WO2022012567A1 - Regend001细胞自体回输制剂治疗特发性肺纤维化的新用途 - Google Patents

Regend001细胞自体回输制剂治疗特发性肺纤维化的新用途 Download PDF

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WO2022012567A1
WO2022012567A1 PCT/CN2021/106176 CN2021106176W WO2022012567A1 WO 2022012567 A1 WO2022012567 A1 WO 2022012567A1 CN 2021106176 W CN2021106176 W CN 2021106176W WO 2022012567 A1 WO2022012567 A1 WO 2022012567A1
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cells
regend001
preparation
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basal
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张婷
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苏州吉美瑞生医学科技有限公司
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Priority to EP21842680.7A priority patent/EP4053263A4/en
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    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/42—Respiratory system, e.g. lungs, bronchi or lung cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
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    • A—HUMAN NECESSITIES
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • C12N2501/30—Hormones
    • C12N2501/33—Insulin

Definitions

  • the application relates to the field of clinical medicine, in particular to a new use of a preparation for autologous reinfusion of REGEND001 cells.
  • Idiopathic pulmonary fibrosis is a chronic progressive fibrotic interstitial pneumonia of unknown etiology. IPF has been included in the National Rare Disease List in 2018, and the number of cases in my country is increasing year by year. IPF occurs mostly in middle-aged and elderly men, and is mainly manifested as progressively worsening dyspnea, accompanied by restrictive ventilation dysfunction and gas exchange disorders, eventually leading to hypoxemia and even respiratory failure. IPF has a poor prognosis, and its lung histology and high-resolution CT (HRCT) of the chest show usual interstitial pneumonia (UIP), the main pathological features include diffuse alveolitis and pulmonary fibrosis, and its lung damage continues to progress , irreversible.
  • HRCT high-resolution CT
  • the traditional way to treat IPF is mainly to take anti-inflammatory drugs and anti-fibrotic drugs.
  • Glucocorticoids suppress inflammatory responses and immune processes.
  • Immunosuppressants cyclophosphamide, azathioprine, methotrexate, etc.
  • glucocorticoids and immunosuppressive/cytotoxic drugs have been used as the basic drugs for the treatment of IPF, but for the treatment of intermediate and advanced fibrosis
  • the curative effect of chemotherapeutic patients is not ideal.
  • pirfenidone and nintedanib are approved and marketed in China for the treatment of IPF.
  • the present application provides an application of a REGEND001 cell autologous reinfusion preparation in the preparation of a product whose function is to improve the lung function of patients with idiopathic pulmonary fibrosis.
  • the product is, for example, a drug or a drug composition for the treatment of idiopathic pulmonary fibrosis.
  • the REGEND001 cell autologous reinfusion preparation is a finished cell preparation prepared from the patient's own bronchial basal layer cells collected, isolated in vitro and cultured and expanded.
  • the preparation process of the REGEND001 cell autologous reinfusion preparation can adopt the method given in the examples of this application, or can adopt the preparation of clinical grade autologous bronchial basal layer cells and the infusion preparation described in Chinese patent application 201910407062.7 (CN111944737A). methods, which are hereby incorporated by reference.
  • the application provides an application of a REGEND001 cell autologous reinfusion preparation in the preparation of a product for improving and/or treating human lung ventilation function.
  • the present application provides the application of a REGEND001 cell autologous infusion preparation in the preparation of a product for improving human lung diffusing function.
  • the application provides an application of a REGEND001 cell autologous infusion preparation in the preparation of a product for improving human motor function.
  • the administration site of the above-mentioned REGEND001 cell autologous infusion preparation is any lung subsegment of the left lingual lobe, the left lower lobe, the right middle lobe, or the right lower lobe.
  • the dosage of the above-mentioned REGEND001 cell autologous infusion preparation is 0.1-10 ⁇ 10 6 bronchial basal layer cells/kg, preferably 1-10 ⁇ 10 6 cells/kg.
  • the application provides an application of a REGEND001 cell autologous infusion preparation in the treatment of idiopathic pulmonary fibrosis.
  • the REGEND001 cell autologous infusion preparation, the bronchial basal layer cells contained therein are suspended in physiological saline for injection, and each 14 mL of the REGEND001 cell autologous infusion preparation contains (4-300) ⁇ 10 6 bronchial basal lamina cells, preferably comprising (2-30) x 10 7 bronchial basal lamina cells.
  • the preparation of the REGEND001 cell autologous infusion preparation comprises the following steps:
  • Tissue preparation Take the in vitro active bronchial brushing tissue from at least one site for future use;
  • Enzymatic hydrolysis digest the above-mentioned tissues with tissue digestion solution, and collect cells after terminating the digestion with stop solution;
  • Plating and expansion culture take part of the digested cells, use the culture plate with trophoblast cells to plate the cells, collect the cells and use the culture plate with trophoblast cells to expand and culture them.
  • the cells grow to 50%-90% of the surface area of the culture plate, or when the cells grow in clonal form until the cells aggregate into clusters, and the cell clones grow to more than 80%, there are 40-100 cells in the clones, and the random observation Subculture was carried out when 3 of the 5 fields of view had A- and B-level clones; among them, A-level clones had regular and smooth outlines, clear clone boundaries, and cells in the clones were compactly arranged and uniform in size; B-level clones had basic outlines , the clone boundary is smooth and clear, most of the cells in the clone are compact and uniform, and a few cells are slightly larger in size and slightly loose in arrangement;
  • the tissue digest comprises 99v% DMEM/F12, 1-20 ng/mL DNase, 0.1-4 mg/mL Protease XIV and 10-200 ng/mL pancreatin; the temperature of the digestion treatment For 37 °C, the time is 0.5-2h.
  • the formulation of the medium used in the plating and expansion culture is: 225mL DMEM, 225mL F12, 20-70mL FBS, 0.2-2mM L-glutamine, 1-14ng/mL insulin, 0.1- 1ng/mL epidermal growth factor, 5-30ug/mL adenine, 2-20ug/mL hydrocortisone.
  • the stop solution comprises 90v% DMEM and 10v% (FBS).
  • the enzymatically hydrolyzed cells and the plated cells are cryopreserved; preferably, before the cells are cryopreserved, bacterial detection and mycoplasma detection are performed; preferably, the cells are thawed rapidly, and plated and/or expanded Increase cultivation.
  • a method for treating idiopathic pulmonary fibrosis comprising administering to a subject with idiopathic pulmonary fibrosis a REGEND001 cell autologous infusion preparation, wherein the REGEND001 cell autologous infusion preparation is collected ,
  • the finished cell preparation obtained from the patient's own bronchial basal layer cells isolated and cultured expanded in vitro.
  • the 14 lung sub-segments are the right middle lobe lateral segment bronchus, the right middle lobe medial segment bronchus, the right lower lobe apical segment bronchus, the right inner basal segment bronchus, Anterior basal segment bronchus of right lung, right extra basal segment bronchus, right posterior basal segment bronchus, left upper lingual lobe bronchus, left lower lingual lobe bronchus, left lower lobe apical segment bronchus, left anterior basal segment bronchus, The left inner basal bronchus, the left outer basal bronchus, and the left posterior basal bronchus.
  • the bronchial basal layer cells contained in the REGEND001 cell autologous infusion preparation are suspended in normal saline for injection, and each 14 mL of the REGEND001 cell autologous infusion preparation contains (4-300) ⁇ 10 6 bronchial basal cells bottom cell, preferably contains (2-30) ⁇ 10 7 bronchi basal cells.
  • the method for treating idiopathic pulmonary fibrosis comprises adding normal saline to 14 mL of the REGEND001 cell autologous infusion preparation and diluting it to 42 mL, and then injecting the diluent into the 14 lung subsections with a syringe one or more of the segments.
  • the idiopathic pulmonary fibrosis subject has moderate or severe diffusing dysfunction.
  • the idiopathic pulmonary fibrosis subject also has one or more diseases or conditions selected from the group consisting of hypertension, type 2 diabetes, coronary heart disease, Hashimoto's thyroiditis, pulmonary emphysema.
  • the dosage of the REGEND001 cell autologous infusion preparation is 0.1-10 ⁇ 10 6 bronchial basal cells/kg, preferably 1-10 ⁇ 10 6 bronchial basal cells/kg.
  • the present application has developed a bronchial basal layer cell product, namely a REGEND001 cell autologous infusion preparation.
  • a bronchial basal layer cell product namely a REGEND001 cell autologous infusion preparation.
  • local administration through the airway and autologous infusion into patients with idiopathic pulmonary fibrosis has the effect of repairing lung damage and improving pulmonary ventilation function.
  • Fig. 1 is the diffusing function (DLCO measured value/predicted value) change before and after the treatment of case 1 in the embodiment 3 of the application;
  • Fig. 3 is the diffusing function (DLCO measured value/predicted value) change before and after the treatment of case 2 in Example 3 of the application;
  • Fig. 4 is the diffusing function (DLCO measured value) change before and after the treatment of case 2 in the embodiment 3 of the application;
  • the predicted value of DLCO in the following examples is calculated by the predicted value (ECSC) equation recommended by the European Thoracic Society.
  • the predicted values are usually correlated with the patient's height (H), weight (W), and age (A).
  • the formula for calculating the predicted value of adult lung function in East China is:
  • the six-minute walk test is an exercise test for the functional status of patients with moderate to severe cardiopulmonary diseases.
  • the patient's response to medical intervention is mainly assessed by the patient's exercise capacity.
  • the guideline recommends using the absolute value.
  • the St. George's Respiratory Questionnaire is one of the most widely used special scales currently used to measure impaired health and quality of life in adult patients with respiratory diseases. Questions were weighted for three aspects: frequency and severity), activity (resulting in shortness of breath or limitation of activities caused by shortness of breath), and impact on daily activities (social impairment and psychological impairment caused by airway disease). Higher scores indicate poorer quality of life (References: Jones PW, Quirk FH, Baveystock CM. The St George's Respiratory Questionnaire. Respir Med. 1991 Sep; 85 Suppl B: 25-31; discussion 33-7). It is currently used by an increasing number of respiratory diseases to assess the quality of life of patients. Clinical trials of nintedanib, a chemotherapeutic drug for IPF approved in 2017, also used this evaluation program. At present, there is no fixed standard, and it is mainly used for the comparison of patients before and after themselves.
  • This embodiment provides a preparation process of clinical-grade autologous bronchial basal layer cells, which specifically includes the following steps:
  • the isolated active bronchial brushing tissue was taken for future use, and in this example, the tissue located in two different parts of the bronchus was selected. The reason for choosing two or more parts is to ensure the success rate of separation.
  • tissue digestion solution Take the tissue digestion solution and stop solution for later use; 99v% of the tissue digestion solution is DMEM/F12, the rest is 1-20ng/mL DNase, 0.1-4mg/mL protease XIV and 10-200ng/mL trypsin; stop 90v% of the liquid is DMEM and 10v% is FBS.
  • the above-mentioned brushed tissues were digested with tissue digestion solution, and cells were collected after termination of digestion of the digested tissues with stop solution.
  • the enzymolyzed cells were partially frozen for bacterial and mycoplasma detection.
  • the trophoblast cells that have been inactivated by irradiation are plated in T25 cell culture flasks and cultured in a cell culture incubator (37°C, 5% CO 2 ), which can be used after 1-2 days, and the cell density is increased after 1-2 days.
  • the cells are preferably spread to 50-70% of the bottom surface of the culture flask.
  • the morphology of clones it can be divided into three grades A, B and C.
  • the A-level clones have regular and smooth outlines, clear clone boundaries, compact cells in the clones, and uniform size
  • B-level clones have basically regular outlines, smooth and clear clone boundaries, most of the cells in the clones are compact and uniform, and a few cells are slightly larger in size , the arrangement is slightly loose
  • C-level clones have irregular outlines, unclear clone boundaries, loose cells in the clones, and hypertrophy.
  • the second passage is carried out. Remove the culture supernatant, add 1 ⁇ DPBS to wash once, add 5 mL of recombinant trypsin to digest at 37°C for 5-10 min, and after most cells become round and bright, pipette the adherent cells down and make a single-cell suspension. Digestion was terminated by an equal volume of DMEM medium, the cell suspension was collected, centrifuged at 1200 rpm for 5 min to remove the supernatant, and the cells were resuspended in medium. The cells were plated on 3 T75 cell culture flasks plated with trophoblast cells at a density of 5-10 ⁇ 10 3 cells/cm 2 , and the medium was changed every other day.
  • the fourth subculture is performed. Remove the culture supernatant from each flask, add 1 ⁇ DPBS to wash once, add 5mL of recombinant trypsin to digest at 37°C for 5-10min, after most cells become round and bright, pipet the adherent cells down and make them into single cells Suspension, the digestion was terminated with an equal volume of DMEM medium, the cell suspension was collected, centrifuged at 1200 rpm for 5 min to remove the supernatant, and the cells were resuspended in the medium. Take 1-10 ⁇ 10 5 cells for biological effect detection HOPX positive rate ⁇ 30%. At the same time, the resuspended cells were plated on 30 T75 cell culture flasks plated with trophoblast cells at a density of 5-10 ⁇ 10 3 cells/cm 2 for culture, and the medium was changed every other day.
  • the trophoblast cells are removed.
  • the operation is to remove the culture supernatant from each culture flask, add 1 ⁇ DPBS for washing once, add 5 mL of recombinant trypsin, place it at 37°C for 1-2 min, blow down the trophoblast cells, wash once with 1 ⁇ DPBS, add 5 mL of recombinant trypsin was digested. After most of the cells became round and bright, the adherent cells were pipetted down and pipetted into a single-cell suspension. The digestion was terminated with an equal volume of DMEM medium, and the cell suspension was collected and centrifuged at 1200 rpm for 5 min. and resuspend the cells in medium. 5-10 ⁇ 10 5 cells were taken for cell purity analysis and the result was that the proportion of KRT5 cells was ⁇ 90%. Finally, all cells were plated in 30 T75 cell culture flasks for culture.
  • This embodiment provides a preparation process of clinical-grade autologous bronchial basal layer cells, which specifically includes the following steps:
  • the isolated active bronchial brushing tissue was taken for future use, and in this example, the tissue located in two different parts of the bronchus was selected. The reason for choosing two or more parts is to ensure the success rate of separation.
  • tissue digestion solution Take the tissue digestion solution and stop solution for later use; 99v% of the tissue digestion solution is DMEM/F12, the rest is 1-20ng/mL DNase, 0.1-4mg/mL protease XIV and 10-200ng/mL trypsin; stop 90v% of the liquid is DMEM and 10v% is FBS.
  • the above-mentioned brushed tissues were digested with tissue digestion solution, and cells were collected after termination of digestion of the digested tissues with stop solution.
  • the enzymolyzed cells were partially frozen for bacterial and mycoplasma detection.
  • the trophoblast cells that have been inactivated by irradiation are plated in T25 cell culture flasks and cultured in a cell culture incubator (37°C, 5% CO 2 ), which can be used after 1-2 days, and the cell density is increased after 1-2 days.
  • the cells are preferably spread to 50-70% of the bottom surface of the culture flask.
  • the morphology of clones it can be divided into three grades A, B and C.
  • the A-level clones have regular and smooth outlines, clear clone boundaries, compact cells in the clones, and uniform size
  • B-level clones have basically regular outlines, smooth and clear clone boundaries, most of the cells in the clones are compact and uniform, and a few cells are slightly larger in size , the arrangement is slightly loose
  • C-level clones have irregular outlines, unclear clone boundaries, loose cells in the clones, and hypertrophy.
  • the product preparation arrangement After the product preparation arrangement is determined, take out the cryopreserved cells, one tube per site, after rapid dissolving in a 37°C water bath, collect all the liquid in a 15mL tube, and add dropwise 2mL of the resuscitation solution that has been incubated at 37°C , centrifuge at 1200 rpm for 5 min, remove the supernatant, resuspend the cells with medium, and culture the cells using a T25 cell culture flask plated with trophoblast cells.
  • the first subculture is performed. Remove the cell culture supernatant, wash once with 1 ⁇ DPBS, add 2 mL of recombinant trypsin to digest at 37°C for 5-10 min, after most cells become round and bright, pipette the adherent cells down and make a single cell suspension with Digestion was terminated with an equal volume of DMEM medium, the cell suspension was collected, centrifuged at 1200 rpm for 5 min to remove the supernatant, and the cells were resuspended in medium, and finally plated on a trophoblast at a density of 5-10 ⁇ 10 3 cells/cm 2 . Cells were cultured in T75 cell culture flasks, and the medium was changed every other day.
  • the second passage is carried out. Remove the culture supernatant, add 1 ⁇ DPBS to wash once, add 5 mL of recombinant trypsin to digest at 37°C for 5-10 min, and after most cells become round and bright, pipette the adherent cells down and make a single-cell suspension. Digestion was terminated by an equal volume of DMEM medium, the cell suspension was collected, centrifuged at 1200 rpm for 5 min to remove the supernatant, and the cells were resuspended in medium.
  • the results are negative, the biological effect test is HOPX positive rate ⁇ 30%, the cells are identified as KRT5 positive, and the cell purity analysis result is KRT5 cell ratio ⁇ 90 %.
  • the resuspended cells were plated on 3 T75 cell culture flasks plated with trophoblast cells at a density of 5-10 ⁇ 10 3 cells/cm 2 for culture, and the medium was changed every other day.
  • the fourth subculture is performed. Remove the culture supernatant from each flask, add 1 ⁇ DPBS to wash once, add 5mL of recombinant trypsin to digest at 37°C for 5-10min, after most cells become round and bright, pipet the adherent cells down and make them into single cells Suspension, the digestion was terminated with an equal volume of DMEM medium, the cell suspension was collected, centrifuged at 1200 rpm for 5 min to remove the supernatant, and the cells were resuspended in the medium. Take 1-10 ⁇ 10 5 cells for biological effect detection HOPX positive rate ⁇ 30%. At the same time, the resuspended cells were plated on 30 T75 cell culture flasks plated with trophoblast cells at a density of 5-10 ⁇ 10 3 cells/cm 2 for culture, and the medium was changed every other day.
  • the trophoblast cells are removed.
  • the operation is to remove the culture supernatant from each culture flask, add 1 ⁇ DPBS for washing once, add 5 mL of recombinant trypsin, place it at 37°C for 1-2 min, blow down the trophoblast cells, wash once with 1 ⁇ DPBS, add 5 mL of recombinant trypsin was digested. After most of the cells became round and bright, the adherent cells were pipetted down and pipetted into a single-cell suspension. The digestion was terminated with an equal volume of DMEM medium, and the cell suspension was collected and centrifuged at 1200 rpm for 5 min. and resuspend the cells in medium. 5-10 ⁇ 10 5 cells were taken for cell purity analysis and the result was that the proportion of KRT5 cells was ⁇ 90%. Finally, all cells were plated in 30 T75 cell culture flasks for culture.
  • the reinfusion sites are the left lingual lobe, left lower lobe and right middle lobe, and 14 lung subsegments (segmental bronchus) of the right lower lobe, which are the lateral segmental bronchus of the right middle lobe, the medial segmental bronchus of the right middle lobe, apical segment bronchus of right lower lobe, right inner basal segment bronchus, right anterior basal segment bronchus, right extra basal segment bronchus, right posterior basal segment bronchus, left upper lingual lobe bronchus, left lower lingual lobe bronchus, The apical segment bronchus of the left lower lobe, the anterior basal segment bronchus of the left lung, the inner basal segment bronchus of the left lung, the outer basal segment bronchus of the left lung, and the posterior basal segment bronchus of the left lung.
  • the use of the REGEND001 cell autologous infusion preparation is planned to be used once, and its treatment is divided into two parts.
  • the first part is the collection, isolation and culture of bronchial basal cells (ie, Example 1 or 2), and the second part is the REGEND001 cell autologous infusion preparation. It takes 4-8 weeks from the collection and isolation of bronchial basal layer cells to administration. After a 24-week follow-up observation, the subjects need to go to the hospital to receive corresponding inspections, including safety inspections and efficacy inspections.
  • Safety check items include blood routine, urine routine, blood biochemistry, electrocardiogram and lung tumor markers.
  • the efficacy test items include pulmonary function test (including the measured value of carbon monoxide diffusing capacity DLCO, the percentage of carbon monoxide diffusing capacity DLCO measured value to the predicted value, forced vital capacity FVC), six-minute walk test, and St. George's Respiratory Questionnaire SGRQ.
  • pulmonary function test including the measured value of carbon monoxide diffusing capacity DLCO, the percentage of carbon monoxide diffusing capacity DLCO measured value to the predicted value, forced vital capacity FVC), six-minute walk test, and St. George's Respiratory Questionnaire SGRQ.
  • IPF is a specific form of chronic progressive fibrotic interstitial pneumonia of unknown etiology confined to the lungs.
  • the diagnostic criteria are: 1. Exclude other interstitial lung diseases (ILDs) (such as ILDs related to home or occupational exposure, ILDs related to connective tissue diseases, and ILDs related to drug toxicity); 2. UIP on HRCT; 3. There is lung disease Patients with tissue samples can be diagnosed by combining HRCT and histological features. (One of the above items 2 and 3 can be satisfied).
  • Pulmonary ventilation function (DLCO), pre-% predicted (that is, the percentage of the measured value to the predicted value), was 50.0% at baseline, 60.7% at 12 weeks after transplantation, an improvement of 21.4% compared with baseline; 61.5% at 24 weeks after treatment, compared with The baseline was improved by 23.0%; the diffusing function was significantly improved after treatment (see Table 1, Figure 1).
  • the measured DLCO value of pulmonary ventilation function (mL ⁇ kPa -1 /s) was 4.40 at baseline, 5.29 at 12 weeks after treatment, and 5.37 at 24 weeks after treatment.
  • the diffusing function was significantly improved after treatment (see Figure 2).
  • the baseline was 330 meters, and after 12 weeks of treatment, it was 523 meters (an increase of 193 meters from the baseline, an increase of 58.48%), and the motor function status improved significantly.
  • Lung ventilation function DLCO pre-% predicted (that is, the percentage of the measured value to the predicted value), 23.1% at baseline, 26% at 4 weeks after treatment, and 28% at 12 weeks after treatment.
  • the diffusing function was significantly improved compared with the baseline (see Table 1 and image 3).
  • the measured DLCO value of pulmonary ventilation function (mL ⁇ kPa -1 /s) was 2.04 at baseline, 2.25 at 4 weeks after treatment, and 2.34 at 12 weeks after treatment.
  • the diffusing function was significantly improved compared with the baseline (see Figure 4).
  • the % predicted before DLCO (that is, the percentage of the measured value to the predicted value), which was not measured at the baseline period, was 23.2% at 4 weeks after treatment and 31% at 12 weeks after treatment, and the diffusion function was significantly improved (see Table 1 and Table 1). Figure 5).
  • the measured value of DLCO (mL ⁇ kPa -1 /s), which was not measured in the baseline period, was 1.64 at 4 weeks after treatment and 2.19 at 12 weeks after treatment, and the diffusing function was significantly improved (see Figure 6).

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Abstract

本发明提供了REGEND001细胞自体回输制剂及其在制备改善特发性肺纤维化患者肺功能的产品中的应用，所述制剂由采集、体外分离和培养扩增的患者自体支气管基底层细胞制备而得到。

Description

REGEND001细胞自体回输制剂治疗特发性肺纤维化的新用途 技术领域
本申请涉及临床医学领域，具体涉及一种REGEND001细胞自体回输制剂的新用途。
背景技术
特发性肺纤维化(IPF)是一种病因不明，慢性进行性纤维化性间质性肺炎。IPF已被列入2018年国家罕见病名录，目前在我国的发病人数呈逐年增加的趋势。IPF好发于中老年男性人群，主要表现为进行性加重的呼吸困难，伴限制性通气功能障碍和气体交换障碍，最终导致低氧血症、甚至呼吸衰竭。IPF预后差，其肺组织学和胸部高分辨率CT(HRCT)表现为普通型间质性肺炎(UIP)，主要病理特征包括弥漫性肺泡炎和肺间质纤维化，其肺部损伤持续进展，不可逆转。肺泡数量的缺乏导致肺部换气功能低下乃至丧失，从而导致患者机体缺氧、活动能力下降乃至死亡。因此IPF诊断后的平均生存期仅2.8年，死亡率高于大多数肿瘤，被称为一种“类肿瘤疾病”。
治疗IPF的传统方法主要是服用抗炎症药物和抗纤维化药物。糖皮质激素能抑制炎症反应和免疫过程。免疫抑制剂(环磷酰胺、硫唑嘌呤、甲氨蝶呤等)也有抑制炎症反应的作用，故糖皮质激素和免疫抑制剂/细胞毒药物曾作为治疗IPF的基本药物，但对于中晚期纤维化患者疗效不理想。目前在中国批准上市的IPF治疗药物有吡非尼酮和尼达尼布等。它们对于患者的肺功能下降有一定的延缓作用，但难以改善肺功能或逆转病程，无法真正阻止纤维化的发生。目前针对IPF，除了开展全肺移植手术之外，全世界范围内都缺乏有效的常规治疗手段，根本的原因在于缺乏有效方法能够再生修复损伤的肺泡结构。
哈佛大学Frank McKeon研究组于2011年率先从小鼠远端支气管中鉴定到支气管基底层细胞这一细胞类群。2018年,在成年人体内成功鉴定并在体外分离扩增到该群支气管基底层细胞。该群细胞属于基底层细胞大类，具有分化形成支气管和肺泡上皮的潜能。
发明内容
本申请提供一种REGEND001细胞自体回输制剂在制备产品中的应用，所述产品的功能为改善特发性肺纤维化患者肺功能。所述产品例如为用于治疗特发性肺纤维化的药物或药物组合物。
所述REGEND001细胞自体回输制剂为采集、体外分离和培养扩增的患者自身支气管基底层细胞制备而得到的细胞制剂成品。所述REGEND001细胞自体回输制剂的制备工艺可以采用本申请实施例中给出的方法，也可以采用中国专利申请201910407062.7(CN111944737A)中所记载的临床级自体支气管基底层细胞及回输制剂的制备方法，在此通过引用并入本文。
本申请提供一种REGEND001细胞自体回输制剂在制备产品中的应用，所述产品的功能为改善特发性肺纤维化患者肺换气功能，所述产品例如为用于改善特发性肺纤维化患者肺换气功能的药物或药物组合物。
本申请提供一种REGEND001细胞自体回输制剂在制备改善和/或治疗人肺换气功能产品中的应用。
本申请提供一种REGEND001细胞自体回输制剂在制备改善人肺弥散功能产品中的应用。
本申请提供一种REGEND001细胞自体回输制剂在制备改善人运动功能产品中的应用。
上述REGEND001细胞自体回输制剂的给药部位为左肺舌叶、左肺下叶与右肺中叶、右肺下叶任一肺亚段。
上述REGEND001细胞自体回输制剂的给药剂量为0.1-10×10 6个支气管基底层细胞/kg，优选为1-10×10 6个细胞/kg。
本申请提供一种REGEND001细胞自体回输制剂在治疗特发性肺纤维化中应用。
在一些实施方式中，所述的REGEND001细胞自体回输制剂，其中包含的支气管基底层细胞悬于注射用生理盐水中，且每14mL REGEND001细胞自体回输制剂中包含(4-300)×10 6个支气管基底层细胞，优选包含(2-30)×10 7个支气管基底层细胞。
在一些实施方式中，所述的REGEND001细胞自体回输制剂的制备包括如下步骤：
组织准备：取来自于至少一个部位的离体活性支气管刷检组织备用；
酶解：用组织消化液对上述组织进行消化处理，用终止液终止消化后收集细胞；
铺板、扩增培养：取部分经消化处理后的细胞，利用铺被滋养层细胞的培养板对细胞进行铺板培养，收集细胞并利用铺被滋养层细胞的培养板对其进行扩增培养，待细胞长满至培养板表面积的50％-90％时，或者待细胞呈克隆状生长至细胞聚集成团，细胞克隆长大至超过80％的克隆中有40-100个细胞，且随机观察的5个视野中有3个视野均有A级和B级克隆时进行传代培养；其中，A级克隆轮廓规则圆滑，克隆边界清晰，克隆 内细胞排列紧致，大小均一；B级克隆轮廓基本规则，克隆边界平滑清晰，克隆内细胞大部分紧致均一，有少量细胞体积稍大，排列略显疏松；
收集细胞：待传代培养细胞长满至培养皿表面积的85％-95％时，消化并收集贴壁细胞，洗涤即可。
在一些实施方式中，所述组织消化液包含99v％的DMEM/F12，1-20ng/mL的DNA酶，0.1-4mg/mL蛋白酶XIV和10-200ng/mL胰酶；所述消化处理的温度为37℃，时间为0.5-2h。
在一些实施方式中，所述铺板、扩增培养采用的培养基的配方为：225mL DMEM、225mL F12、20-70mL FBS、0.2-2mM L-谷氨酰胺、1-14ng/mL胰岛素、0.1-1ng/mL表皮生长因子、5-30ug/mL腺嘌呤、2-20ug/mL氢化可的松。
在一些实施方式中，所述终止液包含90v％DMEM和10v％(FBS)。
在一些实施方式中，对经酶解后的细胞、经铺板培养后的细胞进行冻存；优选在冻存细胞前，进行菌检和支原体检测；优选快速解冻细胞，进行铺板培养和/或扩增培养。
根据本发明的一个方面，提供一种治疗特发性肺纤维化的方法，包括向特发性肺纤维化受试者施用REGEND001细胞自体回输制剂，其中所述REGEND001细胞自体回输制剂为采集、体外分离和培养扩增的患者自身支气管基底层细胞制备而得到的细胞制剂成品。
在一些实施方式中，所述的治疗特发性肺纤维化的方法包括，将所述REGEND001细胞自体回输制剂递送到左肺舌叶、左肺下叶与右肺中叶、右肺下叶的14个肺亚段中的一个或多个，所述14个肺亚段分别为右肺中叶外侧段支气管、右肺中叶内侧段支气管、右肺下叶尖段支气管、右肺内基底段支气管、右肺前基底段支气管、右肺外基底段支气管、右肺后基底段支气管、左肺舌叶上段支气管、左肺舌叶下段支气管、左肺下叶尖段支气管、左肺前基底段支气管、左肺内基底段支气管、左肺外基底段支气管、左肺后基底段支气管。
在一些实施方式中，所述REGEND001细胞自体回输制剂包含的支气管基底层细胞悬于注射用生理盐水中，且每14mL REGEND001细胞自体回输制剂中包含(4-300)×10 6个支气管基底层细胞，优选包含(2-30)×10 7个支气管基底层细胞。
在一些实施方式中，所述的治疗特发性肺纤维化的方法，包括向14mL REGEND001细胞自体回输制剂中加入生理盐水稀释为42mL，然后用注射器将稀释液注射到所述14 个肺亚段中的一个或多个。
在一些实施方式中，所述特发性肺纤维化受试者具有中度或重度弥散功能障碍。
在一些实施方式中，所述特发性肺纤维化受试者还患有选自以下的一种或多种疾病或状况：高血压、2型糖尿病、冠心病、桥本氏甲状腺炎、肺气肿。
在一些实施方式中，所述REGEND001细胞自体回输制剂的给药剂量为0.1-10×10 6个支气管基底层细胞/kg，优选为1-10×10 6个支气管基底层细胞/kg。
本申请技术方案，具有如下优点：本申请开发了支气管基底层细胞产品即REGEND001细胞自体回输制剂。在早期探索性临床研究中，通过气道局部给药，将其自体回输至特发性肺纤维化患者体内，具有修复肺损伤，提高肺脏换气功能的效果。
附图说明
为了更清楚地说明本申请具体实施方式或现有技术中的技术方案，下面将对具体实施方式或现有技术描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图是本申请的一些实施方式，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他的附图。
图1是本申请实施例3中病例1治疗前后弥散功能(DLCO实测值/预计值)变化；
图2是本申请实施例3中病例1治疗前后弥散功能(DLCO实测值)变化；
图3是本申请实施例3中病例2治疗前后弥散功能(DLCO实测值/预计值)变化；
图4是本申请实施例3中病例2治疗前后弥散功能(DLCO实测值)变化；
图5是本申请实施例3中病例3治疗前后弥散功能(DLCO实测值/预计值)变化；
图6是本申请实施例3中病例3治疗前后弥散功能(DLCO实测值)变化；
具体实施方式
下列实施例中的DLCO预计值由欧洲胸科协会推荐的预计值(ECSC)方程计算而得，对我国而言，在ECSC方程的基础上加用校正系数进行校正获得预计值。通常预计值与患者的身高(H)、体重(W)和年龄(A)均具有相关性。我国华东地区成人肺功能预计值计算公式为：
DLCO＝0.3894*H-0.1244*A-0.0790*W-25.3405。
下列实施例中的6分钟步行试验参考标准说明：
据美国胸科协会(ATS)于2002年发布的6分钟步行实验详细指南，六分钟步行试验是一种对中、重度心肺疾病患者功能状态的运动试验，主要适用于测量中到重度心脏或肺疾病患者对于医疗干预的反应，主要评估患者的运动能力。对于获得结果6分钟步行距离的变化用哪种方式(绝对值或百分比或变化占预计值的百分比)表达对临床的意义最大，目前尚无定论，指南给出的建议为使用绝对值。
圣乔治呼吸问卷(SGRQ)参考标准说明：
圣乔治呼吸问卷(SGRQ)是目前用于测量呼吸性疾病成年患者健康受损情况和生活质量的应用最广泛的特殊量表之一,是一种标准化的患者自填问卷,根据症状(症状发生频率和严重程度)、活动(能导致气促或气促引起的活动受限)、对日常活动的影响(气道疾病引起的社会能力损害和心理障碍)三个方面的问题进行权重计分。得分越高，说明生活质量越差(参考文献：Jones PW,Quirk FH,Baveystock CM.The St George's Respiratory Questionnaire.Respir Med.1991 Sep；85 Suppl B:25-31；discussion 33-7)。当前被越来越多的呼吸性疾病采用，用来评估患者的生活质量。2017年获批的治疗IPF的化学药物尼达尼布的临床试验也采用该评估项目。当前并无固定的标准，主要用于患者自身前后的对比。
实施例1 REGEND001细胞自体回输制剂的制备
本实施例提供了一种临床级自体支气管基底层细胞的制备工艺，具体包括如下步骤：
取离体活性支气管刷检组织备用，在本实施例中选择取位于支气管两个不同部位的组织。之所以选择两个或者多个部位，是为了保证分离的成功率。
取组织消化液和终止液备用；其中组织消化液中，99v％为DMEM/F12，其余为1-20ng/mL的DNA酶，0.1-4mg/mL蛋白酶XIV和10-200ng/mL胰酶；终止液中90v％为DMEM，10v％为FBS。
利用组织消化液对上述刷检组织进行消化处理，利用终止液对经消化处理后的组织进行终止消化后收集细胞。经酶解后的细胞进行菌检和支原体检测后部分冻存。
取培养基备用，其中培养基中225mL DMEM，225mL F12，20-70mL FBS，0.2-2mM L-glutamine，1-14ng/mL胰岛素，0.1-1ng/mL表皮生长因子，5-30ug/mL腺嘌呤，2-20ug/mL氢化可的松。
将已经辐照灭活的滋养层细胞铺于T25细胞培养瓶中，放在细胞培养箱中培养(37℃，5％CO 2)，1-2天后可使用，细胞的密度以1-2天后细胞铺展到培养瓶底表面的50-70％为宜。
取部分经消化处理后的细胞，每部位来源细胞重悬并铺于1个铺被滋养层细胞的T25培养瓶中，加入200ng/mL庆大霉素，在37℃，CO 2浓度为4％-8％条件下培养，隔天更换培养基，待细胞呈克隆状生长至细胞聚集成团，细胞克隆长大至超过80％的克隆中有40-100个细胞，且随机观察的5个视野中有3个视野均有A级和B级克隆时，收集细胞进行传代培养。
根据克隆的形态，可以分为A、B和C三级。其中，A级克隆轮廓规则圆滑，克隆边界清晰，克隆内细胞排列紧致，大小均一；B级克隆轮廓基本规则，克隆边界平滑清晰，克隆内细胞大部分紧致均一，有少量细胞体积稍大，排列略显疏松；C级克隆轮廓不规则，克隆边界不清晰，克隆内细胞排列疏松，体态肥大。
对细胞的第一次传代培养，去细胞培养上清，1×DPBS洗涤一次，加入2mL重组胰酶37℃消化5-10min，待大部分细胞变圆变亮后，将贴壁细胞吹打下来并吹打成单细胞悬液，用等体积DMEM培养基终止消化，收集细胞悬液，1200rpm离心5min去上清，并用培养基重悬细胞，最后按照5-10×10 3个细胞/cm 2的密度铺于1个铺被滋养层细胞的T75细胞培养瓶进行培养，隔天换液。
待第一次传代细胞长满至培养瓶底表面积的50％-90％时，进行第二次传代培养。去培养上清，加入1×DPBS洗涤一次，加入5mL重组胰酶37℃消化5-10min，待大部分细胞变圆变亮后，将贴壁细胞吹打下来并吹打成单细胞悬液，用等体积DMEM培养基终止消化，收集细胞悬液，1200rpm离心5min去上清，并用培养基重悬细胞。按5-10×10 3个细胞/cm 2的密度铺于铺被滋养层细胞的3个T75细胞培养瓶上进行培养，隔天换液。
待第二次传代细胞长满至培养瓶底表面积的50％-90％时，进行第三次传代培养。每瓶细胞去培养上清，加入1×DPBS洗涤一次，加入5mL重组胰酶37℃消化5-10min，待大部分细胞变圆变亮后，将贴壁细胞吹打下来并吹打成单细胞悬液，用等体积DMEM培养基终止消化，收集细胞悬液，1200rpm离心5min去上清，并用培养基重悬细胞。取2mL细胞培养上清和3-16×10 5个细胞进行菌检和支原体检测为阴性，庆大霉素残留检测结果低于5.4ppb，生物学效应检测HOPX阳性率≥30％、细胞鉴别为KRT5阳性和细胞纯度分析结果为KRT5细胞比例≥90％。同时重悬细胞按5-10×10 3个细胞/cm 2的密 度铺于铺被滋养层细胞的10个T75细胞培养瓶上进行培养，隔天换液。
待第三次传代细胞长满至培养瓶底表面积的50％-90％时，进行第四次传代培养。每瓶细胞分别去培养上清，加入1×DPBS洗涤一次，加入5mL重组胰酶37℃消化5-10min，待大部分细胞变圆变亮后，将贴壁细胞吹打下来并吹打成单细胞悬液，用等体积DMEM培养基终止消化，收集细胞悬液，1200rpm离心5min去上清，并用培养基重悬细胞。取1-10×10 5个细胞进行生物学效应检测HOPX阳性率≥30％。同时重悬细胞按5-10×10 3个细胞/cm 2的密度铺于铺被滋养层细胞的30个T75细胞培养瓶上进行培养，隔天换液。
待第四次传代培养细胞长满至培养瓶底表面积的50％-90％时，进行滋养层细胞去除。操作为每个培养瓶中去培养上清，加入1×DPBS洗涤一次，加入5mL重组胰酶，在37℃中放置1-2min，将滋养层细胞吹打下来，再用1×DPBS洗涤一次，加入5mL重组胰酶消化，待大部分细胞变圆变亮后，将贴壁细胞吹打下来并吹打成单细胞悬液，用等体积DMEM培养基终止消化，收集细胞悬液，1200rpm离心5min去上清，并用培养基重悬细胞。取5-10×10 5个细胞进行细胞纯度分析结果为KRT5细胞比例≥90％。最后将所有细胞铺于30个T75细胞培养瓶中进行培养。
待细胞培养瓶中细胞长满至培养皿表面积的85％-95％时，每个培养瓶中加入1×DPBS洗涤一次，加入5mL重组胰酶，在37℃中放置5-15min，待大部分细胞变圆变亮后，用1mL枪头轻轻将贴壁细胞吹打下来并吹打成单细胞悬液，用等体积DMEM培养基终止消化，收集细胞悬液，再用5mL DMEM培养基冲洗培养瓶底表面，收集所有的细胞悬液。细胞悬液收集后，1200rpm离心5min，去上清，用注射用生理盐水洗涤3次，每次洗涤步骤为用40mL 1×DPBS重悬细胞沉淀，1200rpm离心5min，去上清。再加入16mL注射用生理盐水重悬细胞，制备成制剂成品，REGEND001细胞自体回输制剂。
实施例2 REGEND001细胞自体回输制剂的制备
本实施例提供了一种临床级自体支气管基底层细胞的制备工艺，具体包括如下步骤：
取离体活性支气管刷检组织备用，在本实施例中选择取位于支气管两个不同部位的组织。之所以选择两个或者多个部位，是为了保证分离的成功率。
取组织消化液和终止液备用；其中组织消化液中，99v％为DMEM/F12，其余为1-20ng/mL的DNA酶，0.1-4mg/mL蛋白酶XIV和10-200ng/mL胰酶；终止液中90v％为 DMEM，10v％为FBS。
利用组织消化液对上述刷检组织进行消化处理，利用终止液对经消化处理后的组织进行终止消化后收集细胞。经酶解后的细胞进行菌检和支原体检测后部分冻存。
取培养基备用，其中培养基中225mL DMEM，225mL F12，20-70mL FBS，0.2-2mM L-glutamine，1-14ng/mL胰岛素，0.1-1ng/mL表皮生长因子，5-30ug/mL腺嘌呤，2-20ug/mL氢化可的松。
将已经辐照灭活的滋养层细胞铺于T25细胞培养瓶中，放在细胞培养箱中培养(37℃，5％CO 2)，1-2天后可使用，细胞的密度以1-2天后细胞铺展到培养瓶底表面的50-70％为宜。
取部分经消化处理后的细胞，每部位来源细胞重悬并铺于1个铺被滋养层细胞的T25培养瓶中，加入200ng/mL庆大霉素，在37℃，CO 2浓度为4％-8％条件下培养，隔天更换培养基，待细胞呈克隆状生长至细胞聚集成团，细胞克隆长大至超过80％的克隆中有40-100个细胞，且随机观察的5个视野中有3个视野均有A级和B级克隆时，收集细胞进行冻存。
根据克隆的形态，可以分为A、B和C三级。其中，A级克隆轮廓规则圆滑，克隆边界清晰，克隆内细胞排列紧致，大小均一；B级克隆轮廓基本规则，克隆边界平滑清晰，克隆内细胞大部分紧致均一，有少量细胞体积稍大，排列略显疏松；C级克隆轮廓不规则，克隆边界不清晰，克隆内细胞排列疏松，体态肥大。
在产品制备安排确定后，取出冻存细胞，每个部位一管，在37℃水浴中快速溶解后，收集所有液体在15mL管中，并逐滴加入2mL已经在37℃温育过的复苏液，1200rpm离心5min，去上清后用培养基重悬细胞后，利用铺被滋养层细胞的T25细胞培养瓶对细胞进行培养。
待解冻细胞长满至细胞培养板表面积的50％-90％时，进行第一次传代培养。去细胞培养上清，1×DPBS洗涤一次，加入2mL重组胰酶37℃消化5-10min，待大部分细胞变圆变亮后，将贴壁细胞吹打下来并吹打成单细胞悬液，用等体积DMEM培养基终止消化，收集细胞悬液，1200rpm离心5min去上清，并用培养基重悬细胞，最后按照5-10×10 3个细胞/cm 2的密度铺于1个铺被滋养层细胞的T75细胞培养瓶进行培养，隔天换液。
待第一次传代细胞长满至培养瓶底表面积的50％-90％时，进行第二次传代培养。去培养上清，加入1×DPBS洗涤一次，加入5mL重组胰酶37℃消化5-10min，待大部 分细胞变圆变亮后，将贴壁细胞吹打下来并吹打成单细胞悬液，用等体积DMEM培养基终止消化，收集细胞悬液，1200rpm离心5min去上清，并用培养基重悬细胞。取2mL细胞培养上清和3-16×10 5个细胞进行菌检和支原体检测为阴性，生物学效应检测HOPX阳性率≥30％、细胞鉴别为KRT5阳性和细胞纯度分析结果为KRT5细胞比例≥90％。同时重悬细胞按5-10×10 3个细胞/cm 2的密度铺于铺被滋养层细胞的3个T75细胞培养瓶上进行培养，隔天换液。
待第二次传代细胞长满至培养瓶底表面积的50％-90％时，进行第三次传代培养。每瓶细胞去培养上清，加入1×DPBS洗涤一次，加入5mL重组胰酶37℃消化5-10min，待大部分细胞变圆变亮后，将贴壁细胞吹打下来并吹打成单细胞悬液，用等体积DMEM培养基终止消化，收集细胞悬液，1200rpm离心5min去上清，并用培养基重悬细胞。按5-10×10 3个细胞/cm 2的密度铺于铺被滋养层细胞的10个T75细胞培养瓶上进行培养，隔天换液。
待第三次传代细胞长满至培养瓶底表面积的50％-90％时，进行第四次传代培养。每瓶细胞分别去培养上清，加入1×DPBS洗涤一次，加入5mL重组胰酶37℃消化5-10min，待大部分细胞变圆变亮后，将贴壁细胞吹打下来并吹打成单细胞悬液，用等体积DMEM培养基终止消化，收集细胞悬液，1200rpm离心5min去上清，并用培养基重悬细胞。取1-10×10 5个细胞进行生物学效应检测HOPX阳性率≥30％。同时重悬细胞按5-10×10 3个细胞/cm 2的密度铺于铺被滋养层细胞的30个T75细胞培养瓶上进行培养，隔天换液。
待第四次传代培养细胞长满至培养瓶底表面积的50％-90％时，进行滋养层细胞去除。操作为每个培养瓶中去培养上清，加入1×DPBS洗涤一次，加入5mL重组胰酶，在37℃中放置1-2min，将滋养层细胞吹打下来，再用1×DPBS洗涤一次，加入5mL重组胰酶消化，待大部分细胞变圆变亮后，将贴壁细胞吹打下来并吹打成单细胞悬液，用等体积DMEM培养基终止消化，收集细胞悬液，1200rpm离心5min去上清，并用培养基重悬细胞。取5-10×10 5个细胞进行细胞纯度分析结果为KRT5细胞比例≥90％。最后将所有细胞铺于30个T75细胞培养瓶中进行培养。
待细胞培养瓶中细胞长满至培养皿表面积的85％-95％时，每个培养瓶中加入1×DPBS洗涤一次，加入5mL重组胰酶，在37℃中放置5-15min，待大部分细胞变圆变亮后，用1mL枪头轻轻将贴壁细胞吹打下来并吹打成单细胞悬液，用等体积DMEM培养基终止消化，收集细胞悬液，再用5mL DMEM培养基冲洗培养瓶底表面，收集所有 的细胞悬液。细胞悬液收集后，1200rpm离心5min，去上清，用注射用生理盐水洗涤3次，每次洗涤步骤为用40mL 1×DPBS重悬细胞沉淀，1200rpm离心5min，去上清。再加入16mL注射用生理盐水重悬细胞，制备成制剂成品，即得REGEND001细胞自体回输制剂。
实施3 REGEND001细胞自体回输制剂用于治疗特发性肺纤维化过程
1)具体步骤：
①回输部位为左肺舌叶、左肺下叶与右肺中叶、右肺下叶的14个肺亚段(段支气管)，分别为右肺中叶外侧段支气管、右肺中叶内侧段支气管、右肺下叶尖段支气管、右肺内基底段支气管、右肺前基底段支气管、右肺外基底段支气管、右肺后基底段支气管、左肺舌叶上段支气管、左肺舌叶下段支气管、左肺下叶尖段支气管、左肺前基底段支气管、左肺内基底段支气管、左肺外基底段支气管、左肺后基底段支气管。左肺上叶与右肺上叶各段支气管均不回输，；
②取实施例1或2制备的16mL REGEND001细胞自体回输制剂中的14mL(经测试14mL REGEND001细胞自体回输制剂中包含(4-300)×10 6个支气管基底层细胞，优选包含(2-30)×10 7个支气管基底层细胞)，加入生理盐水稀释为42mL。将稀释后的REGEND001细胞自体回输制剂转移至注射器中，注射器连接到支气管镜操作孔中的导管上，经纤支镜注入各肺亚段，每个肺段开口处(肺亚段)注入3mL；具体为每个肺段用注射器推进REGEND001细胞自体回输制剂3mL，之后推进5mL空气，用于将REGEND001细胞自体回输制剂推送至肺亚段远端；
③细胞需要约1.5小时时间与肺部炎症和创面区域建立紧密的粘附，完成移植之后要求受试者持平卧状态两小时左右；
④治疗后禁食水2小时，嘱受试者尽量减少咳嗽，必要时可给予可待因口服。
2)治疗疗程
REGEND001细胞自体回输制剂的使用拟一次，其治疗共分为两部分，第一部分为支气管基底层细胞的采集分离和培养(即实施例1或2)，第二部分为REGEND001细胞自体回输制剂的移植回输1次(整个疗程只回输这1次)，从支气管基底层细胞采集分离到给药共需时间4-8周。之后进行为期24周的随访观察，受试者需到医院接受相应的检查，包括安全性检查和疗效性检查。
安全性检查项目有血常规、尿常规、血生化、心电图和肺肿瘤标记物。
疗效性检查项目有肺功能检查(包含一氧化碳弥散量DLCO实测值，一氧化碳弥散量DLCO实测值占预计值的百分比、用力肺活量FVC)、六分钟步行试验、圣乔治呼吸问卷SGRQ。
3)以下为4例IPF患者治疗前后的肺功能数据。
根据2018年9月美国胸科学会(ATS)、欧洲呼吸学会(ERS)、日本呼吸学会(JRS)和拉丁美洲胸科学会(ALAT)四大国际呼吸学会在美国呼吸危重症杂志上联合颁布的有关特发性肺纤维化(IPF)诊断方面的指南(参考文献：1.Raghu G，et al.Diagnosis of Idiopathic Pulmonary Fibross.An Official ATS/ERS/JRS/ALAT Clinical Practice Guideline.Am J Respir Crit Care Med Published Online:Sep 2018；2.Raghu G，et al.ATS/ERS/JRS/ALAT Committee on Idiopathic Pulmonary Fibrosis.An Official ATS/ERS/JRS/ALAT Statement:Idiopathic Pulmonary Fibrosis:Evidence-based Guidelines for Diagnosis and Management.Am J Respir Crit Care Med Published Online:Mar 2011)：IPF是一种原因不明的，局限于肺部的慢性进行性纤维化性间质性肺炎的一种特殊形式。其诊断标准为：1、排除其他间质性肺疾病(ILD)(例如家庭或职业环境暴露相关ILD，结缔组织疾病相关ILD，药物毒性相关ILD)；2、HRCT表现为UIP；3、有肺组织标本的患者可以结合HRCT和组织学特点进行诊断。(其中以上第2条和第3条满足其中一条即可)。特发性肺纤维化患者的肺换气功能(肺弥散功能)受损，一氧化碳弥散量DLCO实测值占预计值的百分比低于80％。(临床肺弥散功能分级：正常：80％≤DLCO占预计值％；轻度：60％≤DLCO占预计值％<80％；中度：40％≤DLCO占预计值％<60％；重度：DLCO占预计值％<40％)
病例1：
男，66岁，身高172cm，体重83kg，反复咳嗽、咳痰伴气促，病程2年，临床诊断为特发性肺纤维化，中度弥散功能障碍，同时患有高血压病7年，2型糖尿病7年。接受实施例1制备的REGEND001细胞自体回输制剂治疗，给药剂量1.13×10 6个支气管基底层细胞/kg。
肺换气功能(DLCO)，前％预(即实测值占预计值百分比)，基线期为50.0％，移植治疗后12周为60.7％，较基线改善21.4％；治疗后24周61.5％，较基线改善23.0％； 治疗后弥散功能明显改善(见表1、图1)。
肺换气功能DLCO实测值(mL·kPa -1/s)，基线期4.40，治疗后12周5.29，治疗后24周5.37，治疗后弥散功能明显改善(见图2)。
6分钟步行试验，基线330米，治疗后12周523米(较基线增加193米，增加58.48％)，运动功能状态改善明显。
圣乔治呼吸问卷(SGRQ)，基线17.39，治疗后12周9.57(较基线下降7.82，44.97％)，治疗对生活质量的影响改善明显。
病例2：
男，64岁，身高172cm，体重86kg，反复咳嗽、咳痰，临床诊断为特发性肺纤维化，重度弥散功能障碍，病程4年，同时患冠心病4年，高血压4年。接受实施例2制备的REGEND001细胞自体回输制剂治疗，给药剂量0.71×10 6个支气管基底层细胞/kg。
肺换气功能DLCO，前％预(即实测值占预计值百分比)，基线期23.1％，治疗后4周26％，治疗后12周28％，弥散功能较基线有明显改善(见表1和图3)。
肺换气功能DLCO实测值(mL·kPa -1/s)，基线期2.04，治疗后4周2.25，治疗后12周2.34，弥散功能较基线有明显改善(见图4)。
尿常规、血生化治疗前后无异常或异常加剧变化。
病例3：
女，60岁，身高156cm，体重55kg，反复咳嗽、咳痰，病程8年余，临床诊断为特发性肺纤维化，病情较重，弥散功能未测出，同时合并桥本氏甲状腺炎。接受实施例2制备的REGEND001细胞自体回输制剂治疗，给药剂量3.84×10 6个支气管基底层细胞/kg。
DLCO前％预(即实测值占预计值百分比)，基线期未测出，治疗后4周该值为23.2％，治疗后12周该值为31％，弥散功能有明显改善(见表1和图5)。
DLCO实测值(mL·kPa -1/s)，基线期未测出，治疗后4周1.64，治疗后12周2.19，弥散功能有明显改善(见图6)。
显然，上述实施例仅仅是为清楚地说明所作的举例，而并非对实施方式的限定。对于所属领域的普通技术人员来说，在上述说明的基础上还可以做出其它不同形式的变化 或变动。这里无需也无法对所有的实施方式予以穷举。而由此所引伸出的显而易见的变化或变动仍处于本申请创造的保护范围之中。

Claims (22)

  1. REGEND001细胞自体回输制剂在制备产品中的应用，所述产品的功能为改善特发性肺纤维化患者肺功能。
  2. REGEND001细胞自体回输制剂在制备产品中的应用，所述产品的功能为改善特发性肺纤维化患者肺换气功能。
  3. REGEND001细胞自体回输制剂在制备改善和/或治疗人肺换气功能产品中的应用。
  4. REGEND001细胞自体回输制剂在制备改善人肺弥散功能产品中的应用。
  5. REGEND001细胞自体回输制剂在制备改善人运动功能产品中的应用。
  6. 根据权利要求1-5中任一一项中的应用，其特征在于，所述REGEND001细胞自体回输制剂的给药部位为左肺舌叶、左肺下叶与右肺中叶、右肺下叶的任一肺亚段。
  7. 根据权利要求1-5中任一一项中的应用，其特征在于，所述REGEND001细胞自体回输制剂的给药剂量为0.1-10×10 6个细胞/kg,优选为1-10×10 6个细胞/kg。
  8. REGEND001细胞自体回输制剂在治疗特发性肺纤维化中应用。
  9. REGEND001细胞自体回输制剂，所述REGEND001细胞自体回输制剂为采集、体外分离和培养扩增的患者自身支气管基底层细胞制备而得到的细胞制剂成品。
  10. 根据权利要求9所述的REGEND001细胞自体回输制剂，其中所述制剂成品中包含的支气管基底层细胞悬于注射用生理盐水中，且每14 mL REGEND001细胞自体回输制剂中包含(4-300)×10 6个支气管基底层细胞，优选包含(2-30)×10 7个支气管基底层细胞。
  11. 根据权利要求9或10所述的REGEND001细胞自体回输制剂，其中所述制备包括如下步骤：
    组织准备：取来自于至少一个部位的离体活性支气管刷检组织备用；
    酶解：用组织消化液对上述组织进行消化处理，用终止液终止消化后收集细胞；
    铺板、扩增培养：取部分经消化处理后的细胞，利用铺被滋养层细胞的培养板对细胞进行铺板培养，收集细胞并利用铺被滋养层细胞的培养板对其进行扩增培养，待细胞长满至培养板表面积的50％-90％时，或者待细胞呈克隆状生长至细胞聚集成团，细胞克隆长大至超过80％的克隆中有40-100个细胞，且随机观察的5个视野中有3个视野均有A级和B级克隆时进行传代培养；其中，A级克隆轮廓规则圆滑，克隆边界清晰，克隆内细胞排列紧致，大小均一；B级克隆轮廓基本规则，克隆边界平滑清晰，克隆内细胞 大部分紧致均一，有少量细胞体积稍大，排列略显疏松；
    收集细胞：待传代培养细胞长满至培养皿表面积的85％-95％时，消化并收集贴壁细胞，洗涤即可。
  12. 根据权利要求11所述的REGEND001细胞自体回输制剂，其特征在于，所述组织消化液包含99v％的DMEM/F12，1-20ng/mL的DNA酶，0.1-4mg/mL蛋白酶XIV和10-200ng/mL胰酶；所述消化处理的温度为37℃，时间为0.5-2h。
  13. 根据权利要求11所述的REGEND001细胞自体回输制剂，其特征在于，所述铺板、扩增培养采用的培养基的配方为：225mL DMEM、225mL F12、20-70mL FBS、0.2-2mM L-谷氨酰胺、1-14ng/mL胰岛素、0.1-1ng/mL表皮生长因子、5-30ug/mL腺嘌呤、2-20ug/mL氢化可的松。
  14. 根据权利要求11所述的REGEND001细胞自体回输制剂，其特征在于，所述终止液包含90v％DMEM和10v％(FBS)。
  15. 根据权利要求11所述的REGEND001细胞自体回输制剂，其特征在于，对经酶解后的细胞、经铺板培养后的细胞进行冻存；优选在冻存细胞前，进行菌检和支原体检测；优选快速解冻细胞，进行铺板培养和/或扩增培养。
  16. 治疗特发性肺纤维化的方法，包括向特发性肺纤维化受试者施用REGEND001细胞自体回输制剂，其中所述REGEND001细胞自体回输制剂为采集、体外分离和培养扩增的患者自身支气管基底层细胞制备而得到的细胞制剂成品。
  17. 根据权利要求16所述的治疗特发性肺纤维化的方法，其中，所述施用包括将所述REGEND001细胞自体回输制剂递送到左肺舌叶、左肺下叶与右肺中叶、右肺下叶的14个肺亚段中的一个或多个，所述14个肺亚段分别为右肺中叶外侧段支气管、右肺中叶内侧段支气管、右肺下叶尖段支气管、右肺内基底段支气管、右肺前基底段支气管、右肺外基底段支气管、右肺后基底段支气管、左肺舌叶上段支气管、左肺舌叶下段支气管、左肺下叶尖段支气管、左肺前基底段支气管、左肺内基底段支气管、左肺外基底段支气管、左肺后基底段支气管。
  18. 根据权利要求17所述的治疗特发性肺纤维化的方法，其中所述REGEND001细胞自体回输制剂包含的支气管基底层细胞悬于注射用生理盐水中，且每14 mL REGEND001细胞自体回输制剂中包含(4-300)×10 6个支气管基底层细胞，优选包含(2-30)×10 7个支气管基底层细胞。
  19. 根据权利要求18所述的治疗特发性肺纤维化的方法，包括向14 mL REGEND001 细胞自体回输制剂中加入生理盐水稀释为42mL，然后用注射器将稀释液注射到所述14个肺亚段中的一个或多个。
  20. 根据权利要求16所述的治疗特发性肺纤维化的方法，其中，所述受试者具有中度或重度弥散功能障碍。
  21. 根据权利要求所述的治疗特发性肺纤维化的方法，其中，所述受试者还患有选自以下的一种或多种疾病或状况：高血压、2型糖尿病、冠心病、桥本氏甲状腺炎、肺气肿。
  22. 根据权利要求16所述的治疗特发性肺纤维化的方法，其中，所述REGEND001细胞自体回输制剂的给药剂量为0.1-10×10 6个支气管基底层细胞/kg，优选为1-10×10 6个支气管基底层细胞/kg。
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