阅读说明：本技术 一种含有生殖干细胞的修复组合物及其制备方法 (Restoration composition containing germ stem cells and preparation method thereof ) 是由 陈则姜 陈忠平 于 2021-01-30 设计创作，主要内容包括：本发明公开了一种含有生殖干细胞的修复组合物及其制备方法,包括子宫内膜干细胞、β-雌二醇、淫羊藿苷、生理盐水；所述人子宫内膜干细胞的含量为2×10～5～1×10～6个/mL；所述β-雌二醇的含量为0.3～0.8mg/ml；所述淫羊藿苷的含量为0.3～0.8mg/ml。所述的含有生殖干细胞的修复组合物具有良好的抑制卵巢早衰作用子宫内膜干细胞与β-雌二醇联用,二者具有良好的协同增效作用,进一步加入淫羊藿苷起到辅助修复卵巢早衰作用,从而三者具有良好的协同增效作用。且所制备得到的子宫内膜干细胞具有良好的活力,细胞活性高、自我更新和分化强。(The invention discloses a restoration composition containing reproductive stem cells and a preparation method thereof, wherein the restoration composition comprises endometrial stem cells, beta-estradiol, icariin and normal saline; the content of the human endometrial stem cells is 2 multiplied by 10 5 ~1×10 6 Per mL; the content of the beta-estradiol is 0.3-0.8 mg/ml; the content of the icariin is 0.3-0.8 mg/ml. The reproductive stem cell-containing repair composition has good ovarian premature senility inhibition effect, combines the endometrial stem cells and the beta-estradiol, has good synergistic effect, and is further added with the icariin to play a role in assisting in repairing the ovarian premature senility, so that the reproductive stem cell-containing repair composition, the endometrial stem cells and the beta-estradiol have good synergistic effect. And are preparedThe obtained endometrial stem cells have good activity, high cell activity, and strong self-renewal and differentiation.)

1. A repairing composition containing germ stem cells is characterized by comprising endometrial stem cells, beta-estradiol, icariin and normal saline;

the content of the human endometrial stem cells is 2 multiplied by 10⁵~1×10⁶Per mL;

the content of the beta-estradiol is 0.3-0.8 mg/ml;

the content of the icariin is 0.3-0.8 mg/ml.

2. The reproductive stem cell-containing repair composition according to claim 1, wherein the content of the endometrial stem cells is 4 x 10⁵one/mL.

3. The reproductive stem cell-containing repair composition according to claim 1 wherein the beta-estradiol is present in an amount of 0.6 mg/ml.

4. The reproductive stem cell-containing repair composition according to claim 1, wherein the icariin is contained in an amount of 0.5 mg/ml.

5. The reproductive stem cell-containing repair composition according to claim 1, wherein the endometrial stem cells are prepared by:

s1, collecting menstrual blood, adding 20-50 parts by weight of PBS buffer solution into 10-20 parts by weight of menstrual blood, adding 10-20 parts by weight of separation solution, centrifuging for the first time, taking the middle layer, adding 0.2-0.6 part by weight of I-type collagenase, mixing uniformly, centrifuging for the second time, and discarding the supernatant to obtain cell sediment;

s2, preparing the cell sediment into 2 x 10 by using normal saline⁴~1× 10⁵The concentration of each/ml, inoculating in a primary culture medium, and culturing at 33-37 deg.C with 4.8-5.2% CO by volume fraction₂Culturing for 10-24 h in the incubator to obtain P0 generation cells;

s3, preparing the P0 generation cells into 2X 10 cells by using normal saline⁴~1× 10⁵Inoculating the strain/ml of the strain in a subculture medium, and performing volume fraction of 4.8-5.2% CO at 33-37 ℃₂Culturing in the incubator until the cell fusion degree is more than 85%, collecting cells, and repeating until P5 generation; thus obtaining the endometrial stem cells.

6. A restoration composition comprising germ stem cells according to claim 5 wherein the separation fluid is a lymphocyte separation fluid.

7. The reproductive stem cell-containing repair composition according to claim 5, wherein the primary culture medium comprises a MEM (minimum essential medium) culture medium and additives, and the additives comprise 5-10 mM/ml D-glucose, 0.5-1 mg/ml sodium beta-glycerophosphate, 5-12 mg/ml trehalose, 0.8-1.5 mg/ml vitamin C, 2-5 ng/ml dexamethasone and 3-6 ng/ml dry cell growth factor.

8. The reproductive stem cell-containing repair composition according to claim 5, wherein the communication medium comprises a base medium and additives, the base medium is an MEM (MEM) medium, and the additives comprise 5-10 mM/ml D-glucose, 0.5-1 mg/ml sodium beta-glycerophosphate, 0.8-1.5 mg/ml 4-hydroxyethylpiperazineethanesulfonic acid, 5-12 mg/ml trehalose, 0.8-1.5 mg/ml vitamin C, 2-5 ng/ml dexamethasone, and 3-6 ng/ml dry cell growth factor.

9. The reproductive stem cell-containing repair composition of claim 5 wherein the communication medium comprises a base medium which is MEM medium and supplements comprising 9mM/ml D-glucose, 0.8mg/ml sodium β -glycerophosphate, 1.2mg/ml 4-hydroxyethylpiperazineethanesulfonic acid, 8mg/ml trehalose, 1mg/ml vitamin C, 4ng/ml dexamethasone, 5ng/ml stem cell growth factor.

10. The method for preparing a reproductive stem cell-containing repairing composition according to any one of claims 1 to 9, wherein the reproductive stem cell-containing repairing composition is obtained by uniformly mixing beta-estradiol, icariin and physiological saline, and resuspending endometrial stem cells.

Technical Field

The invention relates to the technical field of stem cells, in particular to a restoration composition containing germ stem cells and a preparation method thereof.

Background

With the young tumor patients and the improvement of survival rate, iatrogenic damage caused by chemotherapeutic drugs is gradually paid attention. The ovary is the most sensitive part in the reproductive system to chemotherapy, and the ovarian tissue can be damaged by large-dose and long-time chemotherapy, so that the premature ovarian failure is caused. Premature ovarian failure is a common gynecological endocrine disease and is characterized in that after puberty of women, persistent amenorrhea occurs for more than 4 months before the age of 40, the serum estrogen level is reduced, and the follicle-stimulating hormone level is increased. POF causes amenorrhea, infertility and sexual dysfunction of women, and simultaneously greatly increases the risks of osteoporosis, hyperlipidemia, cardiovascular diseases and the like, and seriously harms the physical and mental health and family harmfulness of women.

Disclosure of Invention

The invention provides a restoration composition containing germ stem cells and a preparation method thereof, and the restoration composition has good effect of inhibiting premature ovarian failure.

The invention adopts the following technical scheme for solving the technical problems:

a repairing composition containing reproductive stem cell comprises endometrial stem cell, beta-estradiol, icariin, and normal saline;

the content of the human endometrial stem cells is 2 multiplied by 10⁵~1×10⁶Per mL;

the content of the beta-estradiol is 0.3-0.8 mg/ml;

the content of the icariin is 0.3-0.8 mg/ml.

The inventor of the invention finds that the endometrial stem cells have a repairing effect on ovarian tissues and can improve the endocrine function of ovaries in a large number of researches.

The beta-estradiol can promote normal proliferation of endometrium, improve ovary endocrine, and facilitate pregnancy.

Icariin can be used as an immunopotentiator or a regulator, has the effects of resisting virus, stress, oxidation and the like, improves the immunity of a human body, promotes the recovery of the organism, and has good compatibility.

Meanwhile, the inventor finds that the endometrial stem cells and the beta-estradiol have good synergistic effect when being combined, and the icariin is further added to play a role in assisting in repairing premature ovarian failure, so that the endometrial stem cells, the beta-estradiol and the icariin have good synergistic effect.

Preferably, the content of the endometrial stem cells is 4 x 10⁵one/mL.

Preferably, the beta-estradiol is present in an amount of 0.6 mg/ml.

As a preferable scheme, the content of the icariin is 0.5 mg/ml.

As a preferred embodiment, the preparation method of the endometrial stem cells comprises the following steps:

s1, collecting menstrual blood, adding 20-50 parts by weight of PBS buffer solution into 10-20 parts by weight of menstrual blood, adding 10-20 parts by weight of separation solution, centrifuging for the first time, taking the middle layer, adding 0.2-0.6 part by weight of I-type collagenase, mixing uniformly, centrifuging for the second time, and discarding the supernatant to obtain cell sediment;

s2, preparing the cell sediment into 2 x 10 by using normal saline⁴~1× 10⁵The concentration of each/ml, inoculating in a primary culture medium, and culturing at 33-37 deg.C with 4.8-5.2% CO by volume fraction₂Culturing for 10-24 h in the incubator to obtain P0 generation cells;

s3, preparing the P0 generation cells into 2X 10 cells by using normal saline⁴~1× 10⁵Inoculating the strain/ml of the strain in a subculture medium, and performing volume fraction of 4.8-5.2% CO at 33-37 ℃₂Culturing in the incubator until the cell fusion degree is more than 85%, collecting cells, and repeating until P5 generation; thus obtaining the endometrial stem cells.

The prepared endometrial stem cells have good activity, high cell activity and strong self-renewal and differentiation.

Preferably, the separation liquid is a lymphocyte separation liquid.

As a preferable scheme, the primary culture medium comprises a basic culture medium and additives, wherein the basic culture medium is an MEM culture medium, and the additives comprise 5-10 mM/ml D-glucose, 0.5-1 mg/ml beta-sodium glycerophosphate, 5-12 mg/ml trehalose, 0.8-1.5 mg/ml vitamin C, 2-5 ng/ml dexamethasone and 3-6 ng/ml dry cell growth factors.

As a preferable scheme, the communication medium comprises a basic medium and additives, wherein the basic medium is an MEM medium, and the additives comprise 5-10 mM/ml of D-glucose, 0.5-1 mg/ml of beta-sodium glycerophosphate, 0.8-1.5 mg/ml of 4-hydroxyethyl piperazine ethanesulfonic acid, 5-12 mg/ml of trehalose, 0.8-1.5 mg/ml of vitamin C, 2-5 ng/ml of dexamethasone and 3-6 ng/ml of dry cell growth factors.

As a preferred scheme, the communication medium comprises a basic medium and additives, wherein the basic medium is an MEM medium, and the additives comprise 9mM/ml D-glucose, 0.8mg/ml beta-sodium glycerophosphate, 1.2mg/ml 4-hydroxyethylpiperazine ethanesulfonic acid, 8mg/ml trehalose, 1mg/ml vitamin C, 4ng/ml dexamethasone and 5ng/ml dry cell growth factor.

The invention also provides a preparation method of the restoration composition containing the reproductive stem cells, which comprises the steps of uniformly mixing the beta-estradiol, the icariin and the physiological saline, and re-suspending the endometrial stem cells to obtain the restoration composition containing the reproductive stem cells.

The invention has the beneficial effects that: the reproductive stem cell-containing repair composition has good ovarian premature senility inhibition effect, combines the endometrial stem cells and the beta-estradiol, has good synergistic effect, and is further added with the icariin to play a role in assisting in repairing the ovarian premature senility, so that the reproductive stem cell-containing repair composition, the endometrial stem cells and the beta-estradiol have good synergistic effect.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are a part of the embodiments of the present invention, but not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example 1

A repairing composition containing reproductive stem cell comprises endometrial stem cell, beta-estradiol, icariin, and normal saline;

the content of the human endometrial stem cells is 4 multiplied by 10⁵Per mL;

the content of the beta-estradiol is 0.6 mg/ml;

the content of icariin is 0.5 mg/ml.

The preparation method of the endometrial stem cells comprises the following steps:

the source of menstrual blood is collected during the menstrual cycle in normal healthy women.

S1, collecting menstrual blood, adding 38 parts by weight of PBS buffer solution into 12 parts by weight of menstrual blood, adding 12 parts by weight of lymphocyte separation solution, centrifuging for the first time, taking the middle layer, adding 0.5 part by weight of I-type collagenase, mixing uniformly, centrifuging for the second time, and discarding the supernatant to obtain cell precipitate;

s2, preparing the cell sediment into 5X 10 by using normal saline⁴At a concentration of one/ml, inoculated in a primary culture medium and with a volume fraction of 5% CO at 35 ℃₂Culturing for 18h in the incubator to obtain P0 generation cells;

s3, preparing the P0 generation cells into 5X 10 cells by using normal saline⁴At a concentration of 5% CO/ml, inoculated into subculture medium at 35 deg.C₂Culturing in the incubator until the cell fusion degree is more than 85%, collecting cells, and repeating until P5 generation; thus obtaining the endometrial stem cells.

The primary culture medium comprises a basic culture medium and additives, wherein the basic culture medium is an MEM culture medium, and the additives comprise 7mM/ml D-glucose, 0.9mg/ml beta-sodium glycerophosphate, 10mg/ml trehalose, 0.9mg/ml vitamin C, 3ng/ml dexamethasone and 4ng/ml stem cell growth factors.

The transmission medium comprises a basic medium and additives, wherein the basic medium is an MEM (minimum essential medium), and the additives comprise 9mM/ml D-glucose, 0.8mg/ml beta-sodium glycerophosphate, 1.2mg/ml 4-hydroxyethyl piperazine ethanesulfonic acid, 8mg/ml trehalose, 1mg/ml vitamin C, 4ng/ml dexamethasone and 5ng/ml dry cell growth factors.

The preparation method of the restoration composition containing the germ stem cells comprises the steps of uniformly mixing beta-estradiol, icariin and physiological saline, and re-suspending the endometrial stem cells to obtain the restoration composition containing the germ stem cells.

Example 2

A repairing composition containing reproductive stem cell comprises endometrial stem cell, beta-estradiol, icariin, and normal saline;

the human endometrium is dry and fineThe content of cells is 2 × 10⁵Per mL;

the content of the beta-estradiol is 0.3 mg/ml;

the content of icariin is 0.3 mg/ml.

The preparation method of the endometrial stem cells comprises the following steps:

the source of menstrual blood is collected during the menstrual cycle in normal healthy women.

S1, collecting menstrual blood, adding 38 parts by weight of PBS buffer solution into 12 parts by weight of menstrual blood, adding 12 parts by weight of lymphocyte separation solution, centrifuging for the first time, taking the middle layer, adding 0.5 part by weight of I-type collagenase, mixing uniformly, centrifuging for the second time, and discarding the supernatant to obtain cell precipitate;

s2, preparing the cell sediment into 5X 10 by using normal saline⁴At a concentration of one/ml, inoculated in a primary culture medium and with a volume fraction of 5% CO at 35 ℃₂Culturing for 18h in the incubator to obtain P0 generation cells;

s3, preparing the P0 generation cells into 5X 10 cells by using normal saline⁴At a concentration of 5% CO/ml, inoculated into subculture medium at 35 deg.C₂Culturing in the incubator until the cell fusion degree is more than 85%, collecting cells, and repeating until P5 generation; thus obtaining the endometrial stem cells.

The primary culture medium comprises a basic culture medium and additives, wherein the basic culture medium is an MEM culture medium, and the additives comprise 7mM/ml D-glucose, 0.9mg/ml beta-sodium glycerophosphate, 10mg/ml trehalose, 0.9mg/ml vitamin C, 3ng/ml dexamethasone and 4ng/ml stem cell growth factors.

The transmission medium comprises a basic medium and additives, wherein the basic medium is an MEM (minimum essential medium), and the additives comprise 9mM/ml D-glucose, 0.8mg/ml beta-sodium glycerophosphate, 1.2mg/ml 4-hydroxyethyl piperazine ethanesulfonic acid, 8mg/ml trehalose, 1mg/ml vitamin C, 4ng/ml dexamethasone and 5ng/ml dry cell growth factors.

The preparation method of the restoration composition containing the germ stem cells comprises the steps of uniformly mixing beta-estradiol, icariin and physiological saline, and re-suspending the endometrial stem cells to obtain the restoration composition containing the germ stem cells.

Example 3

A repairing composition containing reproductive stem cell comprises endometrial stem cell, beta-estradiol, icariin, and normal saline;

the content of the human endometrial stem cells is 4 multiplied by 10⁵Per mL;

the content of the beta-estradiol is 0.6 mg/ml;

the content of icariin is 0.5 mg/ml.

The preparation method of the endometrial stem cells comprises the following steps:

the source of menstrual blood is collected during the menstrual cycle in normal healthy women.

S1, collecting menstrual blood, adding 38 parts by weight of PBS buffer solution into 12 parts by weight of menstrual blood, adding 12 parts by weight of lymphocyte separation solution, centrifuging for the first time, taking the middle layer, adding 0.5 part by weight of I-type collagenase, mixing uniformly, centrifuging for the second time, and discarding the supernatant to obtain cell precipitate;

s2, preparing the cell sediment into 5X 10 by using normal saline⁴At a concentration of one/ml, inoculated in a primary culture medium and with a volume fraction of 5% CO at 35 ℃₂Culturing for 18h in the incubator to obtain P0 generation cells;

s3, preparing the P0 generation cells into 5X 10 cells by using normal saline⁴At a concentration of 5% CO/ml, inoculated into subculture medium at 35 deg.C₂Culturing in the incubator until the cell fusion degree is more than 85%, collecting cells, and repeating until P5 generation; thus obtaining the endometrial stem cells.

The primary culture medium comprises a basic culture medium and additives, wherein the basic culture medium is an MEM culture medium, and the additives comprise 5mM/ml D-glucose, 0.5mg/ml beta-sodium glycerophosphate, 5mg/ml trehalose, 0.8mg/ml vitamin C, 2ng/ml dexamethasone and 3ng/ml stem cell growth factors.

The transmission medium comprises a basic medium and additives, wherein the basic medium is an MEM (minimum essential medium), and the additives comprise 5mM/ml D-glucose, 0.5mg/ml beta-sodium glycerophosphate, 0.8mg/ml 4-hydroxyethyl piperazine ethanesulfonic acid, 5mg/ml trehalose, 0.8mg/ml vitamin C, 2ng/ml dexamethasone and 3ng/ml dry cell growth factors.

The preparation method of the restoration composition containing the germ stem cells comprises the steps of uniformly mixing beta-estradiol, icariin and physiological saline, and re-suspending the endometrial stem cells to obtain the restoration composition containing the germ stem cells.

Comparative example 1

Comparative example 1 is different from example 1 in that comparative example 1 does not contain endometrial stem cells, and the rest is the same.

Comparative example 2

Comparative example 2 differs from example 1 in that comparative example 2 does not contain beta-estradiol, and the other is the same.

Comparative example 3

Comparative example 3 differs from example 1 in that comparative example 3 does not contain icariin, and the other is the same.

Comparative example 4

Comparative example 4 differs from example 1 in that the additions to the primary and subculture media described in comparative example 4 differ from example 1, and are otherwise identical.

The primary culture medium comprises a basic culture medium and additives, wherein the basic culture medium is an MEM culture medium, and the additives comprise 7mM/ml D-glucose, 10mg/ml trehalose, 0.9mg/ml vitamin C and 4ng/ml stem cell growth factors.

The communication medium comprises a basic medium and additives, wherein the basic medium is an MEM (minimum essential medium), and the additives comprise 9mM/ml D-glucose, 1.2mg/ml 4-hydroxyethyl piperazine ethanesulfonic acid, 8mg/ml trehalose, 1mg/ml vitamin C and 5ng/ml stem cell growth factor.

To further demonstrate the effect of the present invention, the following test methods were provided:

1. 80 wistar female rats with 2-3 months of age, 200 +/-20 g of body weight and normal estrus cycle are selected and are randomly divided into 8 groups after being adaptively raised for 1 week.

2. Constructing a rat premature ovarian failure model: one-time intraperitoneal injection of 0.1 mL each of cyclophosphamide solution (120mg/kg) and busulfan solution (12mg/kg) is carried out, wherein the dose is 8: 00 lightly taking a vaginal secretion smear by using a cotton swab, carrying out pasteurization, observing the change of vaginal cast-off cells, observing continuous estrus intervals when continuously observing 10d, judging that the premature ovarian failure is successfully modeled, after the modeling is successfully carried out, injecting 60mg/kg of phenobarbital sodium into the abdominal cavity of each experimental rat, fixing the back of the rat upwards on an animal operating table after 15min, disinfecting the skin, holding an ophthalmic forceps to pinch the lower abdominal midline skin tightly, using a surgical scissors to make a small transverse incision, carrying out blunt separation by using the scissors to fully expose the operation visual field, incising the fascia, and using the forceps to pull out the fat pad and the ovary. Then, the ovary is fixed by using forceps, the repairing composition described in examples 1-3 and comparative examples 1-4 is injected into eight groups of rats respectively by using a micro-injector, the other group is a control group, the same amount of normal saline is injected, the injection dose is 20 mu L/g rat body weight, and the index is measured after one week of injection.

3. The expression level of experimental rat ovary Bax and Bcl-2 proteins is detected by adopting an immunohistochemical staining method, the test result is shown in table 1, and the specific process is as follows: taking a continuous section of ovary tissue with the thickness of 6 mu m, performing immunohistochemical staining by adopting an SABC method, and drying the section of the ovary tissue at normal temperature; 20mL of 30% H₂O₂Mixing with 200mL of distilled water, adding into the ovary tissue slice, inactivating endogenous peroxidase at room temperature for 10min, and washing with distilled water for 3 times, each time for 5 min; performing heat repairing on the antigen by using a microwave oven, sealing the antigen for 30min at room temperature by using 5% BSA (bovine serum albumin) sealing solution, and respectively dropwise adding a mixed solution (volume ratio is 1: 200) of Bcl-2 and Bax primary antibody at 4 ℃ overnight; dripping biotinylated goat anti-rabbit IgG at room temperature for 10min, dripping SABC for 30min, DAB developing for 3-10 min, hematoxylin counterstaining, and returning tap water to blue for 15 min; conventional gradient alcohol dehydration, xylene clarity, neutral gum mounting.

TABLE 1 test results

As can be seen from table 1, the reproductive stem cell-containing repair composition of the present invention is effective in inhibiting premature ovarian failure.

Comparing example 1 with example 2, it can be seen that the repair composition prepared in example 1 has better effect of inhibiting premature ovarian failure due to the addition of the endometrial stem cells, the beta-estradiol and the icariin.

Comparing example 1 with example 3, it can be seen that the repairing composition prepared by adding the culture medium in example 3 has better effect of inhibiting premature ovarian failure.

As can be seen from the comparison of example 1 and comparative examples 1 to 3, the endometrial stem cells, the beta-estradiol and the icariin have a synergistic effect in treating premature ovarian failure.

Comparing example 1 with comparative example 4, it can be seen that the addition of beta-sodium glycerophosphate and dexamethasone in the culture medium can improve the activity of endometrial stem cells, thereby improving the effect of inhibiting premature ovarian failure.

In light of the foregoing description of preferred embodiments according to the invention, it is clear that many changes and modifications can be made by the person skilled in the art without departing from the scope of the invention. The technical scope of the present invention is not limited to the contents of the specification, and must be determined according to the scope of the claims.