CN111233996A - Method for promoting engineered cell strain to secrete and express rhIL-24 by using sodium butyrate - Google Patents

Abstract

The invention provides a method for promoting an engineering cell strain to secrete and express rhIL-24 by using sodium butyrate, which belongs to the technical field of gene expression, and is characterized in that the cell strain is treated by sodium butyrate with different final concentrations (0, 0.125, 0.25, 0.5, 1 and 2mmol/L) on the basis of carrying out 10% serum adherence, 0.5% serum adherence and 0.5% serum suspension culture on a fixed-point integration engineering cell strain FCHO/IL-24 secreting and expressing rhIL-24. In the invention, NaBu can improve the level of foreign protein secreted and expressed by CHO cells, promote the level of rhIL-24 secreted and expressed by engineering cells, promote the engineering cells to generate G0/G1 phase block, and change the metabolic activity of the cells under the condition of adherent culture; NaBu is used as an additive of a serum-free culture medium of a cell strain, the level of rhIL-24 secreted and expressed by the cell strain is improved in high-density suspension culture, experimental data are provided for large-scale culture of FCHO/IL-24 cells, and reference can be provided for expression of other genetic engineering proteins by using mammalian cells.

Description

Method for promoting engineered cell strain to secrete and express rhIL-24 by using sodium butyrate

Technical Field

The invention relates to the technical field of gene expression, in particular to a method for promoting an engineering cell strain to secrete and express rhIL-24 by using sodium butyrate.

Background

The melanoma differentiation related gene-7/interleukin 24(mda-7/IL-24) encodes a protein consisting of 206 amino acid residues, the molecular weight is about 23.8 kDa. IL-24 has broad-spectrum and specific anti-tumor activity, can specifically inhibit the growth of various tumors in vitro and in vivo and selectively induce the apoptosis of tumor cells, and has little influence on normal cells⁺CD8⁺The number of T cells can trigger the body to generate immune response for inhibiting tumor cells at the primary part and tumor cells at the metastatic part, which is a characteristic that cancer inhibition genes such as p53 and Rb do not have. Moreover, IL-24 can enhance tumor cell chemoradiotherapy sensitivity, and can produce additive or synergistic antitumor effect when combined with targeted drugs, chemotherapeutic drugs or radiotherapy. Because of the characteristics, the IL-24 is called a magic bullet for treating tumors, and the intensive research on the magic bullet not only has important theoretical significance, but also has important clinical application value.

In the use of IL-24 in clinical tumor therapy studies, IL-24 recombinant adenovirus, "INGN 241", was first developed by Introgen Therapeutics, USA. The results of phase I clinical trials published by the company in 2002 show that INGN241 has good therapeutic effects on 28 patients with solid tumors (breast, lung and colon). However, the development of adenovirus is limited by defects (such as targeting, effective clearance, biological safety and the like), and no report is always available in phase II clinical trials from 2005, which suggests that safety problems may be one of the main reasons. Compared with adenovirus, the protein anti-cancer medicine is safer. Compared with the traditional chemical synthesis medicines, the protein medicines become the main varieties of new biotechnological medicines, and the medicines are very close to normal physiological substances in vivo and have the characteristics of high pharmacological activity, small side effect, small dosage, strong biological activity, good curative effect and the like.

At present, rhIL-24 is expressed in Escherichia coli, yeast, insect and mammal cells, but the biological activity of IL-24 gene engineering protein obtained in different host cells is obviously different. The rhIL-24 expressed by Escherichia coli and yeast has the immunoregulation effect, but the dosage is large (the unit is mu g/mL), while the dosage unit of the rhIL-24 secreted and expressed by HEK293 cells is ng/mL; similarly, in the experiment that rhIL-24 inhibits the proliferation of melanoma cells in vitro, the rhIL-24 expressed by Escherichia coli shows activity at the level of mu g/mL, while the effective concentration of the rhIL-24 secreted and expressed by mammalian cells is only 5 ng/mL.

Mammalian cells are used for expressing rhIL-24, and recombinant plasmids pCEP4-IL-24 are mostly used for constructing a HEK293 cell strain with stable expression, or recombinant adenovirus Ad-IL-24 is used for infecting human immortalized normal cells or tumor cells. A CHO fixed-point integration cell strain FCHO/IL-24 for expressing rhIL-24 is constructed by utilizing a Flp-In system, and the expression level of the rhIL-24 is far higher than that of three randomly integrated HEK293 cell strains (HEK293/pSecTag2A-IL-24, HEK293/pcDNA3.1myc/His-IL-24 and HEK293/pCEP4-IL-24) under the same cell density and culture time. The fixed-point integration system constructs stable and high-yield cell strains, which not only can improve the generation rate of product ratio, but also is beneficial to the optimization of the subsequent large-scale culture process. Usually, the exogenous gene is randomly integrated on the chromosome of the host cell, which is easy to cause the 'position effect' of the inserted chromosome region, thereby influencing the expression level of the gene; the site-directed integration system inserts the exogenous gene into the transcription active region, not only can directly obtain the clone with higher basic expression level, but also can greatly shorten the period and the workload for constructing the recombinant protein high-expression cell strain (the establishment of the cell site-directed integration high-efficiency expression system) because the site is fixed and the repeatability of a pressurizing amplification procedure is better.

However, FCHO/IL-24 cells were adherent cultured with F12 medium containing 10% serum, and their secretory expression rhIL-24 levels were still limited. Addition of a stimulant to the culture medium is also an effective method for increasing the level of the protein expressed by the engineered cell strain, and sodium butyrate is the most commonly used method. Sodium butyrate is a short fatty acid chain that acts as a histone deacetylase inhibitor, altering mammalian cell chromatin structure by reducing enzymatic activity, thereby regulating gene transcription levels through acetylation and deacetylation of histones. In 1973, it was discovered that it could significantly increase the expression level of recombinant proteins in CHO cells, and many successful examples have been shown so far, such as interferon gamma^]The sodium butyrate can stimulate CHO cells and human endothelial cells and improve the expression level of t-PA under different culture conditions (adherent culture and serum-free suspension culture).

As described above, IL-24 has a specific inhibitory effect on in vitro and in vivo proliferation of various tumor cells and has little toxicity to normal cells. The rhIL-24 activity expressed by mammalian cells is much higher than that expressed by bacteria, but the expression level is still very low. In the research aiming at the expression of the rhIL-24 by the mammalian cells, no related technical research for improving the expression level of the rhIL-24 by the engineering cell strain by using the sodium butyrate exists at present.

Disclosure of Invention

The invention aims to provide a method for promoting the engineering cell strain to secrete and express rhIL-24 by using sodium butyrate, which takes a CHO fixed-point integration cell strain FCHO/IL-24 for expressing rhIL-24 as a research object, analyzes the influence of the sodium butyrate on the level of the rhIL-24 secreted and expressed by the engineering cell strain, and provides experimental data for screening serum-free medium additives, so as to solve at least one technical problem in the background technology.

In order to achieve the purpose, the invention adopts the following technical scheme:

the invention provides a method for promoting engineered cell strains to secrete and express rhIL-24 by using sodium butyrate, which comprises the steps of culturing a site-specific integrated engineered cell strain FCHO/IL-24 for a certain time, and then replacing a culture medium with a culture medium containing sodium butyrate NaBu to continue culturing.

Preferably, the culture of the site-directed integration engineering cell strain FCHO/IL-24 is a 10% serum adherent culture.

Preferably, the culture of the site-directed integration engineering cell strain FCHO/IL-24 is a 0.5% serum adherent culture.

Preferably, the culture of the site-directed integration engineering cell strain FCHO/IL-24 is 0.5% serum suspension culture.

Preferably, the culture medium for culturing the site-directed integration engineering cell strain FCHO/IL-24 is DMEM/F12 medium.

Preferably, the medium is supplemented with 100. mu.g/ml of the diabody and 50. mu.g/ml of hygromycin.

Preferably, the concentration of the sodium butyrate in the culture medium is 0.25-2mmol/L, and the serum content in the culture medium after replacement is unchanged.

Preferably, when 10% serum adherent culture is performed, the number of cells seeded per well in a 96-well plate is 650, and the number of cells seeded per well in a 6-well plate is 1X 10⁵And (4) respectively.

Preferably, the number of cells seeded per well in a 96-well plate is 1500 and the number of cells seeded per well in a 6-well plate is 1X 10 in adherent culture with 0.5% serum⁵And (4) respectively.

Preferably, the suspension culture with 0.5% serum is carried out at shaking flask inoculation volume of 20ml, shaking table rotation speed of 119rpm, incubator conditions of 37 deg.C and 5% CO₂Cell seeding density of 5X 10⁵Individual cells/mL.

The invention has the beneficial effects that: the NaBu can improve the level of foreign protein secreted and expressed by CHO cells, promote the level of rhIL-24 secreted and expressed by engineering cells, promote the engineering cells to generate G0/G1 phase block, and change the metabolic activity of the cells under the condition of adherent culture; NaBu is used as an additive of a serum-free culture medium of a cell strain, the level of rhIL-24 secreted and expressed by the cell strain is further improved in high-density suspension culture, experimental data are provided for large-scale culture of FCHO/IL-24 cells, and reference can be provided for expression of other genetic engineering proteins by using mammalian cells.

Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the description of the embodiments are briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings based on these drawings without creative efforts.

FIG. 1 is a microscopic scan of the effect of sodium butyrate with different concentrations on cell growth in 10% serum adherent culture according to the present invention.

FIG. 2 is a graph showing the change of the growth state of cells after adding sodium butyrate to the culture medium according to the embodiment of the present invention.

FIG. 3 is a graph of rhIL-24 concentration (. about.p. <0.01) in the culture supernatant of sodium butyrate cells at various concentrations in adherent 10% and 0.5% serum cultures, in accordance with an example of the present invention.

FIG. 4 is a graph showing the change in specific cell productivity of cells treated with different concentrations of sodium butyrate under 10% and 0.5% serum adherent culture conditions according to the example of the present invention.

FIG. 5 is a graphical representation of the IL-24 concentration profile in the supernatant at 2mM sodium butyrate on day 3 of NaBu treatment under 0.5% serum suspension culture conditions as described in the examples of the present invention.

FIG. 6 is a graph showing the curves of specific productivity as a function of the concentration of sodium butyrate at day 3 of NaBu treatment under 0.5% serum suspension culture conditions, according to the example of the present invention.

FIG. 7 is a graph showing the effect of sodium butyrate on the G0/G1 phase of cells in 10% serum adherent culture, as described in an example of the present invention.

FIG. 8 is a graph showing the effect of sodium butyrate in 0.5% serum adherent culture on the G0/G1 phase of cells, as described in an example of the present invention.

FIG. 9 is a graph showing the effect of different concentrations of sodium butyrate on apoptosis in accordance with the present invention.

FIG. 10 is a graph showing the glucose concentration in the supernatant after treatment with different concentrations of sodium butyrate according to an example of the present invention.

FIG. 11 is a graph showing the concentration of lactic acid in the supernatant after treatment with different concentrations of sodium butyrate according to the example of the present invention.

FIG. 12 is a graph showing the ammonia concentration in supernatants treated with different concentrations of sodium butyrate, according to an example of the present invention.

Detailed Description

Reference will now be made in detail to embodiments of the present invention, examples of which are illustrated in the accompanying drawings, wherein like or similar reference numerals refer to the same or similar elements or elements having the same or similar function throughout. The embodiments described below by way of the drawings are illustrative only and are not to be construed as limiting the invention.

It will be understood by those skilled in the art that, unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. It will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the prior art and will not be interpreted in an idealized or overly formal sense unless expressly so defined herein.

As used herein, the singular forms "a", "an", "the" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. It will be further understood that the terms "comprises" and/or "comprising," when used in this specification, specify the presence of stated features, integers, steps, operations, elements, and/or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and/or groups thereof.

In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, various embodiments or examples and features of different embodiments or examples described in this specification can be combined and combined by one skilled in the art without contradiction.

In the description of this patent, it is noted that the terms "first" and "second" are used for descriptive purposes only and are not to be construed as indicating or implying relative importance or implying any number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one such feature. In the description of the present invention, "a plurality" means two or more unless specifically defined otherwise.

For the purpose of facilitating an understanding of the present invention, the present invention will be further explained by way of specific embodiments with reference to the accompanying drawings, which are not intended to limit the present invention.

It should be understood by those skilled in the art that the drawings are merely schematic representations of embodiments and that the elements shown in the drawings are not necessarily required to practice the invention.

Examples

The embodiment of the invention provides a method for promoting engineered cell strains to secrete and express rhIL-24 by using sodium butyrate, which is characterized in that the cell strains are treated by using sodium butyrate with different final concentrations (0, 0.125, 0.25, 0.5, 1 and 2mmol/L) on the basis of carrying out 10% serum adherence, 0.5% serum adherence and 0.5% serum suspension culture on site-specific integration engineered cell strains FCHO/IL-24 secreting and expressing rhIL-24.

Respectively detecting the expression levels of cell proliferation, cell cycle, apoptosis and rhIL-24 by methods such as MTT, flow detection, ELISA and the like; and the effect of sodium butyrate treatment on glucose, lactate and ammonia concentrations in the supernatants of 10% and 0.5% serum adherent culture cells was examined.

As a result: first, different concentrations of NaBu can increase the level of rhIL-24 secreted and expressed by the engineering cell strain, and after 3 days of treatment with 1mmol/L of NaBu, the level of rhIL-24 expressed by the cell strain is increased by 119.94 +/-1.5% (. p <0.01), 57.49 +/-2.4% (. p <0.01) and 20.17 +/-3.03% (. p <0.05) under the conditions of 10% serum concentration adherent culture, 0.5% serum concentration adherent culture and 0.5% serum suspension culture, respectively. Second, sodium butyrate had a time and dose dependent inhibition of cell line proliferation with 9 ± 3% (. p <0.05), 13 ± 2% (. p <0.01) and 12 ± 2% (. p <0.01) inhibition of cell proliferation under the same culture conditions and sodium butyrate treatment conditions, respectively. But has no obvious promotion effect on the apoptosis, and the 2mmol/L sodium butyrate treatment for 3 days only increases the apoptosis rate of 10 percent of serum adherent culture cells by 1.84 +/-1.06 percent (p > 0.05). However, sodium butyrate caused cell arrest at the G0/G1 phase, 3 days at 1mmol/L, and increased 11.3 ± 0.5% (. about.. about.p <0.01) and 15.0 ± 2.6% (. about.p <0.01) in the G0/G1 phases of 10% and 0.5% serum adherent culture cells, respectively. Finally, with the group without added sodium butyrate as a control, the concentrations of glucose in the supernatants of 10% and 0.5% serum adherent culture cells increased and the concentrations of lactate and ammonia decreased with increasing sodium butyrate, with statistical significance (. about.p < 0.01).

And (4) conclusion: the capacity of the engineering cell strain FCHO/IL-24 to secrete and express rhIL-24 can be obviously improved by treating sodium butyrate.

In the embodiment of the invention, after sodium butyrate is added into a culture medium of a CHO fixed-point integration engineering cell strain, the experimental method for researching the secretion and the growth state of cells specifically comprises the following steps:

experimental materials: sodium butyrate (B5887-250MG) and MTT (M2128-250MG) were purchased from Sigma; ELISA kit for IL-24 (F01531) was purchased from Shanghai West Tang Co; the glucose detection kit (E1010) was purchased from Beijing prilley Gene technology, Inc.; the lactic acid detection kit (A019-2) and the urea nitrogen kit (C013-2) are purchased from Nanjing institute of bioengineering; suspension cultured shake flasks (431143) were purchased from corning.

Cell culture: the culture conditions of the cell strains are three, namely 10% serum adherent culture, 0.5% serum adherent culture and 0.5% serum suspension culture, the latter two are domesticated on the basis of 10% serum adherent conventional culture, the culture medium is DMEM/F12, and 100 mu g/ml double antibody and 50 mu g/ml hygromycin are supplemented into the culture medium. In suspension culture, the volume of the inoculated shake flask is 20ml, and the rotation speed of the shaking table is 119 rpm. The incubator conditions were 37 ℃ and 5% CO₂。

And (3) treating with sodium butyrate: in adherent culture, under the conditions of 10% serum and 0.5% serum, the number of inoculated cells in each well of a 96-well plate is 650 and 1500 respectively; 6 well plates, 1X 10 inoculate per well⁵And (4) cells. After 24h, the medium was changed to medium containing different concentrations of sodium butyrate (0, 0.125, 0.25, 0.5, 1 and 2mmol/L) and the serum concentration was unchanged. If the experiment is over 3 days, the culture medium is changed every 3 days, and the concentration of the sodium butyrate is kept unchanged. In suspension culture, the cell seeding density is 5X 10⁵And (4) adding sodium butyrate for treating each cell per mL, wherein the final concentration is the same as that of the cells, and the solution is not changed in the continuous culture process.

And (3) cell viability detection: in 96-well plates, at least six wells per treatment; after the cells are treated by sodium butyrate with different concentrations for a certain time, 20 mu L of MTT is added into each hole, and after 4 hours, the supernatant in the hole plate is carefully sucked out; add 150. mu.L DMSO to each well, dissolve well, and detect OD490 values with microplate reader. The measured OD was subtracted from the blank well OD, and the maximum and minimum values among the 6 values were deleted and averaged.

Detection of rhIL-24 concentration: the rhIL-24 concentration in the cell culture supernatant was measured according to the ELISA kit instructions. First, 10 × sample dilution and 20 × wash solution were diluted with distilled water. Secondly, 1 times of sample dilution is used to prepare standard solutions (0, 31.25, 62.5, 125, 250, 500, 1000 and 2000ng/mL) of IL-24 protein with different concentrations; cell culture supernatants were diluted 5-20 fold. Then, 100 μ L of standard substance or sample to be detected is added into the enzyme label plate, reaction is carried out for 40min at 37 ℃, washing liquid is fully washed for 3 times, 200 μ L of washing liquid is added into each hole, and the plate is dried on filter paper. Then, 100. mu.L of the first antibody working solution was added to each well, and reacted at 37 ℃ for 20min, followed by washing of the plate. Then, 100. mu.L of the substrate working solution was added to each well, and the reaction was carried out at 37 ℃ in the dark for 15 min. Finally, 100 mu L of stop solution is added into each hole, mixed evenly and used for measuring the light absorption value at 450nm within 30min by using an enzyme-labeling instrument. A standard curve was fitted to the OD values of the standards, and the IL-24 concentration in the sample was calculated. The OD value of the standard curve is between 0.1 and 3.0, and the OD value of the sample is within the range of the standard curve.

Cell cycle detection: 1X 10 inoculations in 6-well plates with 2mL of medium⁵Individual cell, 37 ℃ and 5% CO₂Culturing in an incubator for 24 h; the medium was changed with different final concentrations of NaBu (0, 0.125, 0.25, 0.5, 1 and 2 mmol/L). After 3 days of NaBu treatment, the supernatants from each well were collected, the cells were digested with trypsin without EDTA, the digestion was stopped with the supernatants from each well, and the cells were collected by centrifugation at 1000rpm for 5 min. Add 1mLPBS gently blow to beat the resuspended cells, centrifuge and wash twice. Resuspending the cells with 300. mu.L LPBS, adding 700. mu.L of pre-cooled absolute ethanol dropwise for fixation, mixing well, and storing at-20 ℃. Centrifuging at 1000rpm for 5min before detection, discarding supernatant, and centrifuging and washing twice with PBS; 300. mu.LPI (50. mu.g/mL) and 10. mu.LRNaseA (10mg/mL) were added, and the mixture was incubated at 37 ℃ for 30min in the absence of light and then subjected to on-machine detection.

And (3) detecting cell apoptosis: the number of plated cells and the sodium butyrate treatment are detected in the same cell cycle; after washing the collected cells by PBS centrifugation, the cells were resuspended in 200. mu.L Binding Buffer, and 5. mu.L annexin V and 5. mu.L PI were added and incubated for 30min in the dark. Before flow assay, each sample was supplemented with 300. mu. LBinding Buffer.

And (3) detecting metabolites in the supernatant: the sample is cell culture supernatant collected in MTT experiment, and glucose, lactic acid and ammonia concentration in the supernatant are detected according to the kit instruction. When detecting the glucose concentration, the working solution (reagent R1: reagent R2: 4:1) is used for the same day or stored at 4 ℃ for one week; preparing glucose standard solutions (2000, 1000, 500, 250, 125, 62.5, 31.25 and 15.625 mu mol/L) with different concentrations, and using distilled water as a blank control; adding 5 mul of standard substance or sample to be detected into a 96-well plate, adding 195 mul of working solution, reacting for 20min at 37 ℃, and measuring OD550 value by an enzyme-linked immunosorbent assay. When detecting the ammonia concentration, preparing ammonium chloride standard solutions (2.5, 1.25 and 0.625mmol/L) by a double dilution method; adding 20 mu L of standard substance or sample into a centrifuge tube, then adding 1mL of phenol color developing agent and 1mL of alkaline sodium hypochlorite respectively, uniformly mixing, and carrying out water bath at 37 ℃ for 10 min; mu.L of the reaction solution was taken out and put into a 96-well plate to measure the OD640 value. When the concentration of the lactic acid is detected, the enzyme stock solution diluted by 1:100 is prepared as it is; the prepared color developing agent can be stored for 2 weeks at 4 ℃. Adding 20 mu L of sample to be detected into a centrifuge tube, then adding 1mL of each of the enzyme working solution and the color developing agent, carrying out water bath at 37 ℃ for 10min, adding 2mL of stop solution, taking out 300 mu L of stop solution from each centrifuge tube, adding the stop solution into a 96-well plate, and measuring the OD530 value. The standard curve was fitted to an empirical formula for calculating the sample concentration.

Statistical analysis: performing statistical analysis on the data by using Excel2016 software, calculating an intra-group standard deviation by using a standard deviation function and calculating the significance of difference among groups by using a t-test function in experiments such as MTT, cell counting, cell flow detection and the like; ELISA experimental data and supernatant metabolite detection data were standard curve fit and sample concentration calculations using Origin2018 software. In the significance test, p <0.05 indicated significant differences, and p <0.01 indicated very significant differences.

In the embodiment of the present invention, the experimental detection analysis results are specifically as follows:

sodium butyrate for inhibiting in-vitro proliferation of engineering cells

NaBu can improve the level of foreign protein secreted and expressed by CHO cells, but has an inhibiting effect on cell proliferation. The sodium butyrate inhibits the in vitro proliferation of the engineered cell FCHO/IL-24 under different culture conditions as shown in figure 1 and figure 2. FIG. 1 shows that the higher the NaBu concentration, the worse the cell morphology and the lower the cell density under 10% serum adherent culture conditions. FIGS. 2(a), (c), (e) are cell growth curves for 7 days of treatment with different final concentrations of NaBu (0, 0.125, 0.25, 0.5, 1 and 2mmol/L) under different culture conditions (10% serum adherent culture, 0.5% serum adherent culture and 0.5% serum suspension culture), respectively. FIG. 2(b), (d) and (f) show that NaBu with different concentrations has obvious inhibition effect on the relative activity of FCHO/IL-24 under different culture conditions, which are respectively the same as (a), (c) and (e).

In this experiment, 6 sodium butyrate concentration gradients were set, namely 0, 0.125, 0.25, 0.5, 1 and 2 mmol/L. For FCHO/IL-24 cells cultured in 10% serum adherent culture, the cell density is reduced along with the increase of NaBu concentration, the change of cell morphology is obvious when the concentration of sodium butyrate is 1 or 2mmol/L, and dead cells are increased remarkably, as shown in figure 1. However, even under the action of NaBu, the exponential phase of mass proliferation started at day 4 and reached saturation at day 6 and reached the plateau phase, as shown in FIG. 2 (a). As can be seen from fig. 2(b), at the same NaBu concentration, the relative cell viability gradually decreased with the increase of the culture time; at the same time point, the relative cell viability decreased as the concentration of NaBu increased.

Under adherent culture conditions at 0.5% serum concentration, the inhibition of cell proliferation by NaBu was similar to that of 10% serum, as shown in FIGS. 2(c) and 2 (d). Under the 0.5% serum suspension culture conditions, cells entered exponential growth on day 2, reached a peak on day 4, and then rapidly died off without a change of fluid, as shown in fig. 2 (e). Similarly, NaBu also had an inhibitory effect on the relative viability of suspension cultured cells, as shown in fig. 2 (f). Taking the effect of 1mmol/L of NaBu for 3 days as an example, the inhibition rates of the different culture conditions (10% serum adherent culture, 0.5% serum adherent culture and 0.5% serum suspension culture) on cell proliferation were 9 + -3% (. p <0.05), 13 + -2% (. p <0.01) and 12 + -2% (. p <0.01), respectively. The results show that: NaBu has dose-dependent and time-dependent inhibition on the in vitro proliferation of the engineering cells FCHO/IL-24.

Secondly, promoting the level of engineering cells secreting and expressing rhIL-24 by sodium butyrate

As shown in FIGS. 3 to 6, to determine the effect of NaBu treatment on the secretion and expression of rhIL-24 by engineered cells, the rhIL-24 concentration in the cell culture supernatant was examined under different culture conditions (10% and 0.5% serum adherent culture, 0.5% serum suspension culture). For adherent cultures, culture supernatants from day 3 were examined. In figure 3, varying concentrations of sodium butyrate significantly increased rhIL-24 concentrations in the cell culture supernatant (p <0.01) at 10% and 0.5% serum adherent culture. In figure 4, the specific cell yield increases after different concentrations of sodium butyrate treatment were significant under 10% and 0.5% serum adherent culture conditions (. p < 0.01). In FIG. 5, the IL-24 concentration in the supernatant reached the highest value at day 3 of NaBu treatment under the 0.5% serum suspension culture condition at a sodium butyrate concentration of 2 mM. In FIG. 6, the specific yield is plotted as a function of sodium butyrate concentration at day 3 of NaBu treatment under 0.5% serum suspension culture conditions.

ELISA results show that the secretion expression level of rhIL-24 is obviously improved under the condition of 10% or 0.5% serum adherent culture, although the cell proliferation is inhibited.

Cultured in 10% serum concentration, the rhIL-24 concentration change is positively correlated with NaBu concentration; the rhIL-24 concentrations were 1.62 + -0.05, 1.50 + -0.50, 1.93 + -0.80, 2.67 + -0.55, 3.56 + -0.09% and 4.21 + -0.65 ng/ml at final concentrations of 0, 0.125, 0.25, 0.5, 1 and 2mmol/L, respectively, for sodium butyrate.

When the culture is carried out at 0.5% serum concentration, the highest plateau phase is reached when NaBu is 0.25-2mmol/L, and the average value is 2.34 +/-0.17 ng/ml, as shown in FIG. 3. As the concentration of NaBu increased, the corresponding cell specific yield also increased, the trend with rhIL-24 concentration consistent, as shown in figure 4. Although the expression level of the cells was highest when treated with 2mmol/L sodium butyrate, the inhibition rates of cell proliferation were 18. + -. 4% (10% serum) and 20. + -. 5% (0.5% serum) for 3 days with 2mmol/L NaBu treatment, and 9. + -. 3% and 13. + -. 2% at a NaBu concentration of 1mmol/L, respectively, in combination with the MTT results.

In order to maintain a high relative cell viability for subsequent proliferation, while the rhIL-24 concentration is at its highest, the optimal conditions for NaBu treatment were chosen under adherent culture conditions at 10% and 0.5% serum concentrations: the final concentration is 1mmol/L, and the effect lasts for 3 days.

In the 0.5% serum suspension culture condition, different concentrations of sodium butyrate (0, 0.125, 0.25, 0.5, 1 and 2mmol/L) are used for 3 days, and the rhIL-24 concentration in the supernatant increases along with the increase of the sodium butyrate concentration and is respectively 3.67 +/-0.41, 3.67 +/-0.23, 3.86 +/-0.26, 3.83 +/-0.26, 4.39 +/-0.15 and 4.80 +/-0.30 ng/ml, as shown in figure 5. The inhibition rate of the cells by combining 2mM NaBu for 3 days is only 12 +/-1%, and the treatment conditions of NaBu under the suspension culture condition are selected as follows: 2mmol/L, 3 days of action.

Thirdly, the sodium butyrate promotes the engineering cells to generate G0/G1 phase block

As shown in FIGS. 7 to 8, to analyze the effect of NaBu on the cell cycle under the adherent culture conditions of the engineered cells, 6-well plates were plated at 1X 10 wells per well⁵Cells were cultured in DMEM/F12 medium containing 10% and 0.5% serum, respectively. After 3 days of treatment with NaBu at different final concentrations, flow detection was carried out. At both serum concentrations, cells in the G0/G1 phase increased significantly with increasing NaBu concentration. FIG. 7 shows the flow results under 10% serum conditions, in which the ratio of G0/G1 gradually increased, the ratio of S phase decreased, and the ratio of G2/M phase did not change significantly as the concentration of NaBu increased. FIG. 8 shows the flow results under 0.5% serum conditions, in which the ratio of G0/G1 gradually increased, the ratio of S phase decreased, and the ratio of G2/M phase did not change significantly, as compared with the 10% serum concentration, with increasing NaBu concentration.

At 10% serum concentration, with the increase of NaBu concentration (0.125-2mmol/L), the proportion of cells in G0/G1 gradually increased from 37.60 +/-3.75% of the control group to 55.02 +/-5.46% when the sodium butyrate is 2mmol/L, and the increase rate is 46 +/-8% (. p < 0.01). At 0.5% serum concentration, the proportion of cells in the control group G0/G1 was 46.06 + -1.37%, the proportion of cells in the 2mmol/L NaBu experimental group was 65.46 + -1.97%, and the increase rate was 42 + -8% (. about.p < 0.01).

The change in S phase is contrary to that of G0/G1, i.e., the higher the final concentration of NaBu, the lower the proportion of S phase cells. In the case of a final concentration of NaBu of 2mmol/L, the proportion of S-phase cells in the experimental group is 25.38 +/-5.46% and that in the control group is 41.23 +/-6.76% at a serum concentration of 10%; at 0.5% serum concentration, the experimental group was 16.49 ± 1.16%, the control group was 30.21 ± 3.70%, and the reduction was as high as 45 ± 11% (. about.p < 0.01).

Overall, at the same NaBu concentration, the G0/G1 phase block of the control cells was higher than that of the cells cultured in 10% serum under 0.5% serum culture, indicating that the reduction in serum concentration can promote the G0/G1 phase block; while the block in S phase is significantly lower than that of 10% serum. Such changes in the cell cycle may be an important cause of the inhibition of cell proliferation at low serum concentrations.

In addition, the proportion of G2/M cells was slightly reduced but the change was not large at both serum concentrations with increasing NaBu concentration. Under the serum concentration of 10%, the control group is 21.17 +/-6.85%, and the experimental group with the final concentration of 2mmol/L of NaBu is 19.59 +/-8.78%; the control group and the experimental group were 23.73. + -. 2.97% and 18.05. + -. 1.53%, respectively, at 0.5% serum concentration. In combination with the ELISA results, it was shown that an increased ratio of G0/G1 phases might be associated with an increased level of protein expression.

Fourthly, the promotion effect of the sodium butyrate on the apoptosis of the engineering cells is not obvious

To analyze the effect of NaBu on apoptosis of engineered cells, 6-well plates were plated at 1X 10 wells per well⁵Cells were treated with 10% and 0.5% serum in DMEM/F12 medium for 3 days at different final concentrations of NaBu and then subjected to Annexin-V staining and flow detection. FIG. 9 shows that NaBu promotes apoptosis of the engineered cells, and the concentration of NaBu increases from 0 to 2mmol/L to 4.63 + -2.02%, 4.96 + -1.54%, 5.41 + -2.01%, 6.47 + -3.08% and 7.21 + -3.29% of the control group. But the change was insignificant (p)>0.05) far below the inhibition rate of NaBu on cell proliferation in MTT experiments, indicating that the cell viability is reduced, and more G0/G1 phase block is derived from the cell cycle, but not the cell apoptosis. In FIG. 9, the rate of apoptosis increased with increasing NaBu concentration, but not significantly (p)>0.05)。

Fifthly, the metabolic activity of the cells under adherent culture conditions can be changed by treating the sodium butyrate

After 3 days of treatment with NaBu at different final concentrations, the concentrations of glucose, lactic acid and ammonia in cell culture supernatants were determined using a kit under adherent culture conditions of 10% and 0.5% serum. Under the culture conditions of different serum concentrations, the glucose concentration, the lactic acid concentration and the ammonia concentration respectively have consistent trends, namely, the glucose concentration in the culture supernatant of the experimental group cells is obviously increased (p <0.01) and the lactic acid concentration and the ammonia concentration are both obviously reduced (p <0.01) along with the increase of the NaBu final concentration to 2 mM. Glucose is used as an energy supply molecule for cell metabolism, and the higher the cell proliferation is, the faster the cell consumption is, the lower the concentration in the supernatant is; just because higher NaBu concentration has stronger inhibition effect on the in vitro proliferation capacity of the engineering cell strain, the less the consumption of glucose is, and the higher the glucose concentration in the supernatant is. Lactic acid and ammonia are both products of cell metabolism, and the concentration change rule of cell culture supernatant is just opposite to that of glucose. Compared to the 10% serum culture conditions, the supernatant was at a higher glucose concentration and lower lactate and ammonia concentrations at 0.5% serum concentration for the same NaBu concentration, indicating that both serum reduction and NaBu addition resulted in decreased levels of cellular metabolism.

As shown in fig. 10, the higher the final concentration of sodium butyrate, the higher the glucose concentration in the culture supernatant. As shown in fig. 11, the higher the sodium butyrate concentration, the lower the lactic acid concentration in the supernatant. As shown in fig. 12, the higher the sodium butyrate concentration, the lower the ammonia concentration in the supernatant.

In conclusion, in the embodiment of the invention, on the basis of constructing the site-directed integration engineering cell strain FCHO/IL-24 which stably secretes and expresses rhIL-24, the influence of NaBu (0, 0.125, 0.25, 0.5, 1 and 2mM) with different final concentrations on the cell proliferation capacity, rhIL-24 expression level, cell cycle, cell apoptosis, metabolite concentration and the like of FCHO/IL-24 under different culture conditions (10% serum adherent culture, 0.5% serum adherent culture and 0.5% serum suspension culture) is analyzed.

The NaBu with different concentrations can increase the level of rhIL-24 secreted and expressed by the engineering cell strain, and after being treated with 1mmol/L of NaBu for 3 days, the level of rhIL-24 expressed by the cell strain is increased by 119.94 +/-1.5% (. about.p <0.01), 57.49 +/-2.4% (. about.p <0.01) and 20.17 +/-3.03% (. about.p <0.01) respectively under the conditions of 10% serum concentration adherent culture, 0.5% serum concentration adherent culture and 0.5% serum suspension culture. Under the condition of 10% serum adherent culture, when the final concentration of sodium butyrate is 2mmol/L and the treatment is carried out for 3 days, the inhibition rate on the proliferation of the engineering cell FCHO/IL-24 is 18 +/-4%, and the concentration of rhIL-24 in culture supernatant is 4.2 ng/mL; when the cells were treated with 1mmol/L for 3 days, the inhibition ratio of cell proliferation was 9. + -. 3%, and the rhIL-24 concentration in the culture supernatant was 3.6 ng/mL.

Therefore, treatment with 1mmol/L NaBu for 3 days was the optimal condition for adherent culture. Under the condition of 0.5% serum suspension culture, the change of the viable cell density is related to sodium butyrate and culture time, and the optimum condition of suspension culture is that the viable cell density is treated for 3 days by using 2mmol/L sodium butyrate in combination with the change of the rhIL-24 concentration.

The detection result by a flow cytometer shows that at the same time point, the higher the NaBu concentration is, the higher the proportion of G0/G1 phase block of the cells is, and the proportion of S phase cells is obviously reduced. Sodium butyrate treatment did not cause severe apoptosis, while the relative cell viability of engineered cells was reduced more because of the arrest of the G0/G1 phase of the cycle. Under the adherent culture conditions of 10% and 0.5% serum, the glucose concentration in the supernatant increased and the lactic acid and ammonia concentrations decreased as the sodium butyrate concentration increased. Wherein, sodium butyrate reduces the rate of glucose consumption and lactic acid and ammonia production by cells by inhibiting the proliferation of engineering cells, thereby leading to the increase of the glucose concentration and the reduction of the lactic acid and ammonia concentration in cell culture supernatant.

In conclusion, sodium butyrate inhibited the in vitro proliferation of the engineered cells FCHO/IL-24 in a dose-dependent manner at different serum levels and different culture modes, but at the same time increased the rhIL-24 concentration in the cell culture supernatant and the specific cell productivity. The main reason that NaBu inhibits the in vitro proliferation of the cell strain is to induce G0/G1 phase retardation, and the contribution of apoptosis is limited; at the same time, the reduction in relative viability of the cell lines has an effect on both glucose consumption in the cell culture supernatant and on the production of lactate and ammonia. On the basis of the results, NaBu can be used as an additive of a serum-free culture medium of the cell strain, the level of rhIL-24 secreted and expressed by the cell strain is further improved in high-density suspension culture, experimental data are provided for large-scale culture of FCHO/IL-24 cells, and reference is provided for expression of other genetically engineered proteins by using mammalian cells.

The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope of the present invention are included in the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the claims.

Claims (10)

1. A method for promoting engineered cell strains to secrete and express rhIL-24 by using sodium butyrate is characterized by comprising the following steps: after culturing the site-directed integration engineering cell strain FCHO/IL-24 for a certain time, the culture medium is replaced by a culture medium containing sodium butyrate NaBu to continue culturing.

2. The method for promoting the engineered cell strain to secrete and express rhIL-24 by using the sodium butyrate according to claim 1, wherein the method comprises the following steps: the culture of the fixed-point integration engineering cell strain FCHO/IL-24 is 10 percent serum adherent culture.

3. The method for promoting the engineered cell strain to secrete and express rhIL-24 by using the sodium butyrate according to claim 1, wherein the method comprises the following steps: the culture of the fixed-point integration engineering cell strain FCHO/IL-24 is 0.5 percent serum adherent culture.

4. The method for promoting the engineered cell strain to secrete and express rhIL-24 by using the sodium butyrate according to claim 1, wherein the method comprises the following steps: the culture of the site-directed integration engineering cell strain FCHO/IL-24 is 0.5 percent serum suspension culture.

5. The method of using sodium butyrate for promoting the secretory expression of rhIL-24 by an engineered cell strain according to any one of claims 1-4, characterized in that: the culture medium for culturing the site-specific integration engineering cell strain FCHO/IL-24 is DMEM/F12 culture medium.

6. The method for promoting the engineered cell strain to secrete and express rhIL-24 by using the sodium butyrate according to claim 5, wherein the method comprises the following steps: the medium was supplemented with 100. mu.g/ml of the diabase and 50. mu.g/ml of hygromycin.

7. The method for promoting the engineered cell strain to secrete and express rhIL-24 by using the sodium butyrate according to claim 6, wherein the method comprises the following steps: the concentration of the sodium butyrate in the culture medium is 0.25-2mmol/L, and the serum content in the replaced culture medium is unchanged.

8. The method for promoting the secretion and expression of rhIL-24 by an engineered cell strain by using sodium butyrate according to claim 7, wherein the method comprises the following steps: when 10% serum adherent culture is carried out, fine seeds are inoculated into each hole of a 96-hole plateThe number of cells was 650, and the number of cells seeded per well in 6-well plates was 1X 10⁵And (4) respectively.

9. The method for promoting the secretion and expression of rhIL-24 by an engineered cell strain by using sodium butyrate according to claim 7, wherein the method comprises the following steps: when 0.5% serum adherent culture is performed, the number of cells seeded per well in a 96-well plate is 1500, and the number of cells seeded per well in a 6-well plate is 1 × 10⁵And (4) respectively.

10. The method for promoting the secretion and expression of rhIL-24 by an engineered cell strain by using sodium butyrate according to claim 7, wherein the method comprises the following steps: when 0.5% serum suspension culture is carried out, the volume of shake flask inoculation is 20ml, the rotation speed of shaking table is 119rpm, the incubator condition is 37 ℃ and 5% CO₂Cell seeding density of 5X 10⁵Individual cells/mL.

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