阅读说明：本技术 一种用于肺病治疗的脐带间充质干细胞及其制备方法 (Umbilical cord mesenchymal stem cells for treating lung diseases and preparation method thereof ) 是由 王宇环 罗晓玲 张正涵 于 2020-04-30 设计创作，主要内容包括：本发明提供了一种用于治疗肺部疾病的脐带间充质干细胞及其制备方法。本发明提供的脐带间充质干细胞可用于制备预防或治疗尘肺损伤及纤维化的药物。该药物应用于肺病治疗,尤其是尘肺肺损伤及其纤维化时,安全性高,对对应病症的干预和治疗效果好,通过成熟的细胞培养技术手段可以实现量化生产,具有很好的应用前景。(The invention provides umbilical cord mesenchymal stem cells for treating lung diseases and a preparation method thereof. The umbilical cord mesenchymal stem cells provided by the invention can be used for preparing medicines for preventing or treating pneumoconiosis injury and fibrosis. The medicine is applied to lung disease treatment, particularly pneumoconiosis lung injury and fibrosis thereof, has high safety, good intervention and treatment effects on corresponding diseases, can realize quantitative production by mature cell culture technical means, and has good application prospect.)

1. The application of the mesenchymal stem cells in preparing the medicine for treating the lung diseases is characterized in that the lung diseases are pneumoconiosis, idiopathic pulmonary fibrosis, chronic obstructive pulmonary disease, severe novel coronavirus pneumonia, acute respiratory distress syndrome or lung injury; the mesenchymal stem cells are one or more of umbilical cord mesenchymal stem cells, adipose mesenchymal stem cells, placenta mesenchymal stem cells and bone marrow mesenchymal stem cells.

2. The use of claim 1, wherein the mesenchymal stem cell is an umbilical cord mesenchymal stem cell.

3. The use of claim 2, wherein the umbilical cord mesenchymal stem cells comprise primary cells isolated from human umbilical cord tissue and passaged cells thereof.

4. The use of claim 2, wherein the total injection amount of the umbilical cord mesenchymal stem cells is 1 × 10⁶-36×10⁶One per kg body weight.

5. The application of claim 2, wherein the umbilical cord mesenchymal stem cells are prepared by collecting healthy umbilical cord of fetus produced by caesarean section of full-term pregnancy, and performing isolated culture within 12h by tissue block adherence method, and the culture comprises the following specific steps:

(1) cutting blood vessel along the umbilical vein lumen longitudinally, peeling off umbilical vein intima, cutting the rest tissue into small pieces, transferring into cell culture flask, adding 10mL complete culture medium to make it uniformly distributed, placing in CO₂An incubator;

(2) on day 7, total fluid change, half fluid change on day 12, colony count on day 14, colony count over 50 cells, when the cells reach 70% -80% confluence, passage cells are digested with 0.1% EDTA containing 0.125% trypsin, and passage cells are digested with 2.5 × 10³-5×10³Per cm²Density inoculation, marked as first generation cells, i.e. P1;

(3) taking P1 generation cells to be 3-6 × 10³/cm²Performing density inoculation, performing subculture, adding 20mL of complete culture medium into a culture flask, and placing in CO₂In the incubator, when the cells reach 70% -80% fusion, the subculture cells are digested by 0.1% EDTA containing 0.125% trypsin, and are marked as second generation cells, namely P2;

(4) and sequentially preparing and culturing third to eighth generation secondary cells, namely P3-P8.

6. The use according to claim 5, wherein the complete medium in step (1) is Lonza UltraCULTURE Universal serum-free basal medium containing 2% serum replacement or DMEM/F12 basal medium; the step (A) of1) And CO in step (3)₂The set conditions of the incubator are 37 ℃ and 5% CO₂And saturated humidity.

7. The umbilical cord mesenchymal stem cells are obtained from the umbilical cord of a healthy fetus produced by caesarean section of full-term pregnancy, and are separated and cultured by a tissue block adherence method within 12 hours, wherein the culture method comprises the following specific steps:

(1) cutting blood vessel along the umbilical vein lumen longitudinally, peeling off umbilical vein intima, cutting the rest tissue into small pieces, transferring into cell culture flask, adding 10mL complete culture medium to make it uniformly distributed, placing in CO₂An incubator;

(2) on day 7, total fluid change, half fluid change on day 12, colony count on day 14, colony count over 50 cells, when the cells reach 70% -80% confluence, passage cells are digested with 0.1% EDTA containing 0.125% trypsin, and passage cells are digested with 2.5 × 10³-5×10³/cm²Density inoculation, marked as first generation cells, i.e. P1;

(3) taking P1 generation cells to be 3-6 × 10³/cm²Performing density inoculation, performing subculture, adding 20mL of complete culture medium into a culture flask, and placing in CO₂In the incubator, when the cells reach 70% -80% fusion, the subculture cells are digested by 0.1% EDTA containing 0.125% trypsin, and are marked as second generation cells, namely P2;

(4) and sequentially preparing and culturing third to eighth generation secondary cells, namely P3-P8.

8. A medicament for treating pneumoconiosis, comprising the umbilical cord mesenchymal stem cells according to claim 7.

9. The medicament of claim 8, further comprising other pharmaceutically acceptable carriers, such as: trehalose, anhydrous or aqueous sodium chloride, glucose, sucrose, xylitol, fructose, glycerol, sorbitol, mannitol, potassium chloride, mannose, calcium chloride and magnesium chloride.

10. The drug of claim 9, wherein the drug is an injection solution consisting of 1 × 10⁵-25×10⁵Mesenchymal stem cells per mL, human serum albumin of 0.01-0.05g/mL, and sodium chloride injection of 0.9%.

Technical Field

The invention belongs to the field of biomedicine, and particularly relates to umbilical cord mesenchymal stem cells for treating lung diseases and a preparation method thereof.

Background

The stem cells have the characteristics of self-renewal capacity, multidirectional differentiation potential and the like, and mesenchymal stem cells (mesenchymal stem cells) are one of important members of a stem cell family, originate from early mesoderm and ectoderm, are firstly found in bone marrow, are gradually found in the characteristics of multidirectional differentiation, hematopoietic assistance and immune regulation, and can be induced and differentiated into various tissue cells in vivo or in vitro.

Kotton in 2001 suggested that adult tissue-derived mesenchymal stem cells could home to the lungs, which brought a new opportunity for stem cells to treat respiratory diseases. Burt et al report: the stem cells can be fused in the lung by contacting with somatic cells, show corresponding epithelial-like tissue morphological characteristics and functions, and fulfill the aim of repairing damaged lung tissues. For the mechanism of the stem cells for relieving inflammation and resisting fibrosis, one hypothesis is that the stem cells can replace alveolar epithelial cells and play a role in promoting proliferation, regeneration and repair of damaged alveolar cells; another hypothesis is that stem cells can alter the microenvironment at the damaged site, promoting the secretion of cytokines.

Once pneumoconiosis happens, the pneumoconiosis develops progressively, and currently, the worldwide treatment of the pneumoconiosis is lack of specific drugs, clinically applied drugs mainly inhibit pulmonary fibrosis, and drug therapy can only relieve the ventilation function of patients to a certain extent and cannot effectively cure the diseases. Another direct method is lung lavage, which can physically remove residual dust in alveolar cavities, swallow pneumoconiosis macrophages, inflammation-causing factors, fibrosis-causing factors and other substances, dredge air passages, reverse air passage spasm, increase lung ventilation and blood oxygen ratio and the like, and is considered as the only effective treatment method for the pneumoconiosis at present. The curative effect of the large-capacity whole lung lavage is mainly related to the course of the pneumoconiosis, in the early stage (0-I stage) of the pneumoconiosis, a large amount of dust is still in the alveoli, and the whole lung lavage can wash out the dust in the alveoli, so the curative effect is good; in the late stage (stage iii), a large amount of dust has been transported into the pulmonary interstitium and thus cannot be washed out, the therapeutic effect is far less effective than in the early stage, and the late stage often fails to perform a large volume whole lung lavage due to complications. In addition, studies have demonstrated that there may be loss of alveolar surfactant (PS) during lung lavage.

The application of mesenchymal stem cells in the treatment of various severe lung diseases is a hotspot of research in recent years, and research has proved that the mesenchymal stem cells can improve or reduce lung injury. The self-renewal and multidirectional differentiation potential of the mesenchymal stem cells and the low immunogenicity and immunosuppressive effects of the mesenchymal stem cells provide a reliable theoretical basis for the safety and effectiveness of clinical application of the mesenchymal stem cells; in addition, after being infused into the body, the mesenchymal stem cells can not only migrate to the damaged tissue part, but also inhibit the release of proinflammatory factors and promote the repair of the damaged tissue, thereby providing a more effective treatment means for improving the symptoms of patients with pneumoconiosis and delaying the pulmonary fibrosis of the patients.

Chinese patent CN 106038597A discloses an application of human amniotic mesenchymal stem cells in preparation of a preparation for treating acute lung injury, wherein the mesenchymal stem cells separated from human amniotic tissues are adopted, PQ is injected into a peritoneal cavity to prepare an acute lung injury animal model, and an hAD-MSCs preparation is transplanted through a sublingual vein, so that the acute lung injury treatment effect is good. However, this method is only directed to the treatment of paraquat-induced acute lung injury.

Chinese patent CN 110680833A discloses a set of medicines for treating pneumoconiosis by combining lung lavage, which consists of mesenchymal stem cells and lung lavage liquid. The medicine set can improve serum immunological index, lung ventilation function, serum fibrosis index and CT density histogram of pneumoconiosis patient and improve pulmonary fibrosis. However, the method is complicated in operation and complex in components, and needs to be further optimized.

Disclosure of Invention

The invention aims to provide new application of mesenchymal stem cells, including new application in pneumoconiosis injury and fibrosis thereof.

In one aspect, the invention provides the use of mesenchymal stem cells in the preparation of a medicament for the treatment of a pulmonary disease.

The mesenchymal stem cells include, but are not limited to, one or more of umbilical cord mesenchymal stem cells, adipose mesenchymal stem cells, placenta mesenchymal stem cells and bone marrow mesenchymal stem cells.

The lung diseases include, but are not limited to, pneumoconiosis, Idiopathic Pulmonary Fibrosis (IPF), Chronic Obstructive Pulmonary Disease (COPD), severe novel coronavirus pneumonia, Acute Respiratory Distress Syndrome (ARDS), various acute and chronic lung injuries.

The umbilical cord mesenchymal stem cells comprise primary cells separated from an umbilical cord of a fetus and passage cells thereof.

The passage cell of the umbilical cord mesenchymal stem cell can be 1-50 generations.

The preparation method of the umbilical cord mesenchymal stem cells comprises the steps of taking a healthy fetal umbilical cord produced by caesarean section of gestation at term, collecting the umbilical cord, and then performing isolated culture by using a tissue block wall-adhering method within 12h, wherein the specific culture steps are as follows:

(1) cutting blood vessel along the umbilical vein lumen longitudinally, peeling off umbilical vein intima, cutting the rest tissue into small pieces, transferring into cell culture flask, adding 10mL complete culture medium to make it uniformly distributed, placing in CO₂An incubator;

(2) on day 7, total fluid change, half fluid change on day 12, colony count on day 14, colony count over 50 cells, when the cells reach 70% -80% confluence, passage cells are digested with 0.1% EDTA containing 0.125% trypsin, and passage cells are digested with 2.5 × 10³-5×10³/cm²Density inoculation, marked as first generation cells, i.e. P1;

(3) taking P1 generation cells to be 3-6 × 10³/cm²Performing density inoculation, performing subculture, adding 20mL of complete culture medium into a culture flask, and placing in CO₂When the cells reached 70% -80% confluence in the incubator, the passaged cells were digested with 0.1% EDTA with 0.125% trypsin, and scored as second generation cells, P2.

(4) Sequentially preparing and culturing third to eighth generation secondary cells, namely P3-P8;

the complete culture medium in the step (1) is a Lonza UltraCULTURE general serum-free basal medium or a DMEM/F12 culture medium containing 2% of serum substitute; CO in the step (1) and the step (3)₂The set conditions of the incubator are 37 ℃ and 5% CO₂And saturated humidity.

The umbilical cord mesenchymal stem cells treat lung diseases by means of injection.

The umbilical cord mesenchymal stem cell injection mode is one-time injection or staged injection.

Preferably, theThe injection amount of the umbilical cord mesenchymal stem cells in one-time injection is 1 × 10⁶-6×10⁶Per kg body weight, more preferably 2 × 10⁶One per kg body weight.

Preferably, the umbilical cord mesenchymal stem cells are injected for 1 to 6 times in equal or unequal phases, and the total injection amount is 1 × 10⁶-36×10⁶One injection per kg body weight, preferably 4 times, and total injection amount of 8 × 10⁶One per kg body weight.

Preferably, the cell density of the human umbilical cord mesenchymal stem cells for injection is 2.5 × 10⁵-7.5×10⁵one/mL, preferably 5 × 10⁵one/mL.

The injection methods include but are not limited to: intravenous injection and tracheal administration.

The preparation method of the adipose tissue-derived mesenchymal stem cells comprises the following steps:

(1) aseptically collecting adipose tissue, performing separation culture by collagenase digestion method within 12h, washing adipose tissue, removing blood cells, adding preheated 0.075% collagenase I or collagenase IV working solution with the same amount as the adipose tissue suspension, vigorously shaking the culture bottle for 5-10 s, placing in a vibration gas bath kettle, digesting at 37 deg.C and 70rpm for 50-70min, taking out the culture bottle every 15 min, vigorously shaking the culture bottle for 5-10 s, and continuously placing in the vibration gas bath kettle for digestion;

(2) separation of stromal vascular fraction: filtering the digested tissue by using a blood transfusion device filter screen, then averagely subpackaging the digested tissue into 2 centrifuge tubes with the volume of 50mL, and centrifuging the centrifuge tubes for 10min at the room temperature of 300g-500g to obtain precipitates, namely the matrix blood vessel component;

(3) purifying and precipitating: after centrifugation, the stromal vascular fraction is deposited at the bottom of the centrifuge tube, and the upper layer of oil and the lower layer of collagenase solution are carefully removed from top to bottom by a pipette; resuspending matrix blood vessel component with appropriate amount of normal saline, blowing, centrifuging at room temperature of 300-500 g for 10 min;

(4) after centrifugation, carefully removing the supernatant, and taking care not to directly pour the supernatant; suspending the stromal vascular fraction with 10mL of culture medium, then collecting the stromal vascular fraction into 1 new 50mL centrifuge tube, centrifuging again at room temperature, 200g-400g, 10 min;

(5) a bottle is planted: centrifuging, collecting 15mL supernatant, inspecting, performing sterile detection, resuspending tissue with 20mL culture medium, uniformly distributing 20mL tissue suspension into culture flask, placing the flask at 37 deg.C and 5% CO₂An incubator; performing primary culture for 24 hours, performing full-scale liquid change, and observing the cell state under a mirror; pouring the matrix blood vessel components floating in the culture bottle and having no adherence and the culture solution into a new 50mL centrifuge tube, and centrifuging for 5min at 700g-900 g; pouring out the centrifugal supernatant, adding 8mL of new complete culture solution into each culture bottle again, then adding a proper amount of complete culture solution to a constant volume, blowing and uniformly mixing, and respectively adding 2mL of matrix blood vessel components into each culture bottle; on the fifth day, the liquid is changed fully; collecting cells on the seventh day, carrying out passage or cryopreservation, and culturing for about 7 days, wherein when the area percentage of the cell clone group of the primary culture reaches 70-80%, digesting and harvesting;

(6) after the cell suspension is obtained, the cell suspension is inoculated to a new culture container after constant volume, and the density of passage cells is 5000-²I.e. 3.75 × 10⁵-4.5×10⁵Culture flask according to 4.5 × 10⁵Subculturing in each culture bottle, and adding culture medium to 10mL in each bottle; standing at 37 deg.C for 5% CO₂Culturing in an incubator until the cell fusion degree reaches 85-90 percent to obtain the P1 generation adipose-derived stem cells;

(7) passage: lightly blowing and beating the resuspension cells, inoculating to a new culture container after constant volume, wherein the density of the passage cells is 5000-6000 cells/cm²I.e. 3.75 × 10⁵-4.5×10⁵Culture flask according to 4.5 × 10⁵Subculturing in one/culture flask, adding culture medium to 10 mL/flask, standing at 37 deg.C and 5% CO₂Culturing in an incubator until the cell fusion degree reaches 85-90 percent to obtain the P2 generation adipose-derived stem cells;

(8) and sequentially preparing and culturing third to eighth generation secondary cells, namely P3-P8.

In another aspect, the present invention provides an umbilical cord mesenchymal stem cell.

The umbilical cord mesenchymal stem cells are obtained from the umbilical cord of a fetus produced by healthy full-term gestation cesarean section, and are isolated and cultured by a tissue block adherence method within 12 hours, wherein the culture method comprises the following specific steps:

(1) cutting blood vessel along the umbilical vein lumen longitudinally, peeling off umbilical vein intima, cutting the rest tissue into small pieces, transferring into cell culture flask, adding 10mL complete culture medium to make it uniformly distributed, placing in CO₂An incubator;

(2) on day 7, total fluid change, half fluid change on day 12, and on day 14, counting colonies, counting more than 50 cells as colonies, and when the cells reach 70% -80% confluence, digesting the passage cells with 0.1% EDTA containing 0.125% trypsin, and digesting with 2.5-5 × 10³/cm²Density inoculation, marked as first generation cells, i.e. P1;

(3) taking P1 generation cells to be 3-6 × 10³/cm²Performing density inoculation, performing subculture, adding 20mL of complete culture medium into a culture flask, and placing in CO₂In the incubator, when the cells reach 70% -80% fusion, the subculture cells are digested by 0.1% EDTA containing 0.125% trypsin, and are marked as second generation cells, namely P2;

(4) and sequentially preparing and culturing third to eighth generation secondary cells, namely P3-P8.

The complete culture medium in the step (1) is a Lonza UltraCULTURE general serum-free basal medium or a DMEM/F12 culture medium containing 2% of serum substitute; CO in the step (1) and the step (3)₂The set conditions of the incubator are 37 ℃ and 5% CO₂And saturated humidity.

In still another aspect, the present invention also provides an adipose-derived mesenchymal stem cell.

The preparation method of the adipose tissue-derived mesenchymal stem cells comprises the following steps:

(1) aseptically collecting adipose tissue, performing separation culture by collagenase digestion method within 12h, washing adipose tissue, removing blood cells, adding preheated 0.075% collagenase I or collagenase IV working solution with the same amount as the adipose tissue suspension, vigorously shaking the culture bottle for 5-10 s, placing in a vibration gas bath kettle, digesting at 37 deg.C and 70rpm for 50-70min, taking out the culture bottle every 15 min, vigorously shaking the culture bottle for 5-10 s, and continuously placing in the vibration gas bath kettle for digestion;

(2) separation of stromal vascular fraction: filtering the digested tissue by using a blood transfusion device filter screen, then averagely subpackaging the digested tissue into 2 centrifuge tubes with the volume of 50mL, and centrifuging the centrifuge tubes for 10min at the room temperature of 300g-500g to obtain precipitates, namely the matrix blood vessel component;

(3) purifying and precipitating: after centrifugation, the stromal vascular fraction is deposited at the bottom of the centrifuge tube, and the upper layer of oil and the lower layer of collagenase solution are carefully removed from top to bottom by a pipette; resuspending matrix blood vessel component with appropriate amount of normal saline, blowing, centrifuging at room temperature of 300-500 g for 10 min;

(4) after centrifugation, carefully removing the supernatant, and taking care not to directly pour the supernatant; suspending the stromal vascular fraction with 10mL of culture medium, then collecting the stromal vascular fraction into 1 new 50mL centrifuge tube, centrifuging again at room temperature, 200g-400g, 10 min;

(5) a bottle is planted: centrifuging, collecting 15mL supernatant, inspecting, performing sterile detection, resuspending tissue with 20mL culture medium, uniformly distributing 20mL tissue suspension into culture flask, placing the flask at 37 deg.C and 5% CO₂An incubator; performing primary culture for 24 hours, performing full-scale liquid change, and observing the cell state under a mirror; pouring the matrix blood vessel components floating in the culture bottle and having no adherence and the culture solution into a new 50mL centrifuge tube, and centrifuging for 5min at 700g-900 g; pouring out the centrifugal supernatant, adding 8mL of new complete culture solution into each culture bottle again, then adding a proper amount of complete culture solution to a constant volume, blowing and uniformly mixing, and respectively adding 2mL of matrix blood vessel components into each culture bottle; on the fifth day, the liquid is changed fully; collecting cells on the seventh day, carrying out passage or cryopreservation, and culturing for about 7 days, wherein when the area percentage of the cell clone group of the primary culture reaches 70-80%, digesting and harvesting;

(6) after the cell suspension is obtained, the cell suspension is inoculated to a new culture container after constant volume, and the density of passage cells is 5000-²I.e. 3.75 × 10⁵-4.5×10⁵Culture flask according to 4.5 × 10⁵Subculturing in each culture bottle, and adding culture medium to 10mL in each bottle; standing at 37 deg.C for 5% CO₂Culturing in an incubator until the cell fusion degree reaches 85-90 percent to obtain the P1 generation adipose-derived stem cells;

(7) passage: blowing and beating the re-suspended cells gently, inoculating the re-suspended cells into a new culture container after constant volume, and carrying out passageThe cell density is 5000-²I.e. 3.75 × 10⁵-4.5×10⁵Culture flask according to 4.5 × 10⁵Subculturing in one/culture flask, adding culture medium to 10 mL/flask, standing at 37 deg.C and 5% CO₂Culturing in an incubator until the cell fusion degree reaches 85-90 percent to obtain the P2 generation adipose-derived stem cells;

(8) and sequentially preparing and culturing third to eighth generation secondary cells, namely P3-P8.

In yet another aspect, the invention also provides a medicament for treating a pulmonary disease.

The medicament comprises human mesenchymal stem cells.

The mesenchymal stem cell sources include but are not limited to umbilical cord, fat, placenta and bone marrow.

Preferably, the human mesenchymal stem cells in the medicament are umbilical cord mesenchymal stem cells prepared by the preparation method.

Preferably, the medicament is an injection.

The medicine also comprises other pharmaceutically acceptable carriers, such as: trehalose, anhydrous or aqueous sodium chloride, glucose, sucrose, xylitol, fructose, glycerol, sorbitol, mannitol, potassium chloride, mannose, calcium chloride and magnesium chloride.

In some embodiments, the medicine is an injection solution, and the components of the injection solution comprise mesenchymal stem cells, human serum albumin and 0.9% sodium chloride injection, wherein the content of the mesenchymal stem cells is 1 × 10⁵-25×10⁵The content of human serum albumin is 0.01-0.05 g/mL.

The method provided by the invention is applied to treating lung diseases, particularly pneumoconiosis injury and fibrosis thereof, has high safety and good intervention and treatment effects on corresponding diseases, can realize quantitative production by mature cell culture technical means, and has good application prospect.

Drawings

FIG. 1 is a graph showing the results of the cell colony formation rate of umbilical cord mesenchymal stem cells at the generations P1, P5 and P8. Where A is the total number of formations and B is the formation rate.

FIG. 2 shows the results of flow cytometry detection using umbilical cord mesenchymal stem cells from generations P1, P2, P5 and P8 as research objects.

FIG. 3 is a graph analyzing the percentage of different cell cycles of umbilical cord mesenchymal stem cells of generations P1, P2, P5 and P8.

FIG. 4 shows the expression result of indoleamine 2, 3-dioxygenase after TNF-alpha and INF-gamma stimulation of umbilical cord mesenchymal stem cells.

Detailed Description

The present invention will be further illustrated in detail with reference to the following specific examples, which are not intended to limit the present invention but are merely illustrative thereof. The experimental methods used in the following examples are not specifically described, and the materials, reagents and the like used in the following examples are generally commercially available under the usual conditions without specific descriptions.