 1 were used as the group of the present invention to perform cartilage, osteogenic, and adipogenic differentiation tests. After 3-4 weeks of in vitro culture in the direction of cartilage, acin blue staining showed that the cells cultured in Example 1 had the ability to differentiate into cartilage in vitro (Fig. 1B); in vitro, in vitro cultured in the direction of bone for 3-4 weeks, alizarin red staining It is shown that the cells cultured in Example 1 have the ability to differentiate into bone in vitro (Fig. 1C); the oil red O staining after 3-4 weeks of differentiation into the adipogenic direction in vitro indicates that the cells cultured in Example 1 have a tendency in vitro. The ability to differentiate into fat (Figure 1A).

3.细胞的流式检测3. Flow detection of cells

通过酶消化法将细胞收集到离心管中，细胞悬液调整密度为1×10⁵cells/mL，1,800r/min(120g)离心5min，弃掉上清，用4℃的冷D-Hanks冲洗重悬细胞，再次将细胞悬液以800r/min，离心5min，之后弃去上清。然后用D-Hanks将细胞重悬至1mL，加入抗体5～10μL，避光，冰上放置30min。用D-Hanks冲洗，离心，弃上清，重复该冲洗过程2～3次，确保将未结合抗体除净。最后，加入约200至300μL的D-Hanks制成悬液，用流式细胞仪检测。 The cells were collected into a centrifuge tube by enzymatic digestion. The cell suspension was adjusted to a density of 1×10 ⁵ cells/mL, centrifuged at 1,800 r/min (120 g) for 5 min, the supernatant was discarded, and washed with cold D-Hanks at 4 °C. The cells were resuspended, and the cell suspension was again centrifuged at 800 r/min for 5 min, after which the supernatant was discarded. The cells were then resuspended to 1 mL with D-Hanks, and 5 to 10 μL of the antibody was added, protected from light, and placed on ice for 30 min. Rinse with D-Hanks, centrifuge, discard the supernatant, and repeat the rinsing process 2 to 3 times to ensure that the unbound antibody is removed. Finally, about 200 to 300 μL of D-Hanks was added to make a suspension, which was detected by flow cytometry.

haMPC流式检测结果haMPC flow test results

I型胶原酶消化方法分离、培养的P3代细胞MSCs表面抗原流式鉴定结果如图3所示，流式检测试验图谱如下表2中所示。The results of surface antigen flow identification of P3 cells MSCs isolated and cultured by type I collagenase digestion method are shown in Fig. 3, and the flow detection test pattern is shown in Table 2 below.

表2 流式检测试验图谱Table 2 Flow detection test map

结论：in conclusion:

该结果表明：通过流式细胞仪对脂肪间充质祖细胞进行细胞表面抗原标记表达的分析该细胞纯度高，大部分为脂肪间充质祖细胞，其中CD34、CD45为间充质祖细胞的阴性标记。The results showed that the expression of cell surface antigen marker was detected by flow cytometry on adipose mesenchymal progenitor cells. The purity of the cells was high, most of which were adipose mesenchymal progenitor cells, in which CD34 and CD45 were mesenchymal progenitor cells. Negative mark.

4.脂肪间充质祖细胞表达细胞因子的检测4. Detection of cytokines in adipose mesenchymal progenitor cells

实施例1中所培养的脂肪间充质祖细胞离心后重悬，调整细胞密度接种，48h后收集上清，检测细胞因子TGF-β1，HGF和VEGF、IL6、IL8、IL10，检测结果见下表。The adipose mesenchymal progenitor cells cultured in Example 1 were resuspended after centrifugation, adjusted for cell density, and the supernatant was collected 48 hours later to detect cytokines TGF-β1, HGF and VEGF, IL6, IL8, IL10. table.

结论：该脂肪间充质祖细胞能够表达TGF-β1、HGF、VEGF、IL6、IL8、和IL10。Conclusion: The adipose mesenchymal progenitor cells can express TGF-β1, HGF, VEGF, IL6, IL8, and IL10.

5.脂肪间充质祖细胞的免疫调节作用5. Immunomodulatory effects of adipose mesenchymal progenitor cells

实施例1中的haMPC和激活的PBMC在无血清培养基中共培养5天，以BMC only组为对照，haMPC与PBMC不同细胞比例为各个实验组(1：120、1：240、1：480、1：960、1：1920)。The haMPC and activated PBMC in Example 1 were co-cultured in serum-free medium for 5 days, and the BMC only group was used as control. The ratio of different cells of haMPC and PBMC was experimental group (1:120, 1:240, 1:480, 1:960, 1:1920).

PBMC在共培养前由CFSE荧光染色，并激活。5天共培养之后，采用流式细胞分析CFSE的荧光值在各组之间的差异即可反映PBMC的扩增程度，进而反应haMPC在体外实验中对于PBMC增殖的抑制作用，及其与细胞比例的关系。PBMC were fluorescently stained and activated by CFSE prior to co-culture. After 5 days of co-culture, the difference in fluorescence between the CFSE cells by flow cytometry can reflect the degree of amplification of PBMC, and thus The inhibitory effect of haMPC on PBMC proliferation in vitro and its relationship with cell ratio.

结论：以PBMC only组扩增的荧光值为100％，其余各组与PBMC only组的比值为统计量，(N＝3)。Conclusion: The fluorescence value of the PBMC only group was 100%, and the ratio of the other groups to the PBMC only group was statistically significant (N=3).

haMPC对PBMC增殖抑制活性在比例为1：960时开始减弱，并在此比例组开始呈现剂量相关现象。The proliferation inhibitory activity of haMPC on PBMC began to decrease at a ratio of 1:960, and dose-related phenomena began to appear in this proportional group.

实施例2 haMPC静脉注射/气管滴注联合给药对哮喘的治疗效果Example 2 Therapeutic effect of haMPC intravenous injection/tracheal instillation on asthma

小鼠分别于0、7、14d腹腔注射0.1％OVA 0.1ml致敏，第21d至77d将小鼠放入自制密闭的容器内以2.5％OVA雾化吸入30min/d，3次/周，共8周，建立哮喘模型。The mice were sensitized by intraperitoneal injection of 0.1% OVA 0.1ml at 0, 7, and 14d, and the mice were placed in a self-contained container at 2nd to 77d, and inhaled by 2.5% OVA for 30min/d, 3 times/week. For 8 weeks, an asthma model was established.

实验组(n＝8)于21d分别通过尾静脉注射1×10⁶/100μl和气管内滴注给药1×10⁶/30μl(均为1×10⁶cells+细胞悬液，其中所述的细胞悬液为生理盐水)。The experimental group (n=8) was administered by intravenous injection of 1×10 ⁶ /100 μl and intratracheal instillation of 1×10 ⁶ /30 μl (both 1×10 ⁶ cells+ cell suspension) on 21 days, respectively. The suspension is physiological saline).

注射治疗后第57天(78d)，所有实验组小鼠进行肺功能及气道高反应性检测，炎症细胞分类计数与细胞因子检测，脾CD4+CD25+Foxp3+调节性T细胞；肺泡灌洗液炎症细胞分类计数与细胞因子检测、肺脏组织病理检测。对照组(n＝8)采用PBS致敏及等体积PBS注射治疗。其主要结果如下：On the 57th day after the injection treatment (78d), all the experimental groups were tested for lung function and airway hyperresponsiveness, inflammatory cell classification and cytokine detection, spleen CD4+CD25+Foxp3+ regulatory T cells; alveolar lavage fluid Inflammatory cell differential count and cytokine detection, lung tissue pathology. The control group (n=8) was treated with PBS sensitization and an equal volume of PBS injection. The main results are as follows:

1.尾静脉注射组1. Tail vein injection group

1.1气道高反应性结果如附图4中所示。其中各组气道高反应性值均有随乙酰甲胆碱浓度升高而增加的趋势，M128、M256两个乙酰甲胆碱浓度下，OVA+haMPCs、PBS+PBS组的气道高反应性显著低于OVA+PBS组，P<0.05，其它乙酰甲胆碱浓度下OVA+PBS与OVA+haMPCs组的气道高反应性均无统计学差异。这提示在乙酰甲胆碱诱导小鼠发生了哮喘的情况下，haMPCs对气道高反应性产生了缓解作用。1.1 Airway hyperresponsiveness results are shown in Figure 4. The airway hyperresponsiveness of each group increased with the increase of methacholine concentration. The airway hyperresponsiveness of OVA+haMPCs and PBS+PBS groups under the two methacholine concentrations of M128 and M256. Significantly lower than the OVA+PBS group, P<0.05. There was no significant difference in airway hyperresponsiveness between OVA+PBS and OVA+haMPCs in other methacholine concentrations. This suggests that haMPCs have a mitigating effect on airway hyperresponsiveness in the case of methacholine-induced asthma in mice.

1.2肺组织病理结果如附图5中所示。其中，PBS+PBS组以及PBS+haMPCs组(未给药和给药的正常对照组)的观察结果类似，未出现上皮下纤维化现象。而在OVA处理的模型对照组中可见上皮下胶原纤维沉积明显增厚(见黑色箭头部分)，提示慢性哮喘小鼠出现明显的上皮下纤维化现象。经过haMPCs静脉注射治疗的实验组中，仅观察到轻度的上皮下纤维化，这说明haMPCs静脉给药产生了对哮喘小鼠上皮下纤维化症状的逆转。1.2 Pathological results of lung tissue are shown in Figure 5. Among them, the observation results of the PBS+PBS group and the PBS+haMPCs group (normal control group not administered and administered) were similar, and no subepithelial fibrosis occurred. In the model control group treated with OVA, it was found that the deposition of subepithelial collagen fibers was significantly thickened (see the black arrow), suggesting that there was significant subepithelial fibrosis in mice with chronic asthma. In the experimental group treated with intravenous injection of haMPCs, only mild subepithelial fibrosis was observed, indicating that intravenous administration of haMPCs produced a reversal of subepithelial fibrosis symptoms in asthmatic mice.

2.气管滴注组2. Tracheal instillation group

2.1气道高反应性结果 2.1 airway hyperresponsiveness results

结果如附图6中所示，其中各组气道高反应性值均有随乙酰甲胆碱浓度升高而增加的趋势，M256浓度时，哮喘组与哮喘治疗组、对照组数据有统计学差异，P<0.05。说明haMPCs能缓解哮喘消除的气道高反应性。The results are shown in Fig. 6. Among them, the airway hyperresponsiveness values of all groups increased with the increase of methacholine concentration. At M256 concentration, the asthma group and asthma treatment group and control group had statistical data. Difference, P < 0.05. This indicates that haMPCs can alleviate the airway hyperresponsiveness of asthma elimination.

2.2肺病理检测结果2.2 lung pathology test results

结果如附图7中所示。其中，PBS+PBS组以及PBS+haMPCs组(未给药和给药的正常对照组)的观察结果类似，未出现上皮下纤维化现象。而在OVA处理的模型对照组中可见上皮下胶原纤维沉积明显增厚(见白色箭头部分)，提示慢性哮喘小鼠出现明显的上皮下纤维化现象。经过haMPCs器官内滴注治疗的实验组中，仅观察到轻度的上皮下纤维化，这说明haMPCs静脉给药产生了对哮喘小鼠上皮下纤维化症状的逆转。The result is shown in Figure 7. Among them, the observation results of the PBS+PBS group and the PBS+haMPCs group (normal control group not administered and administered) were similar, and no subepithelial fibrosis occurred. In the model control group treated with OVA, it was found that the deposition of subepithelial collagen fibers was significantly thickened (see the white arrow), suggesting that there was significant subepithelial fibrosis in mice with chronic asthma. In the experimental group treated with intra-hap instillation of haMPCs, only mild subepithelial fibrosis was observed, indicating that intravenous administration of haMPCs produced a reversal of subepithelial fibrosis symptoms in asthmatic mice.

实施例3 haMPC静脉注射/气管滴注联合给药对慢性阻塞性肺病(COPD)的治疗效果Example 3 Therapeutic effect of combined intravenous administration of haMPC/tracheal instillation on chronic obstructive pulmonary disease (COPD)

臭氧暴露建立类似人COPD的实验小鼠(2.5ppm臭氧浓度下暴露3h/d，2次/周，共6周)。对照组空气暴露。臭氧暴露6周后，实验组(n＝8)小鼠接受1×10⁶/100μl haMPCs尾静脉注射或1×10⁶/30μl haMPCs气管内滴注治疗，对照组(n＝8)小鼠以等体积PBS注射治疗。d71(治疗4周后)进行肺功能检测后处死小鼠，收集标本进行炎症细胞分类计数与细胞因子检测，肺泡灌洗液进行炎症细胞分类计数与细胞因子检测，肺组织病理学检测等。其主要结果如下：Ozone exposure established experimental mice similar to human COPD (exposure 3 h/d at 2.5 ppm ozone concentration, 2 times/week for 6 weeks). The control group was exposed to air. After 6 weeks of ozone exposure, the experimental group (n=8) mice received 1×10 ⁶ /100 μl haMPCs tail vein injection or 1×10 ⁶ /30 μl haMPCs intratracheal instillation, and the control group (n=8) mice. Treatment with an equal volume of PBS injection. After d71 (after 4 weeks of treatment), the mice were sacrificed after lung function test. The specimens were collected for inflammatory cell classification and cytokine detection, alveolar lavage fluid for inflammatory cell classification and cytokine detection, and lung histopathology. The main results are as follows:

1.尾静脉注射组1. Tail vein injection group

1.1肺功能检测1.1 lung function test

结果如附图8中所示，其中与正常组相比，COPD模型组O₃+PBS T＝4w与O₃+PBS T＝6w的FEV₂₅/FVC％显著下降，有统计学差异，P<0.01；与COPD模型组O₃+PBS T＝6w相比，6周给药组O₃+haMPCs T＝6w的FEV₂₅/FVC％显著上升，有统计学差异，P<0.01。The results are shown in Fig. 8, in which the FEV ₂₅ /FVC% of the COPD model group O ₃ +PBS T=4w and O ₃ +PBS T=6w was significantly lower than the normal group, and there was a statistical difference, P<0.01; Compared with the COPD model group O ₃ +PBS T=6w, the FEV ₂₅ /FVC% of the O ₃ +haMPCs T=6w in the 6-week administration group increased significantly, and there was a statistical difference, P<0.01.

1.2肺组织病理检测1.2 lung tissue pathology test

结果如附图9和中所示，其中与正常组相比，COPD模型组O₃+PBS T＝4w、O₃+PBS T＝6w肺组织的单位面积/单位视野的MAN、MAA显著下降(P<0.05)；与COPD模型组O₃+PBS T＝4w相比，COPD治疗组O₃+haMPCs T＝4w肺组织的单位面积/单位视野的MAN、MAA显著升高(P<0.05)；与COPD模型组O₃+PBS T＝6w相比，COPD治疗组O₃+haMPCs T＝6w肺组织的单位面积/单位视野的MAN、MAA显著升高(P<0.05)。The results are shown in Figure 9 and in which the MAN, MAA per unit area per unit of field of the lung tissue of the COPD model group was significantly decreased compared with the normal group ( ₃ + PBS T = 4w, O ₃ + PBS T = 6w). P <0.05); compared with model group COPD O ₃ + PBS T = 4w, basis COPD treatment group O ₃ + haMPCs T = 4w lung tissues / vision unit MAN, MAA increased significantly (P <0.05); Compared with CO ₃ model group O ₃ +PBS T=6w, MAN and MAA per unit area/unit field of view of O ₃ +haMPCs T=6w lung tissue in COPD treatment group were significantly increased (P<0.05).

附图10中所示，COPD模型组O₃+PBS T＝4w、O₃+PBS T＝6w肺组织LM明显升高；与COPD模型组O₃+PBS T＝4w相比，COPD治疗组O₃+haMPCs T＝4w肺组织的 LM显著降低(P<0.05)；与COPD模型组O₃+PBS T＝6w相比，COPD治疗组O₃+haMPCs T＝6w肺组织的LM显著降低(P<0.05)。As shown in Fig. 10, the lung tissue LM was significantly increased in the COPD model group O ₃ +PBS T=4w, O ₃ +PBS T=6w; compared with the COPD model group O ₃ +PBS T=4w, COPD treatment group O ₃ +haMPCs T=4w LM of lung tissue was significantly decreased (P<0.05); compared with CO3 model group O ₃ +PBS T=6w, CO ₃ treatment group O ₃ +haMPCs T=6w lung tissue LM significantly decreased (P <0.05).

2.气管内滴注组2. Intratracheal instillation group

2.1肺功能检测2.1 lung function test

结果如附图11中所示，其中与正常组相比，COPD模型组O₃+PBS T＝4w与O₃+PBS T＝6w的FEV₂₅/FVC％显著下降，有统计学差异，P<0.01；与正常组相比，COPD模型组O₃+PBS T＝6w的FEV₂₅/FVC％显著下降，P<0.01；与COPD模型组O₃+PBS T＝4w相比，4周给药组O₃+haMPCs T＝6w的FEV₂₅/FVC％显著上升，有统计学差异，P<0.01；与COPD模型组O₃+PBS T＝6w相比，6周给药组O₃+haMPCs T＝6w的FEV₂₅/FVC％显著上升，结果有统计学差异，P<0.01。The results are shown in Fig. 11, in which the FEV ₂₅ /FVC% of the COPD model group O ₃ +PBS T=4w and O ₃ +PBS T=6w decreased significantly compared with the normal group, and there was a statistical difference, P<0.01; Compared with the normal group, the FEV ₂₅ /FVC% of O ₃ +PBS T=6w was significantly decreased in the COPD model group, P<0.01; compared with the COPD model group O ₃ +PBS T=4w, the 4-week administration group The FEV ₂₅ /FVC% of O ₃ +haMPCs T=6w increased significantly, there was a statistical difference, P<0.01; compared with the COPD model group O ₃ +PBS T=6w, the 6-week administration group O ₃ +haMPCs T= The FEV ₂₅ /FVC% of 6w increased significantly, and the results were statistically different, P <0.01.

2.2肺组织病理学检测2.2 lung histopathology test

结果如附图12和13中所示，其中与正常组相比，COPD模型组O₃+PBS T＝4w、O₃+PBS T＝6w肺组织的单位面积/单位视野的MAN、MAA显著下降(P<0.05)；与COPD模型组O₃+PBS T＝4w相比，COPD治疗组O₃+haMPCs T＝4w肺组织的单位面积/单位视野的MAN、MAA显著升高(P<0.05)；与COPD模型组O₃+PBS T＝6w相比，COPD治疗组O₃+haMPCs T＝6w肺组织的单位面积/单位视野的MAN、MAA显著升高(P<0.05)。附图13中所示，COPD模型组O₃+PBS T＝4w、O₃+PBS T＝6w肺组织LM明显升高；与COPD模型组O₃+PBS T＝4w相比，COPD治疗组O₃+haMPCs T＝4w肺组织的LM显著降低(P<0.05)；与COPD模型组O₃+PBS T＝6w相比，COPD治疗组O₃+haMPCs T＝6w肺组织的LM显著降低(P<0.05)。The results are shown in Figures 12 and 13, wherein the COPD model group O ₃ + PBS T = 4 w, O ₃ + PBS T = 6 w lung tissue per unit area / unit field of view MAN, MAA decreased significantly (P <0.05); COPD model group compared with the _{O 3 + PBS T = 4w,} COPD O ₃ + treatment group basis haMPCs T = 4w lung / vision unit MAN, MAA increased significantly (P <0.05) Compared with the COPD model group O ₃ +PBS T=6w, the MAN/MAA per unit area/unit field of view of O ₃ +haMPCs T=6w lung tissue in the COPD treatment group was significantly increased (P<0.05). As shown in Fig. 13, the lung tissue LM was significantly increased in the COPD model group O ₃ +PBS T=4w, O ₃ +PBS T=6w; compared with the COPD model group O ₃ +PBS T=4w, COPD treatment group O ₃ +haMPCs T=4w LM of lung tissue was significantly decreased (P<0.05); compared with CO3 model group O ₃ +PBS T=6w, CO ₃ treatment group O ₃ +haMPCs T=6w lung tissue LM significantly decreased (P <0.05).

实施例4 雄性小鼠的maMPC静脉注射到雌性小鼠体内的示踪Example 4 Tracing of male mice with maMPC intravenously into female mice

实验过程：experiment procedure:

雌性小鼠分别于第1、3、5、7、9、11、13天皮下注射200ul PBS的2mg Al(OH)₃包被的40ug ovalbumin(OVA)致敏。Female mice were sensitized subcutaneously with 2 mM Al(OH) ₃ coated 40 ug ovalbumin (OVA) on day 1, 3, 5, 7, 9, 11, and 13, respectively.

第20天，尾静脉注射雄性小鼠脂肪间充质祖细胞(细胞量：1×10⁶cells)。On day 20, male mouse adipose mesenchymal progenitor cells were injected into the tail vein (cell volume: 1 × 10 ⁶ cells).

分别于第21、22、23、24、25、26、27天，接受30min 5％雾化OVA，及鼻腔滴注20ul OVA(40mg/ml)激发哮喘(实验组)。On

days

21, 22, 23, 24, 25, 26, and 27, 30% 5% nebulized OVA was administered, and 20ul OVA (40 mg/ml) was administered intranasally to stimulate asthma (experimental group).

分别于第20、21、28、35天4个时间点断颈处死部分小鼠。Part of the mice were sacrificed by cervical dislocation at four time points on the 20th, 21st, 28th and 35th day.

第20天处死小鼠后(即尾静脉注射雄性小鼠脂肪间充质祖细胞后)取肺，用于 FISH检测(检测组织中Y染色体的存在)。After the mice were sacrificed on the 20th day (ie, after injection of male mouse adipose mesenchymal progenitor cells in the tail vein), the lungs were taken for FISH detection (detection of the presence of the Y chromosome in the tissue).

实验结果：Experimental results:

FISH检测初步结果如附图14中所示：The preliminary results of the FISH test are shown in Figure 14:

阳性对照组(未处理的雄性小鼠)中雄性小鼠肺组织的FISH检测结果是阳性的；阴性对照组(未处理的雌性小鼠)中雌性小鼠肺组织的FISH检测结果是阴性的；实验组(接受脂肪间充质祖细胞注射)尾静脉注射雄性小鼠maMPC到雌性小鼠几小时后，肺组织的FISH检测结果是阳性的。上述实验结果说明尾静脉注射雄性小鼠maMPC到雌性小鼠几小时后，maMPC到达肺组织，证明异体来源的本发明的脂肪间充质祖细胞具有与自体来源的脂肪间充质祖细胞相同的效果，且不会产生排异。The FISH test results of the lung tissue of the male mice in the positive control group (untreated male mice) were positive; the FISH test results of the lung tissues of the female mice in the negative control group (untreated female mice) were negative; The experimental group (injected with adipose mesenchymal progenitor cells) was injected into the male mouse maMPC to the female mice for several hours, and the FISH test of the lung tissue was positive. The above experimental results indicate that maMPC reaches lung tissue several hours after the tail vein injection of male mouse maMPC into female mice, demonstrating that the adipose-derived progenitor cells of the invention of the same origin are identical to the adipose-derived progenitor cells of autologous origin. The effect, and will not cause rejection.

在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。 All documents mentioned in the present application are hereby incorporated by reference in their entirety in their entireties in the the the the the the the the In addition, it should be understood that various modifications and changes may be made by those skilled in the art in the form of the appended claims.

Claims

一种脂肪间充质祖细胞的用途，其特征在于，用于制备治疗上呼吸道疾病的药物组合物。Use of a fat mesenchymal progenitor cell for the preparation of a pharmaceutical composition for treating upper respiratory tract diseases.
如权利要求1所述的用途，其特征在于，所述的上呼吸道疾病选自下组：慢性阻塞性肺疾病、哮喘。The use according to claim 1, wherein said upper respiratory tract disease is selected from the group consisting of chronic obstructive pulmonary disease and asthma.
如权利要求1所述的用途，其特征在于，所述的药物组合物包括：脂肪间充质祖细胞，和药学上可接受的载体。The use according to claim 1, wherein said pharmaceutical composition comprises: adipose mesenchymal progenitor cells, and a pharmaceutically acceptable carrier.
如权利要求1所述的用途，其特征在于，所述的治疗包括选自下组的一项或多项指标改善：肺功能、气道高反应性、气道炎症、生活质量评分。The use of claim 1 wherein said treatment comprises one or more indicators selected from the group consisting of lung function, airway hyperresponsiveness, airway inflammation, quality of life score.
如权利要求1所述的用途，其特征在于，所述的脂肪间充质祖细胞具有选自下组的任一或多种特征：The use according to claim 1 wherein said adipose mesenchymal progenitor cells have any one or more of the characteristics selected from the group consisting of:

(i)90％以上的细胞具有表面抗原CD29；(i) more than 90% of the cells have the surface antigen CD29;

(ii)90％以上的细胞具有表面抗原CD90；(ii) more than 90% of the cells have the surface antigen CD90;

(iii)90％以上的细胞具有表面抗原CD73；(iii) more than 90% of the cells have the surface antigen CD73;

(iv)90％以上的细胞具有表面抗原CD49d；(iv) more than 90% of the cells have the surface antigen CD49d;

(v)90％以上的细胞具有表面抗原CD44；(v) more than 90% of the cells have the surface antigen CD44;

(vi)90％以上的细胞具有表面抗原CD105；(vi) more than 90% of the cells have the surface antigen CD105;

(vii)90％以上的细胞具有表面抗原SCA-1。(vii) More than 90% of the cells have the surface antigen SCA-1.
如权利要求1-5任一所述的用途，其特征在于，所述的脂肪间充质祖细胞具有选自下组的任一或多种特征：The use according to any one of claims 1 to 5, wherein the adipose mesenchymal progenitor cells have any one or more of the following characteristics selected from the group consisting of:

(viii)在细胞群中，5％以下的细胞具有表面抗原CD34；(viii) in the cell population, less than 5% of the cells have the surface antigen CD34;

(ix)在细胞群中，5％以下的细胞具有表面抗原CD45；(ix) in the cell population, less than 5% of the cells have the surface antigen CD45;

(x)在细胞群中，5％以下的细胞具有表面抗原Actin；(x) in the cell population, less than 5% of the cells have a surface antigen Actin;

(xi)在细胞群中，5％以下的细胞具有表面抗原HLA-DR；(xi) in the cell population, less than 5% of the cells have the surface antigen HLA-DR;

(xii)在细胞群中，5％以下的细胞具有表面抗原CD14；(xii) in the cell population, less than 5% of the cells have the surface antigen CD14;

(xiv)在细胞群中，5％以下的细胞具有表面抗原CD106；(xiv) in the cell population, less than 5% of the cells have the surface antigen CD106;

(xv)在细胞群中，5％以下的细胞具有表面抗原CD80；(xv) in the cell population, less than 5% of the cells have the surface antigen CD80;

(xvi)在细胞群中，5％以下的细胞具有表面抗原CD86；(xvi) in the cell population, less than 5% of the cells have the surface antigen CD86;

(xvii)在细胞群中，5％以下的细胞具有表面抗原CD31。 (xvii) In the cell population, 5% or less of cells have the surface antigen CD31.
如权利要求1-5任一所述的用途，其特征在于，所述的药物组合物是选自下组的剂型：静脉注射剂，或气管滴注剂。The use according to any one of claims 1 to 5, wherein the pharmaceutical composition is a dosage form selected from the group consisting of an intravenous injection or a tracheal instillation.
如权利要求7所述的用途，其特征在于，所述的静脉滴注剂包括：脂肪间充质祖细胞，及细胞悬液；和/或The use according to claim 7, wherein the intravenous drip agent comprises: adipose mesenchymal progenitor cells, and a cell suspension; and/or

所述的气管滴注剂包括：脂肪间充质祖细胞，及细胞悬液。The tracheal instillation agent includes: adipose mesenchymal progenitor cells, and a cell suspension.
一种用于预防或治疗上呼吸道疾病的药物组合物，其特征在于，所述的药物组合物包括：有效量的脂肪间充质祖细胞，以及药学上可接受的载体。A pharmaceutical composition for preventing or treating an upper respiratory tract disease, characterized in that the pharmaceutical composition comprises an effective amount of adipose mesenchymal progenitor cells, and a pharmaceutically acceptable carrier.
如权利要求9所述的药物组合物，其特征在于，所述的上呼吸道疾病选自下组：慢性阻塞性肺疾病、哮喘。The pharmaceutical composition according to claim 9, wherein said upper respiratory tract disease is selected from the group consisting of chronic obstructive pulmonary disease and asthma.
如权利要求9所述的药物组合物，其特征在于，所述的脂肪间充质祖细胞具有选自下组的任一或多种特征：The pharmaceutical composition according to claim 9, wherein said adipose mesenchymal progenitor cells have one or more characteristics selected from the group consisting of:

(i)90％以上的细胞具有表面抗原CD29；(i) more than 90% of the cells have the surface antigen CD29;

(ii)90％以上的细胞具有表面抗原CD90；(ii) more than 90% of the cells have the surface antigen CD90;

(iii)90％以上的细胞具有表面抗原CD73；(iii) more than 90% of the cells have the surface antigen CD73;

(iv)90％以上的细胞具有表面抗原CD49d；(iv) more than 90% of the cells have the surface antigen CD49d;

(v)90％以上的细胞具有表面抗原CD44；(v) more than 90% of the cells have the surface antigen CD44;

(vi)90％以上的细胞具有表面抗原CD105；(vi) more than 90% of the cells have the surface antigen CD105;

(vii)90％以上的细胞具有表面抗原SCA-1。(vii) More than 90% of the cells have the surface antigen SCA-1.
如权利要求9所述的药物组合物，其特征在于，所述的脂肪间充质祖细胞具有选自下组的任一或多种特征：The pharmaceutical composition according to claim 9, wherein said adipose mesenchymal progenitor cells have one or more characteristics selected from the group consisting of:

(viii)在细胞群中，5％以下的细胞具有表面抗原CD34；(viii) in the cell population, less than 5% of the cells have the surface antigen CD34;

(ix)在细胞群中，5％以下的细胞具有表面抗原CD45；(ix) in the cell population, less than 5% of the cells have the surface antigen CD45;

(x)在细胞群中，5％以下的细胞具有表面抗原Actin；(x) in the cell population, less than 5% of the cells have a surface antigen Actin;

(xi)在细胞群中，5％以下的细胞具有表面抗原HLA-DR；(xi) in the cell population, less than 5% of the cells have the surface antigen HLA-DR;

(xii)在细胞群中，5％以下的细胞具有表面抗原CD14；(xii) in the cell population, less than 5% of the cells have the surface antigen CD14;

(xiv)在细胞群中，5％以下的细胞具有表面抗原CD106；(xiv) in the cell population, less than 5% of the cells have the surface antigen CD106;

(xv)在细胞群中，5％以下的细胞具有表面抗原CD80；(xv) in the cell population, less than 5% of the cells have the surface antigen CD80;

(xvi)在细胞群中，5％以下的细胞具有表面抗原CD86；(xvi) in the cell population, less than 5% of the cells have the surface antigen CD86;

(xvii)在细胞群中，5％以下的细胞具有表面抗原CD31。 (xvii) In the cell population, 5% or less of cells have the surface antigen CD31.
如权利要求9所述的药物组合物，其特征在于，所述的脂肪间充质祖细胞还表达细胞因子，且所述的细胞因子选自下组：TGF-β1，HGF，VEGF、IL6、IL8、IL10，或其组合。The pharmaceutical composition according to claim 9, wherein said adipose mesenchymal progenitor cells further express cytokines, and said cytokines are selected from the group consisting of TGF-β1, HGF, VEGF, IL6, IL8, IL10, or a combination thereof.
如权利要求9所述的药物组合物，其特征在于，所述的药物组合物中，所述的脂肪间充质祖细胞具有分化为骨，软骨和/或脂肪的能力。The pharmaceutical composition according to claim 9, wherein said adipose-derived mesenchymal progenitor cells have the ability to differentiate into bone, cartilage and/or fat.
如权利要求9所述的药物组合物，其特征在于，所述的脂肪间充质祖细胞是通过以下方法制备的：在含有0.0001-20wt％的血清或血清替代品，且不含有抗菌素的培养基中，对分离得到的基质血管组分进行培养和传代，得到P3-P7代的脂肪间充质祖细胞。The pharmaceutical composition according to claim 9, wherein said adipose-derived mesenchymal progenitor cells are prepared by the following method: culture containing 0.0001 to 20% by weight of serum or serum substitute and containing no antibiotic In the base, the isolated stromal vascular components were cultured and passaged to obtain P3-P7 generation adipose mesenchymal progenitor cells.
如权利要求9所述的药物组合物，其特征在于，所述的脂肪间充质祖细胞是在含氧1％-20％(v/v)的环境中培养传代的。The pharmaceutical composition according to claim 9, wherein said adipose mesenchymal progenitor cells are cultured and passaged in an atmosphere containing 1% to 20% (v/v) of oxygen.
如权利要求9所述的药物组合物，其特征在于，所述的药物组合物中，所述的脂肪间充质祖细胞在与单核细胞共培养实验中拟制能力>1:960，优选为1:960-1:120。 The pharmaceutical composition according to claim 9, wherein said adipose mesenchymal progenitor cells have a capacity of >1:960 in a co-culture experiment with monocytes, preferably in said pharmaceutical composition. It is 1:960-1:120.