Method for enhancing the expression of P-selectin in vascular endothelial cells
By treating vascular endothelial cells with shock waves and enhancing P-selectin expression, the problem of stem cells being unable to adhere to vascular endothelial cells was solved, and the effective homing of stem cells to damaged areas was achieved.
Patent Information
- Application Number
- CN201811007661.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2018-08-31
- Publication Date
- 2025-09-09
- Estimated Expiration
- 2038-08-31
AI Technical Summary
In the existing technology, stem cells cannot effectively adhere to vascular endothelial cells, resulting in an inability to effectively home to damaged areas, and there is a lack of simple and effective methods to enhance the expression of P-selectin.
The shock wave device was used to treat vascular endothelial cells, and the pressure, frequency and frequency were set. The expression of P-selectin gene was detected by fluorescence quantitative PCR to enhance its expression.
It significantly enhances the expression of P-selectin in vascular endothelial cells, is simple and does not require drug stimulation, and is particularly effective in vivo, providing a basis for stem cell homing.
Smart Images
Figure BDA0001784323560000041
Abstract
Description
Technical Field
[0001] The present invention relates to the field of biotechnology, and in particular to a method for enhancing the expression of P-selectin in vascular endothelial cells. Background Art
[0002] Stem cells are known in the medical field as "universal cells" because they can differentiate into a variety of functional cells, tissues, and organs. Stem cell transplantation involves the transplantation of healthy stem cells into a patient's body to repair or replace damaged cells or tissues, thereby achieving a cure. Stem cell transplantation has a wide range of therapeutic applications, generally treating neurological and immune disorders, as well as other internal and external medical conditions. However, many technical aspects of stem cell transplantation remain unresolved, significantly impacting the success rate of treatment. A typical issue is the inability of stem cells to effectively home to the damaged area. Numerous studies have shown that P-selectin expression in cells at the damaged site is a key factor in inducing stem cell homing. Without P-selectin, stem cells are unable to locate and adhere to vascular endothelial cells. To enhance stem cell recruitment to the target site, P-selectin expression must be effectively increased. However, there is a lack of technology to simply and effectively regulate P-selectin gene expression in target cells. Summary of the Invention
[0003] A method for enhancing the expression of P-selectin in vascular endothelial cells, comprising the following steps:
[0004] Step (1): setting the shock wave device action mode and action parameters including pressure, frequency, and frequency;
[0005] Step (2): preparing subcultured vascular endothelial cells or vascular endothelial cells from experimental mice, centrifuging them, resuspending them in 10% complete culture medium, and transplanting them into a culture dish, and then subjecting the cells to shock wave treatment using a probe;
[0006] Step (3): After the shock wave treatment, the cells were cultured for another 60 hours, and then the endothelial cells were isolated and the expression of the P-selectin gene was detected by fluorescence quantitative PCR, with the GAPDH gene as the internal standard.
[0007] Preferably, in step (2), the method for preparing the subcultured vascular endothelial cells is as follows: taking out the primary human cardiac microvascular endothelial cell cryopreservation tube from liquid nitrogen, thawing it in a 37°C water bath, transferring it to a culture flask coated with L-polylysine, adding a certain amount of endothelial cell culture medium, culturing it in a 37°C, 5% carbon dioxide incubator, changing the medium every 2 to 3 days, passaged once every 4 to 5 days, digesting the cells with trypsin, and using the fifth generation cells in the experiment.
[0008] Preferably, the primary human cardiac microvascular endothelial cells can be selected from vascular endothelial cells in vivo.
[0009] In any of the above schemes, preferably, in step (1), the shock wave parameter ranges are pressure 1.5-3.0 bar, frequency 8-12 Hz, and frequency 200 times.
[0010] In any of the above schemes, preferably, in step (3), the expression level of the selectin gene is calculated using a relative quantitative method, the expression amount of the target gene is determined by the difference between the Ct values of the target gene and the internal reference gene, GAPDH is used as the internal reference gene, and the quantitative PCR of each RNA sample is repeated three times to eliminate systematic errors.
[0011] In any of the above schemes, preferably, in step (3), the fluorescent quantitative PCR method used for detection is: extracting total RNA with the HighPure RNA Isolation Kit, reverse transcribing the total RNA into cDNA with the TaqMan Reverse Transcription Rengents kit, taking 2.5 μL of the reverse transcription product cDNA template, and performing quantitative PCR detection according to the instructions of the TaqMan Gene Expression Master Mix.
[0012] In any of the above schemes, preferably, in step (3), the continued culturing time is 60 hours.
[0013] In any of the above schemes, preferably, the method for enhancing the expression of P-selectin in vascular endothelial cells is not limited to the expression of P-selectin.
[0014] The present invention enhances P-selectin expression in vascular endothelial cells through shock waves, does not require other drugs or chemical stimulation, is easy to operate, and has significant effects. In particular, it has an absolute advantage in the expression of P-selectin in vascular endothelial cells in vivo, and is the basis for the future development of stem cell homing and stem cell-related technologies. DETAILED DESCRIPTION
[0015] To make the objectives, technical solutions, and advantages of the embodiments of the present invention more clear, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the preferred embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts shall fall within the scope of protection of the present invention.
[0016] Example 1
[0017] 1) Remove cryovials of primary human cardiac microvascular endothelial cells (HCMECs) from liquid nitrogen, rapidly thaw in a 37°C water bath, transfer to poly-L-lysine-coated culture flasks, add an appropriate amount of endothelial cell culture medium, and culture in a 37°C, 5% CO2 incubator. Change the medium every 2–3 days, passage once every 4–5 days, and digest the cells with trypsin. Cells at passage 5 were used in this experiment.
[0018] 2) Passage 5 HCMEC cells were digested and centrifuged, then resuspended in 10% complete culture medium and transplanted into a culture dish. The shock wave probe was disinfected and set to 2.5 bar and 10 Hz. The cells were subjected to shock waves 200 times. The control group consisted of passage 5 HCMEC cells that were not treated with shock waves. The shock wave probe was a product of EMS, Switzerland.
[0019] 3) The cells treated with shock waves were cultured for 60 hours for RNA isolation, and the gene expression levels of P-selectin and E-selectin were detected using fluorescent quantitative real-time PCR technology.
[0020] 4) Calculate the selectin gene expression level using relative quantification. The expression level of the target gene is determined by the difference in Ct values between the target gene and the internal reference gene. In this study, GAPDH was used as the internal reference gene. Quantitative PCR was performed in triplicate for each RNA sample to eliminate systematic errors. The specific steps are as follows:
[0021] Total RNA was extracted using the High Pure RNA Isolation Kit and reverse transcribed into cDNA using the TaqMan Reverse Transcription Reagents kit (25 μL system). Quantitative PCR was performed using 2.5 μL of the reverse-transcribed cDNA template according to the TaqMan Gene Expression Master Mix instructions. Primer information is shown in Table 1. The amplification procedure was: 95°C for 10 min, 95°C for 30 s, 55°C for 30 s, and 72°C for 30 s, for a total of 40 cycles. The quantitative PCR instrument was a Roche 480 II. Primer information is shown in Table 1. At the RNA level, the expression level of the P-selectin gene after shock wave treatment was approximately 24 times that before treatment.
[0022] Table 1: Selectin quantitative PCR primer information
[0023]
[0024] Example 2
[0025] 1) Remove cryovials of primary human cardiac microvascular endothelial cells (HCMECs) from liquid nitrogen, rapidly thaw in a 37°C water bath, transfer to poly-L-lysine-coated culture flasks, add an appropriate amount of endothelial cell culture medium, and culture in a 37°C, 5% CO2 incubator. Change the medium every 2–3 days, passage once every 4–5 days, and digest the cells with trypsin. Cells at passage 5 were used in this experiment.
[0026] 2) HCMEC cells at passage 5 were digested and centrifuged, then resuspended in 10% complete culture medium and transplanted into a culture dish. The shock wave probe was disinfected and the shock wave parameters were set to 1.5 bar and 10 Hz. The cells were subjected to shock waves 200 times. The control group consisted of HCMEC cells at passage 5 that were not treated with shock waves.
[0027] 3) The cells treated with shock waves were cultured for 60 hours for RNA isolation, and the gene expression levels of P-selectin and E-selectin were detected using fluorescent quantitative real-time PCR technology.
[0028] 4) The expression level of the selectin gene was calculated using a relative quantitative method. The expression level of the target gene was determined by the difference in Ct values between the target gene and the internal reference gene. In the present invention, GAPDH was used as the internal reference gene, and quantitative PCR was performed three times for each RNA sample to eliminate systematic errors. From the RNA level, the expression level of the P-selectin gene after shock wave treatment was approximately 15 times that before treatment.
[0029] Example 3
[0030] 1) Remove cryovials of primary human cardiac microvascular endothelial cells (HCMECs) from liquid nitrogen, rapidly thaw in a 37°C water bath, transfer to poly-L-lysine-coated culture flasks, add an appropriate amount of endothelial cell culture medium, and culture in a 37°C, 5% CO2 incubator. Change the medium every 2–3 days, passage once every 4–5 days, and digest the cells with trypsin. Cells at passage 5 were used in this experiment.
[0031] 2) HCMEC cells at passage 5 were digested and centrifuged, then resuspended in 10% complete culture medium and transplanted into a culture dish. The shock wave probe was disinfected and the shock wave parameters were set to 2.0 bar and 10 Hz. The cells were subjected to shock waves 200 times. The control group consisted of HCMEC cells at passage 5 that were not treated with shock waves.
[0032] 3) The cells treated with shock waves were cultured for 60 hours for RNA isolation, and the gene expression levels of P-selectin and E-selectin were detected using fluorescent quantitative real-time PCR technology.
[0033] 4) The expression level of the selectin gene was calculated using a relative quantitative method. The expression level of the target gene was determined by the difference in Ct value between the target gene and the internal reference gene. In the present invention, GAPDH was used as the internal reference gene, and quantitative PCR was performed three times for each RNA sample to eliminate systematic errors. From the RNA level, the expression level of the P-selectin gene after shock wave treatment was approximately 20 times that before treatment.
[0034] Example 4
[0035] 1) Remove cryovials of primary human cardiac microvascular endothelial cells (HCMECs) from liquid nitrogen, rapidly thaw in a 37°C water bath, transfer to poly-L-lysine-coated culture flasks, add an appropriate amount of endothelial cell culture medium, and culture in a 37°C, 5% CO2 incubator. Change the medium every 2–3 days, passage once every 4–5 days, and digest the cells with trypsin. Cells at passage 5 were used in this experiment.
[0036] 2) HCMEC cells at passage 5 were digested and centrifuged, then resuspended in 10% complete culture medium and transplanted into a culture dish. The shock wave probe was disinfected and the parameters were set to 3.0 bar and 10 Hz. The cells were subjected to shock waves 200 times. The control group consisted of HCMEC cells at passage 5 that were not treated with shock waves.
[0037] 3) The cells treated with shock waves were cultured for 60 hours for RNA isolation, and the gene expression levels of P-selectin and E-selectin were detected using fluorescent quantitative real-time PCR technology.
[0038] 4) The expression level of the selectin gene was calculated using a relative quantitative method. The expression level of the target gene was determined by the difference in Ct value between the target gene and the internal reference gene. In the present invention, GAPDH was used as the internal reference gene, and quantitative PCR was performed three times for each RNA sample to eliminate systematic errors. From the RNA level, the expression level of the P-selectin gene after shock wave treatment was approximately 17 times that before treatment.
[0039] Example 5
[0040] 1) Remove cryovials of primary human cardiac microvascular endothelial cells (HCMECs) from liquid nitrogen, rapidly thaw in a 37°C water bath, transfer to poly-L-lysine-coated culture flasks, add an appropriate amount of endothelial cell culture medium, and culture in a 37°C, 5% CO2 incubator. Change the medium every 2–3 days, passage once every 4–5 days, and digest the cells with trypsin. Cells at passage 5 were used in this experiment.
[0041] 2) Passage 5 HCMEC cells were digested and centrifuged, then resuspended in 10% complete culture medium and transplanted into a culture dish. The shock wave probe was disinfected and the parameters were set to 2.5 bar and 12 Hz. The cells were subjected to shock waves 200 times. The control group consisted of passage 5 HCMEC cells that were not subjected to shock wave treatment.
[0042] 3) The cells treated with shock waves were cultured for 60 hours for RNA isolation, and the gene expression levels of P-selectin and E-selectin were detected using fluorescent quantitative real-time PCR technology.
[0043] 4) The expression level of the selectin gene was calculated using a relative quantitative method. The expression level of the target gene was determined by the difference in Ct value between the target gene and the internal reference gene. In the present invention, GAPDH was used as the internal reference gene, and quantitative PCR was performed three times for each RNA sample to eliminate systematic errors. From the RNA level, the expression level of the P-selectin gene after shock wave treatment was approximately 21 times that before treatment.
[0044] Example 6
[0045] 1) Remove cryovials of primary human cardiac microvascular endothelial cells (HCMECs) from liquid nitrogen, rapidly thaw in a 37°C water bath, transfer to poly-L-lysine-coated culture flasks, add an appropriate amount of endothelial cell culture medium, and culture in a 37°C, 5% CO2 incubator. Change the medium every 2–3 days, passage once every 4–5 days, and digest the cells with trypsin. Cells at passage 5 were used in this experiment.
[0046] 2) HCMEC cells at passage 5 were digested and centrifuged, then resuspended in 10% complete culture medium and transplanted into a culture dish. The shock wave probe was disinfected and the parameters were set to 2.0 bar and 8 Hz. The cells were subjected to shock waves 200 times. The control group consisted of HCMEC cells at passage 5 that were not treated with shock waves.
[0047] 3) The cells treated with shock waves were cultured for 60 hours for RNA isolation, and the gene expression levels of P-selectin and E-selectin were detected using fluorescent quantitative real-time PCR technology.
[0048] 4) The expression level of the selectin gene was calculated using a relative quantitative method. The expression level of the target gene was determined by the difference in Ct value between the target gene and the internal reference gene. In the present invention, GAPDH was used as the internal reference gene, and quantitative PCR was performed three times for each RNA sample to eliminate systematic errors. From the RNA level, the expression level of the P-selectin gene after shock wave treatment was approximately 18 times that before treatment.
[0049] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any changes or substitutions that can be easily conceived by a person skilled in the art within the technical scope disclosed in the present invention should be included in the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be based on the scope of protection of the claims.
Claims
1. A method for enhancing the expression of P-selectin in vascular endothelial cells, comprising the following steps: Step (1): setting the shock wave device action mode and action parameters, including pressure, frequency, and frequency; Step (2): preparing subcultured vascular endothelial cells or vascular endothelial cells from experimental mice, centrifuging them, resuspending them in 10% complete culture medium, and transplanting them into a culture dish, and then subjecting the cells to shock wave treatment using a probe; Step (3): After the shock wave treatment, the cells were cultured and the endothelial cells were isolated and the expression of P-selectin gene was detected by fluorescence quantitative PCR, with GAPDH gene as the internal standard. In step (2), the method for preparing the subcultured vascular endothelial cells is as follows: taking out the primary human cardiac microvascular endothelial cell cryopreservation tube from liquid nitrogen, thawing it in a 37°C water bath, transferring it to a culture bottle coated with L-polylysine, placing a certain amount of endothelial cell culture medium, culturing it in a 37°C, 5% carbon dioxide incubator, changing the medium every 2 to 3 days, passaged once every 4 to 5 days, digesting the cells with trypsin, and using the fifth generation cells for the experiment; the primary human cardiac microvascular endothelial cells can be selected from vascular endothelial cells in vivo; In step (1), the shock wave parameters range are pressure 1.5-3.0 bar, frequency 8-12 Hz, and frequency 200 times; In step (3), the expression level of the selectin gene was calculated by relative quantitative method, and the expression amount of the target gene was determined by the difference between the Ct value of the target gene and the internal reference gene. GAPDH was used as the internal reference gene, and the quantitative PCR of each RNA sample was repeated three times to eliminate the systematic error. In step (3), the fluorescence quantitative PCR method used for detection was as follows: total RNA was extracted with High Pure RNA Isolation Kit, and the total RNA was reverse transcribed into cDNA with TaqMan Reverse Transcription Regents kit. 2.5 μL of the reverse transcription product cDNA template was taken and quantitative PCR detection was performed according to the instructions of TaqMan Gene Expression Master Mix. In step (3), the culture time was continued for 60 hours.