WO2018023827A1 - 一种宫内膜干细胞保护液 - Google Patents

一种宫内膜干细胞保护液 Download PDF

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WO2018023827A1
WO2018023827A1 PCT/CN2016/094673 CN2016094673W WO2018023827A1 WO 2018023827 A1 WO2018023827 A1 WO 2018023827A1 CN 2016094673 W CN2016094673 W CN 2016094673W WO 2018023827 A1 WO2018023827 A1 WO 2018023827A1
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fullerene
solution
stem cell
dmem
endometrial stem
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PCT/CN2016/094673
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English (en)
French (fr)
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曹毓琳
林俊堂
李霞云
刘世红
卢承前
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北京臻惠康生物科技有限公司
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N1/00—Preservation of bodies of humans or animals, or parts thereof
    • A01N1/02—Preservation of living parts
    • A01N1/0205—Chemical aspects
    • A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
    • A01N1/0226—Physiologically active agents, i.e. substances affecting physiological processes of cells and tissue to be preserved, e.g. anti-oxidants or nutrients
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N1/00—Preservation of bodies of humans or animals, or parts thereof
    • A01N1/02—Preservation of living parts

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  • the invention belongs to the field of endometrial stem cell preservation, and particularly relates to an endometrial stem cell protection solution.
  • the endometrial cell line has a strong self-renewal ability, and there is a large increase in tissue and blood vessels in each menstrual cycle, and this growth process stops with the end of the menstrual cycle.
  • stromal stem cells mesenchymal stem cells
  • menstrual blood endometrial stem cells
  • these stem cell viability Stronger, stronger self-renewal and value-adding ability to differentiate potential embryonic stem cells. If these stem cells are stored in a scientific way, in the future life of women, once a major disease such as a tumor or serious accidental injury occurs, the transplantation of autologous stem cells can be immediately performed to restore health.
  • the method for storing blood samples is to obtain a blood sample, and after preliminary separation, most of the red blood cells are removed, and then the cultured endometrial stem cells are cryopreserved by directly adding the frozen solution. After collection of endometrial stem cells, it is necessary to store the endometrial stem cells in a protective solution for use.
  • the method for storing endometrial stem cells disclosed in the patent CN201110085328 records that the endometrial stem cells are collected and stored in the collection liquid, and the collection is performed. The liquid is added with antibiotics and heparin to avoid stem cell contamination and blood clotting and increase stem cell viability. But the palace The time during which the endometrial stem cells remain high in the cell preservation solution is still short, and the cell viability decreases with time.
  • the object of the present invention is to overcome the defects in the prior art and design an endometrial stem cell protection solution which can improve the survival rate of stored endometrial stem cells and improve cell quality.
  • the technical solution adopted by the present invention is an endometrial stem cell protection solution, which mainly dissolves a hypoxic protective agent, a mycoplasma inhibitor, and an aminoglycoside antibiotic in a DMEM/F12 medium aqueous solution.
  • the DMEM/F12 medium aqueous solution is dissolved in the DMEM/F12 medium aqueous solution to dissolve 3-15 mg of the hypoxic protective agent, the mycoplasma inhibitor 1-35 mg, and the aminoglycoside antibiotic 100-500 U, respectively, and the DMEM/F12 medium aqueous solution.
  • the concentration is 20-45 mg/ml.
  • the concentration of the DMEM/F12 medium aqueous solution is 33.8 mg/ml.
  • the hypoxic protectant is cobalt chloride and/or sodium lactate.
  • the hypoxic protectant is a mixture of cobalt chloride and sodium lactate in a weight ratio of 5:1.
  • the invention selects cobalt chloride and sodium lactate synergistically as a hypoxic protective agent, and uses cobalt chloride and sodium lactate in combination to induce a hypoxia response, block the degradation of HIF-1, and maintain the effect of maintaining a low oxygen environment.
  • 10-26 mg of mycoplasma inhibitor is dissolved per ml of the aqueous DMEM/F12 medium solution.
  • the mycoplasma inhibitor comprises one or more of tetracycline, a macrolide, a quinolone and a fullerene
  • the fullerene is a fullerene of a hollow fullerene or a hybrid group.
  • the mycoplasmal inhibitor comprises the following components: 10-25 parts by weight of fullerene and 4-10 parts by weight of quinolone, and the fullerene is a fullerene C 60 in a weight ratio of from 1.2 to 3:1.
  • a mixture of fullerene C 84 mycoplasma has a great threat to the storage of cells, it is easy to cause cell infection, and thus apoptosis, the above mixture can effectively maintain the survival rate of endometrial stem cells for cell survival and Growth provides a safe environment.
  • the mycoplasma inhibitor further comprises the following parts by weight: 5-8 parts of Cortex extract, 4-8 parts of Aster extract, and 3-6 parts of oxymatrine.
  • a cortex extract, a purpura extract and oxymatrine are further added to the mycoplasma inhibitor, and the three traditional Chinese medicine components can form a mycoplasma inhibitor through the combination of the traditional Chinese medicine and the western medicine component on the basis of the original western medicine component. It effectively inhibits and kills mycoplasma in the protective solution, has a short action period, has a strong protective effect on cells, and prevents cell infection.
  • the aminoglycoside antibiotic comprises one or more of streptomycin, gentamicin, kanamycin and tobramycin, preferably in a weight ratio of 1:1.5.
  • streptomycin gentamicin
  • kanamycin kanamycin
  • tobramycin preferably in a weight ratio of 1:1.5.
  • aminoglycoside antibiotic 200-400 U is dissolved per ml of the DMEM/F12 medium aqueous solution.
  • the protective liquid provided by the invention is closer to the human body environment, and is suitable for the preservation of endometrial cells, and the added aminoglycoside antibiotics have the effect of sterilization, thereby further improving the quality of the living environment.
  • the endometrial stem cell protection solution of the present invention further increases the survival rate of stem cells and expands the cardinality of the target cells.
  • the protection solution further comprises a growth promoting agent which is different from calf serum.
  • Ascorbic acid, sodium caseinate and egg yolk are mixed, wherein 1-4 mg of erythorbic acid, 1-3 mg of sodium caseinate and 0.1-0.3 ml of egg yolk per ml of calf serum solution are dissolved; /F12 medium water soluble
  • the growth promoting agent is added to the liquid in an amount of 0.05-0.3 ml.
  • the growth promoting agent maintains the osmotic balance in the endometrial stem cells, increases the adherence rate of the stem cells, and increases the viscosity of the protective solution to improve the stability of the protective solution.
  • the protective solution of the present invention further comprises estrone, which dissolves 0.26-0.4 ⁇ 10 -3 mg of estradiol and 0.04-0.08 ⁇ 10 -3 mg of female per ml of the DMEM/F12 medium aqueous solution.
  • estrone dissolves 0.26-0.4 ⁇ 10 -3 mg of estradiol and 0.04-0.08 ⁇ 10 -3 mg of female per ml of the DMEM/F12 medium aqueous solution.
  • Ketones the addition of a small amount of estrogen has a significant effect on the proliferation of endometrial stem cells.
  • the endometrial stem cell protection fluid stored in the endometrial stem cell protection solution of the invention has high survival rate and long storage time, and is sufficient for the next step of proliferation of stem cells, and can increase the cell quality in the proliferative phase, and the cell survival after the increase.
  • the rate is high.
  • Figure 1 is the effect of preservation solution on the growth curve of endometrial stem cells.
  • composition of the endometrial stem cell protection solution is as follows:
  • DMEM/F12 medium aqueous solution 10ml, concentration 20mg/ml;
  • hypoxic protective agent cobalt chloride 20mg, sodium lactate 10mg;
  • Mycoplasma inhibitor quinolone 10mg
  • Aminoglycoside antibiotics streptomycin 1000U;
  • DMEM/F12 is prepared by mixing DMEM and F12 powder in a ratio of 1:1, using double distilled water, adding sodium bicarbonate, adjusting the pH to 7.2 to a constant volume, and diluting with 2 times the amount of water to obtain DMEM/ Aqueous F12 medium.
  • the unit amount of the hypoxic protective agent, the mycoplasma inhibitor, and the aminoglycoside antibiotic are added to the DMEM/F12 medium aqueous solution, and the endometrial stem cell protection solution is obtained by uniformly mixing.
  • composition of the endometrial stem cell protection solution is as follows:
  • DMEM/F12 medium aqueous solution 10ml, concentration 45mg/ml;
  • hypoxic protective agent cobalt chloride 100mg, sodium lactate 20mg;
  • Mycoplasma inhibitor quinolone 200mg
  • Aminoglycoside antibiotics streptomycin 1000 U, tobramycin 1500 streptomycin and tobramycin 2000 U.
  • the preparation method of the protective liquid is as described in Example 1.
  • composition of the endometrial stem cell protection solution is as follows:
  • DMEM/F12 medium aqueous solution 10ml, concentration 33.8mg / ml;
  • hypoxia protective agent cobalt chloride 80mg, sodium lactate 16mg;
  • Mycoplasma inhibitor macrolide 80mg;
  • Aminoglycoside antibiotics gentamicin 2000U.
  • the preparation method of the protective liquid is as described in Example 1.
  • composition of the endometrial stem cell protection solution is as follows:
  • DMEM/F12 medium aqueous solution 10ml, concentration 30mg/ml;
  • hypoxia protective agent cobalt chloride 30mg, sodium lactate 20mg;
  • Mycoplasma inhibitor fullerene C 60 50 mg, fullerene C 84 20 mg, quinolone 25 mg;
  • Aminoglycoside antibiotics streptomycin 700U, tobramycin 800U, gentamicin 1000U;
  • the preparation method of the protective liquid is as described in Example 1.
  • composition of the endometrial stem cell protection solution is as follows:
  • DMEM/F12 medium aqueous solution 10ml, concentration 30mg/ml;
  • hypoxia protective agent cobalt chloride 30mg, sodium lactate 20mg;
  • Mycoplasma inhibitor fullerene C 60 50mg, fullerene C 84 20mg, quinolone 25mg, Phellodendron extract 25mg, Aster extract 20mg, oxymatrine 15mg;
  • Aminoglycoside antibiotics streptomycin 700 U, tobramycin 800 U, gentamicin 1000 U; protective liquid preparation method as described in Example 1.
  • composition of the endometrial stem cell protection solution is as follows:
  • DMEM/F12 medium aqueous solution 10 ml, concentration 30.2 mg / ml;
  • hypoxic protective agent cobalt chloride 50mg, sodium lactate 15mg;
  • Mycoplasma inhibitor fullerene C 60 85mg, fullerene C 70 45mg, quinolone 50mg, Phellodendron extract 40mg, Aster extract 40mg, oxymatrine 30mg;
  • Aminoglycoside antibiotics gentamicin 1000U, kanamycin 1500U;
  • Growth promoter 0.5ml, mixed with calf serum and sodium caseinate, ascorbic acid and egg yolk solution, wherein 1 ml of erythorbic acid, 1 mg of sodium caseinate and 0.1 ml of egg yolk solution are dissolved per milliliter of calf serum solution. ;
  • the unit amount of the hypoxic protective agent, the mycoplasma inhibitor, the aminoglycoside antibiotic and the growth promoting agent are added to the DMEM/F12 medium aqueous solution, and the endometrial stem cell protection solution is obtained by uniformly mixing.
  • composition of the endometrial stem cell protection solution is as follows:
  • DMEM/F12 medium aqueous solution 10ml, concentration 28mg/ml;
  • hypoxia protective agent cobalt chloride 30mg, sodium lactate 20mg;
  • Mycoplasma inhibitor fullerene C 60 100 mg, fullerene C 84 100 mg, quinolone 60 mg, Phellodendron extract 20 mg, purpura extract 20 mg, oxymatrine 20 mg;
  • Aminoglycoside antibiotics tobramycin 1600U, gentamicin 1000U;
  • Growth promoter 3ml, mixed with calf serum and sodium caseinate, ascorbic acid and egg yolk solution, wherein 4ml of erythorbic acid and 3mg of cheese are dissolved per milliliter of calf serum solution. Sodium methoxide and 1.3 ml of egg yolk.
  • the preparation method of the protective liquid is as described in Example 5.
  • composition of the endometrial stem cell protection solution is as follows:
  • DMEM/F12 medium aqueous solution 10ml, concentration 24.5mg / ml;
  • hypoxia protective agent cobalt chloride 46mg, sodium lactate 20mg;
  • Mycoplasma inhibitor fullerene C 60 35 mg, fullerene C 84 24 mg, quinolone 22 mg, cork extract 35 mg, purpura extract 35 mg, oxymatrine 25 mg;
  • Aminoglycoside antibiotics streptomycin 1000U, gentamicin 1000U;
  • Growth promoter 1.8ml, which is made up of calf serum mixed with erythorbic acid, sodium caseinate, ascorbic acid and egg yolk. Each milliliter of calf serum solution dissolves 1.5 mg of isoascorbic acid, 2 mg of sodium caseinate and 0.9. Mg of egg yolk;
  • the preparation method of the protective liquid is as described in Example 5.
  • composition of the endometrial stem cell protection solution is as follows:
  • DMEM/F12 medium aqueous solution 10ml, concentration 33.8mg / ml;
  • hypoxia protective agent cobalt chloride 95mg, sodium lactate 19mg
  • Mycoplasma inhibitor fullerene C 60 35 mg, fullerene C 84 24 mg, quinolone 22 mg, Phellodendron extract 25 mg, purpura extract 22 mg, oxymatrine 20 mg;
  • Aminoglycoside antibiotics streptomycin 800U, tobramycin 1200U, gentamicin 1600U;
  • Growth promoter 2.2ml, from calf serum with erythorbic acid, sodium caseinate and eggs The yellow liquid is mixed, wherein 2.5 ml of erythorbic acid, 1.4 mg of sodium caseinate and 0.2 ml of egg yolk are dissolved per milliliter of calf serum solution;
  • the unit amount of the hypoxic protective agent, the mycoplasma inhibitor, the aminoglycoside antibiotic, the growth promoting agent, and the estrogen are added to the DMEM/F12 medium aqueous solution, and the endometrial stem cell protection solution is obtained by uniformly mixing.
  • composition of the endometrial stem cell protection solution is as follows:
  • DMEM/F12 medium aqueous solution 10 ml, concentration 25.8 mg / ml;
  • hypoxic protective agent cobalt chloride 43mg, sodium lactate 10mg;
  • Mycoplasma inhibitor fullerene C 84 100 mg, quinolone 40 mg, Phellodendron extract 50 mg, Aster extract 40 mg, oxymatrine 30 mg;
  • Aminoglycoside antibiotics gentamicin 1000U, kanamycin 1000U, tobramycin 1000U;
  • Growth promoter 1.8ml, which is made up of calf serum mixed with erythorbic acid, sodium caseinate and egg yolk. Each milliliter of calf serum solution dissolves 3.0 mg of isoascorbic acid, 1 mg of sodium caseinate and 0.4 ml. Yolk liquid;
  • the preparation method of the protective liquid is as described in Example 8.
  • composition of the endometrial stem cell protection solution is as follows:
  • DMEM/F12 medium aqueous solution 10 ml, concentration 20.8 mg / ml;
  • hypoxia protective agent cobalt chloride 50mg, sodium lactate 10mg
  • Mycoplasma inhibitor tetracycline 62 mg, quinolone 50 mg;
  • Aminoglycoside antibiotics streptomycin 2300U, kanamycin 1500U;
  • Growth promoter 2.6ml, which is made up of calf serum mixed with erythorbic acid, sodium caseinate and egg yolk. Each milliliter of calf serum solution dissolves 2.3mg of isoascorbic acid, 1.6mg of sodium caseinate and 0.12ml. Yolk liquid;
  • the preparation method of the protective liquid is as described in Example 8.
  • composition of the endometrial stem cell protection solution is as follows:
  • DMEM/F12 medium aqueous solution 10ml, concentration 45mg/ml;
  • hypoxic protective agent cobalt chloride 100mg, sodium lactate 20mg;
  • Mycoplasma inhibitor quinolone 200mg
  • Aminoglycoside antibiotics penicillin 2000U.
  • composition of the endometrial stem cell protection solution is as follows:
  • DMEM/F12 medium aqueous solution 10ml, concentration 33.8mg / ml;
  • hypoxia protective agent cobalt chloride 80mg
  • Mycoplasma inhibitor macrolide 80mg;
  • Aminoglycoside antibiotics gentamicin 2000U.
  • composition of the endometrial stem cell protection solution is as follows:
  • DMEM/F12 medium aqueous solution 10ml, concentration 30mg/ml;
  • hypoxia protective agent cobalt chloride 30mg, sodium lactate 20mg;
  • Mycoplasma inhibitor fullerene C 60 50mg, fullerene C 84 20mg, quinolone 25mg, Phellodendron extract 25mg, oxymatrine 15mg;
  • Aminoglycoside antibiotics streptomycin 700 U, tobramycin 800 U, gentamicin 1000 U; Comparative Example 4
  • composition of the endometrial stem cell protection solution is as follows:
  • DMEM/F12 medium aqueous solution 10ml, concentration 24.5mg / ml;
  • hypoxia protective agent cobalt chloride 46mg, sodium lactate 20mg;
  • Mycoplasma inhibitor fullerene C 60 35 mg, fullerene C 84 24 mg, quinolone 22 mg, cork extract 35 mg, purpura extract 35 mg, oxymatrine 25 mg;
  • Aminoglycoside antibiotics streptomycin 1000U, gentamicin 1000U;
  • Growth promoter 1.8 ml, mixed with calf serum and erythorbic acid, wherein 1.5 ml of isoascorbic acid was dissolved per ml of calf serum solution.
  • composition of the endometrial stem cell protection solution is as follows:
  • DMEM/F12 medium aqueous solution 10ml, concentration 33.8mg / ml;
  • hypoxia protective agent cobalt chloride 95mg, sodium lactate 19mg
  • Mycoplasma inhibitor fullerene C 60 35 mg, fullerene C 84 24 mg, quinolone 22 mg, Phellodendron extract 25 mg, purpura extract 22 mg, oxymatrine 20 mg;
  • Aminoglycoside antibiotics streptomycin 800U, tobramycin 1200U, gentamicin 1600U;
  • Growth promoter 2.2ml, which is made up of calf serum mixed with erythorbic acid, sodium caseinate and egg yolk. Each milliliter of calf serum solution dissolves 2.5mg of isoascorbic acid, 1.4mg of sodium caseinate and 0.2ml. Yolk liquid;
  • the specific extraction method of the cortex extract and the purpura extract in the hypoxic protectant in the above embodiment is: respectively, formulating the amount of cork and purpura, adding water 8-10 times, soaking for 4 hours, boiling and extracting 2 times, Each time 1.5 hours, combine two boiling liquids, filter, and concentrate the filtrate under reduced pressure to a relative density of 1.05-1.10. After cooling, add 60-80% ethanol, let stand overnight, and take the supernatant to recover ethanol under reduced pressure. The concentrate was free of alcohol and vacuum dried at a temperature of 50-60 ° C and a pressure of -0.05 MPa to obtain a cork extract and a purpura extract, respectively.
  • Test 1 Effect of preservation solution on survival rate of endometrial stem cells during storage
  • the collection tube of test group 1 is provided with the protection liquid described in Example 2;
  • the collection tube of the test group 2 is provided with the protection liquid described in Example 3;
  • the collection tube of test group 3 is provided with the protection liquid described in Example 4;
  • the collection tube of the test group 4 is provided with the protection liquid described in Example 5;
  • the collection tube of test group 5 is provided with the protection liquid described in Example 8;
  • the collection tube of test group 6 is provided with the protection liquid described in Example 9;
  • the collection tube of the test group 7 was filled with the protection liquid described in Comparative Example 1;
  • the collection tube of the test group 8 is provided with the protection liquid described in Comparative Example 2;
  • the collection tube of the test group 9 was filled with the protection liquid described in Comparative Example 3;
  • the collection tube of the test group 10 is provided with the protection liquid described in Comparative Example 4.
  • the collection tube of the test group 11 is provided with the protection liquid described in Comparative Example 5;
  • the collection tube of the test group 12 is equipped with a common graft protection solution, and the commonly used graft protection solution is a phosphate buffer solution containing penicillin/streptomycin and heparin;
  • the collection tube of test group 13 was a blank control.
  • the collection tubes of each test group were placed at 4 ° C for release.
  • the centrifuge of the test group 13 After the centrifuge of the test group 13 is centrifuged, the supernatant is obtained; the supernatant is subjected to anaerobic bacteria and aerobic bacteria examination according to the blood culture method, and identified; if it is a positive result, the whole endometrial stem cells are stored. program.
  • the invention was added in Examples 2-5 and Examples 8-9.
  • the survival rate of the stem cells stored in the protective solution is high, and the storage of Examples 4-5 and 8-9 is not changed for a long time, and the survival rate of the storage 96 hours is still high; there is a highly significant difference compared with the conventional protective solution ( P ⁇ 0.001), the protection of Examples 2-5 and 8-9
  • the protective liquid has a good protective effect on stem cell transplantation, and the protective liquid of Example 9 has the best effect.
  • Comparing Example 2 with Comparative Example 1 it can be seen that after the aminoglycoside antibiotic in the protective solution provided by the present invention is replaced with penicillin antibiotic, the viability of the stem cells is remarkably lowered, and the survival time is short.
  • Comparing Example 3 with Comparative Example 2 it can be seen that the sodium cell lactate is added to the hypoxic protective agent of the protective solution provided by the present invention, and the survival rate of the stem cells is higher.
  • Comparing Example 4, Example 5 and Comparative Example 3 it was found that the dry cell survival rate was higher after the hypoxic protectant was added to the extract of Phellodendron chinense, Aster extract and Oxymatrine.
  • Example 8 Comparing Example 8 with Comparative Example 4, it was found that the growth rate of stem cells was higher after the growth promoting agent was added with sodium caseinate and egg yolk. Comparing Example 8 with Example 9, it can be seen that the activity of the stem cells is higher after the protective solution provided by the present invention is added with estrogen.
  • the individual cells are collected by density gradient centrifugation;
  • the mononuclear cells obtained above were inoculated into Chang complete medium, and the inoculation density of mononuclear cells was 1*10 5 -1*10 6 /ml, and the CO 2 was placed at 37 ° C, saturated humidity, and volume fraction of 5%.
  • the culture is carried out in an incubator.
  • Chang complete medium After 4-5 days from the start of cell culture by inoculation of mononuclear cells in Chang complete medium, Chang complete medium was replaced, and unattached cells were discarded; after that, the whole amount of liquid was changed every 3-4 days (according to cell growth status) The full amount of liquid replacement means replacing all the Chang medium); when the cell growth reaches 80-90% fusion, trypsin is used at a concentration of 0.25% (usually 1 ml per 1-20 ml of cells) The cells were collected by trypsin digestion, and then inoculated and cultured at a density of 5000-6000/cm 2 and recorded as P1 generation.
  • the cells were collected by trypsin digestion with a mass percentage of 0.25%; the cells were resuspended in a pre-cooled cryopreservation solution; the density of the cells was maintained at 1-2*10 6 /ml.
  • the cryopreservation solution was prepared by adding 1 part by volume of DMSO (calbiochem) to 9 parts by volume of Chang Complete Medium (i.e., Chang Complete Medium containing 10% DMSO). Place the cell suspension in a hard plastic cryotube, seal it, and mark it. The cells were simultaneously dropped at a certain distance every 20 minutes, and finally completely frozen in liquid nitrogen.
  • Example 2 Comparing Example 2 with Comparative Example 1, it can be seen that after the aminoglycoside antibiotic in the protective solution provided by the present invention is replaced with a penicillin antibiotic, the activity of the stem cells is remarkably lowered, and the survival time is short, that is, the aminoglycoside antibiotics against the stem cells.
  • the survival rate of the value-added of the offspring has a better effect. Comparing Example 3 with Comparative Example 2, it can be seen that the addition of sodium lactate to the hypoxic protective agent of the protective solution provided by the present invention has a higher survival rate of stem cell progeny. Comparing Example 4, Example 5 and Comparative Example 3, it can be seen that the survival rate of the stem cell progeny increased after the addition of the hypoxic protectant to the cortex extract, the purpura extract and the oxymatrine. Comparing Example 8 with Comparative Example 4, it is known that the growth rate of stem cell progeny is higher after the growth promoting agent is added with sodium caseinate and egg yolk. Comparing Example 8 with Example 9, it can be seen that after the protective solution provided by the present invention is added with estrogen, the survival rate of the stem cell progeny is higher.
  • the collection tube was divided into two groups of the test group, wherein the collection tube of the test group 1 was equipped with the protection liquid described in Example 9; the collection tube of the test group 2 was charged with the protection liquid described in Comparative Example 5.
  • the P1 generation endometrial stem cells of the test group 1 and the test group 2 were inoculated into the 96-well culture at a density of 10 3 /ml. Plate, add Chang complete medium, 20 plates per group of cells, and start MTT test the next day after adherence.
  • MTT colorimetric test cell growth curve 20 ⁇ L of MTT solution (5 mg/ml) was added to each well of the cells to be tested 4 h before the end of the culture. After the culture, the culture solution was aspirated, 150 ⁇ L of DMSO was added, and the absorbance was measured at a wavelength of 492 nm. . Continue for 10 days until the cell proliferation reaches the plateau phase, at the same time every day; plot the cell growth curve, as shown in Figure 1. It can be seen from the figure that the protective solution of estradiol and estrone is added to promote the proliferation of endometrial stem cells. .

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Abstract

提供一种宫内膜干细胞保护液，所述保护液主要是将低氧保护剂、支原体抑制剂以及氨基糖苷类抗生素用DMEM/F12培养基水溶液溶解而成，每毫升所述DMEM/F12培养基水溶液分别溶解所述低氧保护剂3-15mg、所述支原体抑制剂1-20mg以及氨基糖苷类抗生素100-500U，所述DMEM/F12培养基水溶液的浓度为20-45mg/ml。该保护液可提高存储的宫内膜干细胞存活率，提高细胞质量。

Description

一种宫内膜干细胞保护液 技术领域
本发明属于宫内膜干细胞保存领域，具体涉及一种宫内膜干细胞保护液。
背景技术
众所周知，子宫内膜细胞系具有很强的自我更新能力，在每个月经周期都会有组织和血管的大量增长，这一增长过程会随着月经周期的结束而停止。多国科学家最新研究显示在随女性经血脱落的子宫内膜组织中，具有相当数量的间充质干细胞(基质干细胞)--宫内膜(经血)干细胞，数量为骨髓来源的30倍；这些干细胞活力更强，更强的自我更新和增值能力，分化潜能胚胎干细胞。如果把这些干细胞通过科学的方法储存起来，在女性未来的一生中，一旦发生肿瘤等重大疾病或意外严重伤害时，就可立即做自体干细胞的移植，恢复健康。
目前有经血标本的储存方法是获得经血标本后，经过初步分离，去除大部分的红细胞，然后直接添加冻存液冻存将培养后的宫内膜干细胞进行冻存。在宫内膜干细胞采集后需要将宫内膜干细胞保存在保护液中待用，专利CN201110085328中公开的储存宫内膜干细胞的方法中记载采集宫内膜干细胞后将其保存在采集液，其采集液重添加有抗生素和肝素，避免干细胞的污染和凝血，提高干细胞活率。但是，宫 内膜干细胞在该细胞保存液中保持高活率的时间仍然较短，并且随着时间延长，细胞活力下降。
发明内容
本发明的目的在于克服现有技术中的缺陷，设计一种宫内膜干细胞保护液，该保护液可提高存储的宫内膜干细胞存活率，提高细胞质量。
为实现上述目的，本发明所采用的技术方案是一种宫内膜干细胞保护液，所述保护液主要是将低氧保护剂、支原体抑制剂以及氨基糖苷类抗生素用DMEM/F12培养基水溶液溶解而成，每毫升所述DMEM/F12培养基水溶液分别溶解所述低氧保护剂3-15mg、所述支原体抑制剂1-35mg以及氨基糖苷类抗生素100-500U，所述DMEM/F12培养基水溶液的浓度为20-45mg/ml。
优选的，所述DMEM/F12培养基水溶液的浓度为33.8mg/ml。
本发明保护液进一步的方案中，所述低氧保护剂为氯化钴和/或乳酸钠，优选地，所述低氧保护剂为重量份数比为5∶1的氯化钴和乳酸钠的混合物。本发明选用氯化钴和乳酸钠协同作为低氧保护剂，氯化钴和乳酸钠混合使用，诱导低氧应答，阻断HIF-1降解，并维持保持低氧环境的效果极佳。
在其中一个改进方案中，每毫升所述DMEM/F12培养基水溶液溶解支原体抑制剂10-26mg。
进一步的改进，所述支原体抑制剂包括四环素、大环内酯、喹诺酮和富勒烯中的一种或几种，所述富勒烯为空心富勒烯或杂化基团的富勒烯，选自富勒烯C60、富勒烯C70、富勒烯C76、富勒烯C78、富勒烯C80和富勒烯C84中的一种或几种。
优选为所述支原体抑制剂包括以下组分：富勒烯10-25重量份和喹诺酮4-10重量份，所述富勒烯为重量份数比为1.2-3∶1的富勒烯C60和富勒烯C84的混合物，支原体对细胞的存储有很大的威胁作用，很容易造成细胞感染，从而使细胞凋亡，上述混合物能够有效保持宫内膜干细胞的活率，为细胞存活和生长提供安全环境。
进一步地，所述支原体抑制剂还包括以下重量份数的组分：黄柏提取物5-8份、紫菀提取物4-8份、氧化苦参碱3-6份。本发明中进一步在支原体抑制剂中增加了黄柏提取物、紫菀提取物和氧化苦参碱，这三种中药成分能够在原始西药成分的基础上，通过中西医结合的药效成分组成支原体抑制剂，有效抑制和杀死保护液中的支原体，作用周期较短，对细胞具有较强的保护作用，防止细胞感染。
在又一个改进方案中，所述氨基糖苷类抗生素包括链霉素、庆大霉素、卡那霉素以及妥布霉素中的一种或几种，优选为重量份数比为1∶1.5∶2的链霉素、妥布霉素和庆大霉素的混合物。
进一步的改进，每毫升所述DMEM/F12培养基水溶液溶解所述氨基糖苷类抗生素200-400U。
本发明提供的保护液更接近人体体内环境，适合宫内膜细胞的保存，加入的氨基糖苷类抗生素起到杀菌的效果，进一步提高生存环境质量。
本发明宫内膜干细胞保护液为进一步提高干细胞的存活率，扩大目标细胞的基数，在另一个改进方案中，所述保护液还包括促生长剂，所述促生长剂由小牛血清与异抗坏血酸、酪蛋白酸钠和卵黄液混合而成，其中每毫升小牛血清溶液溶解1-4mg的异抗坏血酸、1-3mg的酪蛋白酸钠和0.1-0.3ml的卵黄液；每毫升所述DMEM/F12培养基水溶 液中添加所述促生长剂0.05-0.3ml。该促生长剂维持宫内膜干细胞内的渗透平衡，提高干细胞的贴壁率，同时还能增加保护液的粘度，提高该保护液的稳定性。
在上述基础上，本发明保护液进一步还包括和雌酮，每毫升所述DMEM/F12培养基水溶液中溶解0.26-0.4×10-3mg的雌二醇和0.04-0.08×10-3mg的雌酮；少量雌激素的添加对宫内膜干细胞增值有较明显的促进作用。
在一个对宫内膜干细胞保护效果较佳的具体方案中，每毫升所述DMEM/F12培养基水溶液分别溶解氯化钴9mg、大环内酯8mg以及庆大霉素200U。
在另一个对宫内膜干细胞保护效果更佳的具体方案中，每毫升所述DMEM/F12培养基水溶液分别溶解9.5mg的氯化钴、1.9mg的乳酸钠、3.5mg的富勒烯C60、2mg的富勒烯C84、2.2mg的喹诺酮、2.5mg的黄柏提取物、2.2mg的紫菀提取物、2mg的氧化苦参碱、80U的链霉素、120U的妥布霉素、160U的庆大霉素、0.22ml的促生长剂、0.3×10-3mg雌二醇以及0.05×10-3mg的雌酮；
其中促生长剂中每毫升小牛血清溶液溶解2.5mg的异抗坏血酸、1.4mg的酪蛋白酸钠和0.2ml的卵黄液。
本发明的宫内膜干细胞保护液存储的宫内膜干细胞存活率高，存储时间长，为干细胞下一步的增殖提高足够大的基数；并且，能提高增殖期的细胞质量，增值后的细胞存活率高。
附图说明
图1是保存液对宫内膜干细胞生长曲线的影响。
具体实施方式
下面结合附图和实施例，对本发明的具体实施方式作进一步描述。以下实施例仅用于更加清楚地说明本发明的技术方案，而不能以此来限制本发明的保护范围。
实施例1
宫内膜干细胞保护液的组成如下：
DMEM/F12培养基水溶液10ml，浓度20mg/ml；
低氧保护剂：氯化钴20mg，乳酸钠10mg；
支原体抑制剂：喹诺酮10mg；
氨基糖苷类抗生素：链霉素1000U；
其中，DMEM/F12通过将DMEM和F12的粉末按照1∶1的比例混合，用双蒸水溶解，加入碳酸氢钠后调pH至7.2定容后，用2倍量水稀释，即得DMEM/F12培养基水溶液。
将上述单位量的低氧保护剂、支原体抑制剂和氨基糖苷类抗生素添加至DMEM/F12培养基水溶液，混合均匀即得宫内膜干细胞保护液。
实施例2
宫内膜干细胞保护液的组成如下：
DMEM/F12培养基水溶液10ml，浓度45mg/ml；
低氧保护剂：氯化钴100mg，乳酸钠20mg；
支原体抑制剂：喹诺酮200mg；
氨基糖苷类抗生素：链霉素1000U、妥布霉素1500链霉素和妥布霉素2000U。
保护液的制法如实施例1所述的制法。
实施例3
宫内膜干细胞保护液的组成如下：
DMEM/F12培养基水溶液10ml，浓度33.8mg/ml；
低氧保护剂：氯化钴80mg，乳酸钠16mg；
支原体抑制剂：大环内酯80mg；
氨基糖苷类抗生素：庆大霉素2000U。
保护液的制法如实施例1所述的制法。
实施例4
宫内膜干细胞保护液的组成如下：
DMEM/F12培养基水溶液10ml，浓度30mg/ml；
低氧保护剂：氯化钴30mg、乳酸钠20mg；
支原体抑制剂：富勒烯C6050mg，富勒烯C8420mg，喹诺酮25mg；
氨基糖苷类抗生素：链霉素700U，妥布霉素800U，庆大霉素1000U；
保护液的制法如实施例1所述的制法。
实施例5
宫内膜干细胞保护液的组成如下：
DMEM/F12培养基水溶液10ml，浓度30mg/ml；
低氧保护剂：氯化钴30mg、乳酸钠20mg；
支原体抑制剂：富勒烯C6050mg，富勒烯C8420mg，喹诺酮25mg，黄柏提取物25mg，紫菀提取物20mg，氧化苦参碱15mg；
氨基糖苷类抗生素：链霉素700U，妥布霉素800U，庆大霉素1000U；保护液的制法如实施例1所述的制法。
实施例6
宫内膜干细胞保护液的组成如下：
DMEM/F12培养基水溶液10ml，浓度30.2mg/ml；
低氧保护剂：氯化钴50mg，乳酸钠15mg；
支原体抑制剂：富勒烯C6085mg，富勒烯C7045mg，喹诺酮50mg，黄柏提取物40mg，紫菀提取物40mg，氧化苦参碱30mg；
氨基糖苷类抗生素：庆大霉素1000U、卡那霉素1500U；
促生长剂：0.5ml，由小牛血清与酪蛋白酸钠、抗坏血酸和卵黄液混合而成，其中每毫升小牛血清溶液溶解1mg的异抗坏血酸、1mg的酪蛋白酸钠和0.1ml的卵黄液；
将上述单位量的低氧保护剂、支原体抑制剂、氨基糖苷类抗生素和促生长剂添加至DMEM/F12培养基水溶液，混合均匀即得宫内膜干细胞保护液。
实施例7
宫内膜干细胞保护液的组成如下：
DMEM/F12培养基水溶液10ml，浓度28mg/ml；
低氧保护剂：氯化钴30mg，乳酸钠20mg；
支原体抑制剂：富勒烯C60100mg，富勒烯C84100mg，喹诺酮60mg，黄柏提取物20mg，紫菀提取物20mg，氧化苦参碱20mg；
氨基糖苷类抗生素：妥布霉素1600U，庆大霉素1000U；
促生长剂：3ml，由小牛血清与酪蛋白酸钠、抗坏血酸和卵黄液混合而成，其中每毫升小牛血清溶液溶解4mg的异抗坏血酸、3mg的酪 蛋白酸钠和1.3ml的卵黄液。
保护液的制法如实施例5所述的制法。
实施例8
宫内膜干细胞保护液的组成如下：
DMEM/F12培养基水溶液10ml，浓度24.5mg/ml；
低氧保护剂：氯化钴46mg、乳酸钠20mg；
支原体抑制剂：富勒烯C6035mg，富勒烯C8424mg，喹诺酮22mg，黄柏提取物35mg，紫菀提取物35mg，氧化苦参碱25mg；
氨基糖苷类抗生素：链霉素1000U，庆大霉素1000U；
促生长剂：1.8ml，由小牛血清与异抗坏血酸、酪蛋白酸钠、抗坏血酸和卵黄液混合而成，其中每毫升小牛血清溶液溶解1.5mg的异抗坏血酸、2mg的酪蛋白酸钠和0.9ml的卵黄液；
保护液的制法如实施例5所述的制法。
实施例9
宫内膜干细胞保护液的组成如下：
DMEM/F12培养基水溶液10ml，浓度33.8mg/ml；
低氧保护剂：氯化钴95mg、乳酸钠19mg；
支原体抑制剂：富勒烯C6035mg，富勒烯C8424mg，喹诺酮22mg，黄柏提取物25mg，紫菀提取物22mg，氧化苦参碱20mg；
氨基糖苷类抗生素：链霉素800U，妥布霉素1200U，庆大霉素1600U；
促生长剂：2.2ml，由小牛血清与异抗坏血酸、酪蛋白酸钠和卵 黄液混合而成，其中每毫升小牛血清溶液溶解2.5mg的异抗坏血酸、1.4mg的酪蛋白酸钠和0.2ml的卵黄液；
雌二醇0.3×10-2mg以及雌酮0.05×10-2mg。
将上述单位量的低氧保护剂、支原体抑制剂、氨基糖苷类抗生素、促生长剂以及雌激素添加至DMEM/F12培养基水溶液，混合均匀即得宫内膜干细胞保护液。
实施例10
宫内膜干细胞保护液的组成如下：
DMEM/F12培养基水溶液10ml，浓度25.8mg/ml；
低氧保护剂：氯化钴43mg，乳酸钠10mg；
支原体抑制剂：富勒烯C84100mg，喹诺酮40mg，黄柏提取物50mg，紫菀提取物40mg，氧化苦参碱30mg；
氨基糖苷类抗生素：庆大霉素1000U，卡那霉素1000U、妥布霉素1000U；
促生长剂：1.8ml，由小牛血清与异抗坏血酸、酪蛋白酸钠和卵黄液混合而成，其中每毫升小牛血清溶液溶解3.0mg的异抗坏血酸、1mg的酪蛋白酸钠和0.4ml的卵黄液；
雌二醇0.4×10-2mg以及雌酮0.08×10-2mg。
保护液的制法如实施例8所述的制法。
实施例11
宫内膜干细胞保护液的组成如下：
DMEM/F12培养基水溶液10ml，浓度20.8mg/ml；
低氧保护剂：氯化钴50mg，乳酸钠10mg；
支原体抑制剂：四环素62mg，喹诺酮50mg；
氨基糖苷类抗生素：链霉素2300U，卡那霉素1500U；
促生长剂：2.6ml，由小牛血清与异抗坏血酸、酪蛋白酸钠和卵黄液混合而成，其中每毫升小牛血清溶液溶解2.3mg的异抗坏血酸、1.6mg的酪蛋白酸钠和0.12ml的卵黄液；
雌二醇0.26×10-2mg以及雌酮0.04×10-2mg。
保护液的制法如实施例8所述的制法。
对照实施例1
宫内膜干细胞保护液的组成如下：
DMEM/F12培养基水溶液10ml，浓度45mg/ml；
低氧保护剂：氯化钴100mg，乳酸钠20mg；
支原体抑制剂：喹诺酮200mg；
氨基糖苷类抗生素：青霉素2000U。
对照实施例2
宫内膜干细胞保护液的组成如下：
DMEM/F12培养基水溶液10ml，浓度33.8mg/ml；
低氧保护剂：氯化钴80mg；
支原体抑制剂：大环内酯80mg；
氨基糖苷类抗生素：庆大霉素2000U。
对照实施例3
宫内膜干细胞保护液的组成如下：
DMEM/F12培养基水溶液10ml，浓度30mg/ml；
低氧保护剂：氯化钴30mg、乳酸钠20mg；
支原体抑制剂：富勒烯C6050mg，富勒烯C8420mg，喹诺酮25mg，黄柏提取物25mg，氧化苦参碱15mg；
氨基糖苷类抗生素：链霉素700U，妥布霉素800U，庆大霉素1000U；对照实施例4
宫内膜干细胞保护液的组成如下：
DMEM/F12培养基水溶液10ml，浓度24.5mg/ml；
低氧保护剂：氯化钴46mg、乳酸钠20mg；
支原体抑制剂：富勒烯C6035mg，富勒烯C8424mg，喹诺酮22mg，黄柏提取物35mg，紫菀提取物35mg，氧化苦参碱25mg；
氨基糖苷类抗生素：链霉素1000U，庆大霉素1000U；
促生长剂：1.8ml，由小牛血清与异抗坏血酸混合而成，其中每毫升小牛血清溶液溶解1.5mg的异抗坏血酸。
对照实施例5
宫内膜干细胞保护液的组成如下：
DMEM/F12培养基水溶液10ml，浓度33.8mg/ml；
低氧保护剂：氯化钴95mg、乳酸钠19mg；
支原体抑制剂：富勒烯C6035mg，富勒烯C8424mg，喹诺酮22mg，黄柏提取物25mg，紫菀提取物22mg，氧化苦参碱20mg；
氨基糖苷类抗生素：链霉素800U，妥布霉素1200U，庆大霉素 1600U；
促生长剂：2.2ml，由小牛血清与异抗坏血酸、酪蛋白酸钠和卵黄液混合而成，其中每毫升小牛血清溶液溶解2.5mg的异抗坏血酸、1.4mg的酪蛋白酸钠和0.2ml的卵黄液；
雌二醇0.3×10-2mg。
上述实施例中的低氧保护剂中黄柏提取物和紫菀提取物的具体提取方法为：分别称取配方量的黄柏和紫菀，加水8-10倍量，浸泡4小时，煎煮提取2次，每次1.5小时，合并两次煎煮液，过滤，滤液减压浓缩至相对密度为1.05-1.10，冷却后，加入60-80％乙醇，静置过夜，取上清液减压回收乙醇，至浓缩液无醇味，在温度为50-60℃、压力为-0.05MPa条件下进行真空干燥，分别制得黄柏提取物和紫菀提取物。
试验例 保存液对宫内膜干细胞体外生物学特性的影响
试验一 保存液对宫内膜干细胞存储时的存活率影响
1.采集管分组
将采集管分成试验组13组，其中：
试验组1的采集管装有实施例2所述的保护液；
试验组2的采集管装有实施例3所述的保护液；
试验组3的采集管装有实施例4所述的保护液；
试验组4的采集管装有实施例5所述的保护液；
试验组5的采集管装有实施例8所述的保护液；
试验组6的采集管装有实施例9所述的保护液；
试验组7的采集管装有对照实施例1所述的保护液；
试验组8的采集管装有对照实施例2所述的保护液；
试验组9的采集管装有对照实施例3所述的保护液；
试验组10的采集管装有对照实施例4所述的保护液；
试验组11的采集管装有对照实施例5所述的保护液；
试验组12的采集管装有常用移植保护液，常用移植保护液为含有青霉素/链霉素和肝素的磷酸盐缓冲液；
试验组13的采集管为空白对照。
在开始采集经血以前，将各试验组的采集管均在4℃环境下待放。
2.经血收集
在志愿者月经开始的第二天，采取半蹲或全蹲的姿势，将月经杯对折两次后，放入阴道内。两小时后小心取出，将月事杯中的经血倒入12组采集管中，每支10ml，4℃条件下保存，所述保存期分别为12h、24h和48h。
3.无菌检查
将试验组13采集管离心处理后，得上清液；将上清液按照血培养法进行厌氧菌和需氧菌检查，并鉴定；若为阳性结果，则结束整个储存宫内膜干细胞的程序。
4.计算存活率
计数干细胞上清液中的细胞数。方法：取0.5ml上清液加入等量的0.4％台盼兰染液，充分混匀，取少许滴于血球计数板的计数池内， 静止2分钟，显微镜下观察计数活细胞与死细胞数(凡着兰色的细胞均为不健康或死细胞)，计算细胞存活率。
Figure PCTCN2016094673-appb-000001
结果见表1。
表1宫内膜干细胞存储期不同时段的存活率测定结果的比较
Figure PCTCN2016094673-appb-000002
注：“--”表示没有活率
从表1中看出，在常规保护液进行移植的宫内膜干细胞的随着时间推移，细胞死亡数增大，存活率最低；加入本发明是实施例2-5、实施例8-9的保护液的干细胞存储时存活率高，其中实施例4-5、实施例8-9长时间存储变化不大，存储96h的存活率仍很高；与常规保护液比较都有高度显著性差异(P＜0.001)，实施例2-5、实施例8-9的保 护液对干细胞移植有很好保护作用，其中实施例9的保护液效果最佳。实施例2和对照实施例1的比较可知，本发明提供的保护液中的氨基糖苷类抗生素换成了青霉素抗生素后，干细胞的活率显著降低，且存活时间短。实施例3和对照实施例2的比较可知，本发明提供的保护液的低氧保护剂中加入乳酸钠后，干细胞的活率更高。实施例4、实施例5和对照实施例3的比较可知，低氧保护剂加入黄柏提取物、紫菀提取物和氧化苦参碱后，干细胞活率更高。实施例8和对照实施例4的比较可知，促生长剂加入酪蛋白酸钠和卵黄液后，干细胞的活率更高。而实施例8与实施例9的比较可知，本发明提供的保护液加入雌激素后，干细胞的活率更高。
试验二、保存液对宫内膜干细胞扩增后的存活率影响
1.宫内膜干细胞的分离培养
在获取试验组1-12对应的采集管样品过滤后，采用密度梯度离心法，收集单个可细胞；
将上述所得的单个核细胞接种于Chang完全培养基中，单个核细胞的接种密度为1*105-1*106/ml，置于37℃、饱和湿度、体积分数为5％的CO2培养箱中进行培养。
2.细胞扩增和纯化
待单个核细胞接种于Chang完全培养基中开始细胞培养的4-5天后，更换Chang完全培养基，并弃除未贴壁细胞；以后每3-4天全量换液一次(根据细胞生长状况而定，全量换液即指换掉全部的Chang完全培养基)；待细胞生长达到80-90％融合时，用质量浓度为0.25％的 胰蛋白酶(一般每1-20ml的细胞中使用1ml的所述胰蛋白酶)消化收集细胞，然后按5000-6000个/cm2密度传代接种培养，并记为P1代。
3.细胞冻存：
待步骤3的P1代细胞铺满容器底部后，用质量百分含量为0.25％的胰蛋白酶消化收集细胞；用预冷的冻存液重悬细胞；细胞的密度保持在1-2*106/ml。冻存液为：在9体积份的Chang完全培养基中加入1体积份的DMSO(calbiochem公司)制备而得(即为含有10％DMSO的Chang完全培养基)。将细胞悬液置于硬塑料冻存管中，加盖密封，做好标记。按每20分钟下降一定距离的速度同时降落上述细胞，最终完全投入液氮中冻存。
4.细胞解冻
在冻存两周后将细胞从液氮中取出立即投入42℃水浴中使之迅速完全融化。立即计数融化后的细胞悬液中的细胞数。方法：取0.5ml细胞悬液加入等量的0.4％台盼兰染液，充分混匀，取少许滴于血球计数板的计数池内，静止2分钟，显微镜下观察计数活细胞与死细胞数(凡着兰色的细胞均为不健康或死细胞)，计算细胞存活率。
Figure PCTCN2016094673-appb-000003
结果见表2。
表2宫内膜干细胞扩增后的存活率测定结果的比较
Figure PCTCN2016094673-appb-000004
Figure PCTCN2016094673-appb-000005
从表2中看出，在常规保护液进行移植的宫内膜干细胞扩增后的存活率最低，加入本发明是实施例2-5、实施例8-9的保护液的干细胞扩增后存活率高，与常规保护液比较都有高度显著性差异(P＜0.001)。说明实施例2-5、实施例8-9组分的保护液对干细胞后代增值的有提高存活率的作用，其中实施例9的效果最佳。实施例2和对照实施例1的比较可知，本发明提供的保护液中的氨基糖苷类抗生素换成了青霉素抗生素后，干细胞的活率显著降低，且存活时间短，即氨基糖苷类抗生素对干细胞后代增值的存活率有更好的提高作用。实施例3和对照实施例2的比较可知，本发明提供的保护液的低氧保护剂中加入乳酸钠后，干细胞后代增值的存活率更高。实施例4、实施例5和对照实施例3的比较可知，低氧保护剂加入黄柏提取物、紫菀提取物和氧化苦参碱后，干细胞后代增值的存活率更高。实施例8和对照实施例4的比较可知，促生长剂加入酪蛋白酸钠和卵黄液后，干细胞后代增值的存活率更高。而实施例8与实施例9的比较可知，本发明提供的保护液加入雌激素后，干细胞后代增值的存活率更高。
试验三、保存液对宫内膜干细胞生长曲线的影响
1.将采集管分成试验组2组，其中，试验组1的采集管装有实施例9所述的保护液；试验组2的采集管装有对照实施例5所述的保护液。
按照试验一和试验二的方法采集、分离、纯化、增值宫内膜干细胞后，取试验组1和试验组2的P1代子宫内膜干细胞，以103个/ml的密度接种于96孔培养板，加入Chang完全培养基，每组细胞20个板，待贴壁后第二天开始做MTT试验。
2.MTT比色试验测细胞生长曲线：待定前4h在待测细胞的各孔加20μL MTT液(5mg/ml)，培养结束后，吸弃培养液，加150μLDMSO，在492nm波长处测吸光值。持续10d，直至细胞增殖达到平台期，每天相同时间点进行；绘制细胞生长曲线，见图1；由图中可知，同时添加雌二醇和雌酮的保护液对宫内膜干细胞的增殖有促进作用。
以上所述仅是本发明的优选实施方式，对本技术领域的普通技术人员来说，在不脱离本发明技术原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。

Claims (10)

  1. 一种宫内膜干细胞保护液，其特征在于，所述保护液主要是将低氧保护剂、支原体抑制剂以及氨基糖苷类抗生素用DMEM/F12培养基水溶液溶解而成，每毫升所述DMEM/F12培养基水溶液分别溶解所述低氧保护剂3-15mg、所述支原体抑制剂1-35mg以及氨基糖苷类抗生素100-500U，所述DMEM/F12培养基水溶液的浓度为20-45mg/ml。
  2. 如权利要求1所述的宫内膜干细胞保护液，其特征在于，所述DMEM/F12培养基水溶液的浓度为33.8mg/ml。
  3. 如权利要求1所述的宫内膜干细胞保护液，其特征在于，所述低氧保护剂为氯化钴和/或乳酸钠，优选地，所述低氧保护剂为重量份数比为5∶1的氯化钴和乳酸钠的混合物。
  4. 如权利要求1所述的宫内膜干细胞保护液，其特征在于，所述支原体抑制剂包括四环素、大环内酯、喹诺酮和富勒烯中的一种或几种，所述富勒烯为空心富勒烯或杂化基团的富勒烯，选自富勒烯C60、富勒烯C70、富勒烯C76、富勒烯C78、富勒烯C80和富勒烯C84中的一种或几种。
  5. 如权利要求4所述的宫内膜干细胞保护液，其特征在于，所述支原体抑制剂包括以下组分：富勒烯10-25重量份和喹诺酮4-10重量份，所述富勒烯为重量份数比为1.2-3∶1的富勒烯C60和富勒烯C84的混合物。
  6. 如权利要求5所述的宫内膜干细胞保护液，其特征在于，所述支原体抑制剂还包括以下重量份数的组分：黄柏提取物5-8份、紫菀提取物4-8份、氧化苦参碱3-6份。
  7. 如权利要求1所述的宫内膜干细胞保护液，其特征在于，所述氨基糖苷类抗生素包括链霉素、庆大霉素、卡那霉素以及妥布霉素 中的一种或几种，优选为重量份数比为1∶1.5∶2的链霉素、妥布霉素和庆大霉素的混合物。
  8. 如权利要求1-7中任一项所述的宫内膜干细胞保护液，其特征在于，所述保护液还包括促生长剂，所述促生长剂由小牛血清、酪蛋白酸钠、异抗坏血酸和卵黄液混合而成，其中每毫升小牛血清溶液溶解1-3mg的酪蛋白酸钠、1-4mg的异抗坏血酸和0.1-0.3ml的卵黄液；每毫升所述DMEM/F12培养基水溶液中添加所述促生长剂0.05-0.3ml。
  9. 如权利要求7所述的宫内膜干细胞保护液，其特征在于，所述细胞保护液还包括雌二醇和雌酮，每毫升所述DMEM/F12培养基水溶液溶解0.26-0.4×10-3mg的雌二醇和0.04-0.08×10-3mg的雌酮。
  10. 如权利要求9所述的宫内膜干细胞保护液，其特征在于，每毫升所述DMEM/F12培养基水溶液分别溶解9.5mg的氯化钴、1.9mg的乳酸钠、3.5mg的富勒烯C60、2mg的富勒烯C84、2.2mg的喹诺酮、2.5mg的黄柏提取物、2.2mg的紫菀提取物、2mg的氧化苦参碱、80U的链霉素、120U的妥布霉素、160U的庆大霉素、0.22ml的促生长剂、0.3×10-3mg雌二醇以及0.05×10-3mg的雌酮；
    其中促生长剂中每毫升小牛血清溶液溶解2.5mg的异抗坏血酸、1.4mg的酪蛋白酸钠和0.2ml的卵黄液。
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113826614A (zh) * 2021-09-30 2021-12-24 佛山市第一人民医院（中山大学附属佛山医院） 一种器官保存液
CN115386538A (zh) * 2021-05-24 2022-11-25 上海长一干细胞研究中心有限公司 一种从月经血中分离、提取、培养子宫内膜干细胞的方法和专用培养基

Families Citing this family (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109566597A (zh) * 2018-11-05 2019-04-05 北京世纪劲得生物技术有限公司 一种宫内膜干细胞保护液及其制备方法
CN109609442A (zh) * 2019-01-03 2019-04-12 贵州大学 一种鸭子宫上皮细胞分离及培养方法
CN113812397B (zh) * 2021-09-30 2023-03-14 佛山市第一人民医院（中山大学附属佛山医院） 一种灌注保存液
CN116965403B (zh) * 2023-09-22 2023-12-15 诺莱生物医学科技有限公司 一种干细胞保存液及其制备方法和应用
CN117165534B (zh) * 2023-10-31 2024-01-16 山东大学齐鲁医院 一种永生化人胃癌成纤维细胞株及其构建和增殖强化方法

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102296048A (zh) * 2011-08-24 2011-12-28 杭州易文赛生物技术有限公司 从刮宫样品中获取人宫内膜间充质干细胞的方法
CN103146646A (zh) * 2013-02-22 2013-06-12 浙江大学 一种动员间充质干细胞的制剂及分离方法
CN103289958A (zh) * 2013-07-09 2013-09-11 天津市人民医院 干细胞小球诱导培养液及干细胞小球培养方法
WO2014132100A1 (en) * 2013-03-01 2014-09-04 Mater Medical Research Institute Limited Mobilizing agents and uses therefor
CN105176923A (zh) * 2015-10-12 2015-12-23 浙江生创精准医疗科技有限公司 一种从经血中制备宫内膜间充质干细胞的方法
CN105624099A (zh) * 2016-02-03 2016-06-01 广州赛莱拉干细胞科技股份有限公司 经血源宫内膜干细胞的分离培养方法

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101096653A (zh) * 2006-06-28 2008-01-02 中国人民解放军军事医学科学院基础医学研究所 一种在体外大规模扩增子宫内膜细胞的方法

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102296048A (zh) * 2011-08-24 2011-12-28 杭州易文赛生物技术有限公司 从刮宫样品中获取人宫内膜间充质干细胞的方法
CN103146646A (zh) * 2013-02-22 2013-06-12 浙江大学 一种动员间充质干细胞的制剂及分离方法
WO2014132100A1 (en) * 2013-03-01 2014-09-04 Mater Medical Research Institute Limited Mobilizing agents and uses therefor
CN103289958A (zh) * 2013-07-09 2013-09-11 天津市人民医院 干细胞小球诱导培养液及干细胞小球培养方法
CN105176923A (zh) * 2015-10-12 2015-12-23 浙江生创精准医疗科技有限公司 一种从经血中制备宫内膜间充质干细胞的方法
CN105624099A (zh) * 2016-02-03 2016-06-01 广州赛莱拉干细胞科技股份有限公司 经血源宫内膜干细胞的分离培养方法

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
PIRET, J. P .: "CoC12, a chemical inducer of hypoxia-inducible factor-1, and hypoxia reduce apoptotic cell death in hepatoma cell line HepG2", ANN. N. Y. ACAD. SCI., 31 December 2002 (2002-12-31), pages 443 - 447, XP055603254 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115386538A (zh) * 2021-05-24 2022-11-25 上海长一干细胞研究中心有限公司 一种从月经血中分离、提取、培养子宫内膜干细胞的方法和专用培养基
CN113826614A (zh) * 2021-09-30 2021-12-24 佛山市第一人民医院（中山大学附属佛山医院） 一种器官保存液
CN113826614B (zh) * 2021-09-30 2023-03-14 佛山市第一人民医院（中山大学附属佛山医院） 一种器官保存液

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