WO2022217869A1 - 用于***的壁蜕膜间充质干细胞培养基、共培方法 - Google Patents

用于***的壁蜕膜间充质干细胞培养基、共培方法 Download PDF

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WO2022217869A1
WO2022217869A1 PCT/CN2021/123377 CN2021123377W WO2022217869A1 WO 2022217869 A1 WO2022217869 A1 WO 2022217869A1 CN 2021123377 W CN2021123377 W CN 2021123377W WO 2022217869 A1 WO2022217869 A1 WO 2022217869A1
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cells
mesenchymal stem
decidual
parietal
medium
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French (fr)
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刘小翠
唐淑艳
邓燕莲
蒙燕瑶
雅思敏
杨景利
王进辉
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广东唯泰生物科技有限公司
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    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0662—Stem cells
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    • A01N1/0221—Freeze-process protecting agents, i.e. substances protecting cells from effects of the physical process, e.g. cryoprotectants, osmolarity regulators like oncotic agents
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
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    • C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
    • C12N2509/10—Mechanical dissociation

Definitions

  • the invention relates to the field of biotechnology, in particular to a culture medium and a co-cultivation method for parietal decidual mesenchymal stem cells for treating tumors.
  • MSCs Mesenchymal stem cells
  • PMSCs pariduval Mesenchymal stem cells
  • PMSCs Decidual parietal mesenchymal stem cells
  • PMSCs have the characteristics of simple in vitro proliferation and culture, no ethics, good migration ability, homing to tumor cells and low immunogenicity, and avoid the bone marrow.
  • Mesenchymal stem cells are difficult to obtain and involve risks such as medical ethics and ethics, which make them a new carrier to replace bone marrow-derived mesenchymal stem cells as a new carrier for tumor biotherapy.
  • the effects of maternal-derived mesenchymal stem cells on the biological behavior of malignant tumor cells reported in different studies are different. Studying the adaptability and selectivity of PMSCs to various malignant tumors is the basis for whether PMSCs can be used as carriers for malignant tumor treatment.
  • the technical problem to be solved by the present invention is to provide a culture medium for parietal decidual mesenchymal stem cells for the treatment of tumors for application in tumor treatment, the culture medium for parietal decidual mesenchymal stem cells containing wall Decidual mesenchymal stem cells and selection medium, the parietal decidual mesenchymal stem cells have homing effects on various tumor cells, inhibit proliferation and regulate cell cycle.
  • the technical problem to be solved by the present invention is to provide a co-cultivation method of the parietal decidual mesenchymal stem cell medium and tumor cells, so as to clarify the adaptability and selection effect of parietal decidual mesenchymal stem cells to the homing effect of malignant tumors , the homing effect of mesenchymal stem cells after co-culture is obvious, and the homing effect of decidual mesenchymal stem cells in a specific concentration range on different malignant tumors is clear.
  • the technical problem to be solved by the present invention is to provide a parietal decidual mesenchymal stem cell culture medium for treating tumors for application in tumor treatment, and the parietal decidual mesenchymal stem cells for treating tumors are provided.
  • the medium contains parietal decidual mesenchymal stem cells and a selection medium, and the parietal decidual mesenchymal stem cells have homing effects on various tumor cells, inhibit proliferation and regulate cell cycle.
  • the tumor is cervical tumor, breast tumor or lung tumor.
  • the preparation method of the parietal decidual mesenchymal stem cells comprises the following steps:
  • tissue digestion solution contains 40-60% Tryple-EDTA enzyme and 8-12 mg/ml type II Collagenase in high glucose DMEM medium;
  • the selection medium contains 8-12% serum substitute by volume, 0.5-1mol/ml L-glutamine, 18-25ng/ml basic fibroblast growth factor, 16-22ng /ml epidermal growth factor and 6-12ng/ml stem cell growth factor in DMEM serum-free medium.
  • the tissue washing solution is prepared from the following raw materials by volume percentage: 0.8-1.5% penicillin-streptomycin mixture, 50-55% red blood cell lysate, 44-49% normal saline, The mass fraction of the physiological saline is 0.8-1%.
  • step S12 the tissue digestion solution is vibrated with the tissue block at a temperature of 36 to 39° C. for 1.5 to 4 hours, and the vibration speed is 150 to 200 rpm/min;
  • the centrifugation speed is 1200 ⁇ 1400rpm/min, and the centrifugation time is 5 ⁇ 7min.
  • step S15 when the cell confluence is greater than 80%, the cell surface is washed with PBS buffer at least twice;
  • the centrifugation speed is 1200 ⁇ 1400rpm/min, and the centrifugation time is 5 ⁇ 7min.
  • the method before collecting the parietal decidual mesenchymal stem cells, the method further includes: performing surface antibody marker detection on the parietal decidual mesenchymal stem cells, when the positive indicators of CD73, CD90 and CD105 are >99 at the same time %, the parietal decidual mesenchymal stem cells were collected;
  • the parietal decidual mesenchymal stem cells of the P3 generation were collected, and the parietal decidual mesenchymal stem cells of the P3 generation were digested with trypsin, centrifuged, the supernatant was removed, the cryopreserved solution was added to the pellet, and the cryopreservation was stored in a liquid nitrogen tank. preserved in.
  • the cryopreservation solution is a serum-free complete medium containing 18-25% Cryosure-DEX-40 by volume;
  • the density of cryopreserved cells is 1.5 ⁇ 10 6 to 2.5 ⁇ 10 6 cells/ml.
  • the present invention also provides a co-cultivation method of a parietal decidua mesenchymal stem cell medium and tumor cells, comprising:
  • the above-mentioned wall decidual mesenchymal stem cell culture medium is placed in the upper chamber of the Transwell chamber for cultivation;
  • the tumor cells were cultured in the lower chamber of the Transwell chamber;
  • the upper chamber containing the parietal decidual mesenchymal stem cells was placed in a well plate and co-cultured with the lower chamber tumor cells.
  • the parietal decidual mesenchymal stem cell culture medium of the present invention contains parietal decidual mesenchymal stem cells and a selection medium, wherein the parietal decidual mesenchymal stem cells pass homing, inhibit proliferation and regulate cell cycle to inhibit tumor cells.
  • the present invention adopts serum substitute containing 8-12% volume concentration, 0.5-1mol/ml L-glutamine, 18-25ng/ml basic fibroblast growth factor, 16-22ng/ml epidermal growth factor and 6 DMEM serum-free medium containing ⁇ 12ng/ml stem cell growth factor is used as a selective medium to terminate digestion, and resuspend decidual-derived mesenchymal stem cells from the parietal decidua, which is beneficial to improve the purity of parietal decidual mesenchymal stem cells and accelerate the Mesenchymal stem cell growth to achieve rapid in vitro expansion of parietal decidual mesenchymal stem cells.
  • high-sugar DMEM medium containing 40-60% Tryple-EDTA enzyme and 8-12 mg/ml type II collagenase by volume is used as tissue digestion solution to digest tissue blocks, which is conducive to the crawling of parietal decidua mesenchymal stem cells out of the tissue for adherent growth.
  • the co-cultivation method of the parietal decidual mesenchymal stem cell medium and tumor cells of the present invention clarifies the adaptability and selection effect of parietal decidual mesenchymal stem cells for the homing effect of malignant tumors, so that the mesenchymal stem cells after co-cultivation
  • the homing trajectory of stem cells is obvious, and the homing effect of decidual mesenchymal stem cells in a specific concentration range on different malignant tumors is clarified.
  • Fig. 1 is the cell appearance diagram of the electron microscope after the P5 generation PMSCs are cultured in the upper chamber of the Transwell chamber for 3 days in Example 2 of the present invention
  • Fig. 2 is the cell appearance diagram of the electron microscope after Hela cells are cultured and stabilized in the lower chamber of the Transwell chamber in Example 3 of the present invention
  • Fig. 3 is the cell appearance diagram of the electron microscope after the co-culture of PDB-MSCs and Hela cells in the low concentration group in Example 3 of the present invention
  • Fig. 4 is the cell appearance diagram of the electron microscope after the co-culture of PDB-MSCs in the middle concentration group and Hela cells in Example 3 of the present invention
  • Fig. 5 is the cell appearance diagram of the electron microscope after the co-culture of PDB-MSCs and Hela cells in the high concentration group in Example 3 of the present invention
  • FIG 6 is the cell appearance diagram of the electron microscope after the MCF-7 cells are stabilized in the lower chamber of the Transwell chamber in Example 4 of the present invention.
  • Fig. 7 is the cell appearance diagram of the electron microscope after the co-culture of PDB-MSCs and MCF-7 cells in the low concentration group in Example 4 of the present invention.
  • Fig. 8 is the cell appearance diagram of the electron microscope after the middle concentration group PDB-MSCs and MCF-7 cell co-culture in Example 4 of the present invention.
  • Fig. 9 is the cell appearance diagram of the electron microscope after the co-culture of PDB-MSCs and MCF-7 cells in the high concentration group in Example 4 of the present invention.
  • Figure 10 is the cell appearance diagram of the electron microscope after A549 cells are stabilized in the lower chamber of the Transwell chamber in Example 5 of the present invention.
  • Figure 11 is the cell appearance diagram of the electron microscope after the co-culture of PDB-MSCs in the low concentration group and A549 cells in Example 5 of the present invention.
  • Fig. 12 is the cell appearance diagram of the electron microscope after the co-culture of PDB-MSCs in the middle concentration group and A549 cells in Example 5 of the present invention.
  • Fig. 13 is the cell appearance diagram of the electron microscope after the co-culture of PDB-MSCs and A549 cells in the high concentration group in Example 5 of the present invention
  • Fig. 14 shows that after co-culture of the parietal decidua mesenchymal stem cell medium with different concentrations in Example 3, Example 4, and Example 5 of the present invention, Hela cells, MCF-7 cells, and A549 cells, Hela cells, MCF-7 cells Histogram of inhibition of proliferation of cells and A549 cells;
  • Fig. 15 shows the co-culture of the low-concentration parietal decidua mesenchymal stem cell medium in Example 3, Example 4, and Example 5 of the present invention with Hela cells, MCF-7 cells, and A549 cells. Schematic diagram of the proportion of each cell number in the cycle;
  • Fig. 16 shows that after the medium-concentration parietal decidua mesenchymal stem cell medium in Example 3, Example 4, and Example 5 of the present invention was co-cultured with Hela cells, MCF-7 cells, and A549 cells, the three kinds of cells were in different cells. Schematic diagram of the proportion of each cell number in the cycle;
  • Figure 17 shows the co-culture of the high-concentration parietal decidua mesenchymal stem cell medium in Example 3, Example 4, and Example 5 of the present invention with Hela cells, MCF-7 cells, and A549 cells, and the three types of cells in different cell cycles. Schematic diagram of the proportion of each cell number.
  • the method for preparing parietal decidua mesenchymal stem cells includes the following steps:
  • tissue washing solution to wash blood and blood clots on the placental tissue of healthy full-term neonates, use surgical peeling instruments to separate the parietal decidual tissue, and then use surgical scissors to cut into 1-4 mm 3 tissue blocks And wash with tissue washing solution;
  • the tissue washing solution is prepared from the following raw materials by volume percentage: 1% penicillin-streptomycin mixture, 51.1% red blood cell lysate, 47% physiological saline, and the mass fraction of the physiological saline is 0.9% ;
  • Digestive wall decidua tissue block after adding tissue digestion solution to the chopped tissue blocks, digest with constant temperature shaking at 37° C. for 2 hours, and the rotation speed is 200 rpm/min; the tissue digestion solution contains 50% Tryple- High glucose DMEM medium with EDTA enzyme and 10mg/ml type II collagenase;
  • Termination of digestion add 3 times the volume of selective medium to terminate digestion, filter (filter mesh size is 100 ⁇ m), centrifuge at 1300 rpm/min for 5 min, and retain the precipitate;
  • the selective medium contains 10% serum substitute, DMEM serum-free medium containing 0.8mol/ml L-glutamine, 20ng/ml basic fibroblast growth factor, 20ng/ml epidermal growth factor, and 10ng/ml stem cell growth factor;
  • Cultivate PMSCs inoculate the obtained cell suspension into a T75 culture flask, place it in an incubator at 37°C, 5% CO 2 , and let it stand for primary culture, record it as P0 generation, and observe the change of the medium. , change the fluid every 2-3 days;
  • Cryopreservation use trypsin to digest the P3 generation PMSCs, add the cryopreservation solution to the precipitate after centrifugation, count and calculate the survival rate, program the cooling and cryopreservation in a liquid nitrogen tank for preservation; the S6 cryopreservation solution contains Serum-free complete medium with a volume concentration of 20% Cryosure-DEX-40; cryopreserved cell density is 2 ⁇ 10 6 cells/ml.
  • Methods of recovery of parietal decidual mesenchymal stem cell culture medium including:
  • step S22 Resuspend the decidua mesenchymal stem cells obtained in step S21 in selective medium, centrifuge at 1300 rpm for 6 min, wash the cell surface twice with PBS buffer, centrifuge at 1300 rpm for 6 min, add selective medium to resuspend the pellet for subculture;
  • Co-culture test of parietal decidual mesenchymal stem cell medium and cervical cancer cells including:
  • the complete medium is a high-glucose DMEM serum-free medium containing 10% fetal bovine serum and 1% anti-penicillin by volume;
  • S36 Detection of proliferation inhibition: set a blank group, and collect Hela cells after co-cultivation in different groups to form a low-concentration co-culture group, a medium-concentration co-culture group, and a high-concentration co-culture group; The cells were plated into 96-well plates, and their proliferation was detected by CCK-8 method after 24 hours of growth.
  • Figure 3 is the cell morphology diagram of co-culture of PMSCs and Hela cells in the low concentration group
  • Figure 4 is the cell morphology diagram of the co-culture of PMSCs and Hela cells in the medium concentration group
  • Figure 5 is the cell morphology diagram of the co-culture of PMSCs and Hela cells in the high concentration group ;
  • Figure 3 takes the overlay images from left to right according to the distribution of Hela cells in the well plate (with the upper chamber as the occupied area as the distribution center of Hela cells), and the left one is the low concentration group after co-cultivation for 3 days.
  • Morphology of the outer edge of the well plate is the morphological map of Hela cells in the transition circle of the well plate after co-culture for 3 days in the low concentration group; the third from the left is the morphological map of Hela cells in the center of the well plate after co-culture for 3 days in the low concentration group; Figure 4 from the left To the right, the overlay images are taken in order according to the distribution of Hela cells in the well plate (with the upper chamber as the occupied area as the center of cell distribution). The left one is the morphological map of Hela cells on the outer edge of the well plate after co-cultivation for 3 days in the medium concentration group.
  • the second is the morphological diagram of Hela cells in the transition circle of the well plate after 3 days of co-culture in the medium concentration group;
  • the third from the left is the morphological diagram of Hela cells in the center of the well plate after 3 days of co-culture in the medium concentration group;
  • the distribution in the inner chamber (with the upper chamber as the occupied area as the cell distribution center), the superimposed images were taken sequentially.
  • the left one is the morphological diagram of Hela cells on the outer edge of the well plate after co-cultivation for 3 days in the high concentration group, and the second left is the high concentration group co-cultured for 3 days Morphological diagram of Hela cells in the transition circle of the orifice plate after 3 days; the third from the left is the morphological diagram of Hela cells in the center of the orifice plate after 3 days of co-culture in the high concentration group.
  • Figure 3- Figure 5 shows the cell morphology after co-culture It is obvious that there are significant differences in the inhibition effect of different concentrations of PMSCs on the proliferation of the same Hela cell. Within the concentration range of PMSCs selected in this example, the higher the concentration of PMSCs, the more obvious the inhibition of Hela cell proliferation.
  • the complete medium is a high-glucose DMEM serum-free medium containing 10% fetal bovine serum and 1% anti-penicillin-resistant streptomycin by volume;
  • S32, MCF-7 cell passage observe the cell morphology and medium changes after resuscitation, replace the complete medium every 2 days, when the cell growth and fusion is greater than 80%, collect the cell suspension, count and calculate the survival rate, microbial detection ,pass on;
  • MCF-7 cells observe the growth of MCF-7 cells after co-cultivation for 3 days, and take pictures to record their growth state;
  • S36 Detection of proliferation inhibition: set a blank group, and collect MCF-7 cells after co-cultivation in different groups to form a low-concentration co-cultivation group, a medium-concentration co-cultivation group, and a high-concentration co-cultivation group; 5000 cells were plated into 96-well plates, and their proliferation was detected by CCK-8 method after 24 hours of growth.
  • Figure 7 is the cell morphology diagram of co-culture of PMSCs and MCF-7 cells in the low concentration group
  • Figure 8 is the cell morphology diagram of the co-culture of PMSCs and MCF-7 cells in the medium concentration group
  • Figure 9 is the high concentration group PMSCs and MCF-7 cells
  • the morphological diagram of co-cultured cells; among them, Figure 7 is the order of the distribution of MCF-7 cells in the well plate (with the upper chamber as the occupied area as the center of cell distribution) from left to right, and the left one is the low concentration group.
  • Morphology of MCF-7 cells on the outer edge of the well plate after 3 days of co-culture is the morphological map of MCF-7 cells in the transition circle of the well plate after 3 days of co-culture in the low concentration group; the third from the left is the MCF- 7.
  • Morphological diagram of cells in the center of the well plate Figure 8 is a superimposed image of the distribution of MCF-7 cells in the well plate from left to right (with the upper chamber as the occupied area as the center of cell distribution), and the left one is the medium concentration group.
  • Morphology of MCF-7 cells on the outer edge of the well plate after 3 days of co-culture is the morphological map of MCF-7 cells in the transition circle of the well plate after 3 days of co-culture in the medium concentration group; the third from the left is the MCF-7 cells in the medium concentration group after 3 days of co-culture 7.
  • Morphology of MCF-7 cells on the outer edge of the well plate after 3 days of co-culture is the morphological map of the transition circle of MCF-7 cells in the well plate after 3 days of co-culture in the high-concentration group; the third from the left is the MCF- 7 Morphological diagram of cells in the center of the well plate.
  • Co-culture test of parietal decidual mesenchymal stem cell medium and lung cancer cells including:
  • the complete medium is a high-sugar DMEM serum-free medium containing 10% fetal bovine serum and 1% anti-penicillin-resistant streptomycin by volume;
  • Passaging of S32 and A549 cells observe the cell morphology and medium changes after resuscitation, replace the complete medium every 2 days, and when the cell growth and confluence is greater than 80%, collect the cell suspension, count and calculate the survival rate, microbial detection, passage ;
  • A549 cells in the lower chamber of the Transwell chamber discard the original medium, wash twice with PBS buffer, digest the cells with 1 ml of trypsin for 2 min, use 10 ml of complete medium to terminate the digestion, collect the A549 cells of the P3 generation, add complete
  • the A549 cells were resuspended in the medium, and 1 ⁇ 10 6 cells per well (24-well plate) were plated into the lower chamber of the Transwell chamber of each group in Example 2. After the cells grew steadily (12h), the cell morphology of A549 was shown in the figure. 10 shown;
  • S36 Detection of proliferation inhibition: set a blank group, and collect A549 cells co-cultured from different groups to form a low-concentration co-culture group, a medium-concentration co-culture group, and a high-concentration co-culture group; The cells were plated into 96-well plates, and their proliferation was detected by CCK-8 method after 24 hours of growth.
  • Figure 11 is the cell morphology diagram of co-culture of PMSCs and A549 cells in the low concentration group
  • Figure 12 is the cell morphology diagram of the co-culture of PMSCs and A549 cells in the medium concentration group
  • Figure 13 is the cell morphology diagram of the co-culture of PMSCs and A549 cells in the high concentration group ;
  • the superimposed images were taken in order from left to right according to the distribution of A549 cells in the well plate (with the upper chamber as the occupied area as the cell distribution center), and the left one is the low concentration group after 3 days of co-culture of A549 cells in the well
  • the morphological diagram of the outer edge of the plate, the second left is the morphological diagram of A549 cells in the transition circle of the well plate after co-cultivation for 3 days in the low concentration group;
  • the third left is the morphological diagram of A549 cells in the center of the well plate after co-cultivation for 3 days in the low concentration group;
  • Figure 12 from left to On
  • the left one is the morphological diagram of A549 cells on the outer edge of the well plate after co-cultivation for 3 days in the high concentration group, and the second from the left is the high concentration group after co-cultivation for 3 days.
  • Figure 14 shows the inhibition of proliferation of Hela cells, MCF-7 cells, and A549 cells after co-culture with different concentrations of parietal decidual mesenchymal stem cell medium and Hela cells, MCF-7 cells, and A549 cells.
  • the blank group did not add parietal decidual mesenchymal stem cell medium to co-culture with Hela cells, MCF-7 cells, and A549 cells, so the growth inhibition rate of Hela cells, MCF-7 cells, and A549 cells in the blank group was zero.
  • the higher the concentration of parietal decidual mesenchymal stem cell medium the higher the growth inhibition rate of Hela cells, MCF-7 cells and A549 cells.
  • the proliferation ability of parietal decidual mesenchymal stem cell medium on cervical cancer Hela cells has a significant inhibitory effect.
  • the inhibitory effect of parietal decidual mesenchymal stem cell medium on cervical cancer Hela cells was significantly higher than that of breast cancer MCF-7 cells and lung cancer A549 cells;
  • the inhibitory effect of ability has selectivity and adaptability; among them, the inhibitory effect on parental cervical cancer Hela cells is the strongest, and it has a certain effect on inhibiting proximate cells.
  • the cells were processed according to the steps of the cell cycle test kit (Solarbio Company), and the PMSCs and tumors in different concentration groups in Example 3, Example 4, and Example 5 were detected by PI staining and flow cytometry (Beckman Company). Cell cycle flow cytometry of cell co-cultures.
  • Figure 15 is a schematic diagram of the proportion of the three cells in different cell cycles after co-culturing PMSCs with Hela cells, MCF-7 cells and A549 cells for 3 days in the low concentration group; , MCF-7 cells and A549 cells co-cultured for 3 days, the proportion of the respective cell numbers in the cell cycle of the three cells;
  • Figure 17 is a high concentration group of PMSCs after co-culture with Hela cells, MCF-7 cells and A549 cells for 3 days , a schematic diagram of the proportion of the respective cell numbers in the cell cycle of the three types of cells.
  • Figures 15 to 17 show that a single concentration of parietal decidual mesenchymal stem cells has a significant effect on the apoptosis of Hela cells and MCF-7, and the number of cells in the G1 and S phases is significantly increased, and the number of cells in the G2 phase is significantly increased. The number of cells was reduced; at the same time, it also showed a certain effect on the cell cycle of lung cancer A549, among which the significant reduction of cells in the G2 phase was the most obvious. All of the above indicate that parietal decidual mesenchymal stem cells have the functions of homing, inhibiting proliferation and regulating cell cycle for various tumor cells.
  • parietal decidual mesenchymal stem cells have obvious homing effect on various tumor cell lines, and the strength of homing effect is quite different.
  • parietal decidual mesenchymal stem cells The migration route of the parietal decidual mesenchymal stem cells was still relatively full after 3 days of co-culture.

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Abstract

用于***的壁蜕膜间充质干细胞培养基、共培方法，所述用于***的壁蜕膜间充质干细胞培养基含有壁蜕膜间充质干细胞和选择培养基，所述壁蜕膜间充质干细胞对多种肿瘤细胞具有归巢作用、抑制增殖作用和调节细胞周期作用。

Description

用于***的壁蜕膜间充质干细胞培养基、共培方法 技术领域
本发明涉及生物技术领域，尤其涉及一种用于***的壁蜕膜间充质干细胞培养基、共培方法。
背景技术
间充质干细胞(mesenchymal stem cell，MSC)是具有趋化肿瘤能力的多能干祖细胞，在肿瘤方面的研究有着显著的进展。但子宫来源间充质干细胞对妇科恶性肿瘤细胞生物学行为的影响报道不一，研究壁蜕膜MSCs(Pariduval Mesenchymal stem cell，PMSCs)与肿瘤的适配性和选择性成为临床上***的新方法。
壁蜕膜间充质干细胞(Pariduval Mesenchymal stem cells，PMSCs)具有体外增殖培养简单、不触及道德伦理学、具有良好的迁移能力、向肿瘤细胞归巢和低免疫原性等特点，并规避了骨髓间充质干细胞获取困难、涉及医学道德伦理等风险，使其可以成为替代骨髓间充质干细胞成为肿瘤生物治疗的新载体。但研究报道的母源间充质干细胞对恶性肿瘤细胞生物学行为的影响不一，研究PMSCs与多种恶性肿瘤的适配性和选择性，是PMSCs能否成为恶性肿瘤治疗载体的基础。
现有壁蜕膜间充质干细胞对不同妇科肿瘤的增殖抑制作用的不足有：
1、在相关文献的MSCs与肿瘤的研究方法中，多见MSCs对单种肿瘤的作用，少见MSC的归巢能力与肿瘤类型选择性和适配性的直接分析；
2、在相关文献中的MSCs与肿瘤的研究方法中，常见经改造的间充质干细胞与药物组合后作用于肿瘤的研究，对母源的壁蜕膜间充质干细胞与肿瘤的研究不足；
3、在相关文献中的共培养方法中，PMSCs使用的研究浓度单一，造成对恶性肿瘤细胞生物学行为的影响报道不一。
发明内容
本发明所要解决的技术问题在于，提供了一种用于***的壁蜕膜间充质干细胞培养基，用于在肿瘤治疗中的应用，所述壁蜕膜间充质干细胞培养基含有壁蜕膜间充质干细胞和选择培养基，所述壁蜕膜间充质干细胞对多种肿瘤细胞具有归巢作用、抑制增殖作用和调节细胞周期作用。
本发明还要解决的技术问题在于，提供了壁蜕膜间充质干细胞培养基与肿瘤细胞的共培养方法，明确壁蜕膜间充质干细胞对恶性肿瘤归巢作用的适配性和选择作用，使得共培养后间充质干细胞归巢作用轨迹明显，明确特定浓度范围内壁蜕膜间充质干细胞对不同恶性肿瘤的归巢作用。
本发明还要解决的技术问题在于，提供了用于***的壁蜕膜间充质干细胞培养基，用于在肿瘤治疗中的应用，所述用于***的壁蜕膜间充质干细胞培养基含有壁蜕膜间充质干细胞和选择培养基，所述壁蜕膜间充质干细胞对多种肿瘤细胞具有归巢作用、抑制增殖作用和调节细胞周期作用。
作为上述方案的改进，所述肿瘤为***、乳腺肿瘤或肺肿瘤。
作为上述方案的改进，所述壁蜕膜间充质干细胞的制备方法，包括以下步骤：
S11、将壁蜕膜组织剪成1～4mm 3的组织块，用组织清洗液清洗组织块；
S12、清洗后用组织消化液在恒温振荡下消化组织块，终止消化，过滤，离心，保留沉淀，所述组织消化液为含有体积浓度40～60％Tryple-EDTA酶和8～12mg/mlⅡ型胶原酶的高糖DMEM培养基；
S13、用生理盐水清洗沉淀并将沉淀重悬，离心，去除上清液，加入选择培养基重悬，得到PMSCs悬液；
S14、将PMSCs悬液接种至培养瓶中，在培养箱中进行原代培养，计为P0代；
S15、当细胞融合度大于80％时，消化，过滤、离心，采用选择培养基重悬沉淀进行传代培养；
S6、收集Pn代的壁蜕膜间充质干细胞，消化，离心，去除上清液，将冻存液加入沉淀中，程序降温冻存置于液氮罐中保藏，n≥2。
作为上述方案的改进，所述选择培养基为含有体积浓度8～12％血清替代物、0.5～1mol/ml L-谷氨酰胺、18～25ng/ml碱性成纤维细胞生长因子、16～22ng/ml表皮生长因子和6～12ng/ml干细胞生长因子的DMEM无血清培养基。
作为上述方案的改进，步骤S11中，所述组织清洗液由以下体积百分比原料配制而成：0.8～1.5％青链霉素合剂，50～55％红细胞裂解液，44～49％的生理盐水，所述生理盐水为质量分数为0.8～1％。
作为上述方案的改进，步骤S12中，组织消化液在温度为36～39℃下与组织块振动1.5～4h，振荡速度为150～200rpm/min；
采用选择培养基来终止消化，选择培养基的体积为消化液体积的3～6倍；
采用孔径为100μm的滤网来过滤；
离心速度为1200～1400rpm/min，离心时间为5～7min。
作为上述方案的改进，步骤S15中，当细胞融合度大于80％时，采用PBS缓冲液洗涤细胞表面至少2次；
采用细胞消化液消化3～6min，采用选择培养基来终止消化；
采用孔径为100μm的滤网来过滤；
离心速度为1200～1400rpm/min，离心时间为5～7min。
作为上述方案的改进，步骤S16中，在收集壁蜕膜间充质干细胞之前，还包括：对壁蜕膜间充质干细胞进行表面抗体标记检测，当CD73、CD90和CD105的阳性指标同时>99％时，才收集壁蜕膜间充质干细胞；
收集P3代的壁蜕膜间充质干细胞，采用胰酶消化P3代的壁蜕膜间充质干细胞，离心，去除上清液，将冻存液加入沉淀中，程序降温冻存置于液氮罐中保藏。
作为上述方案的改进，步骤S16中，所述冻存液为含有体积浓度18～25％Cryosure-DEX-40的无血清完全培养基；
冻存细胞密度为1.5×10 6～2.5×10 6个/ml。
相应地，本发明还提供了一种壁蜕膜间充质干细胞培养基与肿瘤细胞的共培养方法，包括：
将上述的壁蜕膜间充质干细胞培养基置于Transwell小室的上室中培养；
将肿瘤细胞置于Transwell小室下室中培养；
将载有壁蜕膜间充质干细胞的上室放入孔板内与下室肿瘤细胞共培养。
实施本发明，具有如下有益效果：
1、本发明壁蜕膜间充质干细胞培养基中含有壁蜕膜间充质干细胞和选择培养基，其中，所述壁蜕膜间充质干细胞通过归巢作用、抑制增殖作用和调节细 胞周期作用来抑制肿瘤细胞。
2、本发明采用含有体积浓度8～12％血清替代物、0.5～1mol/ml L-谷氨酰胺、18～25ng/ml碱性成纤维细胞生长因子、16～22ng/ml表皮生长因子和6～12ng/ml干细胞生长因子的DMEM无血清培养基作为选择培养基来终止消化，以及重悬从壁蜕膜间充质干细胞，有利于提高壁蜕膜间充质干细胞纯度，加速壁蜕膜间充质干细胞生长，实现壁蜕膜间充质干细胞的体外快速扩增。
3、本发明采用含有体积浓度40～60％Tryple-EDTA酶和8～12mg/mlⅡ型胶原酶的高糖DMEM培养基作为组织消化液来消化组织块，有利于壁蜕膜间充质干细胞爬出组织进行贴壁生长。
4、本发明壁蜕膜间充质干细胞培养基与肿瘤细胞的共培养方法，明确壁蜕膜间充质干细胞对恶性肿瘤归巢作用的适配性和选择作用，使得共培养后间充质干细胞归巢作用轨迹明显，明确特定浓度范围内壁蜕膜间充质干细胞对不同恶性肿瘤的归巢作用。
附图说明
图1是本发明实施例2中P5代PMSCs在Transwell小室的上室中培养3天后的电子显微镜的细胞外观图；
图2是本发明实施例3中Hela细胞在Transwell小室下室中培养稳定后的电子显微镜的细胞外观图；
图3是本发明实施例3中低浓度组PDB-MSCs与Hela细胞共培养后的电子显微镜的细胞外观图；
图4是本发明实施例3中中浓度组PDB-MSCs与Hela细胞共培养后的电子显微镜的细胞外观图；
图5是本发明实施例3中高浓度组PDB-MSCs与Hela细胞共培养后的电子显微镜的细胞外观图；
图6是本发明实施例4中MCF-7细胞在Transwell小室下室中培养稳定后的电子显微镜的细胞外观图；
图7是本发明实施例4中低浓度组PDB-MSCs与MCF-7细胞共培养后的电子显微镜的细胞外观图；
图8是本发明实施例4中中浓度组PDB-MSCs与MCF-7细胞共培养后的电 子显微镜的细胞外观图；
图9是本发明实施例4中高浓度组PDB-MSCs与MCF-7细胞共培养后的电子显微镜的细胞外观图；
图10是本发明实施例5中A549细胞在Transwell小室下室中培养稳定后的电子显微镜的细胞外观图；
图11是本发明实施例5中低浓度组PDB-MSCs与A549细胞共培养后的电子显微镜的细胞外观图；
图12是本发明实施例5中中浓度组PDB-MSCs与A549细胞共培养后的电子显微镜的细胞外观图；
图13是本发明实施例5中高浓度组PDB-MSCs与A549细胞共培养后的电子显微镜的细胞外观图；
图14是本发明实施例3、实施例4、实施例5中不同浓度的壁蜕膜间充质干细胞培养基与Hela细胞、MCF-7细胞、A549细胞共培养后，Hela细胞、MCF-7细胞、A549细胞的增值抑制情况柱状图；
图15是本发明实施例3、实施例4、实施例5中低浓度的壁蜕膜间充质干细胞培养基与Hela细胞、MCF-7细胞、A549细胞共培养后，三种细胞在不同细胞周期中各自细胞数量占比示意图；
图16是本发明实施例3、实施例4、实施例5中中浓度的壁蜕膜间充质干细胞培养基与Hela细胞、MCF-7细胞、A549细胞共培养后，三种细胞在不同细胞周期中各自细胞数量占比示意图；
图17是本发明实施例3、实施例4、实施例5中高浓度的壁蜕膜间充质干细胞培养基与Hela细胞、MCF-7细胞、A549细胞共培养后，三种细胞在不同细胞周期中各自细胞数量占比示意图。
具体实施方式
为使本发明的目的、技术方案和优点更加清楚，下面将结合附图对本发明作进一步地详细描述。仅此声明，本发明在文中出现或即将出现的上、下、左、右、前、后、内、外等方位用词，仅以本发明的附图为基准，其并不是对本发明的具体限定。
实施例1
壁蜕膜间充质干细胞的制备方法，包括以下步骤：
S11、分离壁蜕膜组织：使用组织清洗液清洗健康足月新生儿胎盘组织上的血液和血块，使用手术剥离器械分离得到壁蜕膜组织，再用手术剪剪成1～4mm 3的组织块并用组织清洗液清洗；所述组织清洗液由以下体积百分比原料配制而成：1％青链霉素合剂，51.1％红细胞裂解液，47％的生理盐水，所述生理盐水为质量分数为0.9％；
S12、消化壁蜕膜组织块：往剪碎的组织块中加入组织消化液后，于37℃下恒温振荡消化2h，转速为200rpm/min；所述组织消化液为含有体积浓度50％Tryple-EDTA酶和10mg/mlⅡ型胶原酶的高糖DMEM培养基；
S13终止消化：加入3倍体积的选择培养基终止消化，过滤(滤网大小为100μm)，以1300rpm/min转速离心5min，保留沉淀；所述选择培养基为含有体积浓度10％血清替代物、0.8mol/ml L-谷氨酰胺、20ng/ml碱性成纤维细胞生长因子、20ng/ml表皮生长因子、10ng/ml干细胞生长因子的DMEM无血清培养基；
S14、分离得到PMSCs：采用生理盐水清洗并将沉淀重悬，得到重悬液，离心弃去上清液，加入选择培养基重悬，即得到PMSCs悬液；
S15、培养PMSCs：将得到的细胞悬液接种至T75培养瓶中，置于37℃、5％CO 2、饱和湿度的培养箱中静置原代培养，记作P0代，观察培养基变化情况，每隔2-3天换液一次；
S16、换液与传代：观察细胞增殖融合大于80％后，PBS缓冲液洗涤贴壁细胞表面分泌物，使用细胞消化液消化细胞5min，用新鲜完全培养基终止消化，用100μm滤网过滤，离心，细胞培养液重悬沉淀，进行计数和计算存活率，检测无菌等指标，进行传代培养，每隔2～3天传代一次；所述细胞消化液包括质量分数为0.125％的胰蛋白酶和质量分数为0.004％的EDTA；
S17、所述传代培养传至P3时，进行PMSCs阳性指标检测，CD73、CD90、CD105阳性指标均>99％时，停止传代，收集细胞；
S18、冻存：采用胰酶来消化P3代的PMSCs，离心后往沉淀中加入冻存液，计数和计算存活率，程序降温冻存置于液氮罐中保藏；所述S6冻存液为含有体积浓度20％Cryosure-DEX-40的无血清完全培养基；冻存细胞密度为2×10 6个/ml。
实施例2
壁蜕膜间充质干细胞培养基的复苏方法，包括：
S21、将实施例1中冻存的集壁蜕膜间充质干细胞置于37℃的水浴中溶解；
S22、采用选择培养基重悬步骤S21的集壁蜕膜间充质干细胞，1300rpm离心6min，采用PBS缓冲液洗涤细胞表面2次，1300rpm离心6min，加入选择培养基重悬沉淀进行传代培养；
S23、收集P5代的PMSCs，加入选择培养基将细胞重悬成低浓度组、中浓度组和高浓度组，其中，低浓度组的PMSCs浓度为1×10 6个/孔，中浓度组的PMSCs浓度为2×10 6个/孔，高浓度组的PMSCs浓度为4×10 6个/孔，每组以每孔200μL的体积分别铺到不同Transwell小室的上室中，培养3天，PMSCs形态图如图1所示。
实施例3
壁蜕膜间充质干细胞培养基与***细胞的共培试验，包括：
S31、将***细胞株Hela于37℃水浴中快速溶解后，加入完全培养基重悬细胞，离心，用PBS缓冲液洗涤2次，1000rpm离心3min，加入新鲜的完全培养基重悬细胞，转移至T25培养瓶中培养，记作P1代；所述完全培养基为含有体积浓度10％胎牛血清和体积浓度1％抗青链霉素的高糖的DMEM无血清培养液；
S32、Hela细胞传代：观察复苏后的细胞形态与培养基变化情况，2天更换一次完全培养基，待细胞生长融合大于80％，收集细胞悬液，进行计数和计算存活率，微生物检测，传代；
S33、Transwell小室下室中培养Hela细胞：弃去原培养基，PBS缓冲液洗涤2次，采用1ml的胰酶消化细胞2min，用10ml完全培养基终止消化，收集P3代的Hela细胞，加入完全培养基将Hela细胞重悬，按照每孔1×10 6个细胞(24孔板)铺板到实施例2每组的Transwell小室下室中，待细胞生长稳定(12h)，Hela的细胞形态如图2所示；
S34、共同培养：待Transwell小室下室中Hela细胞生长稳定后，将载有PMSCs的上室放入孔板内与下室Hela细胞共培养3天；
S35、Hela细胞的生长：观察共培养3天后的Hela细胞的生长情况，拍照记录其生长状态；
S36、增殖抑制作用检测：设置空白组，以及收集不同组共培养后的Hela细胞，形成低浓度共培组、中浓度共培组和高浓度共培组；具体的，每组按照每孔5000个细胞铺板至96孔板中，生长24h后使用CCK-8法检测其增殖情况。
每组试验至少重复3次，结果取平均值。
图3为低浓度组PMSCs与Hela细胞共培养的细胞形态图，图4为中浓度组PMSCs与Hela细胞共培养的细胞形态图，图5为高浓度组PMSCs与Hela细胞共培养的细胞形态图；其中，图3从左到右按照Hela细胞在孔板内的分布(以小室上室作占据区域作为Hela细胞分布中心)顺序拍摄叠加图，左一是低浓度组共培养3天后Hela细胞在孔板外沿形态图，左二是低浓度组共培养3天后Hela细胞在孔板过渡圈形态图；左三是低浓度组共培养3天后Hela细胞在孔板中心形态图；图4从左到右按照Hela细胞在孔板内的分布(以小室上室作占据区域作为细胞分布中心)顺序拍摄叠加图，左一是中浓度组共培养3天后Hela细胞在孔板外沿形态图，左二是中浓度组共培养3天后Hela细胞在孔板过渡圈形态图；左三是中浓度组共培养3天后Hela细胞在孔板中心形态图；图5从左到右按照Hela细胞在孔板内的分布(以小室上室作占据区域作为细胞分布中心)顺序拍摄叠加图，左一是高浓度组共培养3天后Hela细胞在孔板外沿形态图，左二是高浓度组共培养3天后Hela细胞在孔板过渡圈形态图；左三是高浓度组共培养3天后Hela细胞在孔板中心形态图。
从图3-图5中可以看到，越接近载有PMSCs小室的Hela细胞数量越稀疏，同时出现外缘Hela细胞向小室中心迁移的现象；图3-图5的共培养后的细胞形态图显著表明不同浓度的PMSCs对同一种Hela细胞增殖作用的抑制存在显著差异，在该实施例选取的PMSCs浓度范围内表现出，PMSCs浓度越高，抑制Hela细胞增殖作用越明显。
实施例4
壁蜕膜间充质干细胞培养基与乳腺癌细胞的共培试验，包括：
S31、将乳腺癌细胞株MCF-7于37℃水浴中快速溶解后，加入完全培养基重悬细胞，离心，用PBS缓冲液洗涤2次，1000rpm离心3min，加入新鲜的完全培养基重悬细胞，转移至T25培养瓶中培养，记作P1代；所述完全培养基为，含有体积浓度10％胎牛血清和体积浓度1％抗青链霉素的高糖的DMEM无血清培养液；
S32、MCF-7细胞传代：观察复苏后的细胞形态与培养基变化情况，2天更换一次完全培养基，待细胞生长融合大于80％，收集细胞悬液，进行计数和计算存活率，微生物检测，传代；
S33、Transwell小室下室中培养MCF-7细胞：弃去原培养基，PBS缓冲液洗涤2次，采用1ml的胰酶消化细胞2min，用10ml完全培养基终止消化，收集P3代的MCF-7细胞，加入完全培养基将MCF-7细胞重悬，按照每孔1×10 6个细胞(24孔板)铺板到实施例2每组的Transwell小室下室中，待细胞生长稳定(12h)，MCF-7的细胞形态如图6所示；
S34、共同培养：待Transwell小室下室中MCF-7细胞生长稳定后，将载有PMSCs的上室放入孔板内与下室MCF-7细胞共培养3天；
S35、MCF-7细胞的生长：观察共培养3天后的MCF-7细胞的生长情况，拍照记录其生长状态；
S36、增殖抑制作用检测：设置空白组，以及收集不同组共培养后的MCF-7细胞，形成低浓度共培组、中浓度共培组和高浓度共培组；具体的，每组按照每孔5000个细胞铺板至96孔板中，生长24h后使用CCK-8法检测其增殖情况。
每组试验至少重复3次，结果取平均值。
图7为低浓度组PMSCs与MCF-7细胞共培养的细胞形态图，图8为中浓度组PMSCs与MCF-7细胞共培养的细胞形态图，图9为高浓度组PMSCs与MCF-7细胞共培养的细胞形态图；其中，图7从左到右按照MCF-7细胞在孔板内的分布(以小室上室作占据区域作为细胞分布中心)顺序拍摄叠加图，左一是低浓度组共培养3天后MCF-7细胞在孔板外沿形态图，左二是低浓度组共培养3天后MCF-7细胞在孔板过渡圈形态图；左三是低浓度组共培养3天后MCF-7细胞在孔板中心形态图；图8从左到右按照MCF-7细胞在孔板内的分布(以小室上室作占据区域作为细胞分布中心)顺序拍摄叠加图，左一是中浓度组共培养3天后MCF-7细胞在孔板外沿形态图，左二是中浓度组共培养3天后MCF-7细胞在孔板过渡圈形态图；左三是中浓度组共培养3天后MCF-7细胞在孔板中心形态图；图9从左到右按照MCF-7细胞在孔板内的分布(以小室上室作占据区域作为细胞分布中心)顺序拍摄叠加图，左一是高浓度组共培养3天后MCF-7细胞在孔板外沿形态图，左二是高浓度组共培养3天后MCF-7细胞在孔板过渡圈形态图；左三是高浓度组共培养3天后MCF-7细胞在孔板中心形态图。
从图7-图9中可以看到，越接近载有PMSCs小室的MCF-7细胞数量越稀疏，同时出现外缘MCF-7细胞向小室中心迁移的现象；图7-图9的共培养后的细胞形态图显著表明不同浓度的PMSCs对同一种MCF-7细胞增殖作用的抑制存在显著差异，在该实施例选取的PMSCs浓度范围内表现出，PMSCs浓度越高，抑制MCF-7细胞增殖作用越明显。
实施例5
壁蜕膜间充质干细胞培养基与肺癌细胞的共培试验，包括：
S31、将肺癌细胞株A549于37℃水浴中快速溶解后，加入完全培养基重悬细胞，离心，用PBS缓冲液洗涤2次，1000rpm离心3min，加入新鲜的完全培养基重悬细胞，转移至T25培养瓶中培养，记作P1代；所述完全培养基为，含有体积浓度10％胎牛血清和体积浓度1％抗青链霉素的高糖的DMEM无血清培养液；
S32、A549细胞传代：观察复苏后的细胞形态与培养基变化情况，2天更换一次完全培养基，待细胞生长融合大于80％，收集细胞悬液，进行计数和计算存活率，微生物检测，传代；
S33、Transwell小室下室中培养A549细胞：弃去原培养基，PBS缓冲液洗涤2次，采用1ml的胰酶消化细胞2min，用10ml完全培养基终止消化，收集P3代的A549细胞，加入完全培养基将A549细胞重悬，按照每孔1×10 6个细胞(24孔板)铺板到实施例2每组的Transwell小室下室中，待细胞生长稳定(12h)，A549的细胞形态如图10所示；
S34、共同培养：待Transwell小室下室中A549细胞生长稳定后，将载有PMSCs的上室放入孔板内与下室A549细胞共培养3天；
S35、A549细胞的生长：观察共培养3天后的A549细胞的生长情况，拍照记录其生长状态；
S36、增殖抑制作用检测：设置空白组，以及收集不同组共培养后的A549细胞，形成低浓度共培组、中浓度共培组和高浓度共培组；具体的，每组按照每孔5000个细胞铺板至96孔板中，生长24h后使用CCK-8法检测其增殖情况。
每组试验至少重复3次，结果取平均值。
图11为低浓度组PMSCs与A549细胞共培养的细胞形态图，图12为中浓度组PMSCs与A549细胞共培养的细胞形态图，图13为高浓度组PMSCs与A549 细胞共培养的细胞形态图；其中，图11从左到右按照A549细胞在孔板内的分布(以小室上室作占据区域作为细胞分布中心)顺序拍摄叠加图，左一是低浓度组共培养3天后A549细胞在孔板外沿形态图，左二是低浓度组共培养3天后A549细胞在孔板过渡圈形态图；左三是低浓度组共培养3天后A549细胞在孔板中心形态图；图12从左到右按照A549细胞在孔板内的分布(以小室上室作占据区域作为细胞分布中心)顺序拍摄叠加图，左一是中浓度组共培养3天后A549细胞在孔板外沿形态图，左二是中浓度组共培养3天后A549细胞在孔板过渡圈形态图；左三是中浓度组共培养3天后A549细胞在孔板中心形态图；图13从左到右按照A549细胞在孔板内的分布(以小室上室作占据区域作为细胞分布中心)顺序拍摄叠加图，左一是高浓度组共培养3天后A549细胞在孔板外沿形态图，左二是高浓度组共培养3天后A549细胞在孔板过渡圈形态图；左三是高浓度组共培养3天后A549细胞在孔板中心形态图。
从图11-图13中可以看到，越接近载有PMSCs小室的A549细胞数量越稀疏，同时出现外缘A549细胞向小室中心迁移的现象；图13-图15的共培养后的细胞形态图显著表明不同浓度的PMSCs对同一种A549细胞增殖作用的抑制存在显著差异，在该实施例选取的PMSCs浓度范围内表现出，PMSCs浓度越高，抑制A549细胞增殖作用越明显。
图14是不同浓度的壁蜕膜间充质干细胞培养基与Hela细胞、MCF-7细胞、A549细胞共培养后，Hela细胞、MCF-7细胞、A549细胞的增值抑制情况。其中，空白组没有加入壁蜕膜间充质干细胞培养基与Hela细胞、MCF-7细胞、A549细胞共培，因此空白组对Hela细胞、MCF-7细胞、A549细胞的增长抑制率为零，浓度越高的壁蜕膜间充质干细胞培养基对Hela细胞、MCF-7细胞、A549细胞的增长抑制率越高，因此，壁蜕膜间充质干细胞培养基对***Hela细胞的增殖能力、乳腺癌MCF-7细胞的增殖能力、以及肺癌A549细胞的增殖能力都具有显著的抑制作用。此外，壁蜕膜间充质干细胞培养基对于***Hela细胞的抑制作用明显高于乳腺癌MCF-7细胞和肺癌A549细胞；由此可知，壁蜕膜间充质干细胞培养基对不同肿瘤增殖能力的抑制作用具有选择性和适配性；其中对于亲源的***Hela细胞的抑制作用最强，具有一定的抑制近源细胞的作用。
按照细胞周期试验试剂盒(Solarbio公司)的步骤对细胞进行处理，使用 PI染色法和流式细胞仪(Beckman公司)检测实施例3、实施例4、实施例5中不同浓度组的PMSCs与肿瘤细胞共培养的细胞周期流式图。
图15是低浓度组PMSCs分别与Hela细胞、MCF-7细胞和A549细胞共培3天后，三种细胞在不同细胞周期中各自细胞数量占比示意图；图16是中浓度组PMSCs分别与Hela细胞、MCF-7细胞和A549细胞共培3天后，三种细胞的细胞周期的各自细胞数量占比示意图；图17是高浓度组PMSCs分别与Hela细胞、MCF-7细胞和A549细胞共培3天后，三种细胞的细胞周期的各自细胞数量占比示意图。
图15～图17可知，表明单一浓度的壁蜕膜间充质干细胞对Hela细胞和MCF-7的细胞凋亡有显著影响，表现为G1期和S期的细胞数量均显著增加，G2期的细胞数量减少；同时对肺癌A549的细胞周期也表现出一定的作用，其中以G2期细胞显著减少最为明显。以上均表明壁蜕膜间充质干细胞对于多种肿瘤细胞具有归巢、抑制增殖和调节细胞周期的作用。
从图14～图17可知，壁蜕膜间充质干细胞对多种肿瘤细胞系均有明显的归巢作用，且归巢作用强弱存在较大差异，同时明确了壁蜕膜间充质干细胞的迁移路线，且共培养3天后的壁蜕膜间充质干细胞的生长情况仍旧较为饱满。
以上所揭露的仅为本发明一种较佳实施例而已，当然不能以此来限定本发明之权利范围，因此依本发明权利要求所作的等同变化，仍属本发明所涵盖的范围。

Claims (10)

  1. 用于***的壁蜕膜间充质干细胞培养基，其特征在于，用于在肿瘤治疗中的应用，所述用于***的壁蜕膜间充质干细胞培养基含有壁蜕膜间充质干细胞和选择培养基，所述壁蜕膜间充质干细胞对多种肿瘤细胞具有归巢作用、抑制增殖作用和调节细胞周期作用。
  2. 如权利要求1所述的用于***的壁蜕膜间充质干细胞培养基，其特征在于，所述肿瘤为***、乳腺肿瘤或肺肿瘤。
  3. 如权利要求1所述的用于***的壁蜕膜间充质干细胞培养基，其特征在于，所述壁蜕膜间充质干细胞的制备方法，包括以下步骤：
    S11、将壁蜕膜组织剪成1～4mm 3的组织块，用组织清洗液清洗组织块；
    S12、清洗后用组织消化液在恒温振荡下消化组织块，终止消化，过滤，离心，保留沉淀，所述组织消化液为含有体积浓度40～60％Tryple-EDTA酶和8～12mg/mlⅡ型胶原酶的高糖DMEM培养基；
    S13、用生理盐水清洗沉淀并将沉淀重悬，离心，去除上清液，加入选择培养基重悬，得到PMSCs悬液；
    S14、将PMSCs悬液接种至培养瓶中，在培养箱中进行原代培养，计为P0代；
    S15、当细胞融合度大于80％时，消化，过滤、离心，采用选择培养基重悬沉淀进行传代培养；
    S6、收集Pn代的壁蜕膜间充质干细胞，消化，离心，去除上清液，将冻存液加入沉淀中，程序降温冻存置于液氮罐中保藏，n≥2。
  4. 如权利要求3所述的用于***的壁蜕膜间充质干细胞培养基，其特征在于，所述选择培养基为含有体积浓度8～12％血清替代物、0.5～1mol/ml L-谷氨酰胺、18～25ng/ml碱性成纤维细胞生长因子、16～22ng/ml表皮生长因子和6～12ng/ml干细胞生长因子的DMEM无血清培养基。
  5. 如权利要求3所述的用于***的壁蜕膜间充质干细胞培养基，其特征在于，步骤S11中，所述组织清洗液由以下体积百分比原料配制而成：0.8～1.5％青链霉素合剂，50～55％红细胞裂解液，44～49％的生理盐水，所述生理盐水为质量分数为0.8～1％。
  6. 如权利要求3所述的用于***的壁蜕膜间充质干细胞培养基，其特征在于，步骤S12中，组织消化液在温度为36～39℃下与组织块振动1.5～4h，振荡速度为150～200rpm/min；
    采用选择培养基来终止消化，选择培养基的体积为消化液体积的3～6倍；
    采用孔径为100μm的滤网来过滤；
    离心速度为1200～1400rpm/min，离心时间为5～7min。
  7. 如权利要求3所述的用于***的壁蜕膜间充质干细胞培养基，其特征在于，步骤S15中，当细胞融合度大于80％时，采用PBS缓冲液洗涤细胞表面至少2次；
    采用细胞消化液消化3～6min，采用选择培养基来终止消化；
    采用孔径为100μm的滤网来过滤；
    离心速度为1200～1400rpm/min，离心时间为5～7min。
  8. 如权利要求3所述的用于***的壁蜕膜间充质干细胞培养基，其特征在于，步骤S16中，在收集壁蜕膜间充质干细胞之前，还包括：对壁蜕膜间充质干细胞进行表面抗体标记检测，当CD73、CD90和CD105的阳性指标同时>99％时，才收集壁蜕膜间充质干细胞；
    收集P3代的壁蜕膜间充质干细胞，采用胰酶消化P3代的壁蜕膜间充质干细胞，离心，去除上清液，将冻存液加入沉淀中，程序降温冻存置于液氮罐中保藏。
  9. 如权利要求3所述的用于***的壁蜕膜间充质干细胞培养基，其特征在于，步骤S16中，所述冻存液为含有体积浓度18～25％Cryosure-DEX-40的无血清完全培养基；
    冻存细胞密度为1.5×10 6～2.5×10 6个/ml。
  10. 壁蜕膜间充质干细胞培养基与肿瘤细胞的共培养方法，其特在在于，包括：
    将权利要求1～9所述的壁蜕膜间充质干细胞培养基置于Transwell小室的上室中培养；
    将肿瘤细胞置于Transwell小室下室中培养；
    将载有壁蜕膜间充质干细胞的上室放入孔板内与下室肿瘤细胞共培养。
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