Method for promoting Notch1 protein expression by low-intensity pulsed ultrasound and ultrasound therapeutic device

Abstract

The present invention discloses a method for promoting Notch1 protein expression by low-intensity pulsed ultrasound and an ultrasound therapeutic apparatus, which belongs to the field of biotechnology. The method comprises the following steps: third-generation human umbilical vein endothelial cells (HUVECs) are inoculated on a 96-well plate coated with matrix gel, cultured for 6 hours, and then treated with an ultrasound at an intensity of 0.5-1.5 W / cm 2 The cells were stimulated with low-intensity pulsed ultrasound at a pulse frequency of 1000 Hz and a duty cycle of 50%. This method can effectively promote the expression of Notch1 protein, thereby enhancing the tube-forming ability of HUVEC cells. The ultrasound therapy device can emit an intensity of 0.5~1.5W / cm 2 , ultrasound with a pulse frequency of 1000Hz and a duty cycle of 50%. The use of this ultrasound therapeutic device can stimulate cells, promote the expression of Notch1 protein in cells, and thus enhance the cell's ability to form tubes.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and more particularly to a method for promoting Notch1 protein expression by using low-intensity pulsed ultrasound and an ultrasonic therapeutic apparatus. Background Art

[0002] Human umbilical vein endothelial cells (HUVECs) are a type of stem cell with angiogenic potential that plays a crucial role in embryonic development, particularly in angiogenesis and placenta formation. In the laboratory, researchers may culture HUVECs in a high-glucose environment to mimic the endothelial cell response under diabetic conditions and study the impact of diabetes on vascular health. Studies have found that patients with chronic renal insufficiency experience impaired endothelial function, which may be related to damage or dysfunction of endothelial cells (such as HUVECs). Impaired endothelial cell function can lead to renal hemodynamic disturbances, decreased glomerular filtration rate, and increased inflammatory responses, all of which may exacerbate chronic renal insufficiency. Therefore, angiogenesis is crucial for normal kidney function and repair.

[0003] Notch1 protein is a signaling molecule that plays a key role in the process of angiogenesis. It is involved in regulating the proliferation, migration and differentiation of endothelial cells. These processes are crucial for the formation of new blood vessels. However, the problem of how to effectively promote the expression of Notch1 protein has not been well solved.

[0004] Therefore, it is necessary to provide a method that can effectively promote the expression of Notch1 protein and a device that can be used in this method, and during the application of the device, the device will only promote the expression of Notch1 protein without causing negative effects on the expression of other proteins in the cell. Summary of the Invention

[0005] 1. Problem to be solved

[0006] In response to the problems existing in the prior art, the first purpose of the present invention is to provide a method for promoting the expression of Notch1 protein by using low-intensity pulsed ultrasound, by using low-intensity pulsed ultrasound to stimulate HUVEC cells, thereby promoting the expression of Notch1 protein in HUVEC cells and improving the ability of HUVEC cells to form tubes; the second purpose is to provide an ultrasonic therapeutic device that can be used to promote the expression of Notch1 protein.

[0007] 2. Technical solution

[0008] To solve the above problems, the present invention adopts the following technical solutions.

[0009] A method for promoting Notch1 protein expression by low-intensity pulsed ultrasound, comprising: seeding third-generation human umbilical vein endothelial cells (HUVECs) on a 96-well plate coated with matrigel, culturing for 6 hours, and then irradiating with an intensity of 0.5-1.5 W / cm 2 The rats were stimulated by low-intensity pulsed ultrasound with a pulse frequency of 1000 Hz and a duty cycle of 50%.

[0010] Furthermore, the method for obtaining the third generation human umbilical vein epithelial cells is as follows: the cleaned neonatal umbilical cord is digested with Trypsin enzyme, HUVEC cells are extracted, and the cells are cultured until confluence and then routinely passaged to the third generation.

[0011] Furthermore, in step (2), the cell density of HUVEC cells was about 5×10 3 / hole.

[0012] A low-intensity pulsed ultrasound therapeutic device for alleviating diabetic renal insufficiency. In the method for promoting Notch1 protein expression by low-intensity pulsed ultrasound, the low-intensity pulsed ultrasound therapeutic device is used as a low-intensity pulsed ultrasound transmitter.

[0013] Furthermore, the ultrasonic therapeutic apparatus includes a transmitter box, a signal generating device and an ultrasonic probe. The signal generating device is fixedly connected to the transmitter box and is connected to the ultrasonic probe via a cable. The signal generating device controls the ultrasonic probe to transmit ultrasonic waves.

[0014] Furthermore, the intensity of the ultrasound is 0.5~1.5W / cm 2 , the pulse frequency is 1000Hz and the duty cycle is 50%.

[0015] Furthermore, the specific extraction operation of HUVEC cells is as follows:

[0016] A. Place a 15-20 cm long neonatal umbilical cord in a sterile PBS solution (with double-stranded antibodies and sodium heparin) for storage;

[0017] B. Use a 50mL syringe and fill it with fresh PBS each time. Then clamp the two ends of the umbilical cord with hemostats and gently knead the cord to break up the blood clots and make it easier to flush.

[0018] C. Clamp the lower end of the umbilical cord with surgical forceps, add 15 mL of HUVEC digestive enzyme, and digest at room temperature for 15-20 minutes. During digestion, frequently turn the umbilical cord to allow the enzyme solution to flow within the blood vessels and promote uniform contact between the endothelial cells and the enzyme.

[0019] D. After digestion, first clear the lower outflow port with a blunt needle to prevent blockage. Release the lower surgical forceps and allow the digestion fluid to flow into a 50 mL sterile centrifuge tube. The tube should contain some complete culture medium or pure serum. Rinse the umbilical cord 2-3 times with sterile PBS solution, gently massaging it each time.

[0020] E. Centrifuge the collected liquid at 1000 rpm for 3 minutes;

[0021] F. Pour off the supernatant, add 10 mL of ECM medium, disperse the cells with a Pasteur pipette, transfer all the liquid into a T25 culture flask, and culture at 37°C.

[0022] H. After 24 hours of culture, discard the culture medium and wash 2-3 times with sterile PBS solution to remove red blood cells and dead cells, and add 10 mL of fresh ECM culture medium;

[0023] I. Change the culture medium every 2 days. After 5-7 days of culture, cells can be passaged when they reach 80-90% confluence and form a monolayer.

[0024] 3. Beneficial effects

[0025] Compared with the prior art, the advantages of the present invention are:

[0026] First, the method provided in this protocol promotes Notch1 protein expression in cells without altering β-Actin1 protein, thereby preventing damage to HUVEC cell function. This method is relatively simple to operate, low-cost, and unlikely to produce side effects, helping to improve the safety and effectiveness of treatment.

[0027] Second, this solution also provides a low-intensity pulsed ultrasound therapeutic device for alleviating renal insufficiency caused by diabetes. The ultrasound therapeutic device can emit an intensity of 0.5~1.5W / cm 2 , ultrasound with a pulse frequency of 1000Hz and a duty cycle of 50%; this ultrasound therapeutic device can stimulate cells, promote the expression of Notch1 protein in cells, and thus enhance the cell's tube-forming ability. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is a schematic diagram of the distribution of ultrasonic waves emitted by an ultrasonic therapeutic device;

[0029] Figure 2 Graph showing the identification results of HUVEC cells isolated and extracted from Examples 1 to 3 and Comparative Example 1 (NC is the negative control, i.e., the identification results of Comparative Example 1);

[0030] Figure 3Schematic diagram of protein electrophoresis of Western blot detection of Notch1 and β-Actin1 in Examples 1 to 3 and Comparative Example 1;

[0031] Figure 4 Schematic diagram of the total kidney weight of rats in each component in Example 4;

[0032] Figure 5 Schematic diagram of protein electrophoresis in rats of each group in Example 4;

[0033] Figure 6 Figure 4 is a graph showing changes in blood sugar levels in rats of each group;

[0034] Figure 7 is the blood creatinine content of each group of rats in Example 4;

[0035] Figure 8 is the urea nitrogen content of each group of rats in Example 4;

[0036] Figure 9 Schematic comparison of HE staining of the kidneys of rats in each group in Example 4 ((a) CON group; (b) DN group, (c) LI+CON group, (d) LI+DN group; (1) is the morphology of glomeruli; (2) is the morphology of renal tubules). DETAILED DESCRIPTION

[0037] Example 1:

[0038] An ultrasonic therapeutic device includes a transmitter box, a signal generator, and an ultrasonic probe. The signal generator is fixed inside the transmitter box and connected to the ultrasonic probe via a cable. The signal generator can control the ultrasonic probe's emission intensity to 0.5-1.5W / cm 2 , ultrasonic wave with a pulse frequency of 1000Hz and a duty cycle of 50%.

[0039] The sound intensity distribution diagram of the low-intensity pulsed ultrasonic wave emitted by the ultrasonic probe of the ultrasonic therapeutic device is as follows: Figure 1 As shown in part (I), the sound intensity distribution diagram of the low-intensity pulsed ultrasound emitted by the ultrasonic therapeutic device is as follows Figure 1 As shown in part (II); Figure 1 It can be seen that the low-intensity pulsed ultrasound emitted by the ultrasonic therapeutic device can gradually reduce the range of the low-intensity pulsed ultrasound as the intensity of the sound wave increases, so that the ultrasonic therapeutic device can accurately stimulate the area that needs treatment during use and minimize the impact of the low-intensity pulsed ultrasound on non-treatment areas.

[0040] Example 2:

[0041] HUVEC cells were seeded in 96-well plates at a cell density of approximately 5 × 10 3 / well. The plate was pre-coated with Matrigel (Matrigel, Corning, USA), and 50 μl was added to each well. After culturing for 6 hours in a serum-containing medium, an inverted Olympus microscope was used to obtain images of the duct morphology, and the number of meshes and the degree of duct formation were quantified using Image J image analysis software (National Institutes of Health). The HUVEC cells were stimulated at a high intensity using the ultrasonic therapeutic apparatus described in Example 1. The intensity of the low-intensity pulsed ultrasound was 0.5 W / cm 2 Stimulation was performed for 10 minutes for three consecutive days. The cell culture temperature was maintained at 37°C. On the fourth day, images of ductal morphology were acquired using an inverted Olympus microscope. Image J analysis software (National Institutes of Health) was used to count the number of meshes and quantify the extent of duct formation.

[0042] Example 3:

[0043] HUVEC cells were seeded in 96-well plates at a cell density of approximately 5 × 10 3 / well. The plate was pre-coated with Matrigel (Matrigel, Corning, USA), and 50 μl was added to each well. After culturing for 6 hours in a serum-containing medium, an inverted Olympus microscope was used to obtain images of the duct morphology, and the number of meshes and the degree of duct formation were quantified using Image J image analysis software (National Institutes of Health). The HUVEC cells were stimulated at a high intensity using the ultrasonic therapeutic apparatus described in Example 1. The intensity of the low-intensity pulsed ultrasound was 1.0 W / cm 2 Stimulation was performed for 10 minutes for three consecutive days. The cell culture temperature was maintained at 37°C. On the fourth day, images of ductal morphology were acquired using an inverted Olympus microscope. Image J analysis software (National Institutes of Health) was used to count the number of meshes and quantify the extent of duct formation.

[0044] Example 4:

[0045] HUVEC cells were seeded in 96-well plates at a cell density of approximately 5 × 10 3 / well. The plate was pre-coated with Matrigel (Matrigel, Corning, USA), and 50 μl was added to each well. After culturing for 6 hours in a serum-containing medium, an inverted Olympus microscope was used to obtain images of the duct morphology, and the number of meshes and the degree of duct formation were quantified using Image J image analysis software (National Institutes of Health). The HUVEC cells were stimulated at a high intensity using the ultrasonic therapeutic apparatus described in Example 1. The intensity of the low-intensity pulsed ultrasound was 1.5 W / cm 2 Stimulation was performed for 10 minutes for three consecutive days. The cell culture temperature was maintained at 37°C during the procedure. On the fourth day, images of the ductal morphology were acquired using an inverted Olympus microscope. Image J analysis software (National Institutes of Health) was used to count the number of meshes and quantify the extent of duct formation.

[0046] Comparative Example 1:

[0047] HUVEC cells were seeded in 96-well plates at a cell density of approximately 5 × 10 3 / well. The plate was pre-coated with Matrigel (Matrigel, Corning, USA), and 50 μl was added to each well. After culturing in serum-containing medium for 6 hours, images of the duct morphology were obtained using an inverted Olympus microscope, and the number of meshes and the degree of duct formation were quantified using Image J image analysis software (National Institutes of Health). The cell culture temperature was maintained at 37°C and no manipulation was performed on the cells. On the fourth day, images of the duct morphology were obtained using an inverted Olympus microscope, and the number of meshes and the degree of duct formation were quantified using Image J image analysis software (National Institutes of Health).

[0048] Figure 2 Schematic diagram of angiogenesis of isolated and extracted HUVEC cells; in the figure, (A) is the HUVEC morphology of comparative example 1 under an Olympus microscope (scale = 100um), (B) is the HUVEC morphology of example 2 under an Olympus microscope, (C) is the HUVEC morphology of example 3 under an Olympus microscope, (D) is the HUVEC morphology of example 4 under an Olympus microscope, (E) is the result of the HUVEC cell tube nodes using Image J image analysis software, and (F) is the result of the HUVEC cell tube total length using Image J image analysis software. Figure 1From the morphological diagram of HUVEC under the Olympus microscope, it can be seen that the vascular imaging of Example 2, Example 3 and Example 4 is significantly higher than that of Comparative Example 1; from the analysis results of Image J image analysis software, it can be seen that the total number of HUVEC cells in the embodiment is significantly greater than the total number of HUVEC cells in Comparative Example 1, and the total length of the cell tubes in the embodiment is significantly greater than the total length of the cell tubes in Comparative Example 1. It can be seen that Example 2, Example 3 and Example 4 can enhance the tube-forming ability of HUVEC and significantly increase the number of tube-forming nodes and the length of tubes. Among them, Example 3 has a more significant improvement in the tube-forming ability than Example 2 and Example 1. Figure 3 As shown in the protein electrophoresis diagram of the protein marker Notch1 detected by Western blot, compared with Comparative Example 1, the Notch1 protein expression in HUVEC cells stimulated by low-intensity pulsed ultrasound increased compared with that in HUVEC cells that were not treated. Furthermore, Notch1 protein expression continued to increase with increasing ultrasound intensity. Therefore, low-intensity pulsed ultrasound stimulation of HUVEC cells can promote the tube-forming ability of HUVEC cells by increasing Notch1 protein expression. Furthermore, this method is non-invasive and does not alter the protein expression pattern of HUVEC cells.

[0049] Test Example 1:

[0050] The ultrasonic therapeutic apparatus used in this embodiment is the ultrasonic therapeutic apparatus provided in Example 1.

[0051] Establishment of a diabetic nephropathy (DN) model: 8-week-old male rats weighing approximately 180 g were fed a standard diet for 3 days, then continuously fed a 45% high-fat diet. After 5 weeks of fasting, 50 mg / kg of streptozotocin was injected intraperitoneally. The random blood glucose levels of the rats were continuously monitored, and rats with random blood glucose levels ≥16.7 mmol / L for three consecutive days were considered to have diabetic nephropathy, thus establishing a DN model.

[0052] The DN model was divided into two groups, namely the DN group and the LI+DN (DN rat model after stimulation by ultrasonic therapeutic device) group; and the groups were divided into the CON (normally raised rats, not fed with 45% high-fat feed, and not intraperitoneally injected with 50 mg / kg streptozotocin) group and the LI+CON (CON rat model after stimulation by ultrasonic therapeutic device) group; each group had at least 6 rats.

[0053] The rats in the CON, LI+CON, DN, and LI+DN groups were anesthetized and kept in a stable posture. The kidneys were scanned by Doppler ultrasound to determine the length, width, and thickness of the kidneys. The kidneys in the LI+DN and LI+CON groups were scanned from the dorsal side with an energy intensity of 1.0 W / cm2 Ultrasound stimulation was performed at a frequency of 28 kHz and a pulse rate of 1000 Hz. Each stimulation session lasted 10 minutes, and was repeated once daily for one week. After one week, blood samples were collected by tail clipping, and serum creatinine and urea nitrogen levels were measured using an automated biochemical analyzer. Blood glucose levels were measured every two weeks.

[0054] Figure 4 is a schematic diagram of the total kidney weight of rats in each group after treatment; Figure 4 It can be seen that the kidney weight of the rat model in the DN group was significantly reduced compared with the rat model in the CON group, and the total kidney weight of the rat model in the LI+DN group and the LI+CON group could be restored to the normal range, thereby alleviating the problem of renal insufficiency caused by diabetic lesions.

[0055] like Figure 5 Protein electrophoresis diagrams of the rats in each group show no significant changes in β-Actin protein expression, indicating no change in total renal protein content and comparability among the groups. However, Notch1 protein levels were lower in the DN group compared to the CON group, while those in the LIPUS+DN group were higher compared to the DN group, indicating that low-intensity pulsed ultrasound stimulation can increase Notch1 protein levels in the kidneys of rats with diabetic nephropathy.

[0056] Depend on Figure 6 It can be seen that the blood glucose changes in the LI+CON group rat model and the CON group rat model are similar, with no significant difference, which indicates that the stimulation of low-intensity pulsed ultrasound will not affect kidney function under normal conditions; starting from the 14th day, the blood glucose of the LI+DN group rat model began to show a downward trend compared with the blood glucose of the DN group rat model. By the 42nd day, the blood glucose of the LI+DN group rat model had decreased significantly compared with the blood glucose of the DN group rat model, thereby slowing down the further deterioration of renal function.

[0057] Depend on Figure 7 and Figure 8 The results showed that the serum urea nitrogen and creatinine levels in the CON and LI+CON rat models were both 5.5 mmol / L, indicating that the use of low-intensity pulsed ultrasound did not affect the filtration and excretion functions of the normal kidneys. The serum urea nitrogen and creatinine levels in the LI+DN rat model were 8 mmol / L, and those in the DN rat model were 13 mmol / L. After low-intensity pulsed ultrasound stimulation, the serum urea nitrogen and creatinine levels in the LI+DN rat model were significantly lower than those in the DN rat model, indicating improved renal filtration and excretion functions.

[0058] After the experiment, the kidney tissues of the rats were quickly collected to prepare staining sections. Figure 9As shown, there were no significant changes in the regularity of glomerular collagen fibers, basement membrane, and renal tubules in the LI+CON (c) group and the CON (a) group, indicating that low-intensity pulsed ultrasound stimulation did not cause changes in renal function. Compared with the LI+CON (c) group and the CON (a) group, the DN (b) group showed a significant increase in glomerular collagen fibers, thickened basement membrane, and irregular renal tubules. However, although the LI+DN group, stimulated by low-intensity pulsed ultrasound, had less glomerular collagen fibers, thinner basement membrane, and more regular renal tubules than the LI+CON (c) group and the CON (a) group, the LI+DN group showed improved glomerular and tubular pathology compared to the DN group.

Claims

1. A method for promoting Notch1 protein expression using low-intensity pulsed ultrasound, characterized in that: The method comprises the following steps: seeding third generation human umbilical vein endothelial cells (HUVEC) on a 96-well plate coated with matrix gel, culturing for 6 hours, and applying a 0.5-1.5 W / cm 2 The rats were stimulated by low-intensity pulsed ultrasound with a pulse frequency of 1000 Hz and a duty cycle of 50%.

2. The method for promoting Notch1 protein expression by low-intensity pulsed ultrasound according to claim 1, characterized in that: The method for obtaining the third generation human umbilical vein endothelial cells is as follows: the cleaned neonatal umbilical cord is digested with Trypsin enzyme, HUVEC cells are extracted, and the cells are cultured until confluence and then routinely passaged to the third generation.

3. The method for promoting Notch1 protein expression by low-intensity pulsed ultrasound according to claim 2, characterized in that: The cell density of HUVEC cells was 5 × 10 3 / hole.

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