CN104560870B - A kind of method for preparing decidua mescenchymal stem cell - Google Patents
A kind of method for preparing decidua mescenchymal stem cell Download PDFInfo
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention provides a kind of method for preparing decidua mescenchymal stem cell, including the use of male's term fetus placenta as raw material, aseptic process placenta；Separate decidua tissue；Identify decidua tissue and obtain decidua mescenchymal stem cell whether the identification from maternal tissue；Cultivate decidua mescenchymal stem cell；Decidua mescenchymal stem cell freezes；The recovery of decidua mescenchymal stem cell.The method of the present invention has following technical characterstic：Gather fetus be male placenta, obtain decidua tissue after through sex identification whether the pollution from parent without daughter tissue；Germ contamination method is taken precautions against, pollution probability is reduced from collection source, repeatedly rinses surface, effectively reduce pollution probability；Using single enzymic digestion, flow can be simplified；Animal derived components are reduced using serum free medium culture to use, and cell stable performance, can be maintained decidua mescenchymal stem cell long-term cultivation process in vitro, be maintained cellular morphology, multiplication capacity, the expression of MSC surface markers, differentiation capability etc..
Description
Technical Field
The invention relates to a method for preparing periostracum mesenchymal stem cells, and belongs to the technical field of stem cells.
Background
Mesenchymal Stem Cells (MSCs) are a class of pluripotent stem cells derived from developing mesoderm, have self-renewal and multidirectional differentiation potential, can be differentiated into various types of histiocytes under specific induction conditions, and can form various tissues such as bone, cartilage, fat, cardiac muscle and the like. Mesenchymal stem cells were first mainly derived from bone marrow, but the content of mesenchymal stem cells (BMMSCs) in human bone marrow was extremely low. In recent years, mesenchymal cells having characteristics of stem/progenitor cells have been isolated from various tissues such as peripheral blood, dental pulp, cartilage, muscle, umbilical cord, placenta, and the like. Umbilical cord mesenchymal stem cells, amnion mesenchymal stem cells and chorion mesenchymal stem cells derived from the daughter and decidua mesenchymal stem cells derived from the mother can be separated from the placenta tissue. Adult mesenchymal cells can be differentiated into various cells with specific functions under different conditions, such as osteoblasts, chondrocytes, adipocytes, cardiomyocytes, myocytes and the like, and have great potential medical and storage values. The umbilical cord, amnion and chorion of placenta can be used for extracting mesenchymal stem cells, and can be used for transplanting or cryopreserving cells of children. The mesenchymal stem cells extracted from the decidua of the placenta are from the mother body, can be used for transplanting or cryopreserving the cells of the mother, and have pertinence.
The decidua mesenchymal stem cells are usually obtained by enzyme digestion of decidua tissues, are cultured by using a culture medium containing 10% FBS (fetal bovine serum), and are frozen by using a frozen stock solution containing 10% DMSO (dimethylsulfoxide).
In the prior art, the method for preparing decidua mesenchymal stem cells generally has the following defects:
1) the conventional techniques for separating decidua tissue cannot separate decidua tissue derived from the mother alone, and chorion tissue or amniotic tissue derived from the placenta and derived from the daughter is always mixed.
2) The prior art of placenta collection and decidua separation lacks effective pollution reduction measures, has limited anti-pollution measures, high pollution rate of primary culture, lacks a method for reducing pollution from a collection source, and currently, antibiotics are added in a culture link to inhibit the propagation of pollution bacteria only in the culture process, so that the pollution cannot be effectively reduced;
2) the existing enzyme digestion method is complex and needs multiple times of enzyme digestion;
3) the operations such as enzyme digestion, cryopreservation recovery and the like can cause cell damage to cells;
4) cells are easy to age, the cells are flat, the proliferation is slow or stopped, the differentiation capacity is lost and the like in the stem cell culture process, and no method or technical experience is provided at present for explaining the fact that the mesenchymal stem cell morphology of the periostracum is effectively maintained, the cells are prevented from aging, and the differentiation capacity of the cells is maintained in the proliferation process;
5) after long-term passage of mesenchymal stem cells, telomerase inactivation, increase of oncogene expression, decrease of oncogene expression, change of karyotype and other risks may occur, and currently, research data on the aspect of decidua mesenchymal stem cells are lacked.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides a method for preparing simple decidua mesenchymal stem cells.
The method for preparing the decidua mesenchymal stem cells comprises the following steps:
1) using the placenta of a male full-term fetus discharged in 0-30 min during childbirth as a raw material; washing the placenta with placenta cleaning solution for 1-3 times, and soaking the placenta in the placenta cleaning solution;
2) transporting the placenta soaked in the placenta cleaning solution to a laboratory within 48h, taking out the placenta, cleaning for 1-3 times by using the cleaning solution again, carefully separating decidua from the bottom of the placenta, soaking in the cleaning solution for 0-30 min after cleaning, and shearing decidua tissues into pieces with the diameter of 1-3 mm3A size tissue mass;
3) carrying out sex identification on the decidua tissue sample, and determining that the collected decidua tissue is solely from the mother body;
4) obtaining decidua mesenchymal stem cells from the decidua tissue mass by a collagenase digestion method;
5) culturing decidua mesenchymal stem cells by using a serum-free culture medium, wherein after the decidua mesenchymal stem cells grow to 80% density in the P0 generation, digesting by using 0.25% pancreatin for 3-5min, after the P0 generation, the cells are passaged within 48-72h after passage operation, the cell density is required to be 80% -90% during passage, and the cells are subjected to passage according to the formula (3-8) × 103Per cm2Density inoculation, wherein the change of the pH value of the culture medium is observed during the culture process, once the culture medium turns yellow, a fresh culture medium is added, the same amount of old culture medium in a culture vessel is removed, and cells are passaged from P0 generation to any generation between P1 and P30;
wherein,
the cleaning solution in the step 1) and the step 2) is 0.9% of normal saline;
the collagenase used in the collagenase digestion method in the step 4) is collagenase II with the final concentration of 1-5 mg/ml;
the serum-free culture medium in the step 5) consists of the following components: DMEM/F12, PDGF-BB, bFGF, (TGF) -beta 1, EGF and Transferrin; wherein the PDGF-BB content is 50-100 ng/mL; the bFGF content is 0-50 ng/mL; the content of (TGF) -beta 1 is 0-20 ng/mL; the content of EGF is 0-30 ng/mL; the content of Transferrin is 2.0-4.5 mg/mL.
Preferably, the first and second electrodes are formed of a metal,
the cleaning solution in the step 1) and the step 2) also contains antibiotics, and the antibiotics are penicillin, streptomycin or amphotericin B.
The collagenase digestion method in the step 4) comprises the following steps: putting the decidua tissue block into a temperature of 37 ℃, carrying out oscillatory digestion for 15-60min, adding physiological saline with the volume 1-5 times of that of the tissue block, centrifuging for 5min at the rotating speed of 2500 rpm, removing supernatant, transferring a product obtained after bottom digestion into a culture vessel, adding a serum-free culture medium according to the proportion that 1ml of decidua tissue block is added with 10ml of the serum-free culture medium, wherein the serum-free culture medium in the step 4) comprises the following components: DMEM/F12, PDGF-BB, bFGF, (TGF) -beta 1, EGF and Transferrin; wherein the PDGF-BB content is 50-100 ng/mL; the bFGF content is 0-50 ng/mL; the content of (TGF) -beta 1 is 0-20 ng/mL; the content of EGF is 0-30 ng/mL; the content of Transferrin is 2.0-4.5 mg/mL.
The serum-free culture medium in the step 4) consists of the following components: DMEM/F12+80ng/mL PDGF-BB +20ng/mL bFGF +10ng/mL (TGF) - β 1+10ng/mL EGF +3.0mg/mL transferrin.
The serum-free culture medium in the step 4) is also added with antibiotics, and the antibiotics are penicillin, streptomycin or amphotericin B.
The serum-free culture medium in the step 5) consists of the following components: DMEM/F12+80ng/mL PDGF-BB +20ng/mL bFGF +10ng/mL (TGF) - β 1+10ng/mL EGF +3.0mg/mL transferrin.
The serum-free culture medium for culturing the P0 generation cells in the step 5) is also added with antibiotic, and the antibiotic is penicillin, streptomycin or amphotericin B.
The cryopreservation method of the decidua mesenchymal stem cells comprises the following steps: every 10 th6-107Adding 1ml of freezing solution into the cells, putting the cells into a freezing box, transferring the cells into a refrigerator at the temperature of-80 ℃, and transferring the cells into liquid nitrogen or gas nitrogen at the temperature of-196 ℃ for storage for later use after 12-24 hours; the freezing stock solution consists of base solution and osmotic freezing protective agent, the concentration of the osmotic freezing protective agent is 1-1.4mol/L, and the base solution is culture solution DMEM/F12; the osmotic cryoprotectant is dimethyl sulfoxide.
The method for preparing the decidua mesenchymal stem cells has the following technical effects:
1) the full-term placenta of which the fetus is male is selected as a raw material, so that a pollution prevention method can be realized, the pollution probability is reduced from the source, the surface is washed for multiple times, and the pollution probability is effectively reduced;
2) limiting decidua material taking area, namely gently stripping the most superficial decidua tissue from the bottom of the placenta to reduce the pollution of mixed cells; and meanwhile, sex identification is carried out on the stripped decidua tissue to ensure that the stripped decidua tissue is free from pollution of other tissues of the placenta.
3) According to the method, when the periostracum mesenchymal stem cells are separated, single enzyme digestion is adopted, so that the process can be simplified;
4) serum-free culture medium is used for culture to reduce the use of animal-derived components, the cells can be passaged from the generation P0 to the generation P30 according to the method, the stable performance can be maintained, the in-vitro long-term culture process of the decidua mesenchymal stem cells can be maintained, the cell morphology, the proliferation capacity, the MSC surface marker expression and the differentiation capacity can be maintained, the in-vitro long-term culture process of the decidua mesenchymal stem cells can be maintained, and the telomerase expression, the stable expression of cancer genes and the stable karyotype can be maintained;
5) in the method, enzymatic digestion and cryopreservation recovery are harmless to cells.
Drawings
FIG. 1 is a morphological diagram of prepared decidua mesenchymal stem cells;
FIG. 2-1 is a result of sex determination of the separated decidua tissue and other placental tissues;
FIG. 2-2 is a graph showing the results of surface marker detection of prepared decidua mesenchymal stem cells;
FIG. 3 is a morphological diagram of decidua mesenchymal stem cells after digestion with digestive enzymes;
FIG. 4 is a graph showing the results of measuring the growth curve of decidua mesenchymal stem cells after digestion with digestive enzymes;
FIG. 5 is a morphological diagram of decidua mesenchymal stem cells from generation P0 to generation P10 cultured in a serum-free medium;
FIG. 6 is a graph of decidua mesenchymal stem cells of generations P2, P5 and P10 cultured in serum-free medium, against the morphology of aged and undifferentiated cells;
FIG. 7 is a graph showing the results of measurement of growth curves;
FIG. 8 is a graph showing the results of the differentiation assay induced by osteogenesis, adipogenic cartilage formation;
FIG. 9 is a graph of the results of the flow assay;
FIG. 10 is a graph showing the results of telomerase detection;
FIG. 11 is a graph showing the results of oncogene detection;
FIG. 12 is a graph showing the result of a G banding karyotype analysis.
Detailed Description
Reagents used in the examples were purchased from manufacturers in parentheses following the reagent name.
Example 1 preparation of periostracum mesenchymal Stem cells
The method for preparing decidua mesenchymal stem cells in the embodiment comprises the following steps:
1) using the placenta of a male full-term fetus discharged in 0-30 min during childbirth as a raw material; an operator holds the placenta with sterile gloves to prevent the placenta from contacting objects (such as the ground and the like) which are possibly polluted in the operation process, the placenta is washed for 3 times, the washing liquid is 0.9 percent of normal saline (Guizhou Tiandi), antibiotics can be added or not added into the washing liquid, and the added antibiotics are penicillin (gibco), streptomycin (gibco) and amphotericin B (sigma). Wiping the surface with sterile gauze, removing blood stain on the surface, soaking in a placenta collecting box containing cleaning solution after no liquid drips on the surface of the placenta, and transporting the placenta soaked in the cleaning solution to a laboratory within 48 h.
2) After the placenta is transported to a laboratory, the placenta is placed in a 40cm dish with the length of × 40cm × 20cm (the length of × is the width of × cm), the outer surface of the dish is washed for 3 times by using a sterile cleaning solution, the cleaning solution is 0.9 percent of physiological saline (Guizhou Tiandi), antibiotics can be added or not added into the cleaning solution, the added antibiotics are penicillin (gibco), streptomycin (gibco) and amphotericin B (sigma), the placenta is inverted, the decidua is gently stripped from the outermost tissue at the bottom of the placenta, the decidua tissue which is sheared is cleaned by using the cleaning solution, the cleaning solution is 0.9 percent of physiological saline (Guizhou Tiandi), the cleaning solution is added to soak for 30min, and the decidua tissue is sheared into 1-3 mm3Tissue mass of size.
After the placenta source is treated, the pollution rate is effectively reduced in the subsequent culture process. The placenta source is not disinfected, the subsequent pollution incidence rate is (25.3 +/-7.2%), the pollution incidence rate is reduced to (2.4 +/-1.5%) according to the method, and the pollution incidence is obviously reduced.
3) Performing sex identification on decidua tissue sample, and determining that the collected decidua tissue is derived from mother body
The sex identification method comprises the following steps: extracting a total genome from tissues or cells to be identified according to a method of a kit (solarbio, D1800), adding a reagent of a PCR kit to expand a conserved sequence SYB on a Y chromosome, and performing agarose (takara) gel electrophoresis on the result. From the results, it can be seen that decidua tissue SYB is negative and derived from the maternal tissue, i.e., decidua, while other positive tissues are autologous tissues.
As shown in FIG. 2-1, the decidua tissue derived from the mother body showed negative conserved sequence on the Y chromosome, while the tissue derived from the chorion, amnion, umbilical cord showed positive identification. It can be seen that the decidua tissue is maternal in origin.
4) Isolation of decidua mesenchymal stem cells from decidua tissue mass
Adding 1-5ml of digestive enzyme (gibco) in a volume ratio of 1-5ml of tissue mass, wherein the digestive enzyme is collagenase II (gibco) with a final concentration of 1-5 mg/ml.
The digestion steps are as follows: placing the decidua tissue mass added with digestive enzyme at 37 deg.C, and performing shaking digestion for 15-60min with a shaker. Adding 0.9% physiological saline (Guizhou Tiandi) with volume 1-5 times of the tissue block volume, centrifuging at 2500 rpm for 5min, and removing supernatant. Transferring the product after bottom digestion into a culture vessel, and adding a serum-free culture medium according to the proportion that 1ml of decidua tissue block is added with 10ml of a serum-free culture medium (gibco). And continuing culturing for 5-20 days after the decidua mesenchymal stem cells climb out.
The serum-free medium comprises the following components: DMEM/F12(invitrogen) +80ng/mL PDGF-BB (gibco) +20ng/mLbFGF (invitrogen) +10ng/mL (TGF) - (. beta.) -1 (Peprotech) +10ng/mL EGF (gibco) +3.0mg/mL transferrin (sigma).
Serum-free medium can be supplemented with antibiotics such as penicillin (gibco), streptomycin (gibco), amphotericin B (sigma).
By adopting the method, the operation time of each tissue is saved, and the consumption of consumable reagents such as centrifuge tubes, physiological saline and the like is reduced; the digestion time is shortened from 90min to 60 min.
5) Culturing periostracum mesenchymal stem cells
Culturing the periostracum mesenchymal stem cells by using a serum-free culture medium.
The serum-free medium comprises the following components: DMEM/F12(invitrogen) +80ng/mL PDGF-BB (gibco) +20ng/mLbFGF (invitrogen) +10ng/mL (TGF) - (. beta.) -1 (Peprotech) +10ng/mL EGF (gibco) +3.0mg/mL transferrin (sigma).
The serum-free culture medium used for culturing the decidua mesenchymal stem cells of the generation P0 can be added with antibiotics, wherein the antibiotics are penicillin (gibco), streptomycin (gibco) and amphotericin B (sigma).
Culturing decidua mesenchymal stem cells with serum-free culture medium, growing to 80% density in P0 generation decidua mesenchymal stem cells, and digesting with 0.25% pancreatin (gibco) for 3-5 min. The generations after P0 were cultured in medium without antibiotics.
After the P0 generation, the cells are passaged within 48-72 hours after the passage operation, the cell density is required to reach 80-90% when the passage is carried out, and the cell density is 3 × 10% after the passage3-8×103The seeds are inoculated at the density of 2 per cm. During the cultivation, the pH of the medium was observed and fresh medium was added once the pH turned yellow. Cell morphology was recorded every 24h of photograph observation.
As shown in fig. 1, it can be seen that the obtained cell passage morphology was good;
as shown in fig. 2-2, the surface markers meet ISCT-to-mesenchymal stem cell detection criteria;
as shown in fig. 3, the cells maintained good morphology after the digestive enzymes acted directly on the cells;
as shown in fig. 4, the cell growth capacity was not affected after digestion;
as shown in table 1, the surface markings were unaffected. The formula of the digestive juice does not influence both the decidua mesenchymal stem cells and the decidua mesenchymal stem cells.
TABLE 1
As shown in FIG. 5, the decidua mesenchymal stem cells of P0-P10 generations cultured in serum-free medium have good morphology.
The morphological observation, proliferation test, induced differentiation, flow type, telomerase, oncogene expression and karyotype detection can be carried out at any time in the P0-P30 generations.
Firstly, the morphology of the cells is observed and recorded, and compared with the morphology of aged cells and undifferentiated cells, the detection result is shown in figure 6, and the cell morphology can be well maintained and is not aged after passage;
② the proliferation test comprises: the growth curve is measured, the detection result is shown in figure 7, and it can be seen that the cells maintain strong proliferation activity after multiple passages;
the induced differentiation detection comprises: the detection results of osteogenesis, adipogenesis and chondrogenesis are shown in figure 8, and it can be seen that the cells maintain the differentiation capacity after multiple passages;
the flow detection comprises the following steps: CD73, CD90, CD105, CD14, CD34, CD45, CD 79-alpha and HLA-DR, and the detection result is shown in figure 9, and it can be seen that the cell surface marker expression is maintained stably after multiple passages and accords with the MSC detection standard;
⑤ the telomerase detection method comprisesThe Detection result of the Telomerase Detection Kit (millipore) Kit is shown in FIG. 10, and it can be seen that the cells do not lose Telomerase activity after multiple passages;
the oncogene detection comprises: the Real-time PCR detection of the expression of oncogenes (c-Myc, c-fos, k-ras) and cancer suppressor genes (P53, P21, RB, P16) shows that the expression of the oncogenes and the cancer suppressor genes is relatively stable as shown in FIG. 11;
seventhly, nuclear type detection: the karyotype analysis using the G band is shown in FIG. 12, which shows that the karyotype of the cells after multiple passages is normal, and that the karyotype of the cells should be female karyotype, and that the sex chromosome is XX.
Therefore, the decidua mesenchymal stem cell obtained by the preparation method can maintain the cell morphology, the proliferation capacity, the MSC surface marker expression and the differentiation capacity in the in-vitro passage process, and can also maintain the long-term culture process of the decidua mesenchymal stem cell in vitro, the telomerase expression, the stable expression of oncogene and the stable karyotype.
6) Freezing decidua mesenchymal stem cells:
every 10 th6～107Adding 1ml of the cryopreservation solution into the cells, placing the cells into a cryopreservation box, transferring into a refrigerator at the temperature of-80 ℃, and transferring into liquid nitrogen or gas nitrogen at the temperature of-196 ℃ for preservation after 12-24 h.
The decidua mesenchymal stem cell frozen stock solution consists of a base solution and a permeable cryoprotectant, wherein the concentration of the permeable cryoprotectant is 1mol/L, and the base solution is a culture solution DMEM/F12 (invitrogen); the osmotic cryoprotectant is dimethyl sulfoxide (gibco).
7) Resuscitating decidua mesenchymal stem cells:
taking out decidua mesenchymal stem cells or decidua tissue blocks to be recovered from a liquid nitrogen tank, placing the decidua mesenchymal stem cells or decidua tissue blocks into a water bath kettle at 37 ℃ for incubation for 2-3min, washing the decidua mesenchymal stem cells or decidua tissue blocks with 0.9% pre-cooled at 4 ℃ by using normal saline (Guizhou Tiandi), centrifuging to remove supernatant, and adding a serum-free culture medium.
The serum-free medium used in this step had the same composition as in step 5).
Freezing recovery success rate: the freezing and the storing are recovered for 111 times, and the success rate is 100 percent. The cell morphology is good, the flow detection is qualified, and the differentiation capability is qualified.
The specific method steps involved in the various assays of the present invention are as follows. The parameters involved were determined by the following instruments: OLYMPUS inverted microscope, Leica upright (laica) microscope, FACAIra flow cytometer, UV-visible spectrophotometer, Biorad fluorescent PCR instrument, Biorad PCR instrument, incubator, cryorefrigerator, liquid nitrogen tank, and the like.
OLYMPUS microscope photograph
The cultured P2 generation decidua mesenchymal stem cells are fused to more than 80-90%, and the cells are photographed under an inverted microscope, and are in short fusiform shapes, uniform in size and orderly arranged, as shown in figure 6.
(II) drawing of growth Curve
Digesting the prepared cells to obtain a single cell suspension, and adjusting the concentration of the single cell suspension to 1 × 10 in a serum-free culture medium4Adding the mixture into a 96-well plate in a spot mode; setting 13 groups of 8 multiple wells each with 200 μ l each, culturing in incubator at 37 deg.C containing 5% CO2 and saturated humidity; changing the culture solution every 2 days, adding 20 μ l of 5mg/mL thiazole blue (MTT) (sigma, M5655) into each well after culturing for 1-13 days, and continuing culturing; after 4 hours the culture supernatant was carefully removed; add 100. mu.l of dimethyl sulfoxide (takara,67-68-5) to each well, shake for 5 minutes with a micro-oscillator; and (4) placing the sample on an enzyme-labeling instrument (Thermo) to measure the light absorption value at 570nm or 490nm, and drawing a growth curve after statistical analysis. The cell growth curve after digestion is shown in FIG. 4, and after multiple passages is shown in FIG. 7.
(III) identification of multidirectional differentiation Capacity
1 × 104The P2 generation cell suspension is inoculated in 24-hole cultureIn the plate, 0.5 ml/well; the cells were fused to 60-80% and replaced with osteogenic induction complete medium (gibco), 0.5 ml/well; replacing the induction culture solution for 3-4 days; after 24 days of induction, alizarin red (national drug group) was stained and photographed, and a large number of calcium nodules were observed, as shown in fig. 8.
1 × 104The P2 generation cell suspension of/m is inoculated in a 24-well culture plate, and 0.5 ml/well; cells were fused to 80-90% and replaced with adipogenic induction complete medium (gibco), 0.5 ml/well; replacing the induction culture solution for 3-4 days; after 14 days of induction, photographs were taken after oil red O (gibco) staining, and lipid droplets were produced in all cells, as shown in FIG. 8.
5 × 105The suspension of the P2 generation cells is inoculated in a centrifuge tube of 15ml, and is cultured after centrifugation, and is changed into a complete culture medium for cartilage induction (gibco) at 0.5 ml/hole the next day; replacing the induction culture solution for 3-4 days; after 21 days of induction, sections were stained and photographed with alcian blue (national drug group) and cartilage was produced in all cells, as shown in fig. 8.
(IV) flow cytometry
5 × 106The cells are digested into single cell suspension, the concentration is adjusted to 1 × 105/ml after the cells are washed twice by PBS, a flow cytometer (BD, FACAIra) detects positive indexes CD90, CD73 and CD105 of cell surface markers, negative indexes CD34, CD14, CD45, CD79a and HLA-DR, the positive cell rate exceeds 95 percent, the negative index accords with that below 2 percent, and the characteristics of the mesenchymal stem cells are met, as shown in figure 9.
(V) telomerase detection
Take 1x106Cell based onThe instruction of the Telomerase Detection Kit (mileore) Kit is used for extracting samples for identification, the Detection result is shown in FIG. 10, and it can be seen that the cells do not lose Telomerase activity after multiple passages.
(VI) detection of oncogene and cancer suppressor gene
Taking decidua mesenchymal stem cells 1x106RNA is extracted from cells (refer to QIAGEN RNA extraction kit instruction), reverse transcription is carried out to CDNA (TOYOBO First Strand cDNA Synthesis kit), and fluorescent quantitative PCR (biorad) detection is carried out by adding oncogene and cancer suppressor gene. Use 2-△△CtThe method analyzes experimental data and detects whether the expression of related protooncogenes and cancer suppressor genes changes significantly. The Real-time PCR detection of the expression of the oncogene (c-Myc, c-fos, k-ras) and the cancer suppressor gene (P53, P21, RB, P16) shows that the expression of the oncogene and the cancer suppressor gene is relatively stable as shown in FIG. 11.
(VII) karyotyping
Treating the decidua mesenchymal stem cells in culture with colchicine, digesting, centrifugally collecting, and washing with PBS. Treating with hypotonic KCL (0.075M) for 20-40 min, adding small amount of fixative dropwise, centrifuging at 1500rpm/5min to remove supernatant, retaining 1ml of supernatant, slightly blowing and suspending, slowly adding fixative (methanol: glacial acetic acid in a ratio of 3: 1), and shaking while adding until the tube is full. Centrifuging to remove supernatant, gently patting and suspending, slowly adding fixative while shaking until the tube is full. The supernatant was centrifuged at 4 ℃ overnight. And (4) dripping, namely putting the glass slide in an ice-water bath at 0 ℃, taking out the glass slide, and dripping the glass slide from a high position. After rapid fire-through or air-drying, giemsa staining, and viewing under the mirror (laica).
Claims (8)
1. A method for preparing periostracum mesenchymal stem cells is characterized by comprising the following steps:
1) using the placenta of a male full-term fetus discharged in 0-30 min during childbirth as a raw material; washing the placenta with placenta cleaning solution for 1-3 times, and soaking the placenta in the placenta cleaning solution;
2) transporting the placenta soaked in the placenta cleaning solution to a laboratory within 48h, taking out the placenta, cleaning for 1-3 times with the cleaning solution again, carefully separating the decidua from the bottom of the placenta, cleaning, soaking in the cleaning solution for 0-30 min, and collecting deciduaCutting into pieces of 1-3 mm3A size tissue mass;
3) carrying out sex identification on the decidua tissue sample, and determining that the collected decidua tissue is solely from the mother body;
4) obtaining decidua mesenchymal stem cells from the decidua tissue mass by a collagenase digestion method;
5) culturing decidua mesenchymal stem cells by using a serum-free culture medium, wherein after the decidua mesenchymal stem cells grow to 80% density in the P0 generation, digesting by using 0.25% pancreatin for 3-5min, after the P0 generation, the cells are passaged within 48-72h after passage operation, the cell density is required to be 80% -90% during passage, and the cells are subjected to passage according to the formula (3-8) × 103Per cm2Density inoculation, wherein the change of the pH value of the culture medium is observed during the culture process, once the culture medium turns yellow, a fresh culture medium is added, the same amount of old culture medium in a culture vessel is removed, and cells are passaged from P0 generation to any generation between P1 and P30;
wherein,
the cleaning solution in the step 1) and the step 2) is 0.9% of normal saline;
the collagenase used in the collagenase digestion method in the step 4) is collagenase II with the final concentration of 1-5 mg/ml;
the serum-free culture medium in the step 5) consists of the following components: DMEM/F12, PDGF-BB, bFGF, (TGF) -beta 1, EGF and Transferrin; wherein the PDGF-BB content is 50-100 ng/mL; the bFGF content is 0-50 ng/mL; the content of (TGF) -beta 1 is 0-20 ng/mL; the content of EGF is 0-30 ng/mL; the content of Transferrin is 2.0-4.5 mg/mL.
2. The method for preparing the decidua mesenchymal stem cell according to claim 1, wherein the washing solution in the steps 1) and 2) further contains an antibiotic, and the antibiotic is penicillin, streptomycin or amphotericin B.
3. The method for preparing the periostracum mesenchymal stem cell according to claim 1, wherein the collagenase digestion method in the step 4) comprises the following steps: putting the decidua tissue block into a temperature of 37 ℃, carrying out oscillatory digestion for 15-60min, adding normal saline with the volume 1-5 times of that of the tissue block, centrifuging for 5min at the rotating speed of 2500 rpm, removing supernatant, transferring a product obtained after bottom digestion into a culture vessel, adding a serum-free culture medium according to the proportion that 1ml of decidua tissue block is added with 10ml of the serum-free culture medium, wherein the serum-free culture medium comprises the following components: DMEM/F12, PDGF-BB, bFGF, (TGF) -beta 1, EGF and Transferrin; wherein the PDGF-BB content is 50-100 ng/mL; the bFGF content is 0-50 ng/mL; the content of (TGF) -beta 1 is 0-20 ng/mL; the content of EGF is 0-30 ng/mL; the content of Transferrin is 2.0-4.5 mg/mL.
4. The method for preparing decidua mesenchymal stem cells according to claim 3, wherein the serum-free medium consists of the following components: DMEM/F12+80ng/mL PDGF-BB +20ng/mL bFGF +10ng/mL (TGF) - β 1+10ng/mL EGF +3.0mg/mL transferrin.
5. The method for preparing decidua mesenchymal stem cells according to claim 3 or 4, wherein the serum-free medium is further added with an antibiotic, and the antibiotic is penicillin, streptomycin or amphotericin B.
6. The method for preparing the periostracum-derived mesenchymal stem cell according to claim 1, wherein the serum-free medium in the step 5) is composed of the following components: DMEM/F12+80ng/mL PDGF-BB +20ng/mL bFGF +10ng/mL (TGF) - β 1+10ng/mL EGF +3.0mg/mL transferrin.
7. The method for preparing decidua mesenchymal stem cells according to claim 1 or 6, wherein the serum-free culture medium for culturing the P0 generation cells in the step 5) is further added with an antibiotic, and the antibiotic is penicillin, streptomycin or amphotericin B.
8. The method for preparing decidua mesenchymal stem cell according to claim 1, wherein the method for cryopreserving the decidua mesenchymal stem cell comprises the following steps: every 10 th6-107Adding 1ml of freezing solution into cells, putting the cells into a freezing box, transferring the cells into a refrigerator at the temperature of-80 ℃, transferring the cells into liquid nitrogen or gas nitrogen at the temperature of-196 ℃ for storage for later use after 12-24 hours, wherein the freezing solution consists of base solution and a permeable freezing protective agent, the concentration of the permeable freezing protective agent is 1-1.4mol/L, and the base solution is culture solution DMEM/F12; the osmotic cryoprotectant is dimethyl sulfoxide.
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