CN113616676A - Application of umbilical cord blood stem cell secretion in promoting growth of endometrial cells - Google Patents
Application of umbilical cord blood stem cell secretion in promoting growth of endometrial cells Download PDFInfo
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- C12N5/0602—Vertebrate cells
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0681—Cells of the genital tract; Non-germinal cells from gonads
- C12N5/0682—Cells of the female genital tract, e.g. endometrium; Non-germinal cells from ovaries, e.g. ovarian follicle cells
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- C12N2310/00—Structure or type of the nucleic acid
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- C12N2310/141—MicroRNAs, miRNAs
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- C12N2510/00—Genetically modified cells
Abstract
The invention provides application of an umbilical cord blood stem cell secretion in promoting growth of endometrial cells, and belongs to the technical field of biological medicines. The application provided by the invention comprises the application of the secretion of the cord blood mesenchymal stem cells in preparing an endometrial cell proliferation promoter and the application of the secretion of the cord blood mesenchymal stem cells in preparing an endometrial cell migration promoter, wherein the cord blood mesenchymal stem cells are cord blood mesenchymal stem cells transfected with AC 100830.3-siRNA.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to application of an umbilical cord blood stem cell secretion in promoting growth of endometrial cells.
Background
With the continuous improvement of living standard, the aging of population becomes a common problem in countries all over the world, and the improvement of fertility rate is a necessary choice for effectively slowing down and solving the aging problem of population. At present, the three-birth policy has been opened in our country, but studies show that there are a pair of infertile couples in every 6 couples in our country. Therefore, the infertility is effectively solved, and the fertility rate is effectively improved.
The uterus is an important reproductive organ, which can be divided into 3 layers, namely an intima layer, a muscular layer and a serosa layer from inside to outside. The endometrium is mainly composed of endometrial epithelial cells, endometrial stromal cells, blood vessels and glands. Studies have shown that the risk of female miscarriage is higher when the thickness of the endometrium is thinner, the implantation and pregnancy rates are lower. Thus, an effective increase in endometrial thickness would help to increase fertility. Endometrial stromal cells are the major component of the interior, and their proliferation plays a key role in embryo implantation and pregnancy establishment.
Disclosure of Invention
The invention aims to provide application of an umbilical cord blood stem cell secretion in promoting growth of endometrial cells.
In order to achieve the above-mentioned objects,
the invention provides application of secretion of cord blood mesenchymal stem cells in preparing an endometrial cell proliferation promoter, wherein the cord blood mesenchymal stem cells are cord blood mesenchymal stem cells transfected with AC 100830.3-siRNA.
Preferably, the sequence of the AC100830.3 is shown as SEQ ID NO.1, and the sequence of the AC100830.3-siRNA is shown as SEQ ID NO.2 and SEQ ID NO. 3.
Secondly, the invention provides an application of secretion of cord blood mesenchymal stem cells in preparing an endometrial cell migration promoter, wherein the cord blood mesenchymal stem cells are cord blood mesenchymal stem cells transfected with AC 100830.3-siRNA.
Preferably, the sequence of the AC100830.3 is shown as SEQ ID NO.1, and the sequence of the AC100830.3-siRNA is shown as SEQ ID NO.2 and SEQ ID NO. 3.
Secondly, the invention provides application of AC100830.3-siRNA in promoting the secretion of cord blood mesenchymal stem cells to the proliferation promotion effect of endometrial cells, which is characterized in that the sequence of AC100830.3 is shown as SEQ ID NO.1, and the sequence of AC100830.3-siRNA is shown as SEQ ID NO.2 and SEQ ID NO. 3.
Preferably, the application comprises the application of the AC100830.3-siRNA in promoting the effect of secretion of umbilical cord blood mesenchymal stem cells on promoting the cyclin in the endometrium.
Finally, the invention provides application of the AC100830.3-siRNA in promoting the secretion of the umbilical cord blood mesenchymal stem cells to the promotion effect of migration of endometrial cells, which is characterized in that the sequence of the AC100830.3 is shown as SEQ ID NO.1, and the sequence of the AC100830.3-siRNA is shown as SEQ ID NO.2 and SEQ ID NO. 3.
The invention has the beneficial effects that:
the invention provides application of secretion of umbilical cord blood mesenchymal stem cells transfected with AC100830.3-siRNA in promoting endometrial cell proliferation and endometrial cell migration. Therefore, the secretion of the umbilical cord blood mesenchymal stem cells transfected with the AC100830.3-siRNA can be used for preparing the medicine for promoting the proliferation and migration of endometrial cells, and a foundation is provided for further preparing the medicine for treating infertility.
Drawings
FIG. 1 shows that the inhibition effect of AC100830.3-siRNA to AC100830.3 in umbilical cord blood mesenchymal stem cells is P <0.05, and the difference is significant;
FIG. 2 the effect of secretion of umbilical cord blood mesenchymal stem cells transfected with AC100830.3-siRNA on proliferation of endometrial stromal cells, P <0.05, the difference is significant;
FIG. 3 effect of secretion of cord blood mesenchymal stem cells transfected with AC100830.3-siRNA on Cyclin-D1 and CDK1 of endometrial stromal cells;
figure 4 effect of secretion of umbilical cord blood mesenchymal stem cells transfected with AC100830.3-siRNA on migration of endometrial stromal cells, P <0.05, difference is significant.
Detailed Description
In order to clearly illustrate the technical features of the present solution, the present solution is explained below by way of specific embodiments.
Example 1
(1) The obtained endometrial sample is put into ice-cold Hank's solution added with double antibody to be washed for 3 times under a sterile environment;
(2) placing the tissue on ice, and cutting the tissue into tissue blocks of 1mm × 1mm × 1mm size by using ophthalmic scissors;
(3) transferring the tissue blocks to a triangular flask, adding a proper amount of 0.2% collagenase, and incubating at 37 ℃ for 1 h;
(4) adding 15U/ml DNaseI to continue digesting for 30min, and filtering the obtained cell suspension by using a 50-mesh screen after digestion is finished;
(5) then filtering by using 200-mesh and 400-mesh screens,
(6) acquisition of endometrial stromal cells: collecting filtrate, centrifuging at 900r/min for 5min, collecting cells, performing staining count with dolol blue at 2.5 × 105Inoculating at a density of one cell per ml, removing the culture medium by aspiration after 2h, and adding new culture medium to obtain endometrial stromal cells.
Example 2
(1) Collecting umbilical blood of a healthy caesarean section lying-in woman, diluting and uniformly mixing the umbilical blood with PBS (phosphate buffer solution) in a ratio of 1: 1;
(2) pouring equal volume of Percoll liquid into a centrifuge tube, uniformly mixing, placing the centrifuge tube in a centrifuge, and centrifuging at 2000rpm/min for 20 min;
(3) after the centrifugation is finished, carefully sucking the middle flocculent leucocyte layer and resuspending the cells by using PBS;
(4) placing the centrifugal tube in a centrifuge again, centrifuging at 1000rpm/min for 10min, and collecting cells at the bottom of the centrifugal tube;
(5) adding DMEM/F12 medium containing 10% fetal bovine serum, 0.1% penicillin and streptomycin double antibody to resuspend cells;
(6) after washing the cells again by centrifugation 2 times using DMEM/F12 medium, the cells were counted and the density of the cells was adjustedThe whole is 1 × 106And/ml, inoculating the cells into a cell culture dish, and adding a culture medium for culturing to obtain the umbilical cord blood mesenchymal stem cells.
Example 3
Transgenic improved umbilical blood mesenchymal stem cells
(1) Umbilical blood mesenchymal stem cells are inoculated in a cell culture plate, one group of cells are transfected with NC-siRNA, the other group of cells are inoculated with AC100830.3-siRNA (the sequence of the AC100830.3 gene is shown as SEQ ID NO. 1), and the sequence of the AC100830.3-siRNA is shown as follows:
sense strand: UUUCUUGUCUUUGUAUAUC, SEQ ID NO. 2;
antisense strand: GAUAUACAAAGACAAGAAA, SEQ ID N0.3;
(2) respectively transfecting NC-siRNA and AC100830.3-siRNA into umbilical cord blood mesenchymal stem cells according to the Lipofectamin2000 specification, continuously culturing for 48h, and extracting RNA;
(3) the obtained RNA is reversely transcribed into cDNA according to the instruction of the takara reverse transcription kit;
(4) and (3) detecting the expression level of the gene AC100830.3 in the transfected cells by referring to the specifications of the takara fluorescent quantitative PCR kit, wherein the reaction primers are as follows:
GAPDH upstream primer (5'- >3'): AATGGGCAGCCGTTAGGAAA, SEQ ID NO. 4;
GAPDH downstream primer (5'- >3'): GCGCCCAATACGACCAAATC, SEQ ID NO. 5;
AC100830.3 upstream primer (5'- >3'): GACTGACTTCCCTGGGATGC, SEQ ID NO. 6;
AC100830.3 downstream primer (5'- >3'): GCAGTGGTGTGATCTCAGCT, SEQ ID NO. 7;
(5) by using 2-ΔΔCtThe relative expression level of the AC100830.3 gene was calculated.
The obtained experimental results are shown in FIG. 1, wherein A represents the cells transfected with NC-siRNA, and B represents the cells transfected with AC100830.3-siRNA, and the relative expression level of AC100830.3 in B is 0.142 +/-0.293, which indicates that the AC100830.3-siRNA can effectively inhibit the expression level of AC100830.3 after being transfected with AC 100830.3-siRNA.
Example 3
Effect of secretion of cord blood mesenchymal Stem cells transfected with AC100830.3-siRNA on proliferation of endometrial stromal cells
(1) Inoculating the umbilical cord blood mesenchymal stem cells into a cell culture plate, respectively transfecting NC-siRNA and AC100830.3-siRNA, culturing for 48h by using a serum-free culture medium, and collecting the culture medium, wherein the culture medium is respectively named as a culture medium A and a culture medium B;
(2) the endometrial stromal cells are inoculated in a 96-well cell culture plate, 5000 cells are per well, 100ul of a culture medium A containing 10% FBS is added to the group A cells after the cells are attached to the wall, 100ul of a culture medium B containing 10% FBS is added to the group B cells, and after the cells are cultured for 48 hours respectively, the OD value is detected by using CCK-8.
The results of the experiment are shown in FIG. 2, where A represents endometrial stromal cells cultured in culture medium A, and B represents endometrial stromal cells cultured in culture medium B. Wherein the OD value of the group A is 0.554 +/-0.060, the OD value of the group B is 0.768 +/-0.054, and the difference between the two values has statistical significance. Therefore, it can be concluded that the secretion of umbilical cord blood mesenchymal stem cells transfected with AC100830.3-siRNA has a promoting effect on endometrial stromal cell proliferation.
Example 4
Effect of secretion of cord blood mesenchymal Stem cells transfected with AC100830.3-siRNA on cyclin of endometrial stroma
(1) Inoculating the umbilical cord blood mesenchymal stem cells into a cell culture plate, respectively transfecting NC-siRNA and AC100830.3-siRNA, culturing for 48h by using a serum-free culture medium, and collecting the culture medium, wherein the culture medium is respectively named as a culture medium A and a culture medium B;
(2) inoculating endometrial stromal cells into a 6-well plate, culturing by using a complete culture medium until the cell density is 70%, replacing group A with a culture medium A, replacing group B with a culture medium B, and placing in a cell culture box for continuous culture for 48 h;
(3) after the culture is finished, washing the cells for 3 times by using PBS, adding 100ul of protein lysate containing protease inhibitors and phosphatase inhibitors, and performing lysis for 30min at 4 ℃;
(4) transferring the cell lysate into a 1.5ml EP tube, putting the EP tube into a precooling centrifuge with the temperature of 4 ℃, and centrifuging for 10min at 12000 rom;
(5) after the centrifugation is finished, taking the supernatant into a new centrifuge tube, and determining the protein concentration by using a BCA method, and storing at-20 ℃ for later use;
(6) preparing separation gel with proper concentration, preparing concentrated gel after the separation gel is solidified for 30 minutes at room temperature, and using the concentrated gel after the separation gel is solidified;
(7) adding a Loading buffer into the protein sample, and boiling and denaturing at 95 ℃ for 5 min;
(8) after the sample is loaded, pouring SDS-PAGE electrophoresis buffer solution for electrophoresis, setting the voltage to be 80V, and after about 30min, adjusting the voltage to be 120V until the electrophoresis is finished;
(9) stopping electrophoresis when the bromophenol blue runs out, making a sandwich membrane-rotating clamp, and rotating the membrane for 90min at a constant current of 300mA in an ice-water bath environment;
(10) taking out the membrane, putting the membrane into an antibody incubation box, adding 5% skimmed milk, and sealing at room temperature for 1 h;
(11) Cyclin-D1, CDK1, beta-actin primary antibody were diluted with 2% BSA in the corresponding proportions and incubated overnight at 4 ℃;
(12) the membrane was washed 3 times with 1 × TBST buffer for 30min, the corresponding secondary antibody was added, after incubation for 1h on a shaker, the secondary antibody was removed, and after washing 3 times with 1 × TBST, the membrane was developed.
The results of the experiment are shown in FIG. 3, where A represents endometrial stromal cells cultured in A medium and B represents endometrial stromal cells cultured in B medium. It can be seen that the expression level of Cyclin-D1 and CDK1 in group B is obviously higher than that in group A, and the secretion of cord blood mesenchymal stem cells transfected with AC100830.3-siRNA can be obtained to have the promotion effect on the expression of Cyclin-1 and CDK1 in endometrial stroma cells.
Example 5
Effect of secretion of cord blood mesenchymal Stem cells transfected with AC100830.3-siRNA on migration of endometrial stromal cells
(1) Inoculating the umbilical cord blood mesenchymal stem cells into a cell culture plate, respectively transfecting NC-siRNA and AC100830.3-siRNA, culturing for 48h by using a serum-free culture medium, and collecting the culture medium, wherein the culture medium is respectively named as a culture medium A and a culture medium B;
(2) placing the transwell chamber in a 24-hole cell culture plate, digesting endometrial stromal cells, and preparing a single cell suspension by using a serum-free culture medium;
(3) will be 1 × 105200ul of endometrial stromal cells were seeded in the upper chamber of a transwell chamber;
(4) adding a culture medium A into a lower chamber of a transwell chamber in the group A, and adding a culture medium B into a lower chamber of a transwl chamber in the group B;
(5) placing the cell culture plate in a cell culture box, continuing culturing for 48h, gently cleaning the transwell chamber by using PBS, and wiping off cells on the upper chamber surface of the filter membrane by using a cotton swab;
(6) adding fixing solution for treating for 20min, removing the fixing solution by suction, adding Giemsa staining solution for staining, and staining for 20min at room temperature;
(7) after staining was complete, the chamber was washed with PBS, blotted dry, and photographed under a microscope.
The results of the experiment are shown in FIG. 3, where A represents endometrial stromal cells cultured in A medium and B represents endometrial stromal cells cultured in B medium. It can be seen that the cells of group B passed through the transwell chamber significantly more than group a, and it can be concluded that secretion of umbilical cord blood mesenchymal stem cells transfected with AC100830.3-siRNA has a promoting effect on migration of endometrial stromal cells.
Sequence listing
<110> affiliated Hospital of Qingdao university
Application of <120> umbilical cord blood stem cell secretion in promoting growth of endometrial cells
<130> 7.19
<160> 7
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1609
<212> DNA
<213> Human source (Human)
<400> 1
attacctggg gagctttaaa actatatgct aataatcagg acactctagg ccaatgagac 60
caatctttga agcctgtgag catttccact cgtctgccaa ggcagagaca gccccatcca 120
gaagcaagaa tgagacctcg gggccctggc gcccttgagc caaccccatc tctgttctca 180
caacttgcct tgcctgacct ctggagaaaa acttcagaaa atatcaccct ttcttcccac 240
ctgctcccgc tggccaaaat ccccatcaaa gcctggcttc agcccctcag actgggaact 300
ctatccaaat ggagtgtggg gagtgtgggt gaggaaggac ggaaggaagt aggtcacacg 360
ctggctaccg cacaacgggg aagaggccaa cctcggaaag gcagaagagt ggccgccatg 420
ccgagcttcc agagatggag agaaggtcta ggaagccacc ttggggaagc cgcctgagca 480
ggagcggggc agccagggat cagagcccac ggcctgctgg ggacccatcc tgggcacggg 540
gctctgggct ctgtgcccat tctctgatgt gccacattgt tctgctactt ccttctctgg 600
aagtaataag ggggttgggg gtgggacagc acggccagga gtagatggag cggggtggag 660
ctgccagtgg gggaggaggc tgcggttcat tcccacgacc agctgccctt ctggccgccc 720
tgggactgcg tgggaaaagg tctgggaagt atgggggatg gtgtgtgtgt gtgtgtgtgt 780
gtgtgtgtgt gtgtgtgtgt gtgttttcat gtgtctgact cccttgcttg gcttcagtca 840
caggacaaag ccctttgaga aacagtcacc aacctgtgtt cccactgggc tctgtcctgg 900
gagaggcaca agaacccgcc ctgacgcctg agagaaagac tctgaaggcc tcaggaccaa 960
atccctggag gggtcttggc gtctaatgaa tctagattga ctgacttccc tgggatgctt 1020
ctggaacctt ctgctaagcc ctcagcttct tggcagagct ctattactaa ctggatatca 1080
gaatatgggt gcaaactaga catggggagt gaagtcagcc attctcacag catctaccta 1140
agaaggccag gatcctccac agccacgtag atcgtagagc cagtggcatg ccggggatga 1200
tatacaaaga caagaaactc tgaaacagag aacacaaact aacagcccat aggccaaatg 1260
cagcccgcag gcacatggct tatttggcca acacagcatt taagaaatgt ttgagccgag 1320
gctgggtgag gtggctcagg cctataatcc cagtactttg ggaggccgag gcaggtggat 1380
cacctgaggt caggagttca agaccagcct ggccaacatg acaaaacccc gtctctacta 1440
aaaatacaag aattagccag gtgtggtggt ccatgcctgc agtcccagct acccagctgc 1500
ttggggggct gaggcaggag aatcgcttga acctgggggg cggaggttgc cgtgagctga 1560
gatcacacca ctgcactcca gcctgggcga cagagcgaaa ctccgtctc 1609
<210> 2
<211> 19
<212> RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
uuucuugucu uuguauauc 19
<210> 3
<211> 19
<212> RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
gauauacaaa gacaagaaa 19
<210> 4
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
aatgggcagc cgttaggaaa 20
<210> 5
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
gcgcccaata cgaccaaatc 20
<210> 6
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
gactgacttc cctgggatgc 20
<210> 7
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
gcagtggtgt gatctcagct 20
Claims (7)
1. The application of the secretion of the cord blood mesenchymal stem cells in preparing the endometrial cell proliferation accelerant is characterized in that the cord blood mesenchymal stem cells are the cord blood mesenchymal stem cells transfected with AC 100830.3-siRNA.
2. The use of claim 1, wherein the sequence of the AC100830.3 is shown as SEQ ID No.1, and the sequence of the AC100830.3-siRNA is shown as SEQ ID No.2 and SEQ ID No. 3.
3. The application of the secretion of the cord blood mesenchymal stem cells in preparing the endometrial cell migration promoter is characterized in that the cord blood mesenchymal stem cells are transfected with AC 100830.3-siRNA.
4. The use of claim 3, wherein the sequence of AC100830.3 is shown in SEQ ID NO.1, and the sequence of AC100830.3-siRNA is shown in SEQ ID NO.2 and SEQ ID NO. 3.
The application of the AC100830.3-siRNA in promoting the secretion of cord blood mesenchymal stem cells to the proliferation promotion effect of endometrial cells is characterized in that the sequence of the AC100830.3 is shown as SEQ ID NO.1, and the sequence of the AC100830.3-siRNA is shown as SEQ ID NO.2 and SEQ ID NO. 3.
6. The use of claim 5, wherein the use comprises use of AC100830.3-siRNA in promoting the effect of secretion of cord blood mesenchymal stem cells on the promotion of endometrial cyclin.
The application of the AC100830.3-siRNA in promoting the secretion of cord blood mesenchymal stem cells to the promotion of migration of endometrial cells is characterized in that the sequence of the AC100830.3 is shown as SEQ ID NO.1, and the sequence of the AC100830.3-siRNA is shown as SEQ ID NO.2 and SEQ ID NO. 3.
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| CN114480626A (en) * | 2022-03-14 | 2022-05-13 | 青岛大学附属医院 | Gene related to preeclampsia and application thereof |
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| US20140329704A1 (en) * | 2013-03-28 | 2014-11-06 | President And Fellows Of Harvard College | Markers for mature beta-cells and methods of using the same |
| CN110408673A (en) * | 2019-07-01 | 2019-11-05 | 南通大学附属医院 | Study the method that Hsa_circ_0111659 repairs the mechanism of action in ESC in WJ-MSCs |
| WO2020130800A1 (en) * | 2018-12-21 | 2020-06-25 | Cytonex Sdn. Bhd. | Stem cell conditioned media |
| US20210238683A1 (en) * | 2018-04-20 | 2021-08-05 | The Board Of Regents Of The University Of Texas System | Compositions and Methods for Diagnosis and Treatment of Endometriosis |
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| US20140329704A1 (en) * | 2013-03-28 | 2014-11-06 | President And Fellows Of Harvard College | Markers for mature beta-cells and methods of using the same |
| US20210238683A1 (en) * | 2018-04-20 | 2021-08-05 | The Board Of Regents Of The University Of Texas System | Compositions and Methods for Diagnosis and Treatment of Endometriosis |
| WO2020130800A1 (en) * | 2018-12-21 | 2020-06-25 | Cytonex Sdn. Bhd. | Stem cell conditioned media |
| CN110408673A (en) * | 2019-07-01 | 2019-11-05 | 南通大学附属医院 | Study the method that Hsa_circ_0111659 repairs the mechanism of action in ESC in WJ-MSCs |
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| CN114480626A (en) * | 2022-03-14 | 2022-05-13 | 青岛大学附属医院 | Gene related to preeclampsia and application thereof |
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| CN113616676B (en) | 2022-05-17 |
| CN114788836A (en) | 2022-07-26 |
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