Preparation method of diaphragm satellite cell model for diaphragm dysfunction caused by mechanical ventilation

Abstract

The invention discloses a preparation method of a diaphragm satellite cell model for diaphragm dysfunction caused by mechanical ventilation, and belongs to the technical field of cell biology. Comprising the following steps: (1) after a mouse is anesthetized and mechanically ventilated, cutting diaphragm along the outer edge of the diaphragm, digesting and centrifuging to obtain diaphragm cell suspension I; (2) adding the cell suspension I into an MS column I, centrifuging, re-suspending to obtain a cell suspension II, and then co-incubating with an anti-integrin-alpha7 microbead to obtain a pre-labeled cell suspension III; (3) pre-balancing an MS column II on a magnetic frame; adding the cell suspension III into an MS column II, then taking out the MS column II from the magnetic field, collecting the diaphragm satellite cells, and culturing the diaphragm satellite cells to obtain the diaphragm satellite cells for mechanical ventilation induced diaphragm dysfunction.

Description

Technical Field

[0001] The invention relates to the technical field of cell biology, and in particular to a method for preparing a diaphragm satellite cell model of diaphragm dysfunction caused by mechanical ventilation. Background Art

[0002] Mechanical ventilation (MV) is the main means of clinical rescue of critically ill patients. Ventilator-induced diaphragmatic dysfunction (VIDD) often occurs due to secondary diaphragm dysfunction (DD), which eventually leads to difficulty in weaning from MV.

[0003] In my country, about 73% of ICU patients need MV treatment. After 6 hours of MV, the diaphragm contraction force decreases rapidly; 18 hours of MV intervention can lead to diaphragm atrophy; about 2 / 3 of critically ill patients will have obvious DD on the first day of MV, and the mortality rate of DD patients in the ICU is as high as 50%. MV often leads to difficulty in weaning, and even reintubation within 48 hours of weaning, as well as complications such as cardiovascular disease, barotrauma and ventilator lung injury. In the United States, about 30% of critically ill patients will have difficulty in weaning, and the resulting treatment costs are about 64 billion US dollars a year, accounting for 10% of the total hospital costs. It can be seen that VIDD is a common problem faced by the international medical community.

[0004] At present, there is no recommended drug or effective strategy for the prevention and treatment of VIDD in clinical practice, and relevant research on its pathogenesis and prevention and treatment mechanisms is urgently needed. The bottleneck of scientific research in this field is that there is no stable method to obtain sufficient diaphragm satellite cells, resulting in the lack of cell models for research, which greatly restricts the research progress in this field. Therefore, providing sufficient diaphragm satellite cells is crucial for the study of the pathogenesis and prevention and treatment mechanisms of VIDD. Summary of the invention

[0005] The purpose of the present invention is to provide a method for preparing a diaphragm satellite cell model of diaphragm dysfunction caused by mechanical ventilation, so as to solve the problem that a cell model is urgently needed in current research.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] One of the technical solutions of the present invention is a method for preparing a diaphragm satellite cell model of diaphragm dysfunction induced by mechanical ventilation, comprising the following steps:

[0008] (1) After the mice were anesthetized and mechanically ventilated, the diaphragm was cut along the outer edge of the diaphragm, digested, and centrifuged to obtain diaphragm cell suspension I;

[0009] (2) adding the cell suspension I to MS column I, centrifuging and resuspending to obtain cell suspension II, and then incubating with anti-integrin α7 microbeads to obtain pre-labeled cell suspension III;

[0010] (3) Pre-balancing the MS column II on a magnetic rack; adding the cell suspension III to the MS column II, then removing the MS column II from the magnetic field, collecting and culturing the diaphragm satellite cells, and obtaining the diaphragm satellite cells with mechanical ventilation-induced diaphragm dysfunction.

[0011] Based on the above technical solution, the present invention has the following technical effects:

[0012] The present invention provides a method for preparing a diaphragm satellite cell model of diaphragm dysfunction caused by mechanical ventilation. After optimizing a series of parameters, the present invention finally obtains a method for obtaining diaphragm satellite cells stably and in sufficient quantity. 3×10 6 The diaphragm satellite cells with mechanical ventilation-induced diaphragm dysfunction prepared by the method of the present invention have a series of characteristics: they are more inclined to differentiate ( Figure 7 ), the expression of transcription and proliferation-related factors were significantly changed ( Figure 6 ). BRIEF DESCRIPTION OF THE DRAWINGS

[0013] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0014] Figure 1 Obtained protocol for diaphragm satellite cell isolation.

[0015] Figure 2 This is a diagram showing the diaphragm of a C57 mouse.

[0016] Figure 3 Diaphragm section of C57 mice.

[0017] Figure 4 It is a key link in diaphragmatic digestion.

[0018] Figure 5 This is the magnetic bead separation step for diaphragmatic satellite cells.

[0019] Figure 6In order to isolate the growth characteristics of diaphragmatic satellite cells, a is the expression results of transcription-related factors FoxK1, FoxK2 and ATF4 in different groups (TCM intervention group, mechanical ventilation group and control group); b is the expression results of cell growth cycle-related molecules MyoD and MyoG.

[0020] Figure 7 After mechanical ventilation, the diaphragm satellite cells isolated tend to differentiate; without mechanical ventilation stimulation, the diaphragm satellite cells isolated tend to proliferate. Among them, a is the diaphragm satellite cells of the non-mechanical ventilation group. b is the diaphragm satellite cells of the mechanical ventilation group. c is the BrdU (green) staining result of the diaphragm satellite cells of the non-mechanical ventilation group. d is the BrdU (green) staining result of the diaphragm satellite cells of the non-mechanical ventilation group. DAPI stains the cell nucleus blue. e is the result of staining the diaphragm satellite cells of the non-mechanical ventilation group with the differentiation marker MYH3 (green) antibody. f is the result of staining the diaphragm satellite cells of the mechanical ventilation group with the differentiation marker MYH3 (green) antibody. DAPI stains the cell nucleus blue. DETAILED DESCRIPTION

[0021] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0022] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0023] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0024] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present application description and examples are exemplary only.

[0025] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0026] The technical solutions described in the present invention, unless otherwise specified, are all conventional solutions in the art, and the reagents or raw materials used, unless otherwise specified, are purchased from commercial channels or have been disclosed.

[0027] The embodiment of the present invention provides a method for preparing a diaphragm satellite cell model of diaphragm dysfunction caused by mechanical ventilation, comprising the following steps:

[0028] (1) After the mice were anesthetized and mechanically ventilated, the diaphragm was cut along the outer edge of the diaphragm, digested, and centrifuged to obtain diaphragm cell suspension I;

[0029] (2) adding the cell suspension I to MS column I, centrifuging and resuspending to obtain cell suspension II, and then incubating with anti-integrin α7 microbeads to obtain pre-labeled cell suspension III;

[0030] (3) Pre-balancing the MS column II on a magnetic rack; adding the cell suspension III to the MS column II, then removing the MS column II from the magnetic field, collecting and culturing the diaphragm satellite cells, and obtaining the diaphragm satellite cells with mechanical ventilation-induced diaphragm dysfunction.

[0031] In some specific embodiments, in step (1), the duration of mechanical ventilation is 6 hours.

[0032] In some specific embodiments, in step (1), the centrifugation conditions are: centrifugation at 4°C, 1800 rpm for 15 min.

[0033] In some specific embodiments, in step (2), the concentration of the cell suspension II is 10 6 / 80μL; the volume ratio of the cell suspension II to the anti-integrinα7 microbeads is 4:1.

[0034] In some specific embodiments, in step (2), the co-incubation condition is incubation at 4°C for 15 minutes.

[0035] In some specific embodiments, in step (2), the MS column 1 is pre-equilibrated with a buffer;

[0036] The buffer composition is as follows: 1% FBS is added to 0.1M PBS.

[0037] In some specific embodiments, in step (3), the culture conditions are: culture at 37° C. for 4 days.

[0038] In some specific embodiments, in step (3), the method for collecting diaphragm satellite cells is: washing and collecting diaphragm satellite cells with a buffer;

[0039] The buffer composition is: based on 0.1M PBS, add 1% FBS, 0.7mmol / L CaCl 2 and 0.5mmol / LMgCl 2 .

[0040] The present invention adopts the principle of MACS method to separate diaphragm satellite cells: the immunomagnetic bead method for separating cells is based on the ability of cell surface antigens to bind to specific monoclonal antibodies connected to magnetic beads. In an external magnetic field, cells connected to magnetic beads through antibodies are adsorbed and retained in the magnetic field, while cells without such surface antigens cannot bind to specific monoclonal antibodies connected to magnetic beads and have no magnetism, and do not stay in the magnetic field, thereby allowing the cells to be separated.

[0041] The anti-integrin α7 microbeads in the embodiments of the present invention were purchased from Biopac Biotech. The English name of the product is integerin alpha-7 (avian), and the specification is Supernatant 1ml. The brand of the product is DSHB, and the product number is H1B4-Supernatant 1ml.

[0042] Matrigel matrix gel was purchased from BD Biosciences.

[0043] The buffer composition used in Example 1 of the present invention is as follows: The composition is as follows: 1% FBS is added to 0.1M PBS.

[0044] Example 1

[0045] A method for preparing a sterile diaphragm satellite cell model of diaphragm dysfunction caused by mechanical ventilation, the preparation process is as follows Figure 1 As shown, the following steps are included:

[0046] Ten-week-old C57 male mice were fasted for 6 hours, and then anesthetized by intraperitoneal injection of xylazine (10 mg / kg) and sodium pentobarbital (50 mg / kg) (no neuromuscular blockers were used throughout the process to prevent the effects on muscle cell characteristics). Then, the animals were connected to a ventilator via oral intubation. The important parameters were as follows: the fraction of inspired oxygen was 0.21 (room air), the tidal volume was 10 μL / mg, and the respiratory rate was 150-170 times / min. Mechanical ventilation was implemented for 6 hours. The C57 mice were placed on a constant temperature electric blanket module in a supine position throughout the process; 0.10 mL of lactated Ringer's solution was injected intraperitoneally every hour; and 50 mg / kg of pentobarbital was supplemented every 1.5 hours to ensure adequate anesthesia throughout the process.

[0047] After 6 hours of mechanical ventilation, the diaphragm was cut along the outer edge, the surface liquid was sucked dry, and the cells were transferred to a sterile centrifuge tube. Digestion solution was added and the cells were digested at 37°C for 35 minutes. The cells were filtered with an 80-mesh filter and centrifuged at 4°C, 1800 rpm for 15 minutes to obtain diaphragm cell suspension I.

[0048] The MS column was placed on a magnetic stand and then pre-equilibrated with a buffer containing 1% FBS. Then, the cell suspension I was mixed and added to the pre-equilibrated column. After centrifugation, the column was centrifuged for 10 min. 6 The cells were resuspended in 80 μL of 4% paraformaldehyde and 1% paraformaldehyde to obtain cell suspension II, which was then mixed with 20 μL of anti-integrin α7 microbeads and incubated at 4°C for 15 minutes to obtain pre-labeled cell suspension III.

[0049] Take another MS column and pre-balance it on a magnetic rack. Load the pre-labeled cell suspension III onto the column. Then remove the MS column from the magnetic field, wash it with 1 mL of buffer, collect the diaphragm satellite cells and inoculate them into a 12-well plate pre-coated with Matrigel. Culture at 37°C for 4 days. Then harvest the cells to obtain the diaphragm satellite cell model.

[0050] The final result of this embodiment can obtain 3×10 6 Diaphragm satellite cell models / C57 mice, with a purity of more than 95%.

[0051] Comparative Example 1

[0052] The only difference from Example 1 is that digestion solution is added and digestion is carried out at 37° C. for 25 minutes. The remaining steps are exactly the same as those in Example 1.

[0053] The final result is 0.5×10 5 Diaphragm satellite cell model / C57 mouse.

[0054] Comparative Example 2

[0055] The only difference from Example 1 is that digestion solution is added and digestion is carried out at 37° C. for 50 minutes. The remaining steps are exactly the same as those in Example 1.

[0056] The final result is 0.7×10 5 Diaphragm satellite cell model / C57 mouse.

[0057] Comparative Example 3

[0058] The only difference from Example 1 is that the mixture is mixed with 20 μL of anti-integrin α7 microbeads and incubated at 10° C. for 30 minutes. The remaining steps are exactly the same as in Example 1.

[0059] The final result is 0.3×10 5 A complete diaphragm satellite cell model per C57 mouse.

[0060] Comparative Example 4

[0061] The only difference from Example 1 is that the mixture is mixed with 20 μL of anti-integrin α7 microbeads and incubated at 24° C. for 10 minutes. The remaining steps are exactly the same as in Example 1.

[0062] The final result is 0.5×10 5 Diaphragm satellite cell model / C57 mouse.

[0063] Comparative Example 5

[0064] The only difference from Example 1 is that the buffer in Example 1 is replaced with PBS buffer. The remaining steps are exactly the same as in Example 1.

[0065] The final result is 1.5×10 5 Diaphragm satellite cell model / C57 mouse.

[0066] Effect example

[0067] The diaphragm satellite cell model obtained in Example 1 was used as a control together with the diaphragm cells of the Chinese medicine treatment group and the diaphragm cells of the blank group without any treatment.

[0068] The treatment method of the traditional Chinese medicine treatment group was: the diaphragm satellite cells were treated with the Qiguiyin prescription, which was composed of astragalus, angelica, honeysuckle, artemisia annua and polygonum cuspidatum in a scientific ratio of 12:3:3:2:2.

[0069] The results are as follows Figure 6-7 As shown, the diaphragm satellite cells with mechanical ventilation-induced diaphragm dysfunction prepared by the method of the present invention have a series of characteristics: they are more inclined to differentiate ( Figure 7 ), the expression of transcription and proliferation-related factors were significantly changed ( Figure 6 ).

[0070] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation methods of the present invention. For ordinary technical users in the relevant field, other different forms of changes or modifications can be made on the basis of the above description. It is not necessary and impossible to list all the implementation methods here. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the claims of the present invention.

Claims

1. A method for preparing a diaphragm satellite cell model of mechanical ventilation-induced diaphragm dysfunction, characterized in that: The following steps are involved: (1) After the mice were anesthetized and mechanically ventilated, the diaphragm was cut along the outer edge of the diaphragm, digested, and centrifuged to obtain diaphragm cell suspension I; (2) adding the cell suspension I to MS column I, centrifuging and resuspending to obtain cell suspension II, and then incubating with anti-integrin α7 microbeads to obtain pre-labeled cell suspension III; (3) Take MS column II and pre-equilibrate it on a magnetic rack; The cell suspension III is added to the MS column II, and then the MS column II is taken out from the magnetic field, and the diaphragm satellite cells are collected and cultured to obtain the diaphragm satellite cells with diaphragm dysfunction caused by mechanical ventilation.

2. The preparation method according to claim 1, characterized in that: In step (1), the duration of mechanical ventilation is 6 hours.

3. The preparation method according to claim 1, characterized in that: In step (1), the centrifugation conditions are: centrifugation at 4°C and 1800 rpm for 15 min.

4. The preparation method according to claim 1, characterized in that: In step (2), the concentration of the cell suspension II is 10 6 / 80μL; the volume ratio of the cell suspension II to the anti-integrinα7 microbeads is 4:

1.

5. The preparation method according to claim 1, characterized in that: In step (2), the co-incubation condition is incubation at 4°C for 15 minutes.

6. The preparation method according to claim 1, characterized in that: In step (2), the MS column I is pre-equilibrated with a buffer; The buffer composition is: based on 0.1M PBS, 1% FBS, 0.7mmol / L CaCl2 and 0.5mmol / LMgCl2 are added.

7. The preparation method according to claim 1, characterized in that: In step (3), the culture conditions are: culture at 37°C for 4 days.

8. The preparation method according to claim 1, characterized in that: In step (3), the method for collecting diaphragm satellite cells is: washing and collecting diaphragm satellite cells with a buffer solution; The buffer composition is as follows: 1% FBS is added to 0.1M PBS.

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