阅读说明：本技术 一种用于人月经血的保存液及其制备方法 (Preservation solution for human menstrual blood and preparation method thereof ) 是由 李文东 宋云庆 黎波 卢瑞珊 于 2019-09-26 设计创作，主要内容包括：本发明属于干细胞保存的生物技术领域,提供了一种用于人月经血的保存液及其制备方法。保存液的成分包括生理盐水、水蛭素、葡萄糖、羟乙基淀粉、左氧氟沙星、复合维生素、谷胱甘肽、坏死性凋亡抑制剂Necrostatin-1/RIP1激酶抑制剂,其制备方法如下：1)制备A液：往生理盐水中分别加入葡萄糖、水蛭素以及左氧氟沙星；2)制备B液：往生理盐水中分别加入羟乙基淀粉、复合维生素以及谷胱甘肽；3)制备C液：将A液、B液混合之后加入坏死性凋亡抑制剂Necrostatin-1/RIP1激酶抑制剂,用滤膜过滤后于4℃下保存备用,本发明的保存液能在48小时内维持宫内膜干细胞原有的生物学特性,细胞质量稳定,纯度高。(The invention belongs to the technical field of stem cell preservation, and provides a preservation solution for human menstrual blood and a preparation method thereof. The components of the preservation solution comprise normal saline, hirudin, glucose, hydroxyethyl starch, levofloxacin, multivitamin, glutathione and necrotizing apoptosis inhibitor Necrostatin-1/RIP1 kinase inhibitor, and the preparation method comprises the following steps: 1) preparing a solution A: adding glucose, hirudin and levofloxacin into physiological saline respectively; 2) preparing a solution B: respectively adding hydroxyethyl starch, vitamin complex and glutathione into physiological saline; 3) preparing a solution C: the liquid A and the liquid B are mixed, and then added with necrotizing apoptosis inhibitor Necrostatin-1/RIP1 kinase inhibitor, and the mixture is filtered by a filter membrane and stored at 4 ℃ for later use.)

1. A preserving fluid for human menstrual blood is characterized in that the ingredients of the preserving fluid comprise normal saline, hirudin, glucose, hydroxyethyl starch, levofloxacin, vitamin complex, glutathione and necrotizing apoptosis inhibitor Necrostatin-1/RIP1 kinase inhibitor.

2. The preservation solution according to claim 1, wherein the concentration of hirudin is (1 ~ 5). times.hirudin.

3. The preservation solution according to claim 1, wherein the concentration of hydroxyethyl starch is 1 ~ 5%.

4. The preservation solution according to claim 1, wherein the concentration of levofloxacin is (1 ~ 2) x levofloxacin.

5. The preservation solution according to claim 1, wherein the necroptosis inhibitor Necrostatin-1/RIP1 kinase is inhibited at a concentration of 5 ~ 10 ng/mL.

6. The preservation solution according to claim 1, wherein the concentration of the multivitamin is 1 ~ 5 mg/mL.

7. The preservation solution according to claim 1, wherein the concentration of glutathione is 1 ~ 5 mg/mL.

8. A preparation method of human menstrual blood preservative fluid is characterized by comprising the following steps:

1) preparing a solution A: adding glucose, hirudin and levofloxacin into physiological saline to obtain solution A;

2) preparing a solution B: adding hydroxyethyl starch, vitamin complex and glutathione into normal saline to obtain solution B;

3) preparing a solution C: mixing solution A and solution B, adding necroptosis inhibitor Necrostatin-1/RIP1 kinase inhibitor to obtain solution C, filtering with filter membrane, and storing at 4 deg.C.

9. The method for producing a preservative solution according to claim 8, wherein the volume ratio of the solution A to the solution B in the step 3) is 1: 1.

Technical Field

The invention belongs to the technical field of stem cell preservation, and particularly relates to human menstrual blood preservative fluid and a preparation method thereof.

Background

Menstruation is regulated by the interaction of reproductive hormones of the hypothalamus, pituitary gland and ovary, and in the menstrual period and the proliferation period of the menstrual cycle, the level of estradiol and progesterone in blood is very low, so that the negative feedback effect on the adenohypophysis and the hypothalamus is weakened or eliminated, the secretion of gonadotropin-releasing hormone by the hypothalamus is increased, and then follicle stimulating hormone and luteinizing hormone secreted by the adenohypophysis are increased, so that the follicle develops and the estrogen secretion is gradually increased. At this point, estrogen in turn stimulates the endometrium into the proliferative phase. Luteinizing hormone increases the secretion of progestogen, resulting in ovulation. During this period both estrogen and progestin levels are elevated. This produces an enhanced negative feedback inhibition of the hypothalamus and adenohypophysis, which in turn reduces the levels of ovulation stimulating hormone and luteinizing hormone, resulting in a deterioration of the corpus luteum and thus in a reduction of the estrogen and progestin levels. The endometrium is deprived of the support of these two hormones and is exfoliated and bleeded, i.e. menstruation occurs. At this point, the estrogen and progestin decrease begins the next menstrual cycle again.

The components of menses are mainly blood (3/4 arterial blood, 1/4 venous blood), fragments of endometrial tissue and various active enzymes and biological factors, wherein the fibrinolytic enzyme makes the menstrual blood liquid and non-coagulated, and the prostaglandin acts to contract the uterus. Menstrual blood rapidly coagulates in a short time under the condition of in vitro; the viability decreases, which directly affects the cell quality of endometrial stem cells in menstrual blood. The viability of endometrial stem cells is directly related to menstrual blood preservation; coagulation after the menstrual blood is isolated, influence of hormones in the menstrual blood and the like on the cell quality; how to avoid the above becomes the primary solution. In addition, these problems directly restrict success of in vitro culture of endometrial stem cells derived from menstrual blood; therefore, a menstrual blood preserving fluid needs to be developed, which can meet the anticoagulation and bacteriostasis effects on one hand, and can also meet the requirement that the endometrial stem cells with sufficient quantity and good quality can be extracted after the menstrual blood is preserved in vitro for 48 hours on the other hand.

Disclosure of Invention

In view of the above problems, an object of the present invention is to provide a preservation solution for human menstrual blood and a preparation method thereof, which can maintain the activity of endometrial stem cells from menstrual blood without causing functional variation of the cells, and in the prior art, albumin and the like are often used as a nutrition source for maintaining the activity of placental mesenchymal stem cells.

The technical content of the invention is as follows:

the invention provides a preserving fluid for human menstrual blood, which comprises the components of normal saline, hirudin, glucose, hydroxyethyl starch, levofloxacin, vitamin complex, glutathione and a necrotizing apoptosis inhibitor Necrostatin-1/RIP1 kinase inhibitor.

Furthermore, the concentration of the hirudin is (1-5) x hirudin, the hirudin is one of the most significant and most studied components extracted from leeches and salivary glands thereof, and is a small molecular protein (polypeptide) consisting of 65-66 amino acids, and the hirudin has extremely strong inhibition effect on thrombin and is the strongest natural specific thrombin inhibitor discovered so far;

the buffer solution based on the physiological saline is purchased from a reagent company, so that the stability of a storage solution buffer system can be ensured, and the quality difference caused by artificial configuration is avoided;

the glucose is used as a basic nutrient component of the endometrial stem cells, can be directly absorbed and utilized by the cells, improves the resistance of the cells, and maintains the activity of the cells;

the concentration of the hydroxyethyl starch is 1-5%, and the hydroxyethyl starch can maintain the osmotic pressure of a preservation solution, so that the endometrial stem cells are preserved in an isotonic and isopressure environment for the activity of the placental mesenchymal stem cells;

the concentration of the levofloxacin is (1-2) multiplied by levofloxacin, the levofloxacin quinolone drug is one of medicines, the levofloxacin quinolone drug has broad-spectrum antibacterial action and strong antibacterial action, can effectively inhibit various bacterial communities in the vagina of women, and has strong antibacterial activity on most enterobacteriaceae bacteria, such as escherichia coli, klebsiella, proteus, salmonella, shigella, haemophilus influenzae, legionella pneumophila, neisseria gonorrhoeae and other gram-negative bacteria; it also has antibacterial effect on gram-positive bacteria such as Staphylococcus aureus, Streptococcus pneumoniae, Streptococcus pyogenes, etc., mycoplasma pneumoniae, and chlamydia pneumoniae;

the concentration of the necroptosis inhibitor Necrostatin-1/RIP1 kinase inhibitor is 5-10 ng/mL, the necroptosis inhibitor Necrostatin-1/RIP1 kinase inhibitor can directly and constantly perform apoptosis, preserve the biological characteristics of cells, comprehensively improve the survival rate of the cells and maintain the survival of the cells;

the concentration of the compound vitamin is 1-5 mg/mL;

the concentration of the glutathione is 1-5 mg/mL, the glutathione is a powerful antioxidant and exists in each cell of a body, redundant free radicals can be eliminated, and the antioxidation effect is achieved.

The invention also provides a preparation method of the human menstrual blood preservative fluid, which comprises the following steps:

1) preparing a solution A: adding glucose, hirudin and levofloxacin into physiological saline to obtain solution A;

2) preparing a solution B: respectively adding hydroxyethyl starch, vitamin complex and glutathione into physiological saline to obtain solution B, wherein the combined use of the vitamin complex and the glutathione can comprehensively improve the antioxidant function of cells;

3) preparing a solution C: mixing solution A and solution B, adding necrotizing apoptosis inhibitor Necrostatin-1/RIP1 kinase inhibitor to obtain solution C, filtering with filter membrane, and storing at 4 deg.C, and storing menstrual blood at low temperature to avoid nutritional component denaturation and maintain bioactivity of seed cells.

The volume ratio of the liquid A and the liquid B mixed in the step 3) is 1:1, and the pore size of the filter membrane is 0.22 mu m.

The invention has the following beneficial effects:

the preservation solution for human menstrual blood disclosed by the invention takes glucose as a basic nutrient component, takes physiological saline as a buffer solution, and also comprises different anti-apoptosis preparations, so that the stress resistance of cells can be enhanced, the activity of endometrial stem cells from menses can be well maintained under a proper preservation environment and nutrition supply, the long-time survival rate of in-vitro placenta in the preservation solution is ensured, the functional variation of the cells is not caused, and the in-vitro menses is anticoagulated and bacteriostatic, and the preservation solution disclosed by the invention can preserve in-vitro menstrual blood for 48 hours, can maintain the original biological characteristics of the endometrial stem cells within 48 hours, maintain the quality of the cells, solve the technical problem of in-vitro preservation of menstrual blood, and meanwhile, the separated cells are not differentiated, stable in cell quality and high in purity, and can be produced in a large scale.

The preserving fluid has simple preparation steps, can be prepared in large quantities in a short time, and has the advantages of low cost, stable components, safety and no toxic or side effect.

Drawings

FIG. 1 is a flow identification chart of a control group for extracting endometrial stem cells after the preservation solution of the invention is used for menstrual blood;

FIG. 2 is a flow identification chart of a sample group from which endometrial stem cells are extracted after a preservation solution of the present invention is applied to menstrual blood;

FIG. 3 is a diagram showing the cell morphology of endometrial stem cells extracted after the preservation solution of the invention is used for menstrual blood.

Detailed Description

The present invention is described in further detail in the following description of specific embodiments and the accompanying drawings, it is to be understood that these embodiments are merely illustrative of the present invention and are not intended to limit the scope of the invention, which is defined by the appended claims, and modifications thereof by those skilled in the art after reading this disclosure that are equivalent to the above described embodiments.

All the raw materials and reagents of the invention are conventional market raw materials and reagents unless otherwise specified.