A method and drug combination for enhancing the proliferation ability of type II alveolar epithelial cells cultured in vitro

Abstract

The present invention relates to the technical field of alveolar epithelial cell culture methods, and specifically to a method for enhancing the proliferation ability of type II alveolar epithelial cells cultured in vitro and a drug combination thereof. The drug combination includes atipamezole and dexmedetomidine. Atipamezole and dexmedetomidine are used to treat type II alveolar epithelial cells in succession, which can promote the proliferation of type II alveolar epithelial cells, maintain the stem cell properties of type II alveolar epithelial cells, and maintain the surfactant secretion properties of type II alveolar epithelial cells. The technical solution overcomes the technical problem that the in vitro culture and amplification methods of type II alveolar epithelial cells in the prior art cannot simultaneously avoid cell differentiation and promote cell amplification. The method is simple to operate, low in cost, and has a definite effect. It can be used in combination with other methods, and can maintain the characteristics of type II alveolar epithelial cells for a long time in vitro while promoting their natural proliferation. It has important practical significance and good application prospects.

Description

Technical Field

[0001] The present invention relates to the technical field of alveolar epithelial cell culture methods, and in particular to a method for enhancing the proliferation ability of type II alveolar epithelial cells cultured in vitro and a drug combination thereof. Background Art

[0002] Alveolar epithelial cells are isolated from lung tissue, including type 1 alveolar epithelial cells (AT1) and type 2 alveolar epithelial cells (AT2); type 1 alveolar epithelial cells have a basement membrane at the base and no proliferation ability. Type 2 alveolar epithelial cells secrete surfactant (dipalmitoylphosphatidylcholine) to reduce alveolar surface tension. Type 2 alveolar epithelial cells are cubic or round, with the apex protruding into the alveolar cavity, a round nucleus, and a lightly stained, foamy cytoplasm. Under an electron microscope, the cells are free and have a small number of microvilli. The cytoplasm is rich in mitochondria and lysosomes, with a well-developed rough endoplasmic reticulum and Golgi complex. There are many secretory granules above the nucleus, with high electron density and varying sizes. Type 2 alveolar epithelial cells have the potential to divide, proliferate and differentiate into type 1 alveolar epithelial cells, so they have the function of repairing damaged epithelium. As an important cell population in the lungs, type 2 alveolar epithelial cells play an important role in lung injury repair, inflammation regulation, and lung compliance maintenance.

[0003] The in vitro isolation, culture and expansion of type II alveolar epithelial cells have important practical effects and research value in stem cell therapy and exploration of cell functions. In the prior art, the isolation and culture methods of type II alveolar epithelial cells for mice are mainly as follows: purifying mouse type II alveolar epithelial cells by immunoadhesion method, inoculating lung tissue cell suspension into mouse IgG-coated plates, collecting non-adherent cells, and obtaining mouse type II alveolar epithelial cells by centrifugation purification; then, suspending the purified cells in DMEM / F12 complete medium. At present, the difficulties in isolating type II alveolar epithelial cells for in vitro culture are: (1) The cells are progenitor cells and have the potential to differentiate into type I alveolar epithelial cells. Under natural culture conditions, type II alveolar epithelial cells will differentiate within 48 hours of separation and adherence, and lose their stemness and type II alveolar epithelial cell characteristics; (2) If the culture conditions are controlled to maintain the stemness and self-phenotype of AT2, cell expansion will be limited. There is an urgent need to develop a culture method that can both avoid the differentiation of type II alveolar epithelial cells and promote the expansion of type II alveolar epithelial cells to meet the needs of scientific research and the development of new stem cell drugs. Summary of the invention

[0004] The present invention is intended to provide a combination drug for enhancing the expansion capacity of type II alveolar epithelial cells cultured in vitro, so as to solve the technical problem that the in vitro culture and expansion methods of type II alveolar epithelial cells in the prior art cannot simultaneously avoid cell differentiation and promote cell expansion.

[0005] In order to achieve the above object, the present invention adopts the following technical scheme:

[0006] A combination drug for enhancing the proliferation capacity of type II alveolar epithelial cells cultured in vitro, comprising atipamezole and dexmedetomidine.

[0007] The present scheme also provides an application of a combination drug for enhancing the proliferation capacity of type II pneumopneumoniae cells cultured in vitro in cell culture, wherein the type II pneumopneumoniae cells are first treated with atipamezole, and then the type II pneumopneumoniae cells are cultured using a culture medium containing dexmedetomidine.

[0008] The present invention also provides a method for enhancing the proliferation capacity of type II alveolar epithelial cells cultured in vitro, comprising the following steps performed in sequence:

[0009] S1: Treatment of type II alveolar epithelial cells with atipamezole:

[0010] S2: Type II alveolar epithelial cells were cultured in medium containing dexmedetomidine.

[0011] Furthermore, the dosage ratio of atipamezole to dexmedetomidine is 1-5:1-5.

[0012] Furthermore, the working concentrations of atipamezole and dexmedetomidine were both 1-5 μM.

[0013] Furthermore, the duration of action of atipamezole is 30-45 minutes.

[0014] Furthermore, the combination drug is a proliferation promoter of type II alveolar epithelial cells, a promoter of stem cell performance of type II alveolar epithelial cells, and a promoter of surfactant secretion performance of type II alveolar epithelial cells.

[0015] Furthermore, in S1, type II alveolar epithelial cells were inoculated in a blank culture medium, and after culturing for 48 h, atipamezole was added to the blank culture medium at a final concentration of 1-5 μM, and then cultured for another 30-45 min.

[0016] Furthermore, in S2, the blank culture medium supplemented with atipamezole was replaced with a culture medium containing dexmedetomidine, and the type II alveolar epithelial cells were continuously cultured; the concentration of dexmedetomidine was 1-5 μM.

[0017] Furthermore, in S1 and S2, the cell culture environment was a 37° C., 5% CO 2 incubator; the blank culture medium was DMEM / F12 culture medium containing 10% Australian fetal bovine serum.

[0018] Furthermore, in S2, the inoculum size of type II alveolar epithelial cells was 5×10 5 / mL; in S2, the medium containing dexmedetomidine is a blank medium supplemented with dexmedetomidine.

[0019] In summary, the principles and advantages of this solution are:

[0020] Dexmedetomidine (Dex) is an α2-adrenaline receptor agonist in the prior art. In the process of exploring its mechanism of promoting the proliferation of type II alveolar epithelial cells, the inventors found that after the combined use of α2-adrenaline receptor antagonist Atipamezole (Atip), the overall pro-proliferation effect was enhanced, and the corresponding protein expression characteristics of type II alveolar epithelial cells could be maintained. The above-mentioned combination drug is used to promote the proliferation of type II alveolar epithelial cells, maintain the stem cell properties of type II alveolar epithelial cells, and maintain the surfactant secretion properties of type II alveolar epithelial cells.

[0021] It is generally believed in the prior art that atipamezole is an α2-adrenergic receptor antagonist and dexmedetomidine is an α2-adrenergic receptor agonist. The two have opposite mechanisms of action. If the two are used in combination, the effects will be offset. In experiments on mouse stem cell regeneration, the combined use of atipamezole and dexmedetomidine also failed to promote cell proliferation. However, when the inventors applied the two in combination to the in vitro culture of type II alveolar epithelial cells, the proliferation ability, stemness and secretion ability of AT2 cells were significantly improved, which was not expected by the inventors before the experiment.

[0022] The method proposed in the present invention is simple to operate, low in cost, and effective. It can be used in combination with other methods, and can maintain the characteristics of AT2 for a long time in vitro while promoting its natural proliferation. It has important practical significance and good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is a statistical diagram of AT2 cell proliferation activity under treatment with different concentrations of Dex in Example 1 (MTS method, *** indicates p < 0.001 compared with control).

[0024] Figure 2 This is a statistical graph of AT2 cell proliferation activity, proliferation-related membrane ligand mRNA transcription level and growth factor concentration level after the combined use of Atip and Dex in Example 2 (mean±SD, n=6, ### indicates this group VS. Dex group, p<0.001; #### indicates this group VS. Dex group, p<0.0001; *** indicates compared with the control group, p<0.001; ** indicates compared with the control group, p<0.01).

[0025] Figure 3This is a microscopic picture of AT2 cell monoclonal colonies after the combined use of Atip and Dex in Example 2.

[0026] Figure 4 These are immunofluorescence images of co-staining of AT2 stemness-related marker Nkx2.1 and cell proliferation marker Ki67 under different treatment conditions in Example 2 (red fluorescence Ki67, blue fluorescence DAPI, green fluorescence Nkx2.1).

[0027] Figure 5 Statistical graph of immunofluorescence signals of co-staining of AT2 stemness-related marker Nkx2.1 and cell proliferation marker Ki67 under different treatment conditions of Example 2 (5 fields of view randomly selected at 100×, expressed as the percentage of designated stained cells in total cells (%), mean±SD, n=5, ### indicates this group VS. Dex group, p<0.001; *** indicates the comparison with control group, p<0.001; ns indicates that there is no significant difference among the delineated groups).

[0028] Figure 6 The WB detection results and relative expression statistics of the surfactant sftpc specifically secreted by AT2 under different treatment conditions of Example 2 (the relative expression is expressed as the gray value of the sftpc band / the gray value of the internal reference GAPDH band; mean±SD, n=3; ** indicates p<0.01 compared with other groups).

[0029] Figure 7 These are immunofluorescence images of the proliferation-related nuclear transcription factor β-catenin under different treatment conditions in Example 2 (red fluorescence β-catenin, blue fluorescence DAPI).

[0030] Figure 8 These are immunofluorescence images of the proliferation-related nuclear transcription factor TAZ, the surfactant sftpc specifically secreted by AT2, the cell proliferation marker Ki67, and the AT2 stemness-related marker Nkx2.1 under different treatment conditions in Example 2 (upper row: red fluorescence Ki67, blue fluorescence DAPI, green fluorescence Sftpc; lower row: red fluorescence Nkx2.1, blue fluorescence DAPI, green fluorescence TAZ).

[0031] Fig. 9 This is a statistical chart of the liver / body weight ratio of mice in Comparative Example 1.

[0032] Fig.10 This is a statistical chart of Ki67-positive cells, a cell proliferation marker, under different treatments of Comparative Example 1. DETAILED DESCRIPTION

[0033] The present invention is further described in detail below in conjunction with examples, but the embodiments of the present invention are not limited thereto. Unless otherwise specified, the technical means used in the following examples and experimental examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used can be obtained from commercial sources.

[0034] Example 1: Study on the effect of dexmedetomidine (Dex) on the proliferation activity of type II alveolar epithelial cells

[0035] The acquisition of type II alveolar epithelial cells (AT2) is an existing technology. According to the literature (Jansing NL, McClendon J, Kage H, et al. Isolation of Rat and Mouse Alveolar Type II Epithelial Cells. Methods Mol Biol. 2018; 1809: 69-82. doi: 10.1007 / 978-1-4939-8570-8_6), AT2 cells were isolated from mouse lung tissue. After obtaining AT2 cells, subsequent culture was performed. AT2 cells were cultured at 5×10 5 The cells were inoculated with an inoculum of 10% Australian fetal bovine serum (Gibco) in DMEM / F12 medium (Gibco) at a volume of 100 mL and cultured in a 37°C, 5% CO2 incubator. The cells were given the drug after 48 h of culture (without changing the medium) to make the medium contain different final concentrations of Dex (for specific concentration gradient values, see Figure 1 ). According to the above-mentioned administration method, the proliferation-promoting effect of different concentrations of Dex on AT2 was preliminarily explored. After Dex continued to act for 24 hours (the cells can be cultured and subcultured using the medium containing dexmedetomidine all the time. The reason for taking 24 hours here is that it is a routine cell proliferation observation time point after the drug effect in the industry), the proliferation activity of AT2 was detected using the MTS method. The experimental results are shown in Figure 1 The experimental results show that Dex at a concentration of 1-5μM has a strong proliferation-promoting activity.

[0036] Example 2: Study on the combined effect of atipamezole (Atip) and dexmedetomidine (Dex)

[0037] After confirming the optimal concentration of Dex, the inventors then used the α2-adrenaline receptor antagonist Atip for pretreatment, and then used Dex for treatment to explore the changes in the Dex-proliferation effect. In this scheme, the atipamezole (Atip, Formula I, CAS: 104054-27-5; Chemical formula: C14 H 16 N2) and dexmedetomidine hydrochloride (dexmedetomidine HCL, Dex, formula II, CAS: 145108-58-3; chemical formula: C 13 H 17 ClN2), the specific structural formula is as follows:

[0038]

[0039] In this embodiment, the dosage of Atip refers to the drug instructions and the commonly used scheme in the field, the dosage is the same as that of Dex, and the administration time is 30-45 minutes. That is, the treatment method of AT2 cells is:

[0040] AT2 cells were cultured at 5 × 10 5 / mL of inoculation volume was inoculated in DMEM / F12 medium (gibco) containing 10% Australian fetal bovine serum (gibco), and Atip was administered after 48h of culture (without changing the culture medium), and the final concentration of Atip was controlled at 1-5μM (2.5μM was specifically used in this embodiment), and the cells were cultured in a 37°C, 5% CO2 incubator for 30-45min (40min was specifically used in this embodiment); then the culture medium was replaced with a culture medium containing Dex, and the final concentration of Dex was controlled at 1-5μM (2.5μM was specifically used in this embodiment), and the cells were cultured in a 37°C, 5% CO2 incubator for 24h, and then replaced with a drug-free culture medium for conventional culture (the culture medium containing dexmedetomidine can be used to culture and subculture the cells, and the reason for taking 24h here is that it is a conventional cell proliferation observation time point after the drug effect is used in the industry). 24 hours after Dex administration, cell activity detection, mRNA transcription level detection of proliferation-related membrane ligands Delta1 and Jagged1, and related growth factor concentrations (TGFβ, Wnt5a, PDGF) in the supernatant of cell culture medium were performed. Figure 2, the control group is the group that does not use Atip and Dex in the above culture process (the drug is not added in the corresponding step); the Atip group is the group that uses only 2.5μM Atip to treat the cells in the above culture process (Dex is not added in the corresponding step); the Dex group is the group that uses only 2.5μM Dex to treat the cells in the above culture process (Atip is not added in the corresponding step); the Atip+Dex group is the experimental group that performs Atip and Dex treatment step by step according to the above method. The experimental data show that the combined administration combination can further enhance the proliferation activity of AT2, while increasing the mRNA transcription level of proliferation-related membrane ligands Delta1 and Jagged1, and increasing the concentration of related growth factors (TGFβ, Wnt5a, PDGF) in the supernatant of the cell culture medium. The above experimental results show that: Atip alone basically has no effect on promoting the proliferation of AT2 cells (relative to control), and Dex alone can significantly enhance the proliferation ability of AT2 cells (relative to control). The combined use of Atip and Dex can greatly increase the proliferation activity of AT2 cells. Atip itself does not have a proliferation-promoting effect, but Atip can enhance the cell proliferation-promoting effect of Dex, which shows that there is a synergistic effect between Atip and Dex, which was not expected by the inventors before the experiment.

[0041] 24 hours after Dex administration, AT2 cells were fixed with 4% paraformaldehyde and the proliferation and colony formation of AT2 cells were evaluated by immunofluorescence technique. It was found that AT2 cells could effectively form monoclonal colonies using this culture method (Atip+Dex group). Figure 3 24 hours after Dex administration, cells were fixed with 4% paraformaldehyde and immunofluorescence was used to evaluate the proliferation and stemness of AT2 cells. The AT2 stemness-related marker Nkx2.1 was co-stained with the cell proliferation marker Ki67. The experimental results are shown in Figure 4 For the statistical results of the percentage of Nkx2.1-positive cells and cells positive for both Ki67 and Nkx2.1, see Figure 5. It can be seen from the experimental data that the combined use of Atip and Dex will not affect the stemness of the cells, and type II alveolar epithelial cells will not differentiate into type I alveolar epithelial cells in large quantities during the proliferation culture process. Moreover, in AT2 cells that maintain stemness, the treatment method of Atip+Dex can significantly increase the proliferation potential of the cells, which is specifically manifested in that the proportion of Nkx2.1-positive cells expressing the proliferation marker Ki67 is significantly higher than that of other experimental groups. Whole-cell protein was extracted immediately 24 hours after Dex administration, and the expression level of the surfactant sftpc specifically secreted by AT2 cells was evaluated using western blot technology. It can be observed that the use of this drug combination significantly increases the ability to maintain AT2 characteristics. When Atip or Dex is used alone, the ability of cells to maintain AT2 characteristics does not change significantly relative to the control group. Therefore, the combined treatment method of Atip+Dex can not only promote cell proliferation, but also promote the maintenance of AT2 cells' stemness and the maintenance of AT2 characteristic capabilities.

[0042] 24 hours after Dex administration, immunofluorescence technique was used to evaluate the activation effect of AT2 cell proliferation activity, and proliferation-related nuclear transcription factors TAZ and β-catenin were detected. The experimental results are shown in Figure 7 and Figure 8 It can be seen that in the group using this drug combination, the above transcription factors underwent significant nuclear translocation, and the number of cells was greater, indicating that the proliferation activity was activated.

[0043] Comparative Example 1

[0044] Chinese patent CN113041242A (Application of dexmedetomidine in promoting liver regeneration) studied the role of dexmedetomidine in promoting liver cell proliferation. In its examples, dexmedetomidine and atipamezole were used in combination for an in vivo mouse experimental scheme. The content is roughly as follows: It has been reported in the literature that dexmedetomidine exerts its effects by binding to α2-adrenergic receptors (α2-adrenergic receptor agonist). On this basis, the α2-adrenergic receptor inhibitor atipamezole was used for pretreatment, thereby inhibiting the effect of dexmedetomidine. The saline group was intraperitoneally injected with 500 μl of normal saline 30 minutes before surgery, the DEX group was intraperitoneally injected with 25 μg / kg of dexmedetomidine hydrochloride according to the mouse body weight 30 minutes before surgery, and the ATI group was intraperitoneally injected with 250 μg / kg of atipamezole hydrochloride according to the mouse body weight 10 minutes before dexmedetomidine hydrochloride injection. For some experimental results, see Fig. 9 (Excerpt from A in the fifth figure of CN113041242A) and Fig.10(Excerpt from C in the fifth figure of CN113041242A). The above experimental data show that in mice, the simultaneous use of dexmedetomidine and atipamezole does not promote the proliferation of hepatocytes. The purpose of using atipamezole is to confirm the signaling pathway of dexmedetomidine in stem cell proliferation. The experimental results of this comparative example may cause certain technical biases to researchers, who believe that the combined use of dexmedetomidine and atipamezole will offset the pro-proliferation effect of dexmedetomidine. However, in fact, when the inventors used dexmedetomidine and atipamezole together for in vitro culture of AT2 cells, the combined effect of the two can greatly promote cell proliferation, and at the same time maintain the stemness of the cells and the characteristics of AT2 cells, and obtain unexpected technical effects.

[0045] The above is only an embodiment of the present invention, and the common knowledge such as the known specific technical solutions and / or characteristics in the solution is not described in detail here. It should be pointed out that for those skilled in the art, without departing from the technical solution of the present invention, several modifications and improvements can be made, which should also be regarded as the protection scope of the present invention, and these will not affect the effect of the implementation of the present invention and the practicality of the patent. The scope of protection required by this application shall be based on the content of its claims, and the specific implementation methods and other records in the specification can be used to interpret the content of the claims.

Claims

1. Use of a combination drug for enhancing the proliferation capacity of type II alveolar epithelial cells cultured in vitro in cell culture, characterized in that: The combined drug consists of atipamezole and dexmedetomidine in a dosage ratio of 1-5:1-5; Atipamezole was added to the culture medium inoculated with type II pneumocytes, and after culturing for 30-45 minutes, the type II pneumocytes were cultured in a culture medium containing dexmedetomidine; The working concentrations of atipamezole and dexmedetomidine were both 1-5 μM.

2. Use of a combination drug for enhancing the proliferation ability of type II alveolar epithelial cells cultured in vitro according to claim 1 in cell culture, characterized in that: The combined drug is a proliferation promoter of type II alveolar epithelial cells, a promoter of stem cell performance of type II alveolar epithelial cells, and a promoter of surfactant secretion performance of type II alveolar epithelial cells.

3. A method for enhancing the proliferation capacity of type II alveolar epithelial cells cultured in vitro, characterized in that: The method includes the following steps in sequence: S1: Type II alveolar epithelial cells were inoculated into blank culture medium, and atipamezole was added to the blank culture medium at a final concentration of 1-5 μM after culture, and then cultured for another 30-45 min; S2: Replace the blank medium supplemented with atipamezole with a medium containing 1-5 μM dexmedetomidine, and continue to culture type II alveolar epithelial cells; The dosage ratio of atipamezole to dexmedetomidine is 1-5:1-5.

4. A method for enhancing the proliferation capacity of type II alveolar epithelial cells cultured in vitro according to claim 3, characterized in that: In S1, type II alveolar epithelial cells were seeded in blank culture medium, and atipamezole was added to the blank culture medium after 48 h of culture.

5. A method for enhancing the proliferation capacity of type II alveolar epithelial cells cultured in vitro according to claim 4, characterized in that: In both S1 and S2, the cell culture environment was incubated at 37°C and 5% CO2; the blank culture medium was DMEM / F12 culture medium containing 10% Australian fetal bovine serum; in S1, the inoculation amount of type II alveolar epithelial cells was 5×10 5 / mL; in S2, the medium containing dexmedetomidine is a blank medium supplemented with dexmedetomidine.

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