CN118141900B - Stem cell factor composition and sustained release agent for repairing endometrial injury and preparation method thereof - Google Patents
Stem cell factor composition and sustained release agent for repairing endometrial injury and preparation method thereof Download PDFInfo
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- CN118141900B CN118141900B CN202410574833.2A CN202410574833A CN118141900B CN 118141900 B CN118141900 B CN 118141900B CN 202410574833 A CN202410574833 A CN 202410574833A CN 118141900 B CN118141900 B CN 118141900B
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- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/39—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin, cold insoluble globulin [CIG]
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- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
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- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
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Abstract
The invention belongs to the technical field of biological medicines, and in particular relates to a stem cell factor composition for repairing endometrium injury, a sustained release agent and a preparation method thereof. The invention adopts the 3 rd to 6 th generation human endometrium mesenchymal stem cells, starvation treatment is carried out for 20 to 28 hours by a second culture medium containing Flavine Adenine Dinucleotide (FAD), then the human endometrium mesenchymal stem cell concentrate prepared by filtration and ultrafiltration is compatible with recombinant III type humanized collagen to obtain a stem cell factor composition, which can obviously improve endometrium stromal cell apoptosis induced by mifepristone and repair the thinning of endometrium caused by pathological injury; the stem cell factor composition, hyaluronic acid, slow release material and adhesive are prepared into slow release agent which can play a role in continuous repair treatment.
Description
Technical Field
The invention relates to stem cell factors, in particular to a stem cell factor composition for repairing endometrial damage, a sustained release agent and a preparation method thereof.
Background
Endometrium (endometrium) refers to the layer that forms the inner wall of a mammalian uterus and is part of the female reproductive organ. Is responsive to both estrogens and progestogens and therefore can vary significantly with the sexual cycle (estrus cycle, menstrual cycle). The endometrium is divided into a functional layer and a basal layer 2. The surface 2/3 of the inner membrane is a compact layer and a sponge layer which are collectively called as functional layers, and the functional layers are influenced by the ovarian sex hormone to periodically change and fall off. The basal layer is a 1/3 endomembrane close to the myometrium, is not affected by ovarian sex hormone, and does not change periodically.
Due to the frequent uterine cavity operation, infection, ischemia and other factors, the endometrium injury may cause the female to have amenorrhea, reduced menstrual flow, repeated abortion, infertility, placenta abnormality and the like, and seriously affect the female reproductive health. At the cellular level, endometrial damage occurs due to reduced viability of endometrial stromal cells, and even undergoes apoptosis, thereby inducing endometrial atrophy and disruption of endometrial homeostasis. The apoptosis signal pathway activated by the endometrial injury inhibits endometrial angiogenesis, thereby preventing endometrial regeneration. More importantly, severe endometrial damage is often not reversible.
Clinically, the key factors for successful repair and remodeling of the endometrium are re-epithelialization and revascularization of the endometrium, and secondly, anti-infection and inflammatory control are important environmental guarantees for optimizing endometrial neogenesis. However, there is no ideal clinical treatment method meeting the above requirements at the same time, and the operation and the drug treatment have some side effects or have very little curative effect. Accordingly, scholars are continually exploring therapeutic approaches to repair endometrial lesions to improve the endometrial microenvironment and tolerability status of endometrial lesion patients, thereby increasing the pregnancy rate of these patients.
With the continuous progress of the technology, people attach mesenchymal stem cells to degradable collagen scaffold materials, after the intrauterine adhesion is separated by combining a hysteroscopic technology, the composite of the scaffold materials and the mesenchymal stem cells is transplanted into the intrauterine cavity, so that the mesenchymal stem cells cover the endometrial adhesion surface, the regeneration of endometrial vascular tissues is promoted, the good endometrial blood circulation is found 3 months after the operation, and the functional repair of the damaged endometrium is realized. In the treatment research of endometrial injury, stem cell therapy is widely applied, and has better effects in the aspects of reducing endometrial fibrosis, repairing damaged endometrial, and the like.
While more and more scholars believe that paracrine action of stem cells may be the primary mechanism by which they are involved in therapy, i.e., stem cells rely primarily on their exosomes and secreted growth factors to promote tissue repair. The stem cell exosome contains various chemotactic factors, cytokines, transcription factors and the like, reduces cell death mainly by means of promoting blood vessel regeneration, stimulating host endometrium cell activity, regulating immunity and the like in the aspect of endometrium regeneration, provides nutrition support for host cells and promotes tissue regeneration. Compared with mesenchymal stem cells, the stem cell exosomes or growth factors extracted by centrifugation, filtration and other modes are safer, and have become a new research hotspot.
However, the effect of treating endometrium injury by stem cell factor in the prior art is limited, and the stem cell exosome or stem cell factor in the form of solution is difficult to continuously repair. Therefore, the stem cell factor composition for repairing endometrium injury is prepared, the curative effect is better, and the sustained release preparation can play a role in continuous repair.
Disclosure of Invention
Based on the defects existing in the prior art, the invention aims to provide a stem cell factor composition with better treatment effect by utilizing stem cell factors, and the sustained release preparation prepared from the stem cell factor composition can also play a role in continuously repairing endometrial injury.
In order to achieve the above purpose, the technical scheme of the invention is realized as follows:
in a first aspect, the invention provides an endometrial mesenchymal stem cell concentrate prepared by the following method:
S1, taking 3 rd to 6 th generation human endometrium mesenchymal stem cells, and culturing in a DMEM culture medium containing 8% -10% FBS by volume fraction;
S2, changing the cell into a second culture medium when the cell grows to have the confluence of 60-70%, continuously culturing for 20-28 hours, and collecting a cell culture solution;
S3, filtering the cell culture solution obtained in the step S2 by using a microporous filter membrane with the thickness of 0.22 mu m, and then passing the filtrate through an ultrafiltration membrane with the thickness of 10KD to obtain the endometrium mesenchymal stem cell concentrate.
Preferably, the culture conditions of the step S1 are as follows: the cells were cultured in a cell incubator with a volume fraction of 5% CO 2, a relative humidity of 95% and a temperature of 37 ℃.
Preferably, the second medium in the step S2 is a DMEM/F12 medium containing 2-6 mg/L Flavin Adenine Dinucleotide (FAD) and 1-3% FBS in volume fraction.
In a second aspect, the present invention provides a stem cell factor composition for repairing endometrial damage, comprising the following components: endometrium mesenchymal stem cell concentrate and recombinant III type humanized collagen.
Preferably, the mass ratio of the endometrium mesenchymal stem cell concentrate to the recombinant III type humanized collagen is (8-10): 1.
In a third aspect, the invention provides a stem cell factor sustained release agent for repairing endometrium injury, which comprises the following components: endometrium mesenchymal stem cell concentrate, recombinant III type humanized collagen, hyaluronic acid, slow-release material and adhesive.
Preferably, the stem cell factor sustained release agent for repairing endometrium injury comprises the following components in percentage by mass: 40-50% of endometrium mesenchymal stem cell concentrate, 5% of recombinant III-type humanized collagen, 5-15% of hyaluronic acid, 20-35% of slow release material and 5-15% of adhesive.
Preferably, the stem cell factor sustained release agent for repairing endometrium injury comprises the following components in percentage by mass: 50% of endometrium mesenchymal stem cell concentrate, 5% of recombinant III type humanized collagen, 12% of hyaluronic acid, 25% of slow-release material and 8% of adhesive.
Preferably, the endometrium mesenchymal stem cell concentrate is prepared by the following method:
s1, taking 3 rd to 6 th generation human endometrium mesenchymal stem cells, and culturing in a DMEM (DMEM medium containing 8% -10% FBS);
S2, changing the cell to a second culture medium when the cell grows to have the confluence of 60-70%, continuously culturing for 20-28 hours, and collecting a cell culture solution;
S3, filtering the cell culture solution obtained in the step S2 by using a microporous filter membrane with the thickness of 0.22 mu m, and then passing the filtrate through an ultrafiltration membrane with the thickness of 10KD to obtain endometrium mesenchymal stem cell concentrate;
Preferably, the culture conditions of the step S1 are as follows: culturing in a cell incubator with a volume fraction of CO 2%, a relative humidity of 95% and a temperature of 37 ℃;
Preferably, the second medium in the step S2 is a DMEM/F12 medium containing 2-6 mg/L Flavin Adenine Dinucleotide (FAD) and 1-3% FBS.
Preferably, the molecular weight of the hyaluronic acid is 800-1000 kD.
Preferably, the slow release material may be selected from any one or a mixture of any two or more of ethyl cellulose, hydroxyethyl cellulose, hydroxypropyl methyl cellulose and hydroxypropyl cellulose.
Preferably, the slow release material is hydroxypropyl methylcellulose.
Preferably, the binder may be selected from microcrystalline cellulose and povidone.
Preferably, the binder is povidone.
In a fourth aspect, the invention provides a preparation method of a stem cell factor sustained release agent for repairing endometrium injury, which comprises the steps of taking endometrium mesenchymal stem cell concentrate, recombining III type humanized collagen and hyaluronic acid, stirring and mixing uniformly, adding sustained release materials and adhesives, and mixing uniformly.
Preferably, the preparation method comprises the following steps: and taking 40-50% of endometrium mesenchymal stem cell concentrate, 5% of recombinant III-type humanized collagen and 5-15% of hyaluronic acid according to the mass percentage, stirring and mixing uniformly, adding 20-35% of slow release material and 5-15% of adhesive, and mixing uniformly to obtain the endometrium mesenchymal stem cell.
Preferably, the preparation method comprises the following steps: taking 50% of endometrium mesenchymal stem cell concentrate, 5% of recombinant III-type humanized collagen and 12% of hyaluronic acid according to the mass percentage, stirring and mixing uniformly, adding 25% of slow-release material and 8% of adhesive, and mixing uniformly to obtain the composition.
Preferably, the endometrium mesenchymal stem cell concentrate is prepared by the following method:
s1, taking 3 rd to 6 th generation human endometrium mesenchymal stem cells, and culturing in a DMEM (DMEM medium containing 8% -10% FBS);
S2, changing the cell into a second culture medium when the cell grows to have the confluence of 60-70%, continuously culturing for 20-28 hours, and collecting a cell culture solution;
S3, filtering the cell culture solution obtained in the step S2 by using a microporous filter membrane with the thickness of 0.22 mu m, and then passing the filtrate through an ultrafiltration membrane with the thickness of 10KD to obtain endometrium mesenchymal stem cell concentrate;
Preferably, the culture conditions of the step S1 are as follows: culturing in a cell incubator with a volume fraction of CO 2%, a relative humidity of 95% and a temperature of 37 ℃;
Preferably, the second medium in the step S2 is a DMEM/F12 medium containing 2-6 mg/L Flavin Adenine Dinucleotide (FAD) and 1-3% FBS.
Preferably, the molecular weight of the hyaluronic acid is 800-1000 kD.
Preferably, the slow release material may be selected from any one or a mixture of any two or more of ethyl cellulose, hydroxyethyl cellulose, hydroxypropyl methyl cellulose and hydroxypropyl cellulose.
Preferably, the slow release material is hydroxypropyl methylcellulose.
Preferably, the binder may be selected from microcrystalline cellulose and povidone.
Preferably, the binder is povidone.
Compared with the prior art, the invention has the beneficial effects that:
1. Compared with the concentrated solution prepared by umbilical cord mesenchymal stem cells, bone marrow mesenchymal stem cells and adipose mesenchymal stem cells, the stem cell factor composition prepared by matching recombinant III type humanized collagen can better improve mifepristone-induced endometrium stromal cell apoptosis and better repair endometrium thinning caused by pathological injury. The stem cell factor composition, hyaluronic acid, a slow release material and an adhesive are further prepared into a slow release formulation, and the slow release formulation can also play a role in continuously repairing endometrial injury.
2. The stem cell factor slow release agent takes a stem cell factor composition as an active medicinal component, and the stem cell factor composition mainly comprises endometrium mesenchymal stem cell concentrate and recombinant III type humanized collagen, and the stem cell factor composition and the recombinant III type humanized collagen act synergistically and complement each other to generate a remarkable beneficial effect of improving the healing effect of repairing endometrium injury. The preparation method of the endometrium mesenchymal stem cell concentrate is obtained by a large number of experiments and repeated tests, and the treatment effect of the concentrate containing the stem cell factor can be improved by the step of starving a second culture medium containing Flavine Adenine Dinucleotide (FAD) for 20-28 hours. The cell number selected in the preparation method, the adding amount of flavin adenine dinucleotide in the second culture medium, the adding amount of FBS and the treatment time of the cells in the second culture medium are all key parameters, and compared with the prior art, the preparation method has obvious progress.
Drawings
FIG. 1 cell viability of human endometrial stromal cells
FIG. 2 SD rat endometrium thickness
Detailed Description
The invention will be further illustrated with reference to specific examples, but the invention is not limited to these examples.
The human umbilical cord mesenchymal stem cells, human bone marrow mesenchymal stem cells, and human adipose mesenchymal stem cells used in the following embodiments were all purchased from ATCC.
Recombinant III humanized collagen purchase Shanxi Bo biological medicine Co., ltd., national mechanical injection 20213130488.
Example 1
A stem cell factor composition for repairing endometrial damage comprising the following components: endometrium mesenchymal stem cell concentrate and recombinant III type humanized collagen;
The mass ratio of the endometrium mesenchymal stem cell concentrated solution to the recombinant III type humanized collagen is 10:1, a step of;
the endometrium mesenchymal stem cell concentrate is prepared by the following method:
S1, taking 4 th generation human endometrium mesenchymal stem cells, and culturing in a DMEM medium containing 8% FBS;
S2, changing the cell into a second culture medium when the cell grows to 70% of confluence, continuously culturing for 24 hours, and collecting a cell culture solution;
S3, filtering the cell culture solution obtained in the step S2 by using a microporous filter membrane with the thickness of 0.22 mu m, and then passing the filtrate through an ultrafiltration membrane with the thickness of 10KD to obtain endometrium mesenchymal stem cell concentrate;
The culture conditions of the step S1 are as follows: culturing in a cell incubator with a volume fraction of CO 2%, a relative humidity of 95% and a temperature of 37 ℃;
The second medium in the step S2 is DMEM/F12 medium containing 4mg/L Flavin Adenine Dinucleotide (FAD) and 1% FBS.
Example 2
A stem cell factor composition for repairing endometrial damage comprising the following components: endometrium mesenchymal stem cell concentrate and recombinant III type humanized collagen;
The mass ratio of the endometrium mesenchymal stem cell concentrated solution to the recombinant III type humanized collagen is 10:1, a step of;
the endometrium mesenchymal stem cell concentrate is prepared by the following method:
S1, taking 6 th generation human endometrium mesenchymal stem cells, and culturing in a DMEM medium containing 10% FBS;
s2, changing the cell into a second culture medium when the cell grows to 70% of confluence, continuously culturing for 28 hours, and collecting a cell culture solution;
S3, filtering the cell culture solution obtained in the step S2 by using a microporous filter membrane with the thickness of 0.22 mu m, and then passing the filtrate through an ultrafiltration membrane with the thickness of 10KD to obtain endometrium mesenchymal stem cell concentrate;
The culture conditions of the step S1 are as follows: culturing in a cell incubator with a volume fraction of CO 2%, a relative humidity of 95% and a temperature of 37 ℃;
the second medium in the step S2 is DMEM/F12 medium containing 5mg/L Flavin Adenine Dinucleotide (FAD) and 1% FBS.
Example 3
A stem cell factor composition for repairing endometrial damage comprising the following components: endometrium mesenchymal stem cell concentrate and recombinant III type humanized collagen;
The mass ratio of the endometrium mesenchymal stem cell concentrated solution to the recombinant III type humanized collagen is 10:1, a step of;
the endometrium mesenchymal stem cell concentrate is prepared by the following method:
S1, taking 3 rd generation human endometrium mesenchymal stem cells, and culturing in a DMEM medium containing 8% FBS;
s2, changing the cell into a second culture medium when the cell grows to 60% of confluence, continuously culturing for 20 hours, and collecting a cell culture solution;
S3, filtering the cell culture solution obtained in the step S2 by using a microporous filter membrane with the thickness of 0.22 mu m, and then passing the filtrate through an ultrafiltration membrane with the thickness of 10KD to obtain endometrium mesenchymal stem cell concentrate;
The culture conditions of the step S1 are as follows: culturing in a cell incubator with a volume fraction of CO 2%, a relative humidity of 95% and a temperature of 37 ℃;
The second medium in the step S2 is DMEM/F12 medium containing 3mg/L Flavin Adenine Dinucleotide (FAD) and 2% FBS.
Comparative example 1
A stem cell factor composition for repairing endometrial damage comprising the following components: endometrium mesenchymal stem cell concentrate and recombinant III type humanized collagen;
The mass ratio of the endometrium mesenchymal stem cell concentrated solution to the recombinant III type humanized collagen is 10:1, a step of;
the endometrium mesenchymal stem cell concentrate is prepared by the following method:
S1, taking 4 th generation human endometrium mesenchymal stem cells, and culturing in a DMEM medium containing 8% FBS;
S2, changing the cell into a second culture medium when the cell grows to 70% of confluence, continuously culturing for 24 hours, and collecting a cell culture solution;
S3, filtering the cell culture solution obtained in the step S2 by using a microporous filter membrane with the thickness of 0.22 mu m, and then passing the filtrate through an ultrafiltration membrane with the thickness of 10KD to obtain endometrium mesenchymal stem cell concentrate;
The culture conditions of the step S1 are as follows: culturing in a cell incubator with a volume fraction of CO 2%, a relative humidity of 95% and a temperature of 37 ℃;
The second medium in the step S2 is DMEM/F12 medium containing 4mg/L Flavin Adenine Dinucleotide (FAD) and 8% FBS.
Comparative example 2
A stem cell factor composition for repairing endometrial damage comprising the following components: endometrium mesenchymal stem cell concentrate and recombinant III type humanized collagen;
The mass ratio of the endometrium mesenchymal stem cell concentrated solution to the recombinant III type humanized collagen is 10:1, a step of;
the endometrium mesenchymal stem cell concentrate is prepared by the following method:
S1, taking 6 th generation human endometrium mesenchymal stem cells, and culturing in a DMEM medium containing 10% FBS;
S2, changing the cell into a second culture medium when the cell grows to 70% of confluence, continuously culturing for 36 hours, and collecting a cell culture solution;
S3, filtering the cell culture solution obtained in the step S2 by using a microporous filter membrane with the thickness of 0.22 mu m, and then passing the filtrate through an ultrafiltration membrane with the thickness of 10KD to obtain endometrium mesenchymal stem cell concentrate;
The culture conditions of the step S1 are as follows: culturing in a cell incubator with a volume fraction of CO 2%, a relative humidity of 95% and a temperature of 37 ℃;
the second medium in the step S2 is DMEM/F12 medium containing 5mg/L Flavin Adenine Dinucleotide (FAD) and 1% FBS.
Comparative example 3
A stem cell factor composition for repairing endometrial damage comprising the following components: endometrium mesenchymal stem cell concentrate and recombinant III type humanized collagen;
The mass ratio of the endometrium mesenchymal stem cell concentrated solution to the recombinant III type humanized collagen is 10:1, a step of;
the endometrium mesenchymal stem cell concentrate is prepared by the following method:
S1, taking 3 rd generation human endometrium mesenchymal stem cells, and culturing in a DMEM medium containing 8% FBS;
s2, changing the cell into a second culture medium when the cell grows to 60% of confluence, continuously culturing for 20 hours, and collecting a cell culture solution;
S3, filtering the cell culture solution obtained in the step S2 by using a microporous filter membrane with the thickness of 0.22 mu m, and then passing the filtrate through an ultrafiltration membrane with the thickness of 10KD to obtain endometrium mesenchymal stem cell concentrate;
The culture conditions of the step S1 are as follows: culturing in a cell incubator with a volume fraction of CO 2%, a relative humidity of 95% and a temperature of 37 ℃;
the second medium in the step S2 is DMEM/F12 medium containing 2% FBS.
Comparative example 4
A stem cell factor composition comprising the following components: umbilical cord mesenchymal stem cell concentrate and recombinant type III humanized collagen;
the mass ratio of the umbilical cord mesenchymal stem cell concentrate to the recombinant III type humanized collagen is 10:1, a step of;
the umbilical cord mesenchymal stem cell concentrate is prepared by the following method:
s1, taking 4 th generation human umbilical cord mesenchymal stem cells, and culturing in a DMEM medium containing 8% FBS;
S2, changing the cell into a second culture medium when the cell grows to 70% of confluence, continuously culturing for 24 hours, and collecting a cell culture solution;
S3, filtering the cell culture solution obtained in the step S2 by using a microporous filter membrane with the thickness of 0.22 mu m, and then passing the filtrate through an ultrafiltration membrane with the thickness of 10KD to obtain an umbilical cord mesenchymal stem cell concentrate;
The culture conditions of the step S1 are as follows: culturing in a cell incubator with a volume fraction of CO 2%, a relative humidity of 95% and a temperature of 37 ℃;
The second medium in the step S2 is DMEM/F12 medium containing 4mg/L Flavin Adenine Dinucleotide (FAD) and 1% FBS.
Comparative example 5
A stem cell factor composition comprising the following components: bone marrow mesenchymal stem cell concentrate and recombinant III type humanized collagen;
the mass ratio of the bone marrow mesenchymal stem cell concentrated solution to the recombinant III type humanized collagen is 10:1, a step of;
the bone marrow mesenchymal stem cell concentrate is prepared by the following method:
s1, taking 4 th generation human bone marrow mesenchymal stem cells, and culturing in a DMEM medium containing 8% FBS;
S2, changing the cell into a second culture medium when the cell grows to 70% of confluence, continuously culturing for 24 hours, and collecting a cell culture solution;
S3, filtering the cell culture solution obtained in the step S2 by using a microporous filter membrane with the thickness of 0.22 mu m, and then passing the filtrate through an ultrafiltration membrane with the thickness of 10KD to obtain a mesenchymal stem cell concentrate;
The culture conditions of the step S1 are as follows: culturing in a cell incubator with a volume fraction of CO 2%, a relative humidity of 95% and a temperature of 37 ℃;
The second medium in the step S2 is DMEM/F12 medium containing 4mg/L Flavin Adenine Dinucleotide (FAD) and 1% FBS.
Comparative example 6
A stem cell factor composition comprising the following components: adipose-derived stem cell concentrate and recombinant type III humanized collagen;
the mass ratio of the adipose-derived stem cell concentrate to the recombinant III type humanized collagen is 10:1, a step of;
The adipose-derived mesenchymal stem cell concentrate is prepared by the following method:
s1, taking 4 th generation human adipose-derived mesenchymal stem cells, and culturing the human adipose-derived mesenchymal stem cells in a DMEM medium containing 8% FBS;
S2, changing the cell into a second culture medium when the cell grows to 70% of confluence, continuously culturing for 24 hours, and collecting a cell culture solution;
S3, filtering the cell culture solution obtained in the step S2 by using a microporous filter membrane with the thickness of 0.22 mu m, and then passing the filtrate through an ultrafiltration membrane with the thickness of 10KD to obtain a concentrated solution of the adipose-derived mesenchymal stem cells;
The culture conditions of the step S1 are as follows: culturing in a cell incubator with a volume fraction of CO 2%, a relative humidity of 95% and a temperature of 37 ℃;
The second medium in the step S2 is DMEM/F12 medium containing 4mg/L Flavin Adenine Dinucleotide (FAD) and 1% FBS.
Experimental example 1 isolation and culture of human endometrial mesenchymal cells
1. Cutting off endometrium whole layer and myometrium layer with thickness of about 5mm by operation, rapidly placing into D-Hanks solution pre-cooled to 0deg.C;
2. rinsing 3 times with sterile D-Hanks solution containing the green streptomycin to remove surface blood and impurities;
3. the tissue was minced with an ophthalmic scissors into small pieces about 1 mm 3 a in size. Then rapidly placing the tissues into a centrifuge tube filled with digestive enzyme solution, so that the tissues are always completely immersed in the digestive enzyme solution; the digestive enzyme solution consists of 400mg/mL collagenase III and 40mg/mL DNase I;
4. Shaking and digesting for 40min at 37 ℃, and lightly blowing bubbles to the bottom of the centrifuge tube by using a pipette in the middle of the process so as to keep continuous bubbling;
5. Carefully sucking digestive juice, reserving tissue fragments, filtering the digestive juice by a 200-mesh filter screen, and collecting filtrate;
6. centrifuging the filtrate at 800rpm for 5min, and discarding the supernatant;
7. washing the cell pellet with D-Hanks pre-cooled to 0deg.C for 2 times to remove digestive enzymes;
8. the cell pellet was resuspended in DMEM medium containing 10% fbs and then inoculated into a flask and cultured in a cell incubator with 5% CO 2 volume fraction, 95% relative humidity and 37 ℃;
9. The first liquid change is carried out on the 4 th day after inoculation, most of non-adherent cells and residual red blood cells are removed, the morphology and arrangement mode of the cells are observed, and the photographing record is carried out. Half liquid exchange is carried out every 2 days in the period, and when the cell fusion reaches about 80%, 2.5% trypsin is used for digestion and passage;
10. And carrying out subculture purification for 2 generations to obtain the human endometrium mesenchymal stem cells with higher purity, wherein the human endometrium mesenchymal stem cells of 3-6 generations can be used for subsequent experiments.
Experimental example 2 Effect on endometrial stromal cells
Endometrium is mainly divided into functional layer and basal layer. The functional layer of endometrium is periodically changed under the regulation of ovarian sex hormone; the basal layer mainly consists of endometrial stromal cells (endometrial stromal cells), is not affected by sex hormone and does not change periodically. Endometrial damage can lead to an imbalance in endometrial homeostasis, resulting in reduced endometrial stromal cell viability and even death. Thus, endometrial stromal cells play a vital role in repairing endometrial lesions.
Human endometrial stromal cells HESCs are purchased from MEISENCTCC, are cultured in a high-sugar DMEM medium containing 12% FBS and 1% diabody as a complete medium under the conditions that the volume fraction of CO 2 is 5%, the relative humidity is 95% and the temperature is 37 ℃, are fully plated on a culture dish, are subjected to digestion treatment, are inoculated into 96-hole cell culture plates, and are used for subsequent experiments, the cell inoculation density is 5×10 4/mL, the inoculation volume of each hole is 100 mu L, and the culture time is 24 hours;
Blank groups, negative control groups, examples 1-3, comparative examples 1-7, 5 duplicate wells per group were set.
Blank group: removing the old culture medium, and adding the complete culture medium;
Negative control group: the old medium was removed and complete medium containing 70. Mu. Mol/L mifepristone (mifepristone) was added;
Example 1 group: removing the old medium, adding a medium consisting of 10 v/v% of the stem cell factor composition prepared in example 1 and 90 v/v% of the complete medium, and simultaneously adding 70. Mu. Mol/L mifepristone;
Example 2 group: removing the old medium, adding a medium consisting of 10 v/v% of the stem cell factor composition prepared in example 2 and 90 v/v% of the complete medium, and simultaneously adding 70. Mu. Mol/L mifepristone;
Example 3 group: removing the old medium, adding a medium consisting of 10 v/v% of the stem cell factor composition prepared in example 3 and 90 v/v% of the complete medium, and simultaneously adding 70. Mu. Mol/L mifepristone;
Comparative example 1 group: removing the old medium, adding a medium composed of 10 v/v% of the stem cell factor composition prepared in comparative example 1 and 90 v/v% of the complete medium, and simultaneously adding 70. Mu. Mol/L mifepristone;
Comparative example 2 group: removing the old medium, adding a medium composed of 10 v/v% of the stem cell factor composition prepared in comparative example 2 and 90 v/v% of the complete medium, and simultaneously adding 70. Mu. Mol/L mifepristone;
Comparative example 3 group: removing the old medium, adding a medium composed of 10 v/v% of the stem cell factor composition prepared in comparative example 3 and 90 v/v% of the complete medium, and simultaneously adding 70. Mu. Mol/L mifepristone;
Comparative example 4 group: the old medium was removed, and a medium consisting of 15 v/v% of the stem cell factor composition prepared in comparative example 4 and 85 v/v% of the complete medium was added, while 70. Mu. Mol/L mifepristone was added;
Comparative example 5 group: the old medium was removed, and a medium consisting of 15 v/v% of the stem cell factor composition prepared in comparative example 5 and 85 v/v% of the complete medium was added, while 70. Mu. Mol/L mifepristone was added;
comparative example 6 group: the old medium was removed, and a medium consisting of 15 v/v% of the stem cell factor composition prepared in comparative example 6 and 85 v/v% of the complete medium was added, while 70. Mu. Mol/L mifepristone was added.
Concentrated solution group: the old medium was removed and a medium consisting of 10 v/v% of the endometrial mesenchymal stem cell concentrate prepared in example 1 and 90 v/v% of complete medium was added, while 70. Mu. Mol/L mifepristone was added.
After each group is subjected to the culture treatment for 24 hours, the cell viability of each group is detected by adopting an MTT method, specifically, the absorbance (A) value of each hole is detected at the wavelength of 492 nm by adopting an ELISA detector, and the cell viability of each group is calculated, and the experimental result is shown in figure 1.
Mifepristone treatment can induce apoptosis of human endometrial stromal cells HESCs, and the stem cell factor composition prepared in examples 1-3 can remarkably reduce the influence of mifepristone and reduce the occurrence of apoptosis. The endometrium mesenchymal stem cell concentrate and the recombinant III type humanized collagen have synergistic effect and better curative effect. Under the same preparation conditions, compared with umbilical cord mesenchymal stem cell concentrate, bone marrow mesenchymal stem cell concentrate and adipose mesenchymal stem cell concentrate, the endometrium mesenchymal stem cell concentrate can more effectively reduce apoptosis of human endometrium stromal cells, and has better effect of treating endometrium injury.
Experimental example 3 in vivo effects of treating endometrial injury in rats
Selecting a female SD rat with the age of 6-8 weeks and the weight of 180-220 g, and performing intraperitoneal injection of pentobarbital sodium for anesthesia; the skin is cut at the position of about 1cm above the urethra of the rat and enters the abdominal cavity, the uterus is exposed, the endometrium with the length of about 1.0cm and the width of about 0.5cm is scraped off by a scraping blade on the right uterine wall, the pathological endometrium injury caused by the clinical human endometrium scraping operation is simulated, and the operation incision is sutured after operation. After the rats are anesthetized and revived, the rats are placed in an animal house for feeding, the feeding environment is a clean cage and single cage for feeding, the pads are replaced and kept dry, and free drinking and feeding are realized and nutrition is enhanced. The daily routine injection of 200,000U/kg penicillin sodium into thigh muscle can prevent wound infection within three days after molding.
Applying the rat endometrium injury model established above, injecting the stem cell factor composition prepared in examples 1-3 and comparative examples 1-6 or the concentrated solution prepared in example 1 onto injured endometrium in situ at a dose of 2.0ml/kg, injecting the same amount of physiological saline into the sham operation group and the modeling group in situ once every 2 days, setting 3-5 SD rats in each group, and comparing and analyzing the repair effect of each group on endometrium injury.
On day 33 after molding, rats were perfused with normal saline, then replaced with 4% paraformaldehyde for rapid perfusion fixation of tissues, uterine tissues at the uterine injury site and at 0.25cm above and below were removed, fixed in 4% paraformaldehyde for 24h, paraffin was used to embed the tissues, and tissue sections were performed at 5 μm thickness. The experimental results are shown in FIG. 2.
Clinical treatment effects can be simulated by applying a rat endometrium injury model. The experimental results in fig. 2 show that the stem cell factor composition prepared in embodiments 1-3 of the invention can effectively relieve pathological endometrial damage, and has better repairing effect than the concentrated solution of mesenchymal stem cells derived from umbilical cord, bone marrow or fat.
The foregoing has outlined the basic principles, features, and advantages of the present invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, and that the above embodiments and descriptions are merely illustrative of the principles of the present invention, and various changes and modifications may be made without departing from the spirit and scope of the invention, which is defined in the appended claims. The scope of the invention is defined by the appended claims and equivalents thereof.
Claims (8)
1. A stem cell factor composition for repairing endometrial damage, comprising the following components: endometrium mesenchymal stem cell concentrate and recombinant III type humanized collagen;
the endometrium mesenchymal stem cell concentrate is prepared by the following method:
S1, taking 3 rd to 6 th generation human endometrium mesenchymal stem cells, and culturing in a DMEM culture medium containing 8% -10% FBS by volume fraction;
S2, changing the cell into a second culture medium when the cell grows to have the confluence of 60-70%, continuously culturing for 20-28 hours, and collecting a cell culture solution;
S3, filtering the cell culture solution obtained in the step S2 by using a microporous filter membrane with the thickness of 0.22 mu m, and then passing the filtrate through an ultrafiltration membrane with the thickness of 10KD to obtain endometrium mesenchymal stem cell concentrate;
The second culture medium in the step S2 is a DMEM/F12 culture medium containing 2-6 mg/L flavin adenine dinucleotide and 1-3% FBS in volume fraction;
The mass ratio of the endometrium mesenchymal stem cell concentrated solution to the recombinant III type humanized collagen is (8-10): 1.
2. The stem cell factor sustained release agent for repairing endometrium injury is characterized by being prepared from the following components: the stem cell factor composition of claim 1, hyaluronic acid, a slow release material and a binder.
3. The stem cell factor sustained release agent for repairing endometrium injury as defined in claim 2, wherein: the composite material is prepared from the following components in percentage by mass: 40-50% of endometrium mesenchymal stem cell concentrate, 5% of recombinant III-type humanized collagen, 5-15% of hyaluronic acid, 20-35% of slow release material and 5-15% of adhesive.
4. A stem cell factor sustained release agent for repairing endometrium injury as defined in claim 3, wherein: the slow release material can be any one or a mixture of more than two of ethyl cellulose, hydroxyethyl cellulose, hydroxypropyl methyl cellulose and hydroxypropyl cellulose.
5. A stem cell factor sustained release agent for repairing endometrium injury as defined in claim 3, wherein: the binder may be selected from microcrystalline cellulose and povidone.
6. A stem cell factor sustained release agent for repairing endometrium injury as defined in claim 3, wherein: the molecular weight of the hyaluronic acid is 800-1000 kD.
7. A preparation method of a stem cell factor sustained release agent for repairing endometrium injury, which is characterized in that the stem cell factor composition as defined in claim 1 and hyaluronic acid are taken, stirred and mixed uniformly, and then sustained release material and adhesive are added and mixed uniformly to obtain the composition;
The endometrium mesenchymal stem cell concentrate in the stem cell factor composition is prepared by the following method:
S1, taking 3 rd to 6 th generation human endometrium mesenchymal stem cells, and culturing in a DMEM culture medium containing 8% -10% FBS by volume fraction;
S2, changing the cell into a second culture medium when the cell grows to have the confluence of 60-70%, continuously culturing for 20-28 hours, and collecting a cell culture solution;
S3, filtering the cell culture solution obtained in the step S2 by using a microporous filter membrane with the thickness of 0.22 mu m, and then passing the filtrate through an ultrafiltration membrane with the thickness of 10KD to obtain endometrium mesenchymal stem cell concentrate;
The second culture medium in the step S2 is a DMEM/F12 culture medium containing 2-6 mg/L flavin adenine dinucleotide and 1-3% FBS in volume fraction;
The molecular weight of the hyaluronic acid is 800-1000 kD.
8. The method for preparing the stem cell factor sustained release agent for repairing endometrium injury as set forth in claim 7, wherein: and taking 40-50% of endometrium mesenchymal stem cell concentrate, 5% of recombinant III-type humanized collagen and 5-15% of hyaluronic acid according to the mass percentage, stirring and mixing uniformly, adding 20-35% of slow release material and 5-15% of adhesive, and mixing uniformly to obtain the endometrium mesenchymal stem cell.
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