Method for treating pulmonary arterial hypertension by miR-142-3p and application of miR-142-3p

Abstract

The invention discloses a method for treating pulmonary arterial hypertension through miR-142-3p and application, and belongs to the field of biological medicines.The treatment method specifically comprises the following steps that firstly, lentivirus is prepared, and a pre-transfection experiment is conducted; iI, detecting the influence of the lentivirus on the proliferation efficiency and migration activity of the hPASMCs cells; iII, selecting and culturing multiple groups of male SD rats to prepare PH model rats; iV, carrying out a hemodynamic experiment on the PH model rat; v, performing histomorphological analysis on the PH model rats after the experiment; vI, collecting and recording hemodynamic experiment and histomorphological analysis results; the miR-142-3p has good treatment and prevention effects, can well reduce right ventricular pressure, has no obvious systemic toxic effect, can be administered in an injection or nasal drop mode according to different purposes, is harmless to a human body, is easy to synthesize and administrate, is simple and feasible, and has low production cost.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a method and application of miR-142-3p in treating pulmonary hypertension. Background Art

[0002] Pulmonary hypertension is a pulmonary vascular disease that affects the arterioles, which can lead to increased pulmonary artery pressure and vascular resistance. According to the standards of the World Health Organization (WHO), pulmonary hypertension is diagnosed when the mean pulmonary artery pressure (mPAP) measured by right cardiac catheter at sea level and resting state is ≥25 mmHg. Pulmonary hypertension can be divided into primary pulmonary hypertension and secondary pulmonary hypertension. The former has a low incidence rate of about 1 to 2 per million people, while the latter is very common. The main causes include connective tissue diseases, congenital heart disease, HIV infection, pulmonary venous disease, respiratory diseases, thromboembolic diseases, etc. The continuous deterioration and progression of pulmonary hypertension can lead to pathological conditions such as right heart failure, venous congestion, and limited venous return of important organs. It is very easy to form cachexia and has a very high mortality rate. At present, the treatment of pulmonary hypertension is still very difficult. Secondary pulmonary hypertension mainly corrects the cause, while primary pulmonary hypertension has few treatment methods. The commonly used targeted drugs in clinical practice are often very expensive, and the overall clinical effect is not ideal. Therefore, actively exploring the prevention and treatment methods of pulmonary hypertension has become a clinical problem that needs to be solved urgently. In view of the shortcomings of the current diagnosis and treatment of pulmonary arterial hypertension, the purpose of the present invention is to provide a method and application of miR-142-3p in treating pulmonary arterial hypertension. Summary of the invention

[0003] The purpose of the present invention is to solve the defects in the prior art and to propose a method and application of miR-142-3p for treating pulmonary hypertension.

[0004] In order to achieve the above object, the present invention adopts the following technical solutions:

[0005] The method of treating pulmonary arterial hypertension with miR-142-3p, the specific steps of the treatment method are as follows:

[0006] Ⅰ. Prepare lentivirus and conduct pre-transfection experiments;

[0007] II. Detect the effect of lentivirus on the proliferation efficiency and migration activity of hPASMCs;

[0008] III. Select and culture multiple groups of male SD rats to prepare PH model rats;

[0009] IV. Conduct hemodynamic experiments using PH model rats;

[0010] V. Conduct histological morphological analysis on PH model rats after the experiment;

[0011] VI. Collect and record the results of hemodynamic experiments and tissue morphological analysis.

[0012] As a further solution of the present invention, the specific steps of preparing the lentivirus in step I are as follows:

[0013] S1.1: Sequence alignment was performed using the Blast public database, and the hs-miR-142-3p sequence that was consistent with the rat rno-miR-142-3p sequence was selected. Specific primers were designed for the selected hs-miR-142-3p sequence, and then the GV260 vector was linearized using restriction enzymes;

[0014] S1.2: Detect the linearization effect by agarose gel electrophoresis, cut and purify the desired fragment, and configure the PCR reaction system, then set the PCR cycle conditions, and place has-miR-142-3p in a PCR instrument for reaction, then detect the PCR product by agarose gel electrophoresis, and purify the target fragment;

[0015] S1.3: Mix the linearized GV260 vector with the miRNA fragment obtained by PCR amplification, add ligase for ligation reaction, and then use chemical method or electroporation method to transform the recombinant plasmid into Escherichia coli, culture the transformed cells on LB agar plates containing antibiotics, and screen positive clones by PCR or enzyme digestion identification;

[0016] S1.4: Use a plasmid extraction kit to extract plasmid DNA from positive clones, use restriction enzymes to cut the extracted plasmid to confirm the presence and correctness of the inserted fragment, and then submit the recombinant plasmid for sequencing to verify the correct sequence of the miRNA fragment. Transfect the identified recombinant plasmid into a suitable packaging cell line, add the viral packaging plasmid, collect the cell supernatant after standing for 48 hours, and obtain the purified LV-miR-142-3p virus by centrifugation and filtration.

[0017] As a further embodiment of the present invention, the raw materials of the PCR reaction system described in S1.2 specifically include 32.5 μL of ddH2O, 10 μL of 5xPS Buffer, 4 μL of dNTP Mix (2.5 mM each), 1 μL of upstream amplification primer (10 μM), 1 μL of downstream amplification primer (10 μM), 1 μL of template (10 ng / μL) and 0.5 μL of PrimeSTAR HSDNA polymerase;

[0018] The template is sourced from plasmid or bacterial solution, and the amount of plasmid template is less than 200 ng, and the amount of bacterial solution template is 1 μL.

[0019] As a further solution of the present invention, the specific steps of the pre-transfection experiment in step I are as follows:

[0020] S2.1: Use 3-8 passage hPASMCs as experimental cells and inoculate 1×10 cells per well in a 96-well plate. 4 The cells were plated according to the standard of 100 cells, and the 96-well plate was placed in the incubator for culture until the cell fusion reached 20% to 30%. According to the requirements of six groups of different MOI values ​​of 1, 5, 10, 15, 50 and 100, the control virus with the corresponding virus concentration was added to each well;

[0021] S2.2: Incubate the cells in the virus solution for 12 hours, and gently shake the culture plate during the culture period to ensure that the virus is in full contact with the cells. After 12 hours, remove the virus solution in the wells and replace it with regular culture medium to continue culturing for 72 hours;

[0022] S2.3: After 72 hours, observe the cells using a fluorescence microscope to confirm the virus infection effect, take pictures and record the fluorescence signals of the infected cells. According to the results of fluorescence microscope observation, select the best MOI value and use T25 culture flasks. At the same time, inoculate 5×10 5 Cells were transfected;

[0023] S2.4: When the cells are fused to 20%-30%, add LV-miR-142-3p virus for transfection. After 12 hours of transfection, change the culture medium and continue culturing for 72 hours. After the culture is completed, use flow cytometry to screen the successfully transfected cells.

[0024] As a further solution of the present invention, the specific steps of detecting the effect of lentivirus on the proliferation efficiency and migration activity of hPASMCs in step II are as follows:

[0025] S3.1: hPASMCs cells were selected as experimental cells for CCK-8 experiment. The adherent cells in logarithmic growth phase were digested with trypsin, and the culture flask was gently shaken to observe the cell shedding. When the cell shedding rate was greater than 70%, serum was added to terminate the digestion, and the liquid containing cells was transferred to a centrifuge tube. The centrifugation condition was set to 1200 rpm and centrifuged for 3 minutes.

[0026] S3.2: After centrifugation, discard the supernatant, retain the cell pellet, resuspend the cells in serum-containing medium, and count the cells. 4 The cells were inoculated into a 96-well plate according to the standard of cells / 100ul cells. A blank control group without cells and 3 experimental groups were set up, and each experimental group had 5 replicate wells. 200μL of PBS buffer was added to each well around the plate.

[0027] S3.3: After the cells were inoculated, they were placed in an incubator. When the cells had attached to the wall for 12 hours, the culture medium of each well was aspirated, and serum-free culture medium without drugs was added to the blank group, and serum-free culture medium containing 10 μg / mL lipopolysaccharide (LPS) was added to the experimental group. After culturing for 12 hours, 10 μL of CCK-8 stock solution was added to each well, and the wells were placed in an incubator for 2 hours. The absorbance of each well was detected at a wavelength of 450 nm using an enzyme reader;

[0028] S3.4: hPASMCs cells were selected as experimental cells for the Transwell experiment. When the growth density of hPASMCs cells reached 90%, the cells were digested with trypsin, and serum-free medium was added to resuspend the cells after digestion to prepare a cell suspension and count them. In a 24-well plate, 750 μL of complete medium was first added to each well, and then the Transwell chamber was placed in the 24-well plate, and 200 μL of cell suspension was added to each chamber;

[0029] S3.5: When the cells grow to a density of 30-40%, add LPS to treat the cells. After 12 hours of LPS treatment, use a cotton swab to gently remove the non-migrated cells in the membrane, then put the chamber into crystal violet staining solution and stain at room temperature for 15 minutes. Then, wash the crystal violet staining solution with PBS, and then use a syringe needle to gently remove the membrane of the Transwell chamber;

[0030] S3.6: Place a drop of neutral gum on the glass slide, place the film upside down on the neutral gum, and then cover the slide with a specified amount of neutral gum. Carefully press to remove bubbles, turn on the inverted phase contrast microscope, and collect images of cell migration in each group.

[0031] S3.7: Collect and record the results of CCK-8 experiments and Transwell experiments, and based on the recorded experimental data, evaluate the effect of miR-142-3p on the proliferation efficiency and migration activity of human smooth muscle cells.

[0032] As a further embodiment of the present invention, the specific steps of preparing PH model rats in step III are as follows:

[0033] S4.1: 30 male SD rats weighing 180-250 g were selected and divided into group A, group B and group C with 10 rats in each group. The selected male SD rats were then adaptively fed in an SPF animal experimental center for three days;

[0034] S4.2: Male SD rats in group A that had been fed for three days were injected with normal saline at 60 mg / kg, male SD rats in group B that had been fed for three days were injected with monocrotaline (MCT) at 60 mg / kg, male SD rats in group C that had been fed for three days were injected with MCT at 60 mg / kg, and 5 μL of LV-miR-142-3p was dripped into the nasal cavity of each male SD rat in group C.

[0035] As a further solution of the present invention, the specific steps of the hemodynamic experiment in step IV are as follows:

[0036] S5.1: Select one or more PH model rats from Group A, Group B, and Group C, respectively, and anesthetize and kill the PH model rats with 20% urethane at a standard of 7 mL / kg. Under sterile conditions, cut the neck skin with ophthalmic scissors, isolate the right external jugular vein, connect a polyethylene catheter pre-filled with 5% sodium heparin to the pressure sensor, and then slowly insert the catheter into the right external jugular vein until it enters the right ventricle;

[0037] S5.2: Connect the catheter through the BL-420F biological function experimental system to monitor the right ventricular systolic pressure (RVSP) of the three groups of PH model rats in real time. After the catheter is stable, record the basal RVSP value, compare the changes in RVSP of the three groups of PH model rats before and after treatment, and use statistical software to analyze the data to confirm whether the intranasal administration of miR-142-3p overexpression lentivirus can significantly improve the increase in right ventricular systolic pressure caused by monocrotaline;

[0038] S5.3: Select one or more PH model rats from Group A, Group B, and Group C, respectively, anesthetize and kill the PH model rats with 20% urethane at a standard of 7 mL / kg, wash the blood outside the heart and lungs with PBS, immediately remove the left lung lobe and freeze it in a -80°C refrigerator, and then fix the right lung lobe in 4% paraformaldehyde solution;

[0039] S5.4: The hearts of the three groups of PH model rats were dissected and divided into the right ventricle, left ventricle and ventricular septum, and the weight of each part was weighed using a balance. The Fulton index, i.e., the right ventricular hypertrophy index RVHI value, was calculated based on the weighed weight. The changes in RVHI of the three groups of PH model rats were compared, and it was confirmed whether intranasal administration of miR-142-3p overexpressing lentivirus could reduce the increase in right heart index of rats caused by monocrotaline;

[0040] S5.5: Randomly select one or more PH model rats from group B, and inject NC (negative control) or mimic (miR-142-3p analog) at 5 mg / kg through the tail vein of the selected rats at 1, 3, 5, and 7 days after the selected rats were injected with MCT at the standard of 60 mg / kg. One week later, inject NC or miR-142-3p mimic every 6 days, for a total of 2 injections. After 28 days, the PH model rats were dissected and the RVSP value was measured. The RVSP of the miR-142-3p mimic group was compared with that of the NC group to evaluate whether the tail vein administration of miR-142-3pmimic can improve the increase in right ventricular systolic pressure in rats caused by monocrotaline;

[0041] S5.6: Randomly select one or more PH model rats from group B, and inject NC or mimic at 5 mg / kg through the rat tail vein on the 1st, 3rd, 5th, and 7th days after the selected rats were injected with MCT at the standard of 60 mg / kg. One week later, inject NC or miR-142-3p mimic every 6 days, for a total of 2 injections. After 28 days, anesthetize and kill the PH model rats with 20% urethane at the standard of 7 mL / kg, wash the blood outside the heart and lungs with PBS, and immediately remove the left lung lobe and freeze it in a -80℃ refrigerator, and then fix the right lung lobe in 4% paraformaldehyde solution;

[0042] S5.7: The hearts of PH model rats were dissected and divided into the right ventricle, left ventricle and ventricular septum. The weight of each part was weighed using a balance. The Fulton index, i.e., RVHI value, was calculated based on the weighed weight. The changes in RVHI of PH model rats were compared, and it was confirmed whether the tail vein administration of miR-142-3p overexpressing lentivirus could reduce the increase in right heart index of rats caused by monocrotaline.

[0043] S5.8: Perform histomorphological analysis on the right lung lobes fixed in 4% paraformaldehyde solution, and compare the analysis results.

[0044] As a further solution of the present invention, the specific steps of the tissue morphology analysis in step V are as follows:

[0045] S6.1: After the right lung lobe was fixed in 4% paraformaldehyde solution for 24 hours, the lung tissue was washed with running water for 3 hours to remove the paraformaldehyde, and then the washed lung tissue was dehydrated with ethanol of different concentrations, and then the dehydrated lung tissue was transparentized with xylene, and the treated lung tissue was immersed in paraffin wax. After immersion in wax, the lung tissue was embedded in paraffin wax and then placed on a low temperature platform for solidification. When the wax block was completely solidified, the tissue was processed into 5 μm thick slices using a slicer;

[0046] S6.2: After baking the lung tissue slices on a slide baking machine at 60°C for 2 hours, the lung tissue slices were dewaxed with xylene, and the dewaxed lung tissue slices were hydrated with ethanol of different concentrations, and the treated lung tissue slices were placed in deionized water;

[0047] S6.3: After dewaxing and hydration, perform H&E staining on lung tissue sections. Add hematoxylin staining solution to the tissue sections, stain for 3 minutes, and then rinse with running water for 5 minutes. Check the degree of staining. When the degree of staining is appropriate, use 1% hydrochloric acid alcohol to differentiate the tissue sections for 1 second, rinse with tap water for 20 minutes, then add eosin staining solution to the tissue sections, stain for 8 minutes, and rinse with running water for 2 seconds.

[0048] S6.4: After H&E staining, different concentrations of ethanol were used to dehydrate the stained lung tissues, and then the dehydrated lung tissues were transparentized with xylene. After transparency, the tissue sections were sealed with neutral gum and coverslips, and the staining was observed under a microscope, and photos were taken for preservation. Image J image analysis software was used for analysis to obtain the results of miR-142-3p overexpression lentivirus intranasal administration to improve the stenosis of the pulmonary arteries and pulmonary arterioles caused by monocrotaline in rats;

[0049] S6.5: For the lung tissues of each group of rats after staining, 5 sections were randomly selected, and 10 fields of view were randomly selected from each section to detect the thickness of the pulmonary artery wall and the lumen area, and to observe the pulmonary artery stenosis. Then, 10-12 pulmonary arterioles with a diameter of 100±50μm were randomly selected, and the ratio of the cross-sectional area of ​​the wall to the lumen area was calculated using Image J software. It was also analyzed whether the tail vein administration of miR-142-3p mimic could improve the stenosis of the pulmonary artery and pulmonary arteriole lumen caused by monocrotaline in rats.

[0050] Application of miR-142-3p in the treatment of pulmonary hypertension, wherein the miR-142-3p expression in the lung tissue of a rat pulmonary hypertension model was significantly reduced, and human pulmonary artery smooth muscle cells (hPASMCs) were used to observe the effect of has-miR-142-3p on the proliferation and migration of hPASMCs, and the results showed that has-miR-142-3p overexpression lentivirus could significantly inhibit the abnormal proliferation and migration of hPASMCs induced by lipopolysaccharide; MCT was used to establish a rat pulmonary hypertension model, and the miR-142-3p overexpression lentivirus was dripped into the pulmonary hypertension model rats by nose, and after 28 days of observation, the right ventricular pressure, right heart index and pulmonary vascular remodeling of the rats were significantly improved compared with the initial model rats; MCT was used to establish a rat pulmonary hypertension model, and the synthesized miR-142-3p The mimic was injected into the tail vein of rats with pulmonary hypertension model. After 28 days of observation, the right ventricular pressure, right heart index and pulmonary vascular remodeling of the rats were significantly improved compared with the initial model rats. MCT was used to establish a rat pulmonary hypertension model. After 14 days of modeling, the right ventricular pressure was measured and the right ventricular size was observed. The synthetic miR-142-3p mimic was injected into the tail vein of rats with pulmonary hypertension model. After 28 days of observation, the right ventricular pressure, right heart index and pulmonary vascular remodeling of the rats were significantly improved compared with the initial model rats.

[0051] Compared with the prior art, the present invention has the following beneficial effects:

[0052] The miR-142-3p in the method and application for treating pulmonary hypertension with miR-142-3p enters the body through two ways, namely, nasal drops and mouse tail vein injection, and has good therapeutic and preventive effects. The method of detecting the right ventricular pressure of pulmonary hypertension is used to determine that the therapeutic effect of miR-142-3p is good. The experimental results show that compared with the pulmonary hypertension in the control group, miR-142-3p can effectively reduce the right ventricular pressure and has no obvious systemic toxicity, including viral vectors. According to the effects of nasal and systemic administration, except for the different administration methods and dosages, no difference in treatment is observed. According to different purposes, injection or nasal drops can be selected for administration. The miR-142-3p of the present invention is a biological component in the body, a microRNA in epigenetics, harmless to the human body, easy to synthesize, easy to administer, simple and easy to operate, and low in production cost. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] The accompanying drawings are used to provide further understanding of the present invention and constitute a part of the specification. They are used to explain the present invention together with the embodiments of the present invention and do not constitute a limitation of the present invention.

[0054] Figure 1A flowchart of the method and application of miR-142-3p for treating pulmonary arterial hypertension proposed by the present invention;

[0055] Figure 2 A schematic diagram of a ring structure of miR-142-3p constructed into a lentivirus in the method and application of miR-142-3p for treating pulmonary arterial hypertension proposed by the present invention;

[0056] Figure 3 A flowchart of the lentivirus preparation process in the method and application of miR-142-3p for treating pulmonary arterial hypertension proposed in the present invention;

[0057] Figure 4 A comparison diagram of miR-142-3p inhibiting the increase in hPASMCs activity caused by LPS in the method and application of miR-142-3p for treating pulmonary arterial hypertension proposed by the present invention;

[0058] Figure 5 A comparison diagram of miR-142-3p inhibiting hPASMCs migration in the method and application of miR-142-3p for treating pulmonary arterial hypertension proposed by the present invention;

[0059] Figure 6 A comparison diagram of RVSP waveforms of miR-142-3p in the method for treating pulmonary hypertension and application of miR-142-3p in the present invention to alleviate MCT PH model rats;

[0060] Figure 7 A comparison diagram of miR-142-3p in the method for treating pulmonary hypertension and application of miR-142-3p in the present invention to alleviate the increase in RVSP in MCT PH model rats;

[0061] Figure 8 A comparison diagram of miR-142-3p in the method and application of miR-142-3p for treating pulmonary hypertension proposed by the present invention to alleviate the increase in right heart index in MCT PH model rats;

[0062] Fig. 9 This is a comparison of images of the improvement of monocrotaline-induced stenosis of the pulmonary artery and pulmonary arteriole lumen in rats after nasal administration of miR-142-3p overexpressing lentivirus in the method and application of the present invention for treating pulmonary hypertension;

[0063] Fig.10 A comparison diagram of the improvement of monocrotaline-induced stenosis of the pulmonary artery and pulmonary arteriole lumen in rats after nasal administration of miR-142-3p overexpressing lentivirus in the method and application of the present invention for treating pulmonary hypertension;

[0064] Fig.11A comparison diagram of miR-142-3p in the method for treating pulmonary hypertension and application of miR-142-3p in the present invention to alleviate the increase in right ventricular systolic pressure in MCT PH model rats;

[0065] Fig.12 A comparison diagram of miR-142-3p in the method and application of miR-142-3p for treating pulmonary hypertension proposed by the present invention to alleviate the increase in right heart index in MCT PH model rats;

[0066] Fig.13 This is an image comparison of miR-142-3p in the method and application of the present invention for treating pulmonary hypertension, which improves the stenosis of the pulmonary artery and pulmonary arteriole lumen in rats induced by monocrotaline after tail vein administration of miR-142-3p mimic;

[0067] Fig.14 This is a comparison chart of the improvement of monocrotaline-induced stenosis of the pulmonary artery and pulmonary arteriole lumen in rats after tail vein administration of miR-142-3p mimic in the method and application of miR-142-3p for treating pulmonary hypertension proposed in the present invention. DETAILED DESCRIPTION

[0068] The technical solutions in the embodiments of the present invention will be described clearly and completely below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments.

[0069] Example 1

[0070] Reference Figure 1 This embodiment discloses a method for treating pulmonary hypertension using miR-142-3p. The specific steps of the treatment method are as follows:

[0071] Prepare lentivirus and perform pre-transfection experiments.

[0072] Specifically, refer to Figures 1 to 3, sequence alignment was performed using the Blast public database, has-miR-142-3p that was consistent with the rat rno-miR-142-3p sequence was selected, and specific primers were designed for the selected has-miR-142-3p sequence. The GV260 vector was then linearized using restriction enzymes, the linearization effect was detected by agarose gel electrophoresis, the desired fragments were cut and purified, and a PCR reaction system was configured. The PCR cycle conditions were then set, and has-miR-142-3p was placed in a PCR instrument for reaction. The PCR product was then detected by agarose gel electrophoresis, and the target fragment was purified. The linearized GV260 vector was then PCR-amplified with the m The iRNA fragments are mixed, and ligase is added for ligation reaction. The recombinant plasmid is then transformed into Escherichia coli by chemical or electroporation. The transformed cells are cultured on LB agar plates containing antibiotics, and positive clones are screened by PCR or enzyme digestion identification. Plasmid DNA is extracted from positive clones using a plasmid extraction kit, and the extracted plasmid is cut with a restriction enzyme to confirm the presence and correctness of the inserted fragment. The recombinant plasmid is then submitted for sequencing to verify the correct sequence of the miRNA fragment. The identified recombinant plasmid is transfected into a suitable packaging cell line, and the viral packaging plasmid is added. After standing for 48 hours, the cell supernatant is collected and centrifuged and filtered to obtain the purified LV-miR-142-3p virus.

[0073] It should be further explained that the raw materials for the PCR reaction system specifically include 32.5 μL of ddH2O, 10 μL of 5xPS Buffer, 4 μL of dNTP Mix (2.5 mM each), 1 μL of upstream amplification primer (10 μM), 1 μL of downstream amplification primer (10 μM), 1 μL of template (10 ng / μL) and 0.5 μL of PrimeSTAR HSDNA polymerase;

[0074] The template is sourced from plasmid or bacterial solution, and the amount of plasmid template is less than 200 ng, and the amount of bacterial solution template is 1 μL.

[0075] Specifically, hPASMCs cells of passage 3-8 were used as experimental cells. In a 96-well plate, cells were plated according to the standard of inoculating 1×104 cells per well, and the 96-well plate was placed in an incubator for culture until the cells were fused to 20% to 30%. According to the requirements of six groups of MOI values ​​of 1, 5, 10, 15, 50 and 100, control viruses with corresponding virus concentrations were added to each well. The cells were cultured in the virus solution for 12 hours, and the culture plate was gently shaken during the culture period to ensure that the virus was fully in contact with the cells. After 12 hours, the virus solution in the well was removed and the culture medium was replaced. The culture was continued for 72 hours until the virus was fully infected. After 72 hours, the cells were observed using a fluorescence microscope to confirm the virus infection effect, and the fluorescence signals of the infected cells were photographed and recorded. According to the results of fluorescence microscope observation, the best MOI value was selected, and T25 culture flasks were used. 5×105 cells were inoculated in each flask for transfection. When the cells were fused to 20%-30%, LV-miR-142-3p virus was added for transfection. After 12 hours of transfection, the culture medium was changed to regular culture medium and cultured for another 72 hours. After the culture was completed, the successfully transfected cells were screened using flow cytometry.

[0076] Detect the effect of lentivirus on the proliferation efficiency and migration activity of hPASMCs.

[0077] Specifically, refer to Figure 1 , Figure 4 as well as Figure 5 hPASMCs were selected as experimental cells for CCK-8 experiments. Adherent cells in the logarithmic growth phase were digested with trypsin, and the culture flask was gently shaken to observe the cell shedding. When the cell shedding was greater than 70%, serum was added to terminate the digestion, and the liquid containing cells was transferred to a centrifuge tube. The centrifugation condition was set to 1200 rpm for 3 minutes. After the centrifugation was completed, the supernatant was discarded, the cell pellet was retained, and the cells were resuspended in a serum-containing culture medium. The cells were counted and 1×10 4The cells were inoculated into a 96-well plate according to the standard of cells / 100ul cells. A blank control group without cells and 3 experimental groups were set up, and each experimental group had 5 replicate wells. 200μL PBS buffer was added to each well around the plate. After the cells were inoculated, they were placed in an incubator. After the cells adhered to the wall for 12 hours, the culture medium of each well was aspirated, and serum-free culture medium without drugs was added to the blank group, and serum-free culture medium containing 10μg / mL LPS was added to the experimental group. After culturing for 12 hours, 10μL of LPS was added to each well. CCK-8 stock solution was added and placed in an incubator for 2 hours. The absorbance of each well was detected at a wavelength of 450nm using an enzyme reader. hPASMCs cells were selected as experimental cells for the Transwell experiment. When the growth density of hPASMCs cells reached 90%, the cells were digested with trypsin. After digestion, serum-free medium was added to resuspend the cells to make a cell suspension and count them. In a 24-well plate, 750 μL of complete medium was first added to each well. Then the Transwell chamber was placed in a 24-well plate, and 200 μL of cell suspension was added to each chamber. When the cells grew to a density of 30-40%, LPS was added to treat the cells. After 12 hours of LPS treatment, , gently remove the non-migrated cells in the membrane with a cotton swab, then place the chamber in crystal violet staining solution and stain at room temperature for 15 minutes, then clean the crystal violet staining solution with PBS, and then gently remove the film of the Transwell chamber with a syringe needle, drop a drop of neutral gum on the slide, turn the film upside down on the neutral gum, then take a specified dose of neutral gum to cover it and seal it, and carefully press to remove bubbles, turn on the inverted phase contrast microscope, collect images of cell migration in each group, collect and record the results of CCK-8 experiments and Transwell experiments, and based on the recorded experimental data, evaluate the effect of miR-142-3p on the proliferation efficiency and migration activity of human smooth muscle cells.

[0078] Several groups of male SD rats were selected and cultured to prepare PH model rats.

[0079] Specifically, 30 male SD rats weighing 180-250 g were selected and divided into group A, group B and group C with 10 rats in each group. The selected male SD rats were then adaptively fed for three days in an SPF-level animal experimental center. Normal saline was injected intraperitoneally into the male SD rats in group A that had been fed for three days at a standard of 60 mg / kg, MCT was injected intraperitoneally into the male SD rats in group B that had been fed for three days at a standard of 60 mg / kg, and MCT was injected intraperitoneally into the male SD rats in group C that had been fed for three days at a standard of 60 mg / kg, and 5 μL of LV-miR-142-3p was dripped into the nasal cavity of each male SD rat in group C.

[0080] Hemodynamic experiments were performed using PH model rats.

[0081] Specifically, refer to Figure 1 , Figures 6 to 14, one or more PH model rats were selected from group A, group B and group C respectively, and the PH model rats were anesthetized and killed with 20% urethane at a standard of 7 mL / kg. Under sterile conditions, the neck skin was cut with ophthalmic scissors, and the right external jugular vein was isolated. A polyethylene catheter pre-filled with 5% sodium heparin was connected to the pressure sensor, and then the catheter was slowly inserted into the right external jugular vein until it entered the right ventricle. The catheter was connected through the BL-420F biological function experimental system to monitor the right ventricular systolic pressure of the three groups of PH model rats in real time. After the catheter was stable, the basal RVSP value was recorded, and the changes in RVSP of the three groups of PH model rats before and after treatment were compared. The data were analyzed using statistical software to confirm whether the intranasal administration of miR-142-3p overexpression lentivirus significantly improved the increase in right ventricular systolic pressure caused by monocrotaline. One or more PH model rats were selected from group A, group B and group C respectively, and the 20% urethane was used for anesthesia at a standard of 7 mL / kg. The PH model rats were anesthetized and killed with 20% urethane as standard, and the blood outside the heart and lungs was washed with PBS, and the left lung lobe was immediately removed and frozen in a -80°C refrigerator, and the right lung lobe was fixed in a 4% paraformaldehyde solution. The hearts of the three groups of PH model rats were dissected and divided into the right ventricle, left ventricle and ventricular septum, and the weight of each part was weighed using a balance. The Fulton index, i.e., the RVHI value, was calculated based on the weighed weight. The changes in RVHI of the three groups of PH model rats were compared, and it was confirmed whether the intranasal administration of miR-142-3p overexpressing lentivirus could reduce the increase in the right heart index of rats caused by monocrotaline. One or more PH model rats in group B were randomly selected. On the 1st, 3rd, 5th, and 7th days after the selected rats were injected with MCT at a standard of 60 mg / kg, NC or mimic was injected through the rat tail vein at a standard of 5 mg / kg. One week later, NC or miR-142-3p was injected every 6 days. mimic, and injected twice. After 28 days, the PH model rats were dissected and the RVSP value was measured. The RVSP of the miR-142-3p mimic group was compared with that of the NC group to evaluate the expression of miR-142-3p To investigate whether the administration of mimic into the tail vein could improve the increase of right ventricular systolic pressure in rats caused by monocrotaline, one or more PH model rats in group B were randomly selected. After the selected rats were injected with MCT at a standard of 60 mg / kg, NC or mimic was injected into the tail vein at a standard of 5 mg / kg on the 1st, 3rd, 5th, and 7th days. One week later, NC or miR-142-3pmimic was injected every 6 days, for a total of 2 injections. After 28 days, the PH model rats were anesthetized and killed with 20% urethane at a standard of 7 mL / kg. The blood outside the heart and lungs was washed with PBS, and the left lung lobe was immediately removed and frozen in a -80°C refrigerator. The right lung lobe was then fixed in a 4% paraformaldehyde solution. The heart of the PH model rats was dissected and dissected into the right ventricle, left ventricle, and ventricular septum. The mass of each part was weighed using a balance.The Fulton index, i.e., RVHI value, was calculated based on the weighed mass. The changes in RVHI of PH model rats were compared, and it was confirmed whether the tail vein administration of miR-142-3p overexpressing lentivirus could reduce the increase in right heart index of rats caused by monocrotaline. The right lung lobes fixed in 4% paraformaldehyde solution were subjected to histomorphometric analysis, and the analysis results were compared.

[0082] The histomorphological analysis was performed on the PH model rats after the experiment.

[0083] Specifically, refer to Figure 1 , Fig. 9 , Fig.10 , Fig.13 as well as Fig.14After the right lung lobe was fixed in a 4% paraformaldehyde solution for 24 hours, the lung tissue was washed with running water for 3 hours to remove the paraformaldehyde, and then the washed lung tissue was dehydrated with ethanol of different concentrations, and then the dehydrated lung tissue was transparentized with xylene, and the treated lung tissue was immersed in paraffin. After immersion in wax, the lung tissue was embedded in paraffin and then placed on a low-temperature platform for solidification. When the wax block was completely solidified, the tissue was processed into 5μm thick slices using a slicer, and the lung tissue slices were baked on a baking machine at 60°C for 2 hours, and then the lung tissue slices were dewaxed with xylene, and the dewaxed lung tissue slices were hydrated with ethanol of different concentrations, and then the processed lung tissue slices were sliced. Place in deionized water, after dewaxing and hydration, perform H&E staining on lung tissue sections, add hematoxylin staining solution on the tissue sections, stain for 3 minutes, and then rinse with running water for 5 minutes, check the degree of staining, after the degree of staining is appropriate, use 1% hydrochloric acid alcohol to differentiate the tissue sections for 1 second, rinse with tap water for anti-blue for 20 minutes, then add eosin staining solution on the tissue sections, stain for 8 minutes, rinse with running water for 2 seconds, after H&E staining is completed, use different concentrations of ethanol to dehydrate the stained lung tissue, and then use xylene to make the dehydrated lung tissue transparent. After the transparency is completed, use neutral gum and coverslip to seal the tissue sections, and observe the staining under a microscope, take pictures and save them, and use Image J image analysis software was used for analysis to obtain the results of improving the stenosis of the pulmonary artery and pulmonary arteriole lumen in rats induced by monocrotaline after intranasal administration of miR-142-3p overexpression lentivirus. For the lung tissue of PH model rats induced by tail vein administration of miR-142-3p overexpression lentivirus after staining, 5 sections were randomly selected, and 10 fields of view were randomly selected for each section to detect the thickness of the pulmonary artery wall and the lumen area, and the pulmonary artery stenosis was observed. Then 10-12 pulmonary arterioles with a diameter of 100±50μm were randomly selected, and the ratio of the cross-sectional area of ​​the wall to the lumen area was calculated using Image J software, and it was analyzed whether the stenosis of the pulmonary artery and pulmonary arteriole lumen in rats induced by monocrotaline was improved after tail vein administration of miR-142-3p mimic.

[0084] The results of hemodynamic experiments and tissue morphological analysis were collected and recorded.

[0085] Example 2

[0086] This embodiment discloses the application of miR-142-3p in the treatment of pulmonary hypertension. In a rat pulmonary hypertension model, the expression of miR-142-3p in lung tissue was significantly reduced. Human pulmonary artery smooth muscle cells were used to observe the effect of has-miR_142-3p on the proliferation and migration of hPASMCs. The results showed that has_miR-142-3p overexpression lentivirus could significantly inhibit the abnormal proliferation and migration of hPASMCs induced by lipopolysaccharide. MCT was used to establish a rat pulmonary hypertension model, and the miR-142-3p overexpression lentivirus was dripped into the pulmonary hypertension model rats by nose. The results were observed after 28 days. Compared with the initial model rats, the right ventricular pressure, right heart index and pulmonary vascular remodeling of the rats were significantly improved. MCT was used to establish a rat pulmonary hypertension model, and the synthesized miR-142-3p The mimic was injected into the tail vein of pulmonary hypertension model rats. After 28 days of observation, the right ventricular pressure, right heart index and pulmonary vascular remodeling of the rats were significantly improved compared with the initial model rats. MCT was used to establish a rat pulmonary hypertension model. After 14 days of modeling, the right ventricular pressure was measured and the right ventricular size was observed. The synthetic miR-142-3p mimic was injected into the tail vein of pulmonary hypertension model rats. After 28 days of observation, the right ventricular pressure, right heart index and pulmonary vascular remodeling of the rats were significantly improved compared with the initial model rats.

Claims

1. A method for treating pulmonary arterial hypertension using miR-142-3p, characterized in that: The specific steps of this treatment method are as follows: Ⅰ. Prepare lentivirus and conduct pre-transfection experiments; II. Detect the effect of lentivirus on the proliferation efficiency and migration activity of hPASMCs; III. Select and culture multiple groups of male SD rats to prepare PH model rats; IV. Conduct hemodynamic experiments using PH model rats; V. Conduct histological morphological analysis on PH model rats after the experiment; VI. Collect and record the results of hemodynamic experiments and tissue morphological analysis.

2. The method for treating pulmonary hypertension with miR-142-3p according to claim 1, characterized in that: The specific steps of preparing lentivirus in step I are as follows: S1.1: Sequence alignment was performed using the Blast public database, and the hs-miR-142-3p sequence that was consistent with the rat rno-miR-142-3p sequence was selected. Specific primers were designed for the selected hs-miR-142-3p sequence, and then the GV260 vector was linearized using restriction enzymes; S1.2: Detect the linearization effect by agarose gel electrophoresis, cut and purify the desired fragment, and configure the PCR reaction system, then set the PCR cycle conditions, and place has-miR-142-3p in a PCR instrument for reaction, then detect the PCR product by agarose gel electrophoresis, and purify the target fragment; S1.3: Mix the linearized GV260 vector with the miRNA fragment obtained by PCR amplification, add ligase for ligation reaction, and then use chemical method or electroporation method to transform the recombinant plasmid into Escherichia coli, culture the transformed cells on LB agar plates containing antibiotics, and screen positive clones by PCR or enzyme digestion identification; S1.4: Use a plasmid extraction kit to extract plasmid DNA from positive clones, use restriction enzymes to cut the extracted plasmid to confirm the presence and correctness of the inserted fragment, and then submit the recombinant plasmid for sequencing to verify the correct sequence of the miRNA fragment. Transfect the identified recombinant plasmid into a suitable packaging cell line, add the viral packaging plasmid, collect the cell supernatant after standing for 48 hours, and obtain the purified LV-miR-142-3p virus by centrifugation and filtration.

3. The method for treating pulmonary hypertension with miR-142-3p according to claim 2, characterized in that: S1.2 The PCR reaction system is configured with raw materials including 32.5 μL of ddH2O, 10 μL of 5xPS Buffer, 4 μL of dNTP Mix (2.5 mM each), 1 μL of upstream amplification primer (10 μM), 1 μL of downstream amplification primer (10 μM), 1 μL of template (10 ng / μL) and 0.5 μL of PrimeSTAR HSDNA polymerase; The template is sourced from plasmid or bacterial solution, and the amount of plasmid template is less than 200 ng, and the amount of bacterial solution template is 1 μL.

4. The method for treating pulmonary hypertension with miR-142-3p according to claim 2, characterized in that: The specific steps of the pre-transfection experiment described in step Ⅰ are as follows: S2.1: Use 3-8 passage hPASMCs as experimental cells and inoculate 1×10 cells per well in a 96-well plate. 4 The cells were plated according to the standard of 100 cells, and the 96-well plate was placed in the incubator for culture until the cell fusion reached 20% to 30%. According to the requirements of six groups of different MOI values ​​of 1, 5, 10, 15, 50 and 100, the control virus with the corresponding virus concentration was added to each well; S2.2: Incubate the cells in the virus solution for 12 hours, and gently shake the culture plate during the culture period to ensure that the virus is in full contact with the cells. After 12 hours, remove the virus solution in the wells and replace it with regular culture medium to continue culturing for 72 hours; S2.3: After 72 hours, observe the cells using a fluorescence microscope to confirm the virus infection effect, take pictures and record the fluorescence signals of the infected cells. According to the results of fluorescence microscope observation, select the best MOI value and use T25 culture flasks. At the same time, inoculate 5×10 5 Cells were transfected; S2.4: When the cells are fused to 20%-30%, add LV-miR-142-3p virus for transfection. After 12 hours of transfection, replace the culture medium and continue culturing for 72 hours. After the completion of the culture, use flow cytometry to screen the successfully transfected cells.

5. The method for treating pulmonary hypertension with miR-142-3p according to claim 4, characterized in that: The specific steps of detecting the effect of lentivirus on the proliferation efficiency and migration activity of hPASMCs described in step II are as follows: S3.1: hPASMCs cells were selected as experimental cells for CCK-8 experiment. The adherent cells in logarithmic growth phase were digested with trypsin, and the culture flask was gently shaken to observe the cell shedding. When the cell shedding rate was greater than 70%, serum was added to terminate the digestion, and the liquid containing cells was transferred to a centrifuge tube. The centrifugation condition was set to 1200 rpm and centrifuged for 3 minutes. S3.2: After centrifugation, discard the supernatant, retain the cell pellet, resuspend the cells in serum-containing medium, and count the cells. 4 The cells were inoculated into a 96-well plate according to the standard of cells / 100ul cells. A blank control group without cells and 3 experimental groups were set up, and each experimental group had 5 replicate wells. 200μL of PBS buffer was added to each well around the plate. S3.3: After the cells were inoculated, they were placed in an incubator. When the cells had attached to the wall for 12 hours, the culture medium of each well was aspirated, and serum-free culture medium without drugs was added to the blank group, and serum-free culture medium containing 10 μg / mL lipopolysaccharide (LPS) was added to the experimental group. After culturing for 12 hours, 10 μL of CCK-8 stock solution was added to each well, and the wells were placed in an incubator for 2 hours. The absorbance of each well was detected at a wavelength of 450 nm using an enzyme reader; S3.4: hPASMCs cells were selected as experimental cells for the Transwell experiment. When the growth density of hPASMCs cells reached 90%, the cells were digested with trypsin, and serum-free medium was added to resuspend the cells after digestion to prepare a cell suspension and count them. In a 24-well plate, 750 μL of complete medium was first added to each well, and then the Transwell chamber was placed in the 24-well plate, and 200 μL of cell suspension was added to each chamber; S3.5: When the cells grow to a density of 30-40%, add LPS to treat the cells. After 12 hours of LPS treatment, use a cotton swab to gently remove the non-migrated cells in the membrane, then put the chamber into crystal violet staining solution and stain at room temperature for 15 minutes. Then, wash the crystal violet staining solution with PBS, and then use a syringe needle to gently remove the membrane of the Transwell chamber; S3.6: Place a drop of neutral gum on the glass slide, place the film upside down on the neutral gum, and then cover the slide with a specified amount of neutral gum. Carefully press to remove bubbles, turn on the inverted phase contrast microscope, and collect images of cell migration in each group. S3.7: Collect and record the results of CCK-8 experiments and Transwell experiments, and based on the recorded experimental data, evaluate the effect of miR-142-3p on the proliferation efficiency and migration activity of human smooth muscle cells.

6. The method for treating pulmonary hypertension with miR-142-3p according to claim 5, characterized in that: The specific steps of preparing PH model rats in step III are as follows: S4.1: 30 male SD rats weighing 180-250 g were selected and divided into group A, group B and group C with 10 rats in each group. The selected male SD rats were then adaptively fed in an SPF animal experimental center for three days; S4.2: Male SD rats in group A that had been fed for three days were injected with normal saline at 60 mg / kg, male SD rats in group B that had been fed for three days were injected with monocrotaline (MCT) at 60 mg / kg, male SD rats in group C that had been fed for three days were injected with MCT at 60 mg / kg, and 5 μL of LV-miR-142-3p was dripped into the nasal cavity of each male SD rat in group C.

7. The method for treating pulmonary hypertension with miR-142-3p according to claim 5, characterized in that: The specific steps of the hemodynamic experiment described in step IV are as follows: S5.1: Select one or more PH model rats from Group A, Group B, and Group C, respectively, and anesthetize and kill the PH model rats with 20% urethane at a standard of 7 mL / kg. Under sterile conditions, cut the neck skin with ophthalmic scissors, isolate the right external jugular vein, connect a polyethylene catheter pre-filled with 5% sodium heparin to the pressure sensor, and then slowly insert the catheter into the right external jugular vein until it enters the right ventricle; S5.2: Connect the catheter through the BL-420F biological function experimental system to monitor the right ventricular systolic pressure (RVSP) of the three groups of PH model rats in real time. After the catheter is stable, record the basal RVSP value, compare the changes in RVSP of the three groups of PH model rats before and after treatment, and use statistical software to analyze the data to confirm whether the intranasal administration of miR-142-3p overexpression lentivirus can significantly improve the increase in right ventricular systolic pressure caused by monocrotaline; S5.3: Select one or more PH model rats from Group A, Group B, and Group C, respectively, anesthetize and kill the PH model rats with 20% urethane at a standard of 7 mL / kg, wash the blood outside the heart and lungs with PBS, immediately remove the left lung lobe and freeze it in a -80°C refrigerator, and then fix the right lung lobe in 4% paraformaldehyde solution; S5.4: The hearts of the three groups of PH model rats were dissected and divided into the right ventricle, left ventricle and ventricular septum, and the weight of each part was weighed using a balance. The Fulton index, i.e., the right ventricular hypertrophy index (RVHI) value, was calculated based on the weighed weight. The changes in RVHI of the three groups of PH model rats were compared, and it was confirmed whether intranasal administration of miR-142-3p overexpressing lentivirus could reduce the increase in right heart index of rats caused by monocrotaline; S5.5: Randomly select one or more PH model rats from group B, and inject NC (negative control) or mimic (miR-142-3p analog) at 5 mg / kg through the tail vein of the selected rats at 1, 3, 5, and 7 days after the selected rats were injected with MCT at the standard of 60 mg / kg. One week later, inject NC or miR-142-3p mimic every 6 days, for a total of 2 injections. After 28 days, the PH model rats were dissected and the RVSP value was measured. The RVSP of the miR-142-3p mimic group was compared with that of the NC group to evaluate whether the tail vein administration of miR-142-3pmimic can improve the increase in right ventricular systolic pressure in rats caused by monocrotaline; S5.6: Randomly select one or more PH model rats from group B, and inject NC or mimic at 5 mg / kg through the rat tail vein on the 1st, 3rd, 5th, and 7th days after the selected rats were injected with MCT at the standard of 60 mg / kg. One week later, inject NC or miR-142-3p mimic every 6 days, for a total of 2 injections. After 28 days, anesthetize and kill the PH model rats with 20% urethane at the standard of 7 mL / kg, wash the blood outside the heart and lungs with PBS, and immediately remove the left lung lobe and freeze it in a -80℃ refrigerator, and then fix the right lung lobe in 4% paraformaldehyde solution; S5.7: The hearts of PH model rats were dissected and divided into the right ventricle, left ventricle and ventricular septum. The weight of each part was weighed using a balance. The Fulton index, i.e., RVHI value, was calculated based on the weighed weight. The changes in RVHI of PH model rats were compared, and it was confirmed whether the tail vein administration of miR-142-3p overexpressing lentivirus could reduce the increase in right heart index of rats caused by monocrotaline. S5.8: Perform histomorphological analysis on the right lung lobes fixed in 4% paraformaldehyde solution, and compare the analysis results.

8. The method for treating pulmonary hypertension with miR-142-3p according to claim 5, characterized in that: The specific steps of the tissue morphology analysis in step V are as follows: S6.1: After the right lung lobe was fixed in 4% paraformaldehyde solution for 24 hours, the lung tissue was washed with running water for 3 hours to remove the paraformaldehyde, and then the washed lung tissue was dehydrated with ethanol of different concentrations, and then the dehydrated lung tissue was transparentized with xylene, and the treated lung tissue was immersed in paraffin wax. After immersion in wax, the lung tissue was embedded in paraffin wax and then placed on a low temperature platform for solidification. When the wax block was completely solidified, the tissue was processed into 5 μm thick slices using a slicer; S6.2: After baking the lung tissue slices on a slide baking machine at 60°C for 2 hours, the lung tissue slices were dewaxed with xylene, and the dewaxed lung tissue slices were hydrated with ethanol of different concentrations, and the treated lung tissue slices were placed in deionized water; S6.3: After dewaxing and hydration, perform H&E staining on lung tissue sections. Add hematoxylin staining solution to the tissue sections, stain for 3 minutes, and then rinse with running water for 5 minutes. Check the degree of staining. When the degree of staining is appropriate, use 1% hydrochloric acid alcohol to differentiate the tissue sections for 1 second, rinse with tap water for 20 minutes, then add eosin staining solution to the tissue sections, stain for 8 minutes, and rinse with running water for 2 seconds. S6.4: After H&E staining, different concentrations of ethanol were used to dehydrate the stained lung tissues, and then the dehydrated lung tissues were transparentized with xylene. After transparency, the tissue sections were sealed with neutral gum and coverslips, and the staining was observed under a microscope, and photos were taken for preservation. Image J image analysis software was used for analysis to obtain the results of miR-142-3p overexpression lentivirus intranasal administration to improve the stenosis of the pulmonary arteries and pulmonary arterioles caused by monocrotaline in rats; S6.5: For the lung tissues of each group of rats after staining, 5 sections were randomly selected, and 10 fields of view were randomly selected from each section to detect the thickness of the pulmonary artery wall and the lumen area, and to observe the pulmonary artery stenosis. Then, 10-12 pulmonary arterioles with a diameter of 100±50μm were randomly selected, and the ratio of the cross-sectional area of ​​the wall to the lumen area was calculated using Image J software. It was also analyzed whether the tail vein administration of miR-142-3p mimic could improve the stenosis of the pulmonary artery and pulmonary arteriole lumen caused by monocrotaline in rats.

9. Use of miR-142-3p in treating pulmonary arterial hypertension, for implementing the method for treating pulmonary arterial hypertension with miR-142-3p according to any one of claims 1 to 8, characterized in that: The miR-142-3p expression in the lung tissue of the rat pulmonary hypertension model was significantly reduced. Human pulmonary artery smooth muscle cells (hPASMCs) were used to observe the effect of has-miR-142-3p on the proliferation and migration of hPASMCs. The results showed that has-miR-142-3p overexpression lentivirus could significantly inhibit the abnormal proliferation and migration of hPASMCs induced by lipopolysaccharide. MCT was used to establish a rat pulmonary hypertension model, and the miR-142-3p overexpression lentivirus was nasally dripped into the pulmonary hypertension model rats. After 28 days, the right ventricular pressure, right heart index and pulmonary vascular remodeling of the rats were significantly improved compared with the initial model rats. MCT was used to establish a rat pulmonary hypertension model, and the synthesized miR-142-3p The mimic was injected into the tail vein of pulmonary hypertension model rats. After 28 days of observation, the right ventricular pressure, right heart index and pulmonary vascular remodeling of the rats were significantly improved compared with the initial model rats. MCT was used to establish a rat pulmonary hypertension model. After 14 days of modeling, the right ventricular pressure was measured and the right ventricular size was observed. The synthetic miR-142-3p mimic was injected into the tail vein of pulmonary hypertension model rats. After 28 days of observation, the right ventricular pressure, right heart index and pulmonary vascular remodeling of the rats were significantly improved compared with the initial model rats.

Images