Method for promoting Notch1 protein expression through low-intensity pulse ultrasonic waves and ultrasonic therapeutic apparatus

Abstract

The invention discloses a method for promoting Notch1 protein expression through low-intensity pulse ultrasonic waves and an ultrasonic therapeutic apparatus, and belongs to the technical field of biology. The method comprises the following steps: inoculating third-generation human umbilical vein endothelial cells (HUVEC) on a 96-well plate coated with matrigel, culturing for 6 hours, and performing ultrasonic stimulation on the third-generation human umbilical vein endothelial cells by using low-intensity pulse ultrasonic waves with the intensity of 0.5-1.5 W / cm < 2 >, the pulse frequency of 1000Hz and the duty ratio of 50%. The method can effectively promote the protein expression of Notch1, thereby enhancing the tubulation ability of HUVEC cells. The ultrasonic therapeutic apparatus can emit ultrasonic waves with the intensity of 0.5-1.5 W / cm < 2 >, the pulse frequency of 1000Hz and the duty ratio of 50%, and the ultrasonic therapeutic apparatus can stimulate cells and promote protein expression of Notch1 in the cells, so that the tube forming capability of the cells is enhanced.

Description

Technical Field

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

[0002] HUVEC (Human Umbilical Vein Endothelial Cells) is a type of stem cell with angiogenic potential, which plays an important role in embryonic development, especially in angiogenesis and placenta formation. In the laboratory, researchers may culture HUVEC cells in a high-sugar environment to simulate the response of vascular endothelial cells under diabetic conditions and study the impact of diabetes on vascular health. Studies have found that patients with chronic renal insufficiency have impaired endothelial function, which may be related to damage or dysfunction of endothelial cells (such as HUVEC cells). When endothelial cell function is impaired, it may lead to renal hemodynamic disorders, decreased glomerular filtration rate, and increased inflammatory response, all of which may aggravate the condition of chronic renal insufficiency. Therefore, angiogenesis ability is crucial for the normal physiological function and repair of the kidneys.

[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 the 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 In view of 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 stimulating HUVEC cells with low-intensity pulsed ultrasound, 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.

[0006] 2. Technical solution

[0007] To solve the above problems, the present invention adopts the following technical solutions.

[0008] A method for promoting Notch1 protein expression by low-intensity pulsed ultrasound, the method comprising: inoculating third-generation human umbilical vein endothelial cells HUVEC on a 96-well plate coated with matrix gel, culturing for 6 hours, and using 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%.

[0009] Furthermore, the method for obtaining the third generation human umbilical vein epithelial cells is: digesting the cleaned neonatal umbilical cord with Trypsin enzyme, extracting HUVEC cells, culturing them to confluence, and then routinely passaged to the third generation.

[0010] Furthermore, in step (2), the cell density of HUVEC cells was about 5×10 3 / hole.

[0011] A low-intensity pulsed ultrasound therapeutic device for relieving 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 transmitting device.

[0012] 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 emit ultrasonic waves.

[0013] Furthermore, the intensity of the ultrasound is 0.5~1.5W / cm 2 , pulse frequency is 1000Hz, and duty cycle is 50%.

[0014] Furthermore, the specific extraction operation of HUVEC cells is as follows: A. Place 15-20 cm long newborn umbilical cord in sterile PBS solution (with double antibody and heparin sodium) for storage; B. Use a 50mL syringe and fill it with fresh PBS each time. Clamp the two ends of the umbilical cord with hemostats and gently squeeze to dissolve the blood clots in the umbilical cord for easy flushing. C. Clamp the lower end of the umbilical cord with surgical forceps, add 15 mL of HUVEC-specific digestive enzyme, and digest at room temperature for 15 to 20 minutes. During digestion, frequently turn the umbilical cord to allow the enzyme solution to flow in the blood vessels, promoting uniform contact between endothelial cells and the enzyme. D. After digestion, first use a blunt needle to clear the outlet at the lower end to prevent blockage, loosen the surgical forceps at the lower end, and let the digestion fluid flow into a 50mL sterile centrifuge tube. There should be some complete culture medium or pure serum in the tube. Rinse the umbilical cord 2 to 3 times with sterile PBS solution, and gently rub it with your hands each time; E. Centrifuge the collected liquid at 1000 rpm for 3 minutes; F. Pour off the supernatant, add 10 mL of ECM medium, blow off the cells with a Pasteur pipette, transfer all the liquid into a T25 culture flask, and culture at 37°C; 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; I. Change the culture medium every 2 days. After 5-7 days of culture, cells can be subcultured when they are confluent to 80-90% monolayer.

[0015] 3. Beneficial effects

[0016] Compared with the prior art, the advantages of the present invention are: 1. The method provided in this scheme is conducive to promoting the expression of Notch1 protein in cells, while not changing the β-Actin1 protein, thereby not causing damage to the function of HUVEC cells. The operation process of this method is relatively simple, low-cost, and not prone to side effects, which helps to improve the safety and effectiveness of treatment.

[0017] Second, this solution also provides a low-intensity pulsed ultrasonic therapeutic device for alleviating renal insufficiency caused by diabetes. The ultrasonic 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

[0018] Figure 1 A schematic diagram of the distribution of ultrasonic waves emitted by an ultrasonic therapeutic device; Figure 2 This is a diagram showing the identification results of HUVEC cells isolated and extracted from Examples 1 to 3 and Comparative Example 1 (NC is a negative control, i.e., the identification results of Comparative Example 1); Figure 3 Schematic diagram of protein electrophoresis of Western blot detection of Notch1 and β-Actin1 in Examples 1 to 3 and Comparative Example 1; Figure 4 Schematic diagram of the total kidney weight of rats in each component in Example 4; Figure 5 Schematic diagram of protein electrophoresis in rats of each group in Example 4; Figure 6 The graphs are blood sugar changes of rats in each group in Example 4; Figure 7is the blood creatinine content of rats in each group in Example 4; Figure 8 is the urea nitrogen content of each group of rats in Example 4; Fig. 9 Schematic comparison of HE staining of 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

[0019] Embodiment 1:

[0020] An ultrasonic therapeutic device includes a transmitter box, a signal generator and an ultrasonic probe, wherein the signal generator is fixed inside the transmitter box and connected to the ultrasonic probe through a cable. The signal generator can control the ultrasonic probe to emit an intensity of 0.5~1.5W / cm 2 , ultrasonic wave with pulse frequency of 1000Hz and duty cycle of 50%.

[0021] The sound intensity distribution diagram of the low-intensity pulsed ultrasonic wave emitted by the ultrasonic probe of the ultrasonic therapeutic apparatus 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 range of the low-intensity pulsed ultrasound emitted by the ultrasonic therapeutic device will gradually decrease as the intensity of the sound waves increases, so that the ultrasonic therapeutic device can accurately stimulate the parts that need treatment during use and minimize the impact of the low-intensity pulsed ultrasound on non-treatment parts.

[0022] Embodiment 2:

[0023] 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 a serum-containing medium for 6 hours, 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 calculated using Image J image analysis software (National Institutes of Health). The HUVEC cells were stimulated at a certain intensity using the ultrasonic therapeutic apparatus described in Example 1. The intensity of the low-intensity pulsed ultrasound was 0.5 W / cm 2The stimulation time was 10 minutes and the stimulation was continued for 3 days. During the operation, the cell culture temperature was maintained at 37°C. On the fourth day, images of the tube morphology were obtained using an inverted Olympus microscope, and the number of meshes and the degree of tube formation were calculated using Image J image analysis software (National Institutes of Health).

[0024] Embodiment 3:

[0025] 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 a serum-containing medium for 6 hours, 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 calculated using Image J image analysis software (National Institutes of Health). The HUVEC cells were stimulated at a certain intensity using the ultrasonic therapeutic apparatus described in Example 1. The intensity of the low-intensity pulsed ultrasound was 1.0 W / cm 2 The stimulation time was 10 minutes and the stimulation was continued for 3 days. During the operation, the cell culture temperature was maintained at 37°C. On the fourth day, images of the tube morphology were obtained using an inverted Olympus microscope, and the number of meshes and the degree of tube formation were calculated using Image J image analysis software (National Institutes of Health).

[0026] Embodiment 4:

[0027] 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 a serum-containing medium for 6 hours, 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 calculated using Image J image analysis software (National Institutes of Health). The HUVEC cells were stimulated at a certain intensity using the ultrasonic therapeutic apparatus described in Example 1. The intensity of the low-intensity pulsed ultrasound was 1.5 W / cm 2The stimulation time was 10 minutes and the stimulation was continued for 3 days. During the operation, the cell culture temperature was maintained at 37 °C. On the fourth day, images of the tube morphology were obtained using an inverted Olympus microscope, and the number of meshes and the degree of tube formation were calculated using Image J image analysis software (National Institutes of Health).

[0028] Comparative Example 1: 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 calculated using Image J image analysis software (National Institutes of Health). The cell culture temperature was maintained at 37 °C and no operations were performed on it. 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 calculated using Image J image analysis software (National Institutes of Health).

[0029] 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 tube nodes of HUVEC cells using the Image J image analysis software, and (F) is the result of the total length of tubes of HUVEC cells using the Image J image analysis software. Figure 1 From the morphological diagram of HUVEC under the Olympus microscope, it can be seen that the vascular imaging of Examples 2, 3 and 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 example is significantly greater than the total number of HUVEC cells in Comparative Example 1, and the total length of the cell tubes in the example is significantly greater than the total length of the cell tubes in Comparative Example 1. It can be seen that Examples 2, 3 and 4 can all 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 3From the protein electrophoresis diagram of the protein marker Notch1 detected by Western blot, it can be seen that, compared with Example 1, the Notch1 protein expression of HUVEC cells stimulated by low-intensity pulsed ultrasound increased compared with that of HUVEC cells not treated; and as the ultrasound intensity increased, the Notch1 protein expression continued to increase. Therefore, after stimulating HUVEC cells with low-intensity pulsed ultrasound, the tube-forming ability of HUVEC cells can be promoted by increasing Notch1 protein expression. And this method is a non-invasive method and will not change the protein expression pattern of HUVEC cells.

[0030] Test Example 1: The ultrasonic therapeutic device used in this embodiment is the ultrasonic therapeutic device provided in Embodiment 1.

[0031] Establishment of diabetic nephropathy (DN) model: 8-week-old male rats weighing about 180 g were fed with ordinary feed for 3 days, and then continuously fed with 45% high-fat feed. After 5 weeks of fasting overnight, 50 mg / kg streptozotocin was injected intraperitoneally once; the random blood glucose levels of the rats were continuously tested, and rats with random blood glucose ≥16.7 mmol / L for three consecutive days were regarded as diabetic nephropathy rats, that is, the DN model was obtained.

[0032] The DN model was divided into 2 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.

[0033] The rats in the CON, LI+CON, DN, and LI+DN groups were anesthetized with breathing to keep them in a stable posture. The kidneys were scanned with Doppler ultrasound to determine the length, width, and thickness of the kidneys. The kidneys in the LI+DN and LI+CON groups were scanned with an energy intensity of 1.0 W / cm 2 , ultrasound stimulation with a frequency of 28kHz and a pulse frequency of 1000Hz. Single stimulation lasted for 10min, once a day, for 1 week. One week later, the rats' tails were cut to collect blood samples, and the creatinine and urea nitrogen content in the rats' serum was determined using an automatic biochemical analyzer. The blood glucose changes of the rats were measured every two weeks.

[0034] Figure 4 is a schematic diagram of the total kidney weight of rats in each group after treatment; Figure 4It 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.

[0035] like Figure 5 The schematic diagram of protein electrophoresis in each group of rats shows that there is no significant change in the expression of β-Actin protein in each group of rats, indicating that the total protein content in the kidney has not changed and the groups are comparable. The Notch1 protein in the DN group is lower than that in the CON group, and the Notch1 protein in the LIPUS+DN group is higher than that in the DN group, indicating that low-intensity pulsed ultrasound stimulation can increase the Notch1 protein content in the kidneys of rats with diabetic nephropathy.

[0036] Depend on Figure 6 It can be seen that the blood sugar 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 sugar of the LI+DN group rat model began to decrease compared with the blood sugar of the DN group rat model. On the 42nd day, the blood sugar of the LI+DN group rat model had decreased significantly compared with the blood sugar of the DN group rat model, thereby slowing down the further deterioration of renal function.

[0037] Depend on Figure 7 and Figure 8 It can be seen that the urea nitrogen content and creatinine content in the serum of the CON group rat model and the LI+CON group rat model were both 5.5mmol / L, and the use of low-intensity pulsed ultrasound would not affect the filtration and excretion function of normal kidneys. The urea nitrogen content and creatinine content in the serum of the LI+DN group rat model were 8mmol / L, and the urea nitrogen content and creatinine content in the serum of the DN rat model were 13mmol / L. After the use of low-intensity pulsed ultrasound stimulation, the urea nitrogen content and creatinine content in the serum of the LI+DN group rat model were significantly lower than those of the DN group rats, and the filtration and excretion function of the kidneys were improved.

[0038] After the experiment, the kidney tissues of the rats were quickly collected to prepare staining sections. Fig. 9As shown, there was no significant change in the regularity of glomerular collagen fibers, basement membrane and renal tubules between the LI+CON (c) group rat model and the CON (a) group rat model, so the stimulation of low-intensity pulsed ultrasound did not cause changes in renal function. Compared with the LI+CON (c) group rat model and the CON (a) group rat model, the DN (b) group rat model had significantly more glomerular collagen fibers, thickened basement membrane and irregular renal tubules; however, although the LI+DN group rat model stimulated by low-intensity pulsed ultrasound had less glomerular collagen fibers, thin basement membrane and regular renal tubules than the LI+CON (c) group rat model and the CON (a) group rat model, the glomerular and tubular pathology of the LI+DN group rat model stimulated by low-intensity pulsed ultrasound was improved compared with the DN group.

Claims

1. A method for promoting Notch1 protein expression by low-intensity pulsed ultrasound, characterized in that: The method comprises the following steps: inoculating the third generation human umbilical vein endothelial cells HUVEC on a 96-well plate coated with matrix gel, culturing for 6 hours, and applying a strength 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%.

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 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.

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

4. A low-intensity pulsed ultrasonic therapeutic device for relieving diabetic renal insufficiency, characterized by: In the method for promoting Notch1 protein expression by low-intensity pulsed ultrasound as described in any one of claims 1 to 3, the ultrasonic therapeutic apparatus is used as a transmitter of low-intensity pulsed ultrasound.

5. A low-intensity pulsed ultrasonic therapeutic device for relieving diabetic renal insufficiency according to claim 4, characterized in that: It includes a transmitter box, a signal generating device and an ultrasonic probe. The signal generating device is fixedly connected in the transmitter box and connected to the ultrasonic probe through a cable. The signal generating device controls the ultrasonic probe to emit ultrasonic waves.

6. A low-intensity pulsed ultrasonic therapeutic device for relieving diabetic renal insufficiency according to claim 5, characterized in that: The intensity of ultrasound is 0.5~1.5W / cm 2 , pulse frequency is 1000Hz, and duty cycle is 50%.

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