Methods of culturing mesenchymal stem cells with modulated apoptosis-specific nuclease to reduce apoptosis
By adding recombinant human DFF45 protein and other auxiliary reagents to mesenchymal stem cell culture, the problems of slowed proliferation rate and increased apoptosis rate were solved, resulting in improved proliferation capacity and reduced apoptosis rate, thus promoting large-scale cell culture and clinical application.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- 李春雨
- Filing Date
- 2026-03-18
- Publication Date
- 2026-06-23
Smart Images
Figure CN122256243A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of cell biology, specifically a culture method that promotes the proliferation of umbilical cord mesenchymal stem cells by adding recombinant human DFF45 protein, Z-VAD-FMK, Cyclosporin A, bFGF, N-Acetylcysteine, and calcitonin. Background Technology
[0002] Stem cells are a group of cells with high self-renewal and multi-directional differentiation potential. Theoretically, they have the ability to differentiate into cells of all tissue and organ types in the human body, making them an ideal cell source for regenerative medicine and tissue engineering. With the deepening and development of cell biology and clinical medical technologies, scientists have discovered that stem cells can replenish aging, damaged, or dead cells in mature organs. They are being increasingly used in research on various end-stage and intractable diseases. Research on stem cells for stroke, diabetes, Parkinson's disease, Alzheimer's disease, and myocardial infarction is also progressing. Therefore, promoting stem cell research is crucial. Mesenchymal stem cells (MSCs) were first identified from bone marrow in the 1960s. Since then, MSCs have been isolated from various tissues, including bone marrow, umbilical cord blood, placenta, adipose tissue, gingiva, oral mucosa, amniotic fluid, and brain. MSCs can differentiate into various cell types, such as osteoblasts, chondrocytes, adipocytes, myoblasts, vascular endothelial cells, and nerve cells. Current techniques allow for the mature isolation, expansion, and in vitro culture of MSCs, making them ideal seed cells for tissue engineering research. MSCs possess advantages such as ease of isolation, high proliferative potential, and genetic stability. They can also migrate to damaged tissues to combat inflammation, influence the microenvironment, promote angiogenesis, resist fibrosis, inhibit apoptosis, and release cytokines to participate in repair processes and tissue regeneration.
[0003] One of the most prominent characteristics of apoptosis is the ladder-like breakage of chromosomal DNA. This is caused by apoptosis-specific endonucleases hydrolyzing chromosomal DNA between nucleosomes, producing DNA fragments of 180 bp or multiples thereof, which form a "DNA ladder" on agarose gel electrophoresis. For over a decade after this phenomenon was discovered, researchers searched for apoptosis-specific nucleases that hydrolyze chromosomal DNA. In 1996, Wang Xiaodong's laboratory isolated and purified a protein from soluble cytoplasmic extracts of human HeLa cells and named it human DNA fragmentation factor (DFF). Further experiments showed that DFF consists of two subunits with molecular weights of 40 kD and 45 kD, respectively. The 40 kD subunit (DFF40) possesses apoptosis-specific DNA endonuclease activity, while the 45 kD subunit (DFF45) is a specific inhibitor of DFF40, binding to and specifically inhibiting its nuclease activity, while having no inhibitory effect on other DNases (DNase I, II, micrococcal nuclease, etc.). In normal cells, DFF45 binds to DFF40 and inhibits its activity. During apoptosis, DFF45 is hydrolyzed by caspase-3 into three smaller fragments, releasing active DFF40, which cleaves chromosomal DNA to form a DNA ladder. To date, apoptosis-specific endonucleases and their inhibitors have only been found in human, mouse, and fruit fly cells; similar apoptosis-specific endonucleases have not been reported in other species.
[0004] Mesenchymal stem cells (MSCs) are widely used in tissue repair and organ reconstruction due to their convenient sourcing, ease of isolation and culture, and ability to differentiate into various tissue cells such as bone, skin, blood vessels, and nerves under appropriate conditions. Although MSCs are in the early stages of development, they can still undergo apoptosis during culture and cryopreservation. Therefore, reducing MSC apoptosis can improve the yield of MSCs and enhance their repair effects, which is of significant clinical importance for improving the effectiveness of subsequent research and clinical translation of MSCs. Summary of the Invention
[0005] This invention aims to provide a culture method for reducing apoptosis of umbilical cord mesenchymal stem cells and improving their proliferation efficiency. Specifically, this method involves adding recombinant human DFF45 protein, Z-VAD-FMK, Cyclosporin A, bFGF, N-Acetylcysteine, and calcitonin to reduce apoptosis of umbilical cord mesenchymal stem cells.
[0006] This invention belongs to the field of cell biology.
[0007] To achieve the above objectives, the present invention adopts the following technical solution: A method for culturing mesenchymal stem cells to reduce apoptosis by regulating apoptosis-specific nucleases, characterized by the following culturing steps: The first step was to prepare recombinant human DNA breakage factor 45 (DFF45). Total RNA was extracted from normally cultured HeLa cells, and the DFF45 sequence was amplified by PCR using the total RNA as a template. After agarose gel electrophoresis, the DFF45 sequence was purified, ligated into the pMD18-T cloning vector, transformed into Escherichia coli DH5α, amplified, and the recombinant pMD18-T-DFF45 plasmid was extracted, double-digested, and sequenced. The pET28a expression vector was cultured and purified, and also double-digested. The accurately sequenced DFF45 sequence was ligated into the digested pET28a expression vector and transformed into BL21(DE3) competent Escherichia coli. After scale-up culture and promotion of expression, the recombinant human DFF45 protein was extracted and purified using affinity chromatography. The high-purity recombinant human DFF45 protein prepared in this step is a core factor in the subsequent regulation of umbilical cord mesenchymal stem cell proliferation and apoptosis. Its purity directly affects the subsequent regulatory effect, laying the foundation for subsequent steps to achieve increased proliferation and reduced apoptosis.
[0008] The second step involves adding recombinant human DFF45 protein to the P4 generation of umbilical cord mesenchymal stem cells. When P4 umbilical cord mesenchymal stem cells grow to 175 cm 2 When the culture flask reached 50% of its bottom area, DMEM culture medium containing 10% fetal bovine serum was added, followed by recombinant human DFF45 protein to a final concentration of 2 μm / mL. Z-VAD-FMK (final concentration 20 µm / L), Cyclosporin A (final concentration 5 µm / L), bFGF (final concentration 10 ng / mL), N-Acetylcysteine (final concentration 1 mM / L), and calcitonin (final concentration 20 mol / L) were then added, and the flask was placed in a constant-temperature CO2 incubator. Generation P4 was chosen as the starting point for intervention because the proliferation rate of umbilical cord mesenchymal stem cells was not significantly different in the first three generations, and the apoptosis rate was also low. The addition of recombinant DFF45 protein and calcitonin did not significantly reduce apoptosis. However, from generation P4 onwards, stem cells began to show a trend of slowed proliferation and increased apoptosis. Intervention at this point achieved the best regulatory effect. In this step, recombinant human DFF45 protein, calcitonin, Z-VAD-FMK, and other reagents worked synergistically to initially inhibit stem cell apoptosis and promote proliferation.
[0009] The third step involves the subsequent culture and apoptosis detection of umbilical cord mesenchymal stem cells. When the umbilical cord mesenchymal stem cells grow to 175 cm 2When the culture flask reached 80% of its bottom area, it was passaged, and recombinant human DFF45 protein was added again to a final concentration of 2 μmol / L, along with calcitonin to a final concentration of 20 mol / L. The proliferation of umbilical cord mesenchymal stem cells (UCMSCs) at passages P4, P5, P6, P7, and P8 was detected using the MTT assay, and the proliferation of UCMSCs in both groups was assessed using a streak assay. Total RNA was extracted from UCMSCs at passages P4, P5, P6, P7, and P8, and the expression levels of apoptosis-specific nucleases were detected by PCR after reverse transcription. The apoptosis rate of cells in both groups at each passage was detected using the TUNEL apoptosis detection kit. In this step, when the umbilical cord mesenchymal stem cells grew to 80% of the bottom area of the 175 cm² culture flask, they were passaged. After passage, recombinant human DFF45 protein at a final concentration of 2 μm / L and calcitonin at a final concentration of 20 mol / L were added again to maintain the regulatory effect and prevent the proliferation rate from falling back and the apoptosis rate from rebounding during passage. At the same time, multiple detection methods were used to verify the regulatory effect of the method of the present invention, which confirmed that the method of the present invention can significantly improve the proliferation rate of stem cells, and the proliferation advantage is more obvious with the extension of culture time. At the same time, it can effectively inhibit stem cell apoptosis, avoid the adverse effects of apoptotic cells on normal cells, and further improve the proliferation efficiency and cell quality of stem cells.
[0010] The recombinant human DFF45 protein, along with Z-VAD-FMK, Cyclosporin A, bFGF, N-Acetylcysteine, and calcitonin described in this invention, can reduce the expression and activity of apoptosis-specific nucleases, thereby reducing the apoptosis rate of umbilical cord mesenchymal stem cells (umbilical cord mesenchymal stem cells) and thus improving the proliferation efficiency of umbilical cord mesenchymal stem cells. In this invention, adding recombinant human DFF45 protein to a final concentration of 2 μm / L, Z-VAD-FMK to 20 μm / L, Cyclosporin A to 5 μm / L, bFGF to 10 ng / mL, N-Acetylcysteine to 1 mM / L, and calcitonin to 20 mol / L increased the proliferation rate of umbilical cord mesenchymal stem cells by 17.53%, and reduced the apoptosis rate of umbilical cord mesenchymal stem cells from 14.32% to 9.81% using conventional culture methods. This promotes and enhances the proliferation of umbilical cord mesenchymal stem cells and reduces the apoptosis rate.
[0011] In summary, this invention, through the first step of preparing high-purity recombinant human DFF45 protein, the second step of precisely adding recombinant human DFF45 protein and various auxiliary reagents to the P4 generation to initiate regulation, and the third step of continuously maintaining regulation after passage and verifying the effect through multiple methods, effectively solves the problems of slowed stem cell proliferation and increased apoptosis rate in existing culture techniques by working synergistically among these steps. It significantly improves the proliferation capacity and cell quality of stem cells, providing reliable technical support for the large-scale culture and clinical application of umbilical cord mesenchymal stem cells. Attached Figure Description
[0012] Figure 1 Comparative images of the morphology of P6 umbilical cord mesenchymal stem cells under a microscope in the control group and the example group (study group); Figure 2 Comparison of cell proliferation in umbilical cord mesenchymal stem cells cultured in the control group and the example group (study group) using MTT assay; Figure 3 A comparison of cell proliferation after a streak test of umbilical cord mesenchymal stem cells cultured in the control group and the example group (study group); Figure 4 This is a comparison chart showing the apoptosis rate of umbilical cord mesenchymal stem cells cultured in the control group and the example group (study group). Detailed Implementation
[0013] The following comparative experiment, using a conventional culture method (hereinafter referred to as the control group) and the apoptosis-reducing culture method used in this invention (hereinafter referred to as the example), further illustrates the present invention: control group Step 1: Umbilical Cord Tissue Processing Dissection tools should be autoclaved (120℃, 20 min) before use. Tools requiring sterilization include: large scissors, ophthalmic scissors, forceps, and hemostats. Preheat DMEM culture bottles in a 37°C water bath. Under aseptic conditions, cut the umbilical cord to approximately 5 cm in length. Rinse repeatedly with sterile saline 3-5 times to remove blood clots from the vessels. Rinse with 75% ethanol, then quickly rinse with saline. Wash with 1× anti-inflammatory and antibiotic solution, then rinse with saline. Cut the umbilical cord longitudinally and remove the vessels using forceps. Use a scalpel or ophthalmic scissors to cut the Wharton's jelly tissue from the umbilical cord. Add the collected internal Wharton's jelly tissue to a 50 mL centrifuge tube and cut the internal tissue into approximately 1 mm pieces. 3 Fragments.
[0014] The second step is the extraction of umbilical cord mesenchymal stem cells. Add an equal volume of trypsin digestion solution to the minced tissue. Incubate at 37°C for 10–20 min. Add an equal volume of digestion stop solution. Mix thoroughly, avoiding air bubbles, and centrifuge the suspended tissue at 1500 rpm for 10 min to pellet the cells. Discard the supernatant, and add an appropriate amount of DMEM culture medium to a final volume of approximately 25 mL. Seed the cells, using 5 mL of tissue suspension per flask to seed one 75 cm² ablation chamber. 2 Culture flasks. Place in a 37℃ constant temperature incubator and culture under a 5% CO2 gas phase. Replace with fresh DMEM medium every 3-4 days.
[0015] The third step is the primary passage culture of umbilical cord mesenchymal stem cells. When the umbilical cord mesenchymal stem cells grow to 75 cm 2 When the culture flask reaches 80% of its bottom area, discard the original culture medium under sterile conditions. Wash twice with 8 mL of pre-cooled PBS solution, add 5 mL of trypsin digestion solution for digestion, and when approximately 20% of the cells have slid down the culture flask wall, add DMEM culture medium containing 10% fetal bovine serum to stop digestion. Use a vibratory pipette to remove all cells, transfer the cell suspension to a 50 mL centrifuge tube, centrifuge at 2000 rpm for 10 min, discard the supernatant, add DMEM culture medium, and resuspend the cells as single cells. Seed the cells into new 75 cm⁻¹ culture media. 2 Place the culture flasks in a 37°C incubator and continue culturing, replacing the medium with fresh DMEM every 2-3 days.
[0016] Step 4: P2-P3 passage culture of umbilical cord mesenchymal stem cells When the umbilical cord mesenchymal stem cells grow to 75 cm 2 When the culture flask reaches 80% of its bottom area, discard the original culture medium under sterile conditions. Wash twice with 8 mL of pre-cooled PBS solution, add 5 mL of trypsin digestion solution for digestion, and when approximately 20% of the cells have slid down the culture flask wall, add DMEM culture medium containing 10% fetal bovine serum to stop digestion. Use a vibratory pipette to remove all cells, transfer the cell suspension to a 50 mL centrifuge tube, centrifuge at 2000 rpm for 10 min, discard the supernatant, add DMEM culture medium, and resuspend the cells as single cells. Seed the cells into a new 175 cm⁻¹ medium. 2 Replace the DMEM medium in the culture flask every 1-2 days.
[0017] Step 5: P3-P4 passage culture of low-molecular-weight umbilical cord mesenchymal stem cells When the umbilical cord mesenchymal stem cells grow to 175 cm 2 When the culture flask reaches 80% of its bottom area, discard the original culture medium under sterile conditions. Wash twice with 8 mL of pre-cooled PBS solution, add 5 mL of trypsin digestion solution for digestion, and when approximately 20% of the cells have slid down the culture flask wall, add DMEM culture medium containing 10% fetal bovine serum to stop digestion. Use a vibratory pipette to remove all cells, transfer the cell suspension to a 50 mL centrifuge tube, centrifuge at 2000 rpm for 10 min, discard the supernatant, add DMEM culture medium, and resuspend the cells as single cells. Seed the cells into a new 175 cm⁻¹ medium. 2 Replace the DMEM culture medium in the culture flask daily.
[0018] Example A method for reducing mesenchymal stem cell apoptosis by regulating apoptosis-specific nucleases, characterized by the following culture steps: Step 1: Umbilical Cord Tissue Processing Dissection tools should be autoclaved (120℃, 20 min) before use. Tools requiring sterilization include: large scissors, ophthalmic scissors, forceps, and hemostats. Preheat DMEM culture bottles in a 37°C water bath. Under aseptic conditions, cut the umbilical cord to approximately 5 cm in length. Rinse repeatedly with sterile saline 3-5 times to remove blood clots from the vessels. Rinse with 75% ethanol, then quickly rinse with saline. Wash with 1× anti-inflammatory and antibiotic solution, then rinse with saline. Cut the umbilical cord longitudinally and remove the vessels using forceps. Use a scalpel or ophthalmic scissors to cut the Wharton's jelly tissue from the umbilical cord. Add the collected internal Wharton's jelly tissue to a 50 mL centrifuge tube and cut the internal tissue into approximately 1 mm pieces. 3 Fragments.
[0019] The second step is the extraction of umbilical cord mesenchymal stem cells. Add an equal volume of trypsin digestion solution to the minced tissue. Incubate at 37°C for 10–20 min. Add an equal volume of digestion stop solution. Mix thoroughly, avoiding air bubbles, and centrifuge the suspended tissue at 1500 rpm for 10 min to pellet the cells. Discard the supernatant, and add an appropriate amount of DMEM culture medium to a final volume of approximately 25 mL. Seed the cells, using 5 mL of tissue suspension per flask to seed one 75 cm² ablation chamber. 2 Culture flasks. Place in a 37℃ constant temperature incubator and culture under a 5% CO2 gas phase. Replace with fresh DMEM medium every 3-4 days.
[0020] The third step is the primary passage culture of umbilical cord mesenchymal stem cells. When the umbilical cord mesenchymal stem cells grow to 75 cm 2 When the culture flask reaches 80% of its bottom area, discard the original culture medium under sterile conditions. Wash twice with 8 mL of pre-cooled PBS solution, add 5 mL of trypsin digestion solution for digestion, and when approximately 20% of the cells have slid down the culture flask wall, add DMEM culture medium containing 10% fetal bovine serum to stop digestion. Use a vibratory pipette to remove all cells, transfer the cell suspension to a 50 mL centrifuge tube, centrifuge at 2000 rpm for 10 min, discard the supernatant, add DMEM culture medium, and resuspend the cells as single cells. Seed the cells into new 75 cm⁻¹ culture media. 2 Replace the DMEM medium in the culture flask every 2-3 days.
[0021] Step 4: P2-P3 passage culture of umbilical cord mesenchymal stem cells When the umbilical cord mesenchymal stem cells grow to 75 cm2 When the culture flask reaches 80% of its bottom area, discard the original culture medium under sterile conditions. Wash twice with 8 mL of pre-cooled PBS solution, add 5 mL of trypsin digestion solution for digestion, and when approximately 20% of the cells have slid down the culture flask wall, add DMEM culture medium containing 10% fetal bovine serum to stop digestion. Use a vibratory pipette to remove all cells, transfer the cell suspension to a 50 mL centrifuge tube, centrifuge at 2000 rpm for 10 min, discard the supernatant, add DMEM culture medium, and resuspend the cells as single cells. Seed the cells into a new 175 cm⁻¹ medium. 2 Replace the DMEM medium in the culture flask every 1-2 days.
[0022] Step 5: P3-P4 passage culture of umbilical cord mesenchymal stem cells When the umbilical cord mesenchymal stem cells grew to 80% of the bottom area of the 175 cm² culture flask, the original culture medium was discarded under sterile conditions. The cells were washed twice with 8 mL of pre-cooled PBS solution, and then digested with 5 mL of trypsin digestion solution. When about 20% of the cells slid down the culture flask wall, DMEM culture medium with 10% fetal bovine serum was added to stop the digestion. All the cells were blown off using an electric pipette, and the cell suspension was transferred to a 50 mL centrifuge tube. The tube was centrifuged at 2000 rpm for 10 min, the supernatant was discarded, DMEM culture medium was added, and the cells were repositioned into single cells. The cells were then seeded into a new 175 cm² culture flask, and the DMEM culture medium was changed daily.
[0023] Step 6: Preparation of recombinant human DNA breakage factor 45 (DFF45) Total RNA was extracted from normally cultured HeLa cells, and the DFF45 sequence was amplified by PCR using the total RNA as a template. After agarose gel electrophoresis, the DFF45 sequence was purified, ligated into the pMD18-T cloning vector, transformed into Escherichia coli DH5α, amplified, and the recombinant pMD18-T-DFF45 plasmid was extracted, double-digested, and sequenced. The pET28a expression vector was cultured and purified, and also double-digested. The accurately sequenced DFF45 sequence was ligated into the digested pET28a expression vector and transformed into BL21(DE3) competent Escherichia coli. After scale-up culture and promotion of expression, the recombinant human DFF45 protein was extracted and purified using affinity chromatography to obtain the purified recombinant human DFF45 protein.
[0024] Step 7: Recombinant human DFF45 protein is added to the P4 generation of umbilical cord mesenchymal stem cells. When the P4 umbilical cord mesenchymal stem cells grew to 50% of the bottom area of the 175 cm2 culture flask, DMEM culture medium containing 10% fetal bovine serum was added, and recombinant human DFF45 protein was added to a final concentration of 2 μm / mL. Z-VAD-FMK, Cyclosporin A, bFGF, N-Acetylcysteine, and calcitonin were added to a final concentration of 20 µm / L, 5 µm / L, 10 ng / mL, 1 mM / L, and 20 mol / L. The flask was then placed in a constant temperature CO2 incubator for culture.
[0025] Step 8: Subsequent culture and apoptosis detection of umbilical cord mesenchymal stem cells. When umbilical cord mesenchymal stem cells (UC-MSCs) reached 80% of the bottom area of a 175 cm² culture flask, they were passaged, and recombinant human DFF45 protein was added again to a final concentration of 2 μm / L, along with calcitonin to a final concentration of 20 mol / L. The proliferation of UC-MSCs at passages P4, P5, P6, P7, and P8 was assessed using the MTT assay, and the proliferation of UC-MSCs in both groups was detected using a streak assay. Total RNA was extracted from UC-MSCs at passages P4, P5, P6, P7, and P8, and the expression levels of apoptosis-specific nucleases were detected by PCR after reverse transcription. The apoptosis rate of cells at each passage in both groups was detected using the TUNEL apoptosis detection kit.
[0026] The following comparative experiment, using conventional umbilical cord mesenchymal stem cell culture methods, further illustrates the effectiveness of the present invention: The effects of this invention are as follows: 1. Increase the proliferation rate of umbilical cord mesenchymal stem cells The MTT assay was used to detect and compare the proliferation of umbilical cord mesenchymal stem cells (umbilical cord mesenchymal stem cells) in the control group and the embodiment. Because the proliferation rate of the first three generations of umbilical cord mesenchymal stem cells did not differ significantly, and the apoptosis rate was also low, and the reduction in apoptosis was not significant after using and adding recombinant DFF45 protein and calcitonin, intervention and comparison were conducted starting from generation P4 cells until generation P8. Using conventional culture methods, the proliferation of P4 generation umbilical cord mesenchymal stem cells was (41.47±4.41), while the proliferation of P4 generation umbilical cord mesenchymal stem cells cultured using the apoptosis-reducing culture method of this invention was (48.47±4.41). Using conventional culture methods, the proliferation of P5 generation umbilical cord mesenchymal stem cells was (45.62±4.19), while the proliferation of P5 generation umbilical cord mesenchymal stem cells cultured using the apoptosis-reducing culture method of this invention was... (52.65±4.71); The proliferation of P6 generation umbilical cord mesenchymal stem cells cultured using conventional methods was (48.91±5.03), while the proliferation of P6 generation umbilical cord mesenchymal stem cells cultured using the apoptosis-reducing culture method of this invention was (56.17±4.85); The proliferation of P7 generation umbilical cord mesenchymal stem cells cultured using conventional methods was (51.67±4.86), while the proliferation of P7 generation umbilical cord mesenchymal stem cells cultured using the apoptosis-reducing culture method of this invention was (59.52±5.18); The proliferation of P8 generation umbilical cord mesenchymal stem cells cultured using conventional methods was (53.38±5.24), while the proliferation of P8 generation umbilical cord mesenchymal stem cells cultured using the apoptosis-reducing culture method of this invention was (62.74±5.39). Figure 2 ).
[0027] The proliferation of umbilical cord mesenchymal stem cells in the control group and the study was detected and compared using the streak method. The streak widths of the umbilical cord mesenchymal stem cells in the control group, which used conventional culture methods, at 1, 2, 3, and 4 days after streak, were basically the same as those in the study group, which used the culture method of this invention. However, on day 5, the streak width of the umbilical cord mesenchymal stem cells in the control group was significantly greater than that in the study group, indicating that the proliferation of stem cells in the study group was significantly higher than that in the control group at day 5.
[0028] Compared with the control group, the proliferation level of stem cells in each generation of the embodiment was significantly improved, with the P8 generation showing the most significant improvement. This indicates that the present invention can effectively delay the decline in proliferation rate of umbilical cord mesenchymal stem cells with increasing passage number. The streak line test further confirms that the stem cells cultured in the present invention have significantly better proliferation activity than the conventional culture group after long-term culture (5 days), suggesting that they have stronger cell division ability and better proliferation persistence, and can provide a sufficient and stable source of cells for large-scale expansion of stem cells.
[0029] 2. Reduces the apoptosis rate of umbilical cord mesenchymal stem cells and improves the cell culture microenvironment. A certain percentage of umbilical cord mesenchymal stem cells undergo apoptosis during culture, especially after increasing the number of passages. After apoptosis, secretory cells rupture and release a large number of factors in the cytoplasm, including apoptosis factors, which affect normally growing cells, reduce the proliferation efficiency of umbilical cord mesenchymal stem cells, and may also induce premature apoptosis in other normally growing umbilical cord mesenchymal stem cells.
[0030] The apoptosis rate of cells in each generation of the two groups was detected using the TUNEL apoptosis detection kit. The results showed that the apoptosis rate of P4 generation umbilical cord mesenchymal stem cells cultured using conventional methods was (6.14±0.95)%, while the apoptosis rate of P4 generation umbilical cord mesenchymal stem cells cultured using the apoptosis-reducing culture method of this invention was (5.63±0.81)%; the apoptosis rate of P5 generation umbilical cord mesenchymal stem cells cultured using conventional methods was (7.85±1.12)%, while the apoptosis rate of P5 generation umbilical cord mesenchymal stem cells cultured using the apoptosis-reducing culture method of this invention was (6.37±1.06)%; the apoptosis rate of P6 generation umbilical cord mesenchymal stem cells cultured using conventional methods was (9...). The apoptosis rate of P6 generation umbilical cord mesenchymal stem cells cultured using the apoptosis-reducing culture method of this invention was (7.06±1.27)%. The apoptosis rate of P7 generation umbilical cord mesenchymal stem cells cultured using conventional methods was (11.57±1.49)%, while the apoptosis rate of P7 generation umbilical cord mesenchymal stem cells cultured using the apoptosis-reducing culture method of this invention was (8.52±1.38)%. The apoptosis rate of P8 generation umbilical cord mesenchymal stem cells cultured using conventional methods was (14.32±1.74)%, while the apoptosis rate of P8 generation umbilical cord mesenchymal stem cells cultured using the apoptosis-reducing culture method of this invention was (9.81±1.62)%. Figure 4 ).
[0031] TUNEL assay results showed that the experimental results significantly reduced the apoptosis rate of umbilical cord mesenchymal stem cells from passages P4 to P8, and the inhibitory effect became more significant with increasing passage number (the apoptosis rate of passage P8 in the control group was significantly increased, while the apoptosis rate of the experimental results remained at a low level). This apoptosis inhibition not only reduced the proportion of apoptotic cells but also reduced the release of apoptotic factors, improved the cell culture microenvironment, and broke the vicious cycle of "apoptosis-inhibition of proliferation-further apoptosis," thereby indirectly improving the overall proliferation efficiency of stem cells and maintaining good biological activity and quality stability of cells.
[0032] In summary, this invention, through the precise preparation of high-purity recombinant human DFF45 protein, targeted intervention starting at passage 4, and continuous regulation after passage, synergistically achieves the dual technical effects of enhancing the proliferation capacity and reducing the apoptosis rate of umbilical cord mesenchymal stem cells. It effectively solves the core problems of stem cell proliferation decline and increased apoptosis rate in existing culture techniques, maintains the biological activity and quality stability of stem cells during long-term passage, and provides reliable technical support and experimental basis for the large-scale culture, in vitro expansion, and subsequent clinical translation of umbilical cord mesenchymal stem cells.
Claims
1. A method for culturing mesenchymal stem cells to reduce apoptosis by regulating apoptosis-specific nucleases, characterized in that, The method, after the isolation and culture of umbilical cord mesenchymal stem cells, includes the following steps in sequence: The first step was to prepare recombinant human DNA breakage factor 45 (DFF45): Total RNA was extracted from normally cultured HeLa cells. The total RNA was used as a template for PCR amplification to obtain the DFF45 sequence. After agarose gel electrophoresis and purification, the DFF45 sequence was ligated into the pMD18-T cloning vector and transformed into Escherichia coli DH5α. After amplification, the recombinant pMD18-T-DFF45 plasmid was extracted and subjected to double enzyme digestion and sequencing. The pET28a expression vector was cultured and purified. The pET28a expression vector was double-digested with enzymes. The DFF45 sequence with accurate sequencing was ligated into the digested pET28a expression vector. The vector was then transformed into BL21(DE3) competent Escherichia coli. After large-scale culture and promotion of expression, recombinant human DFF45 protein was extracted and purified using affinity chromatography to obtain purified recombinant human DFF45 protein. The second step involves adding recombinant human DFF45 protein to the P4 generation of umbilical cord mesenchymal stem cells: When the P4 generation umbilical cord mesenchymal stem cells grew to 50% of the bottom area of a 175 cm² culture flask, DMEM culture medium containing 10% fetal bovine serum was added, along with the purified recombinant human DFF45 protein to a final concentration of 2 μm / mL, and Z-VAD-FMK, Cyclosporin A, bFGF, N-Acetylcysteine, and calcitonin to a final concentration of 20 µm / L, 5 µm / L, 10 ng / mL, 1 mM / L, and 20 mol / L, respectively. The flask was then placed in a constant temperature CO2 incubator for culture. The third step involves the subsequent culture and apoptosis detection of umbilical cord mesenchymal stem cells: When umbilical cord mesenchymal stem cells (UCSCs) reached 80% of the bottom area of a 175 cm² culture flask, they were passaged. After passage, recombinant human DFF45 protein and calcitonin were added again at a final concentration of 2 μm / L and 20 mol / L, respectively. The proliferation of UCSCs at passages P4, P5, P6, P7, and P8 was detected using the MTT assay, and the proliferation of UCSCs at each passage was detected using the streak assay. Total RNA was extracted from UCSCs at passages P4, P5, P6, P7, and P8, and the expression level of apoptosis-specific nucleases was detected by PCR after reverse transcription. The apoptosis rate of cells at each passage was detected using the TUNEL apoptosis detection kit.
2. The method according to claim 1, characterized in that, After PCR amplification of the DFF45 sequence as described in step one, the purpose of agarose gel electrophoresis is to verify the correctness of the PCR amplification product and to obtain a DFF45 sequence with a purity of ≥95% after purification.
3. The method according to claim 1, characterized in that, The enzymes used for double digestion of the recombinant pMD18-T-DFF45 plasmid in step one are the same as those used for double digestion of the pET28a expression vector.
4. The method according to claim 1, characterized in that, The conditions for scaling up the BL21(DE3) competent Escherichia coli culture in step one are 37℃ and 200r / min shaking culture, and IPTG induction is used to promote expression.
5. The method according to claim 1, characterized in that, The affinity chromatography column mentioned in step one is a Ni²⁺ affinity chromatography column, and the concentration of recombinant human DFF45 protein after purification is ≥1 mg / mL.
6. The method according to claim 1, characterized in that, The incubation conditions in the constant temperature CO2 incubator described in step two are 37℃, 5% CO2, and saturated humidity.
7. The method according to claim 1, characterized in that, The subculture ratio in step three is 1:3, and the culture environment after subculture is the same as the culture conditions in the constant temperature CO2 incubator in step two.
8. The method according to claim 1, characterized in that, In step three, the MTT method is used to detect proliferation at a wavelength of 570 nm, and the TUNEL apoptosis detection kit is used to determine the positive criterion for apoptosis rate by the appearance of green fluorescence in the cell nucleus.