Application of dovitinib lactate in upregulating lactoferrin expression in cells

Abstract

The invention provides an application of dovitinib lactate in up-regulating lactoferrin expression in cells, relates to the field of biotechnology, and proposes for the first time an application of dovitinib lactate in up-regulating lactoferrin expression in cells. Experiments have shown that dovitinib lactate can activate the LF promoter, significantly enhance the expression of lactoferrin in cells, and promote lactoferrin synthesis.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to application of dovitinib lactate in up-regulating lactoferrin expression in cells. Background Art

[0002] Lactoferrin (LF) is an important non-heme iron-binding glycoprotein in breast milk, which is mainly expressed and secreted by mammary epithelial cells. Lactoferrin not only participates in the transport of iron, but also has powerful biological functions such as broad-spectrum antibacterial, antioxidant, anti-cancer, and immune system regulation. It is considered to be a new type of antibacterial and anti-cancer drug and a food and feed additive with great development potential. Lactoferrin has broad market prospects, especially in the field of infant formula.

[0003] Dovitinib Lactate monohydrate is the lactate salt of Dovitinib, a multi-target PTK inhibitor that effectively inhibits class III (FLT3 / c-Kit), class IV (FGFR1 / 3), and class V (VEGFR1-4) RTKs. Dovitinib has been used as a therapy in combination with other drugs to treat multiple types of cancer and is being evaluated in preclinical and clinical trials. In medical studies, Dovitinib sensitizes resistant HCC cells to TRAIL and tegazine-induced apoptosis by inhibiting STAT3. Studies have shown that mammary epithelial cells (MECs) perform lactation functions after delivery, and acute phase involution of the mammary gland is characterized by a decrease in signal transducer and activator of transcription (STAT) 5, followed by an increase in STAT3 signaling, leading to a downregulation of milk production. In mice lacking STAT3, mammary gland involution (the return of the lactating mammary gland to a quiescent state after weaning) is delayed, and in the absence of lactogenic stimulation, secretory alveolar epithelial cells remain fully functional for 6 days after weaning. This suggests that inhibiting STAT3 expression may help maintain the function of mammary epithelial cells and improve lactation capacity. However, there are currently no studies on the promotion of lactoferrin expression in mammary epithelial cells by dovitinib lactate monohydrate.

[0004] In view of this, the present invention is proposed. Summary of the invention

[0005] One of the purposes of the present invention is to provide a new use of dovitinib lactate, namely, use of dovitinib lactate in up-regulating lactoferrin expression in cells.

[0006] The second object of the present invention is to provide a method for regulating the synthesis of lactoferrin by cells in vitro.

[0007] The third object of the present invention is to provide a method for promoting lactoferrin production in mammals.

[0008] In order to achieve the above-mentioned purpose of the present invention, the following technical solutions are particularly adopted:

[0009] In a first aspect, the present invention provides a use of dovitinib lactate in up-regulating lactoferrin expression in cells.

[0010] Furthermore, dovitinib lactate upregulates the expression of lactoferrin by activating the LF promoter.

[0011] Furthermore, the cells include cells containing a lactoferrin gene controlled by a LF promoter.

[0012] Furthermore, the cells include mammary epithelial cells or host cells transfected with a lactoferrin gene expression vector containing a LF promoter.

[0013] Furthermore, the mammary epithelial cells are from any one of humans, cattle, sheep, camels, horses, donkeys, reindeer, alpacas or llamas.

[0014] In a second aspect, the present invention provides a method for regulating in vitro cell synthesis of lactoferrin, comprising adding dovitinib lactate during in vitro bovine mammary epithelial cell culture to obtain lactoferrin through culture;

[0015] The concentration of dovitinib lactate in the culture medium is ≤100 nM and >0.

[0016] Furthermore, the concentration of dovitinib lactate in the culture medium is 1-100 nM.

[0017] Furthermore, the culturing time is at least 24 hours.

[0018] In a third aspect, the present invention provides a method for promoting lactoferrin production in mammals, comprising administering dovitinib lactate to an animal containing a lactoferrin gene controlled by a LF promoter.

[0019] Furthermore, the method of administering dovitinib lactate includes oral administration, injection or external application.

[0020] The application of dovitinib lactate in up-regulating the expression of lactoferrin in cells provided by the present invention is the first to propose the application of dovitinib lactate in up-regulating the expression of lactoferrin in cells. Experiments have shown that dovitinib lactate can activate the LF promoter, significantly enhance the expression of lactoferrin in cells, and promote the synthesis of lactoferrin. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0022] Figure 1 The specific structure of the LF promoter-copGFP expression vector provided in Example 1 of the present invention;

[0023] Figure 2 This is a picture of fluorescent reporter cells observed under a fluorescence microscope after being treated with dovitinib lactate provided in Example 1 of the present invention;

[0024] Figure 3 The chemical structural formulas of dovitinib lactate and dovitinib provided in Example 2 of the present invention;

[0025] Figure 4 This is a diagram of the cell state observation under an inverted microscope after bovine mammary epithelial cells were treated with different concentrations of dovitinib lactate for 24 hours as provided in Example 2 of the present invention;

[0026] Figure 5 This is a diagram of the cell state observation under an inverted microscope after bovine mammary epithelial cells were treated with different concentrations of dovitinib for 24 hours as provided in Example 2 of the present invention;

[0027] Figure 6 The statistical graph of LF expression after bovine mammary epithelial cells were treated with different concentrations of dovitinib lactate for 24 hours provided in Example 2 of the present invention;

[0028] Figure 7 The statistical graph of LF expression after bovine mammary epithelial cells were treated with different concentrations of dovitinib for 24 hours provided in Example 2 of the present invention;

[0029] Figure 8 This is a statistical graph of LF expression after human mammary epithelial cells were treated with different concentrations of dovitinib for 24 hours as provided in Example 4 of the present invention. DETAILED DESCRIPTION

[0030] Unless otherwise defined herein, scientific and technical terms used in conjunction with the present invention shall have the meanings commonly understood by those of ordinary skill in the art. The meaning and scope of the terms should be clear, however, in the case of any potential ambiguity, the definitions provided herein take precedence over any dictionary or external definitions. In this application, unless otherwise stated, the use of "or" means "and / or". In addition, the use of the term "including" and other forms is non-limiting.

[0031] The methods and techniques of the present invention are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification unless otherwise indicated.

[0032] In one aspect, the present invention provides the use of dovitinib lactate in up-regulating the expression of lactoferrin in cells.

[0033] The present invention proposes for the first time the use of dovitinib lactate in up-regulating the expression of lactoferrin in cells. Experiments have shown that dovitinib lactate can activate the LF promoter, significantly enhance the expression of lactoferrin in cells, and promote lactoferrin synthesis.

[0034] In some specific embodiments, dovitinib lactate upregulates the expression of lactoferrin by activating the LF promoter.

[0035] In some specific embodiments, the cells include cells containing a lactoferrin gene controlled by a LF promoter. Dovitinib lactate can promote the expression of lactoferrin in cells containing a lactoferrin gene controlled by a LF promoter by activating the LF promoter.

[0036] In some specific embodiments, the cell comprises a mammary epithelial cell or a host cell transfected with a lactoferrin gene expression vector containing a LF promoter.

[0037] Erdafitinib can be applied to mammary epithelial cells or host cells transfected with a lactoferrin gene expression vector containing the LF promoter to upregulate lactoferrin expression in the cells.

[0038] In some specific embodiments, the mammary epithelial cells are from any one of humans, cows, sheep, camels, horses, donkeys, reindeer, alpacas or llamas.

[0039] According to another aspect of the present invention, a method for regulating in vitro cell synthesis of lactoferrin is also provided, comprising adding dovitinib lactate during in vitro bovine mammary epithelial cell culture to obtain lactoferrin through culture;

[0040] The concentration of dovitinib lactate in the culture medium is ≤100 nM and >0.

[0041] Bovine mammary epithelial cells were treated with dovitinib lactate. The concentration of dovitinib lactate could significantly increase the expression of LF. The data showed that when the concentration of dovitinib lactate was ≤100nM and >0, the expression of LF increased significantly.

[0042] In some specific embodiments, the concentration of dovitinib lactate in the culture medium is 1-100 nM.

[0043] The concentration of dovitinib lactate in the culture medium may be, but is not limited to, 1 nM, 10 nM, 20 nM, 30 nM, 40 nM, 50 nM, 60 nM, 70 nM, 80 nM, 90 nM or 100 nM, or any value between 1 and 100 nM.

[0044] When the concentration of dovitinib lactate in the culture medium is 1-10 nM, it has a significant effect on promoting the synthesis of lactoferrin by bovine mammary epithelial cells in vitro, among which, the effect is most significant when the concentration is 1 nM.

[0045] Specifically, in practical applications, Erdafitinib can be directly added to the culture medium or used as a reagent to prepare a culture medium that promotes the expression of cell lactoferrin.

[0046] In some specific embodiments, the culturing time is at least 24 hours.

[0047] According to another aspect of the present invention, a method for promoting lactoferrin production in mammals is provided, comprising administering dovitinib lactate to an animal containing a lactoferrin gene controlled by a LF promoter.

[0048] In some specific embodiments, the administration of dovitinib lactate includes oral administration, injection or external application.

[0049] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0050] Cell culture

[0051] MAC-T cells: purchased from Shanghai Cell Bank, culture conditions: 90% DMEM + 10% FBS.

[0052] Bovine mammary epithelial cells (BMEC): The mammary tissue of Holstein cows from the Dachang slaughterhouse in Hebei Province was processed by enzyme digestion, and the primary bovine mammary epithelial cells were purified by differential adhesion and differential digestion methods. The culture conditions were 90% DMEM / F12 + 10% FBS. The cells were digested with 0.25% EDTA-trypsin for subculture, and digestion was terminated with complete medium.

[0053] Example 1 Verification of the activation of LF promoter by dovitinib lactate to promote lactoferrin synthesis

[0054] 1. Vector construction and plasmid amplification

[0055] pGreenZeo lenti reporter vector, enzyme digestion to obtain linearized vector, synthesize LF promoter sequence, use homologous recombination to construct LF promoter-copGFP expression vector, transform E. coli, screen positive colonies on resistance medium, and after identification and sequencing, perform bacterial liquid expansion and extract plasmid for use. The specific structure of LF promoter-copGFP expression vector is as follows: Figure 1 As shown, Cell-specific Promoter refers to LF promoter, and the nucleotide sequence of LF promoter is shown in SEQ ID NO.1.

[0056] 2. Preparation of LF promoter-copGFP promoter reporter cells

[0057] (1) The LF promoter-copGFP expression vector plasmid was transfected into 293T cells together with the packaging vector. After 48 hours, the supernatant containing the virus was collected and the lentivirus was concentrated, purified and titered.

[0058] (2) When the MAC-T cell fusion rate reaches about 70%, virus infection is performed. After 24 hours, the culture medium is replaced with fresh complete culture medium and culture is continued in a carbon dioxide incubator.

[0059] (3) 48 hours after adding the virus, add Zeocin to screen out cell lines with stable expression.

[0060] 3. Verification of dovitinib lactate

[0061] pGreenZeo lenti reporter (LF promoter-copGFP) cells were cultured in 96-well plates. When the cells were attached and fused to 80%, they were treated with 1 μM dovitinib lactate. Cell fluorescence was observed under a fluorescence microscope after 24 hours. The specific results are shown in the figure. Figure 2 As shown, by constructing a fluorescent reporter cell line (LF promoter drives copGFP expression), fluorescence microscopy observation revealed obvious fluorescence production under dovitinib lactate treatment.

[0062] Example 2 Optimization of the amount of dovitinib lactate added

[0063] The 11th generation BMECs were cultured in 6-well plates. When the cells were attached and fused to 80%, they were treated with dovitinib lactate and dovitinib, respectively. 0.1% DMSO was used to replace dovitinib lactate and dovitinib as a control group. The chemical structures of dovitinib lactate and dovitinib are as follows: Figure 3 As shown, Figure 3 (a) is the chemical structural formula of dovitinib lactate, Figure 3(b) is the chemical structure of dovitinib. The concentrations of dovitinib lactate and dovitinib were 1 nM, 10 nM, 100 nM and 1000 nM, respectively. After 24 hours, the cells were collected and the cell status was observed under an inverted microscope. The observation results are shown in FIG. Figure 4 and Figure 5 shown.

[0064] At the same time, RNA was extracted and RT-qPCR was performed to detect the expression of LF. The primers used were shown in Table 1, and GAPDH was used as the internal reference. Figure 6 and Figure 7 shown.

[0065] Table 1 List of qPCR specific primers used

[0066]

[0067] like Figure 4 and Figure 6 It can be seen that the treatment of BMEC with 1nM, 10nM and 100nM Dovitinib Lactate monohydrate can increase the expression of LF, among which the 1nM and 10nM concentrations have a significant effect. However, there is no difference between the 1000nM treatment and the control group.

[0068] like Figure 5 and Figure 7 It can be seen that different concentrations of Dovitinib treated BMECs all inhibited the expression of LF, while Dovitinib Lactate monohydrate could enhance the expression of LF.

[0069] Example 3 Lactic acid can help you increase the expression of lactoferrin in human mammary epithelial cells

[0070] Referring to the method of Example 2, dovitinib lactate at concentrations of 1 nM and 10 nM was applied to human mammary epithelial cells (MCF-10A), and cells were collected 24 hours later, RNA was extracted, and RT-qPCR was performed to detect the expression of LF. The primers used were shown in Table 2, and GAPDH was used as an internal reference. The specific results are shown in Table 2. Figure 8 As shown. It can be seen that dovitinib lactate has the effect of upregulating the expression of lactoferrin in human mammary epithelial cells.

[0071] Table 2 List of specific primers used in qPCR in human mammary epithelial cells (MCF-10A)

[0072]

[0073] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. The application of dovitinib lactate in upregulating lactoferrin expression in cells in vitro; The cells include mammary epithelial cells or host cells transfected with a lactoferrin gene expression vector containing a LF promoter; The nucleotide sequence of the LF promoter is shown in SEQ ID NO.1; The mammary epithelial cells are from any one of humans, cattle, sheep, camels, horses, donkeys, reindeers, alpacas or llamas; The concentration of dovitinib lactate in the culture medium is 1-10 nM.

2. The use according to claim 1, characterized in that: The dovitinib lactate upregulates the expression of lactoferrin by activating the LF promoter.

3. A method for regulating the synthesis of lactoferrin by cells in vitro, characterized in that: It includes adding dovitinib lactate during in vitro cell culture to obtain lactoferrin; The concentration of dovitinib lactate in the culture medium is 1-10 nM; The culturing time is at least 24 hours; The cells include mammary epithelial cells or host cells transfected with a lactoferrin gene expression vector containing a LF promoter; The mammary epithelial cells are from any one of humans, cattle, sheep, camels, horses, donkeys, reindeers, alpacas or llamas; The nucleotide sequence of the LF promoter is shown in SEQ ID NO.1.

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