Method for rapidly inducing bone marrow-derived macrophages to transform into myofibroblasts

Abstract

The invention discloses a method for rapidly inducing bone marrow-derived macrophages to be transformed into myofibroblasts. The method for rapidly inducing the bone marrow-derived macrophages to be transformed into the myofibroblasts comprises the following steps: adding the bone marrow-derived macrophages BMDM into the improved 1640 culture medium, carrying out cell culture on the BMDM, replacing the BMDM once every two days, and completing the transformation of the macrophages into the myofibroblasts within five days. Compared with an existing scheme, the method has the advantage that the speed of inducing the BMDM cells to be converted into the myofibroblasts is higher.

Description

Technical Field

[0001] The invention belongs to the field of cell biology and relates to a method for rapidly inducing the transformation of bone marrow-derived macrophages into myofibroblasts. Background Art

[0002] Fibrosis is scarring and tissue hardening caused by excessive deposition of extracellular matrix (ECM) proteins by myofibroblasts in chronic inflammatory responses, which can affect multiple organs. If not controlled, it can lead to organ damage or even failure. Therefore, myofibroblasts are an important research subject in the study of organ fibrosis.

[0003] Classical theory holds that myofibroblasts are derived from fibroblasts under various stimuli. However, recent studies have found that macrophages also have the ability to transform into myofibroblasts, a phenomenon called macrophage-myofibroblast transition (MMT). Current studies have found that the MMT process is widely present in macrophages in tumors, kidney macrophages, lung macrophages, and cardiac macrophages, and exacerbates the fibrosis process of the corresponding organs. Intervention targeting the MMT process can slow down the rate of organ fibrosis and improve symptoms.

[0004] In 2014, Nikolic-Paterson et al. first described that bone marrow-derived macrophages can directly transdifferentiate into myofibroblasts, leading to renal interstitial fibrosis. This process is called MMT, and transdifferentiated macrophages are called MMT cells. MMT cells show co-expression of macrophage (CD68+ or F4 / 80+) and myofibroblast (α-SMA+) markers, accompanied by spindle-shaped changes in cell morphology.

[0005] The results of a study on biopsy (biopsy) found co-expression of macrophage (CD68+) and myofibroblast (α-SMA+) markers, and MMT cells were present in active fibrotic lesions, but were basically absent in samples of acute inflammation without fibrosis or without fibrotic lesions, suggesting that MMT is involved in active renal fibrosis. At the same time, the study also confirmed through lineage tracing that in the animal model of unilateral ureteral obstruction of renal fibrosis, there were a large number of F4 / 80+ myeloid macrophages expressing α-SMA and type I collagen, indicating that the macrophages that developed MMT were bone marrow-derived, and most of them co-expressed CD206, indicating that the cells that developed MMT were mainly M2 type. The results of another study on chronic rejection injury of allogeneic renal transplants showed that in the transplanted kidney biopsy specimens of patients with chronic rejection, MMT cells (CD68+ / α-SMA+) accounted for approximately 50% of the total number of myofibroblasts.

[0006] At the same time, the study also confirmed through lineage tracing that in the unilateral ureteral obstruction animal model of renal fibrosis, there were a large number of F4 / 80+ myeloid macrophages expressing α-SMA and type I collagen, suggesting that the macrophages that developed MMT were bone marrow-derived, and most of them co-expressed CD206, indicating that the cells that developed MMT were mainly M2 type.

[0007] Before the present invention was proposed, the existing scheme mainly relied on Tgf-β (10 ng / mL) alone to stimulate BMDM to achieve the purpose of MMT transformation. However, the existing scheme relies on the transdifferentiation effect of Tgf-β on macrophages, and the stimulation time is relatively long, often requiring more than 5 to 7 days to complete the entire differentiation process. Summary of the invention

[0008] The purpose of the present invention is to provide an improved 1640 culture medium for rapidly inducing the transformation of bone marrow-derived macrophages into myofibroblasts in view of the above-mentioned deficiencies in the prior art.

[0009] Another object of the present invention is to provide application of improved 1640 culture medium.

[0010] Another object of the present invention is to provide a method for rapidly inducing the transformation of bone marrow-derived macrophages into myofibroblasts.

[0011] The purpose of the present invention can be achieved through the following technical solutions:

[0012] A modified 1640 medium for rapidly inducing transformation of bone marrow-derived macrophages into myofibroblasts is prepared by using 1640 medium as a basic medium and adding IL-4 with a final concentration of 10-30 ng / mL, IL-13 with a final concentration of 10-30 ng / mL, and Tgf-β with a final concentration of 8-15 ng / mL.

[0013] As a preferred embodiment of the present invention, the improved 1640 medium is obtained by using 1640 medium as the base medium, and adding IL-4 with a final concentration of 20 ng / mL, IL-13 with a final concentration of 20 ng / mL, and Tgf-β with a final concentration of 10 ng / mL.

[0014] The improved 1640 culture medium of the present invention is used for rapidly inducing transformation of bone marrow-derived macrophages into myofibroblasts.

[0015] A method for rapidly inducing the transformation of bone marrow-derived macrophages into myofibroblasts comprises adding bone marrow-derived macrophages BMDM into the modified 1640 culture medium, culturing the BMDM cells at 37° C., replacing the culture medium every two days, and completing the transformation of macrophages into myofibroblasts within five days.

[0016] As a preferred embodiment of the present invention, the transformation of macrophages into myofibroblasts is completed within 5 days.

[0017] As a preferred embodiment of the present invention, the transformation of macrophages into myofibroblasts is completed within 4 days.

[0018] As a preferred embodiment of the present invention, the transformation of macrophages into myofibroblasts is completed within 3 days.

[0019] Beneficial effects:

[0020] Compared with the existing scheme, the invention can induce BMDM cells to transform into myofibroblasts faster.

[0021] from Figure 1 It can be seen that after 5 days of induction, compared with the existing scheme, the cell spindle shaped induced by the present invention is longer and closer to the morphological characteristics of myofibroblasts. Figure 3 It can be seen that at 3 days of induction treatment, the mRNA levels of the three main molecular markers of myofibroblasts in the present invention increased, showing significant statistical differences compared with before treatment. In the existing scheme, there was no statistical difference in the Cd206 mRNA level compared with before treatment.

[0022] from Figure 4 and Figure 5 It can be seen that at 5 days of induction treatment, the protein expression levels of the three main molecular markers of myofibroblasts in the present invention increased, while the α-SMA of the existing scheme did not increase at each time point within 5 days. By quantifying the protein gray value, after adopting this scheme, the expression levels of α-SMA, CD206, and CD90 proteins increased at 3 days and 5 days compared with before treatment, and reached the statistically significant threshold.

[0023] In summary, based on molecular markers and morphological evidence, the present invention can complete the transformation step of BMDM into myofibroblasts more quickly than existing solutions. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Cell morphological changes of bone marrow-derived macrophages after 5 days of treatment with this protocol and the existing protocol

[0025] Figure 2 PCA analysis of transcriptome sequencing of bone marrow-derived macrophages after 5 days of this protocol

[0026] Figure 3 The bone marrow-derived macrophages were treated with this protocol and the existing protocol for 0.5, 1, 2, and 3 days, respectively, and the mRNA content of MMT markers α-sma, Cd206, and Cd90 was detected using qPCR technology

[0027] Figure 4Bone marrow-derived macrophages were treated with this protocol and the existing protocol for 0.5, 1, 2, 3, and 5 days, respectively, and the protein content of MMT markers α-SMA, CD206, and CD90 was detected using Western Blot technology.

[0028] Figure 5 Bone marrow-derived macrophages were treated with this protocol and the existing protocol for 5 days, respectively, and the in situ expression of MMT markers α-SMA (Figure A), CD206 (Figure B), and CD90 (Figure C) in the cells was detected using immunofluorescence technology. DETAILED DESCRIPTION

[0029] Terminology explanation:

[0030] 1. Macrophage-Myofibroblast Transition (MMT): There are many different types of cells in the human body. Under certain conditions, different cell types can transform into each other. MMT refers to the cell biological process of macrophages transforming into fibroblasts.

[0031] 2. Mouse bone marrow-derived macrophages (BMDM): In scientific research, a series of experiments that simulate in vivo processes in an in vitro culture dish are called in vitro experiments. BMDM is a commonly used research subject in in vitro experiments conducted by macrophage-related research institutes. It is derived from monocytes in the bone marrow of mouse long bones. After 5 days of induction in 1640 culture medium containing L929 supernatant, monocytes are transformed into macrophages, namely BMDM.

[0032] 3. PCA (principle component analysis) is an unsupervised machine learning algorithm and a technique for exploring high-dimensional data structures. It is mainly used to reduce the dimensionality of data to identify differences between different samples. In this case, it is mainly used to distinguish and identify differences between groups and within groups.

[0033] Example 1

[0034] (1) Obtaining BMDM2: Obtain BMDM according to the steps in Trouplin, V., Boucherit, N., Gorvel, L., Conti, F., Mottola, G., Ghigo, E. Bone Marrow-derived Macrophage Production. J. Vis. Exp. (81), e50966, doi: 10.3791 / 50966 (2013).

[0035] (2) Complete the transformation from BMDM to myofibroblasts:

[0036] Based on the BMDM obtained in step (1), the following cytokines were additionally added to the 1640 culture medium (the concentrations in brackets are the final concentrations in the culture medium):

[0037] IL-4: 20ng / mL, IL-13: 20ng / mL, Tgf-β: 10ng / mL,

[0038] The 1640 culture medium and the above cytokines can be purchased through commercial channels, such as: (1640:

[0040] http: / / boster.com / index / products / productsDetail? goods_sn=PY G0049

[0041] IL-4: https: / / abclonal.com.cn / catalog / RP01161

[0042] IL-13:https: / / abclonal.com.cn / catalog / RP01596

[0043] Tgf-β: https: / / abclonal.com.cn / catalog / RP00671)

[0044] BMDMs were cultured using the above culture medium, which was replaced every two days. The transformation of macrophages into myofibroblasts was completed within five days. The specific characteristics include:

[0045] 1. The cell morphology changes from round to long spindle-shaped with fibroblast characteristics ( Figure 1 ).

[0046] 2. After culture, through transcriptome sequencing, the cells showed different gene transcription characteristics from before culture, and PCA dimensionality reduction could be used to effectively distinguish the cells before and after culture. ( Figure 2 )

[0047] 3. After culture, myofibroblast-related molecular markers (α

[0048] -SMA,CD90,CD206) expression increased significantly. This increase also included mRNA levels (qPCR detection, Figure 3 ) and protein levels (Western Blot detection, Figure 4 , immunofluorescence detection, Figure 5 )

[0049] Note: All statistical tests in the figures were performed using the Oneway-ANOVA test method.

[0050] *:p<0.05; **:p<0.01; ***:p<0.001.

[0051] Example 2

[0052] A modified 1640 medium, wherein the following cytokines are additionally added to the 1640 medium (the concentrations in brackets are the final concentrations in the medium):

[0053] IL-4: 18ng / mL, IL-13: 18ng / mL, Tgf-β: 12ng / mL.

[0054] Example 3

[0055] A modified 1640 medium, wherein the following cytokines are additionally added to the 1640 medium (the concentrations in brackets are the final concentrations in the medium):

[0056] IL-4: 22ng / mL, IL-13: 18ng / mL, Tgf-β: 10ng / mL.

[0057] Example 4

[0058] A modified 1640 medium, wherein the following cytokines are additionally added to the 1640 medium (the concentrations in brackets are the final concentrations in the medium):

[0059] IL-4: 20ng / mL, IL-13: 22ng / mL, Tgf-β: 10ng / mL.

[0060] Example 5

[0061] A modified 1640 medium, wherein the following cytokines are additionally added to the 1640 medium (the concentrations in brackets are the final concentrations in the medium):

[0062] IL-4: 17ng / mL, IL-13: 18ng / mL, Tgf-β: 19ng / mL.

Claims

1. A modified 1640 medium for rapidly inducing transformation of bone marrow-derived macrophages into myofibroblasts, characterized in that: The 1640 medium is used as the basic medium, and IL-4, IL-13 and 8-15 ng / mL Tgf-β are added to the final concentration of 10-30 ng / mL.

2. The improved 1640 medium according to claim 1, characterized in that The improved 1640 medium is based on the 1640 medium, and is added with IL-4 with a final concentration of 20 ng / mL, IL-13 with a final concentration of 20 ng / mL, and Tgf-β with a final concentration of 10 ng / mL.

3. Use of the improved 1640 medium according to claim 1 or 2 in rapidly inducing transformation of bone marrow-derived macrophages into myofibroblasts.

4. A method for rapidly inducing transformation of bone marrow-derived macrophages into myofibroblasts, characterized in that: The method comprises adding bone marrow-derived macrophages BMDM to the improved 1640 culture medium described in claim 1 or 2, culturing the BMDM at 37° C., replacing the culture medium every two days, and completing the transformation of macrophages into myofibroblasts within five days.

5. The method according to claim 4, characterized in that The transformation of macrophages into myofibroblasts was completed in 5 days.

6. The method according to claim 4, characterized in that The transformation of macrophages into myofibroblasts was completed in 4 days.

7. The method according to claim 4, characterized in that The transformation of macrophages into myofibroblasts was completed in 3 days.

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