CN111662876B - Method for inducing human fibroblast to reprogram to Leydig-like cell - Google Patents
Method for inducing human fibroblast to reprogram to Leydig-like cell Download PDFInfo
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- CN111662876B CN111662876B CN201910284275.5A CN201910284275A CN111662876B CN 111662876 B CN111662876 B CN 111662876B CN 201910284275 A CN201910284275 A CN 201910284275A CN 111662876 B CN111662876 B CN 111662876B
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- C12N5/0681—Cells of the genital tract; Non-germinal cells from gonads
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Abstract
The invention provides a method for inducing human fibroblast to be reprogrammed into Leydig-like cells, which utilizes the expression of NR5A1 (also known as sterogenic factor 1, SF1) gene and micromolecular compounds Forskolin, DAPT and Purmorphamine to induce the human fibroblast to be reprogrammed into the Leydig-like cells. The Leydig-like cells induced by the invention are obtained by directly reprogramming human skin fibroblasts through a transcription factor NR5A1 and a small molecular compound, do not pass through a stem cell stage, and avoid the risk of tumor formation during transplantation. The method can simply and quickly reprogram the human skin fibroblasts into functional Leydig-like cells, can secrete testosterone, and is beneficial to treating male hypogonadism.
Description
Technical Field
The invention relates to the field of cell biology and tissue engineering, in particular to a method for inducing human fibroblasts to reprogram Leydig-like cells by using NR5A1 and a small molecular compound.
Background
Leydig cell transplantation technology transplants Leydig cells or functional Leydig-like cells induced in vivo and in vitro into the body to play a role, and hopefully replaces the traditional hormone replacement method to treat the male primary hypogonadism. Therefore, the Leydig cell transplantation technique requires a large number of safe and effective seed cells. Traditionally used seed cells include stem cell-induced Leydig-like cells and fibroblast reprogrammed Leydig-like cells.
Compared with the traditional androgen replacement therapy, the Leydig cell transplantation technology can simulate the normal hormone secretion in vivo and avoid the side effects of hemoglobin, hematocrit, coronary calcified plaque volume increase, high-density lipoprotein cholesterol reduction and the like brought by hormone therapy. Transplantation of functional Leydig cells in vitro has been shown to treat castrate mice and restore serum testosterone levels. However, for clinical purposes, human Leydig cells are not only of small origin but also difficult to ethically comply. Both of these problems are solved by Leydig-like cells induced by stem cells or reprogrammed by fibroblasts.
Stem cell induction or fibroblast reprogramming techniques have been used to differentiate or directly transform cells into a variety of different types of functional cells, such as cardiomyocytes, neurons, islet β cells, and the like. In addition, fibroblasts can be reprogrammed to pluripotent stem cells prior to indirect conversion into various somatic cells. These functional somatic cells derived from stem cell induction or fibroblast direct/indirect transdifferentiation acquire various characteristics and functions typical of target cells, and no longer maintain molecular and cellular characteristics such as gene expression profile of original cells. These induced functional cells can be used not only to mimic the associated disease, but also for therapeutic purposes by transplantation into animal models of the disease.
Currently, ZHANG Z Y et al and XING X et al found that stem cells can be induced to differentiate into functional Leydig-like cells in the presence of insulin-transferrin-selenium (insulin-transferase-selenium) by in vivo induction or in vitro induction with Luteinizing Hormone (LH), thyroid hormone (thyroid hormone), insulin-like growth factor 1 (igf-1), platelet-derived growth factor-BB (PDGF-BB), respectively. HOU Y P et al and YANG Y et al, respectively, found that fibroblasts could be reprogrammed to functional Leydig-like cells by transfection with SF-1, GATA4, NGFI-B or Dmrt1, gata4, nr5A1, verifying the key role of NR5A 1. However, both stem cell induction and gene manipulation have potential tumorigenicity, and the latter also involves the risk of viral infection, which is detrimental to clinical use.
In recent years, small molecule compounds (< 900 Da) have attracted attention from researchers due to their characteristics of high efficiency, safety, stability, easy storage, etc., and have a tendency to gradually replace the conventional gene manipulation methods. It can directly permeate cell membrane, does not need any virus vector, has no immunogenicity, and is easy to quantify, standardize and regulate. Unlike traditional gene manipulation, it mainly acts by regulating specific signal pathways, or epigenetic and metabolic related processes, and is more beneficial to the research of potential mechanisms. Since 2010, small molecule compounds are completely utilized, and human beings successfully reprogram fibroblasts into pluripotent stem cells, cardiac muscle cells, nerve cells and the like, so that the huge potential value of the small molecule compounds in the field of regenerative medicine is proved. In addition, small molecule compounds are born based on biosynthetic chemical design, and thus have infinite possibilities and optimized properties in terms of functional structure.
Disclosure of Invention
The invention aims to provide a method for inducing human fibroblasts to reprogram into Leydig-like cells.
In order to achieve the above object, the present invention provides a method for inducing human fibroblasts to reprogram Leydig-like cells, which comprises inducing human fibroblasts to reprogram Leydig-like cells by using expression of NR5A1 gene and a small molecule compound, wherein the small molecule compound is a mixture of Forskolin, DAPT and Purmorphamine.
As a preferable scheme, the method comprises the steps of constructing a slow virus transfected human skin fibroblast carrying NR5A1 to enable the human skin fibroblast to express NR5A1, culturing the virus transfected skin fibroblast with a common culture solution for 3-4 days, replacing a signal molecule induction solution after the NR5A1 is stably expressed, continuing culturing, and inducing to be a Leydig-like cell after 7-14 days, wherein the signal molecule induction solution is formed by adding three substances of Forskolin, DAPT and Purmorphamine into the common culture solution.
As a preferred embodiment, the general culture medium formulation is 10% fetal bovine serum +100U/mL penicillin + 100. Mu.g/mL streptomycin + high glucose DMEM medium at 37 ℃ 5% CO 2 And culturing the cells under the environment.
As a preferable scheme, the signal molecule inducing solution is formed by adding 10 mu M Forskolin, 5 mu M DAPT and 0.1 mu M Purmorphamine into a common culture solution.
As a preferred scheme, the detailed steps of the method are as follows:
(I) plasmid construction and cell culture
1.1 cell culture
Firstly, collecting common human skin fibroblasts, culturing the human skin fibroblasts in a common culture solution consisting of 10% fetal calf serum, 100U/mL penicillin, 100 mu g/mL streptomycin and high-glucose DMEM culture medium, and adhering to a 6-well plate; inoculation cell density (3-5). Times.10 4 /cm 2 Cultured at 37 deg.C, 5% 2 The incubator of (1);
1.2 plasmid construction
Inserting human NR5A1 into a pGMLV vector containing GFP, and constructing a pGMLV no-load plasmid containing GFP;
(II) Virus-transfected cells
Lentivirus packaging and transfection: the target gene NR5A1 plasmid and the virus packaging auxiliary plasmid are cotransfected with HEK293T cells by a calcium chloride method, after high-titer viruses are obtained, the NR5A1 lentiviruses and no-load viruses are respectively transfected to common human skin fibroblasts, and the transfection efficiency can respectively reach 60-90% and 90-100%;
(III) Small molecule Induction
Culturing the skin fibroblasts transfected by the virus for 3-4 days by using a common culture solution, wherein the formula is as follows: 10% fetal bovine serum +100U/mL penicillin + 100. Mu.g/mL streptomycin + high-sugar DMEM medium;
then the culture solution is replaced by a signal molecule inducing solution formed by adding small molecular compounds into the common culture solution, and the culture is continued for 7 to 14 days. The formula of the signal molecule inducing liquid is as follows: 10% fetal calf serum, 100U/mL penicillin, 100 mug/mL streptomycin, high-sugar DMEM culture medium, 10 mug M Forskolin, 5 mug M DAPT and 0.1 mug M Purmorphamine, and then replacing the signal molecule induction liquid every 2-3 days for continuous induction for more than 7 days; then, the cells are cultured and maintained by using a signal molecule inducing solution;
(IV) Testosterone secretion function identification
The induced cell culture supernatant was taken and the testosterone concentration was determined by chemiluminescence.
The method has the beneficial effects that the method can simply and quickly transdifferentiate the human skin fibroblasts into the functional Leydig-like cells, can secrete testosterone, and is beneficial to treating male hypogonadism. Furthermore, the Leydig-like cells induced by the invention are obtained by human skin cells through the expression of a transcription factor NR5A1 and direct reprogramming of small molecular compounds, do not pass through a stem cell stage, and avoid the risk of tumor formation during transplantation.
Drawings
FIG. 1 is a schematic diagram of the method for reprogramming dermal fibroblasts into Leydig-like cells according to the present invention.
Fig. 2 identification of reprogramming of the inventors' skin fibroblasts into Leydig-like cells, a. Culture morphology of normal human skin fibroblasts; B. transdifferentiation into a cell morphology of Leydig-like cells; C. and (5) identifying testosterone secretion function.
Detailed Description
Hereinafter, the technique of the present invention will be described in detail with reference to specific embodiments. It should be understood that the following detailed description is only for the purpose of assisting those skilled in the art in understanding the present invention, and is not intended to limit the present invention.
The process of inducing the transdifferentiation of human fibroblasts into Leydig-like cells by using the expression of NR5A1 gene and micromolecular compounds Forskolin, DAPT and Purmorphamine comprises the following steps:
(1) Collecting skin fibroblasts of ordinary people, and culturing the skin fibroblasts of the ordinary people;
(2) Constructing an NR5A1 lentiviral vector;
(3) Cell density (3-5). Times.10 4 /cm 2 In the case of (1), the lentivirus transfects human skin fibroblasts, and determines that the fibroblasts begin to express NR5A1;
(4) 3-4 days after infection, culturing cells for 7-14 days by using a signal molecule induction solution containing 10 mu M Forskolin, 5 mu M DAPT and 0.1 mu M Purmorphamine;
(5) Leydig-like cells were obtained and identified.
Example 1.
(I) plasmid construction and cell culture
1.1 cell culture
Firstly, collecting common human skin fibroblasts, culturing the human skin fibroblasts in a common culture solution consisting of 10% fetal bovine serum (Hyclone) +100U/mL penicillin (Hyclone) +100 mu g/mL streptomycin (Hyclone) + high-sugar DMEM medium (Hyclone), and adhering to a 6-well plate; inoculation cell density (3-5). Times.10 4 /cm 2 Cultured at 37 deg.C, 5% 2 The incubator of (1);
1.2 plasmid construction
Inserting human NR5A1 into a pGMLV vector containing GFP, and constructing a pGMLV no-load plasmid containing GFP;
(II) Virus-transfected cells
Lentivirus packaging and transfection: co-transfecting HEK293T cells by a target gene NR5A1 plasmid and a virus packaging auxiliary plasmid through a calcium chloride method to obtain high-titer viruses, and respectively transfecting NR5A1 lentiviruses and no-load viruses to common human skin fibroblast cells;
(III) Small molecule Induction
The virus-transfected skin fibroblasts (which express NR5A1-GFP or GFP, respectively) were cultured for 3 days using a common culture medium according to the following formulation: 10% fetal bovine serum (Hyclone) +100U/mL penicillin (Hyclone) + 100. Mu.g/mL streptomycin (Hyclone) + high-glucose DMEM medium (Hyclone);
then, the culture is continued for 7 to 14 days by using a signal molecule inducing solution, and the formula of the signal molecule inducing solution is as follows: 10% fetal bovine serum (Hyclone) +100U/mL penicillin (Hyclone) +100 μ g/mL streptomycin (Hyclone) + high-sugar DMEM medium (Hyclone) +10 μ M Forskolin +5 μ M DAPT +0.1 μ M Purmorphamine, and then replacing the signal molecule inducing solution every 2-3 days for continuous induction for more than 7 days; the subsequent cell culture is maintained by using a signal molecule inducing solution;
(IV) Testosterone secretion function identification
The induced cell culture supernatant was taken and the Testosterone concentration (Access Testosterone Assay, beckman COULTER) was determined by chemiluminescence.
The method uses Forskolin + DAPT + Purmorphamine as signal molecule inducing liquid, so that human skin fibroblasts expressing NR5A1 are reprogrammed to Leydig-like cells, and the testosterone secretion capacity of the method is far better than that of single micromolecule Forskolin or single DAPT or single Purmorphamine. Therefore, the invention selects the mixture of Forskolin + DAPT + Purmorphamine as the inducing molecule.
Example 2 to example 8: the culture and transdifferentiation methods in examples 2 to 8 are the same as in example 1, and are not described in detail, but the formulations and the processing times are different, as shown in the following table.
Leydig-like cells obtained in the different examples had no significant difference in morphology and were able to secrete testosterone. The testosterone secretion capacity of the obtained cells is stronger than that of the cells cultured in a signal molecule induction solution for 14 days than that of the cells cultured in the signal molecule induction solution for 7-10 days.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (5)
1. A method for inducing human fibroblasts to reprogram Leydig-like cells is characterized in that the expression of NR5A1 gene and a small molecular compound are used for inducing the human fibroblasts to reprogram Leydig-like cells, wherein the small molecular compound is a mixture of Forskolin, DAPT and Purmorphamine.
2. The method as claimed in claim 1, wherein the method comprises constructing slow virus carrying NR5A1 to transfect human dermal fibroblasts, allowing normal human dermal fibroblasts to express NR5A1, culturing the virus-transfected dermal fibroblasts in a common culture solution for 3-4 days, stably expressing NR5A1, culturing the virus-transfected dermal fibroblasts in a signal molecule inducing solution, and inducing Leydig-like cells after 7-14 days, wherein the signal molecule inducing solution is prepared by adding Forskolin, DAPT and Purmorphamine into the common culture solution.
3. The method of claim 2, wherein the common medium formulation is 10% fetal bovine serum +100U/mL penicillin +100 μ g/mL streptomycin + high glucose DMEM medium at 37 ℃ and 5% co 2 And culturing the cells under the environment.
4. The method for inducing human fibroblasts to reprogram Leydig-like cells according to claim 2, wherein said signal molecule inducing solution is prepared by adding 10. Mu.M Forskolin, 5. Mu.M DAPT and 0.1. Mu.M Purmorphamine to a common culture solution.
5. The method of claim 1, wherein the steps of the method are as follows:
(I) plasmid construction and cell culture
1.1 cell culture
Firstly, collecting common human skin fibroblasts, culturing the human skin fibroblasts in a common culture solution consisting of 10% fetal calf serum, 100U/mL penicillin, 100 mu g/mL streptomycin and high-glucose DMEM culture medium, and adhering to a 6-well plate; inoculation cell density (3-5). Times.10 4 /cm 2 Cultured at 37 deg.C, 5% 2 The incubator of (1);
1.2 plasmid construction
Inserting human NR5A1 into a pGMLV vector containing GFP, and constructing a pGMLV no-load plasmid containing GFP;
(II) Virus-transfected cells
Lentivirus packaging and transfection: the target gene NR5A1 plasmid and the virus packaging auxiliary plasmid transfect HEK293T cells by a calcium chloride method, and after high-titer viruses are obtained, the NR5A1 viruses and no-load viruses are respectively transfected to common human skin fibroblasts, wherein the transfection efficiency can respectively reach 60-90% and 90-100%;
(III) Small molecule Induction
Culturing the skin fibroblast after virus transfection for 3-4 days by using a common culture solution, wherein the formula is as follows: 10% fetal bovine serum +100U/mL penicillin + 100. Mu.g/mL streptomycin + high-sugar DMEM medium;
then the culture solution is replaced by a signal molecule inducing solution formed by adding small molecular compounds into the common culture solution, and the culture is continued for 7 to 14 days. The formula of the signal molecule inducing liquid is as follows: 10% fetal bovine serum +100U/mL penicillin + 100. Mu.g/mL streptomycin + high-sugar DMEM medium + 10. Mu.M Forskolin + 5. Mu.M DAPT + 0.1. Mu.M Purmorphamine, and then replacing the signal molecule induction liquid every 2-3 days for continuous induction for more than 7 days; then, the cells are cultured and maintained by using a signal molecule inducing solution;
(IV) Testosterone secretion function identification
The induced cell culture supernatant was taken and the testosterone concentration was determined by chemiluminescence.
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