Construction method and application of hypertrophy heart disease-pulmonary arterial hypertension model

Abstract

The invention discloses a construction method and application of a hypercongenital heart disease-pulmonary arterial hypertension model, which better simulates children with the congenital heart disease in Yunnan by adopting the conditions of lateral anastomosis of aorta abdominalis and inferior vena cava and hypoxia feeding, and makes a new contribution to the research of pulmonary arterial hypertension. In the experimental shunt + hypoxia group and the monocrotaline + hypoxia group, the heart of the SD rat has the change of pulmonary arterial hypertension, four cases in the shunt + hypoxia group have tricuspid regurgitation, and the rest experimental rats have no tricuspid regurgitation.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a method for constructing a high-altitude congenital heart disease-pulmonary hypertension model and an application thereof. Background Art

[0002] Pulmonary arterial hypertension (PAH) is a group of pathophysiological syndromes characterized by increased pulmonary vascular resistance and pulmonary artery pressure. High-flow pulmonary hypertension is the most common complication of left-to-right shunt congenital heart disease and one of the important causes of perioperative death. Yunnan is located in the plateau, and the inherent oxygen content in the air is lower than that in the plains. Hypoxia has become a high-risk factor for children with congenital heart disease-related pulmonary hypertension in Yunnan compared with other plains. Therefore, the construction of a plateau congenital heart disease-pulmonary hypertension model can better simulate children with congenital heart disease in Yunnan, thereby making up for the shortcomings of the traditional pulmonary hypertension model. Summary of the invention

[0003] The purpose of the present invention is to provide a method for constructing a high-altitude congenital heart disease-pulmonary hypertension model and its application. A rat pulmonary hypertension model is established by adopting the conditions of side-to-side anastomosis of the abdominal aorta and the inferior vena cava and feeding in a hypoxic environment, so as to better simulate the pathological and physiological state of children with left-to-right congenital heart disease in the Yunnan plateau, thereby providing a reliable animal model scheme for further basic research.

[0004] The above technical objectives of the present invention are achieved through the following technical solutions:

[0005] A method for constructing a high-altitude congenital heart disease-pulmonary hypertension model comprises the following steps:

[0006] S1, SD rats were adaptively raised for 3 days after arrival;

[0007] S2. Preoperative preparation: fasting for 6 hours before surgery, but no drinking water;

[0008] S3, anesthesia induction: air pump flow rate maintained at 250-300 ml / min, anesthetic concentration 5%, about 1-2 minutes, observe the respiratory rate, preferably down to 20 times / min;

[0009] S4. After the hair removal treatment, lie on your back and maintain the room temperature at 26 degrees Celsius.

[0010] S5. Anesthesia maintenance: air pump flow rate is maintained at 250-300 ml / min, anesthetic concentration is maintained at 1.5-2%, and respiratory rate is observed at 20 times / min;

[0011] S6. After anesthesia takes effect, disinfect and lay the drape;

[0012] Cut the skin in the middle of the abdomen;

[0013] Cut the rectus abdominis muscle;

[0014] Wrap the abdominal organs with moist gauze and place them outside the right side of the rat;

[0015] The retroperitoneal fat was bluntly dissected with a cotton swab to expose the abdominal aorta and the parallel inferior vena cava.

[0016] The pug clamps were used to occlude the aorta and the inferior vena cava 0.8 cm above and below the proposed shunt, respectively;

[0017] Puncture the inner side of the aorta and the inner side of the inferior vena cava, and trim the shunt to an appropriate size;

[0018] One side of the inferior vena cava is suspended with one needle of 8-0 Pro l ene suture;

[0019] 8-0Pro l ene suture was used for side-to-side anastomosis;

[0020] After the anastomosis is completed, the pug clamp is opened and the blood can be seen to be shunted from the arterial side to the venous side;

[0021] After the anastomosis is completed, check carefully for bleeding, count the instruments, and use 5-0 Pro l ene suture to close the rectus abdominis and skin layer by layer.

[0022] Turn off anesthesia and wait for the animal to wake up naturally. Do not leave the animal before it wakes up.

[0023] Observe the animal's condition for 30 minutes, and feed it in a separate feeding box for 3 days after confirming that the animal has recovered well.

[0024] S7. After three days, the fish enter the hypoxia chamber, where the conditions are 10% oxygen concentration, 0.2% carbon dioxide, 22-26°C temperature, 60-70% humidity, 12 hours a day, and 4 weeks of breeding.

[0025] Preferably, S6 is specifically as follows: cutting the skin in the middle of the abdomen from the xiphoid process to 1 cm above the bladder.

[0026] Preferably, S7 specifically includes: puncturing the inner side of the aorta and the inner side of the inferior vena cava, and trimming the shunt opening to a size of 2 mm.

[0027] The present invention also discloses the application of the plateau congenital heart disease-pulmonary hypertension animal model prepared by the construction method of the plateau congenital heart disease-pulmonary hypertension animal model in basic research on pulmonary hypertension.

[0028] In summary, the present invention has the following beneficial effects:

[0029] The present invention establishes a high-altitude congenital heart disease-pulmonary hypertension model, adopts the conditions of abdominal aorta-inferior vena cava side-to-side anastomosis and hypoxic feeding, so as to better simulate the children with congenital heart disease in the Yunnan plateau, and make new contributions to the basic research of pulmonary hypertension. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 is the pulmonary artery soil of SD rats in the control group of the present invention;

[0031] Figure 2 This is the pulmonary artery diagram of SD rats in the body-to-lung shunt + hypoxia group in the present invention;

[0032] Figure 3 is a graph showing the T test result of pulmonary artery media thickening in the present invention;

[0033] Figure 4 This is a diagram of the mitochondrial morphology of macrophages in SD rats in the body-lung shunt + hypoxia group of the present invention;

[0034] Figure 5 This is a picture of cardiac color Doppler ultrasound examination of three groups of SD rats in the present invention;

[0035] Figure 6 It is a comparison diagram of PAD in the present invention;

[0036] Figure 7 It is a comparison diagram of PAAT in the present invention;

[0037] Figure 8 is the right ventricular mean pressure map in the present invention;

[0038] Fig. 9 It is a WB experimental verification diagram in the present invention;

[0039] Fig.10 It is the whole film diagram in the present invention. DETAILED DESCRIPTION

[0040] The present invention is further described in detail below in conjunction with the accompanying drawings.

[0041] Embodiment, a method for constructing a high-altitude congenital heart disease-pulmonary hypertension model, comprising the following steps:

[0042] S1 and SD rats were adaptively housed for 3 days after arrival without any manipulation to acclimate to human activities.

[0043] S2. Preoperative preparation: Do not eat or drink for 6 hours before surgery.

[0044] S3. Anesthesia induction: Maintain the air pump flow at 250-300 ml / min, anesthetic concentration at 5% for about 1-2 minutes, observe the respiratory rate, and it is best to reduce it to 20 times / min.

[0045] S4. After the hair removal treatment, lie on your back. You can put two pieces of gauze on your back to elevate the abdominal blood vessels. Use a heating lamp to maintain the room temperature at 26 degrees Celsius.

[0046] S5. Anesthesia maintenance: air pump flow is maintained at 250-300 ml / min, anesthetic concentration is 1.5-2%, and respiratory rate is observed at 20 times / min.

[0047] It should be noted that strict aseptic operation should be performed during the operation and the use of antibiotics should be avoided after the operation.

[0048] S6. After anesthesia takes effect, disinfect and lay the drape;

[0049] Cut the skin in the middle of the abdomen;

[0050] Cut the rectus abdominis muscle;

[0051] Wrap the abdominal organs with moist gauze and place them outside the right side of the rat;

[0052] The retroperitoneal fat was bluntly dissected with a cotton swab to expose the abdominal aorta and the parallel inferior vena cava.

[0053] The pug clamps were used to occlude the aorta and the inferior vena cava 0.8 cm above and below the proposed shunt, respectively;

[0054] Puncture the inner side of the aorta and the inner side of the inferior vena cava, and trim the shunt to 2 mm;

[0055] One side of the inferior vena cava is suspended with a 8-0 Pro l ene suture needle. The needle is a double-ended needle. The purpose is to expose the anastomosis on the venous side, and then perform a side-to-side anastomosis of the abdominal aorta and the inferior vena cava.

[0056] After the anastomosis is completed, check carefully for bleeding, count the instruments, and use 5-0 Pro l ene suture to close the rectus abdominis and skin layer by layer.

[0057] Turn off anesthesia and wait for the animal to wake up naturally. Do not leave the animal before it wakes up.

[0058] Observe the animal's condition for 30 minutes. After confirming that the animal has recovered well, feed it in a separate feeding box for 3 days.

[0059] S7. After three days, the fish enter the hypoxia chamber, where the conditions are 10% oxygen concentration, 0.2% carbon dioxide, 22-26°C temperature, 60-70% humidity, 12 hours a day, and 4 weeks of breeding.

[0060] 1. Experimental Materials

[0061] Fifteen male SD rats, 8 weeks old, weighing 300-350 g; Reward R500 general-purpose small animal anesthesia machine; gas concentration controller; biological signal acquisition and processing system; soda lime; sodium heparin injection; isoflurane; monocrotaline; surgical magnifying glass.

[0062] 2. Animal model establishment

[0063] 2.1. Shunt + hypoxia group: 5 SD rats were subjected to side-to-side anastomosis of the abdominal aorta and inferior vena cava and hypoxic feeding to establish a rat pulmonary hypertension model, as described above.

[0064] 2.2. Crotaline + hypoxia group: 5 SD rats were treated with Crotaline and hypoxia feeding conditions to establish a rat pulmonary hypertension model. The operation method was: after a single subcutaneous injection of Crotaline (60 mg / kg), the rats were placed in an oxygen concentration of 10%, carbon dioxide 0.2%, temperature 22-26°C, humidity 60-70%, 12 hours a day, and raised for 3-4 weeks.

[0065] 2.3. Control group: 5 SD rats were not treated.

[0066] 2.4 Test Results

[0067] like Figure 1 Shown is the pulmonary artery of SD rats in the control group, and the arrow points to the normal tunica media.

[0068] like Figure 2 As shown, the pulmonary arteries of SD rats in the systemic-pulmonary shunt + hypoxia group showed obvious pathological changes. The arrows point to the areas with narrowed lumen and obvious hyperplasia of the intima and media layers.

[0069] like Figure 3 As shown, the T test results of pulmonary artery media thickness were different, and "**" represents P < 0.01E.

[0070] like Figure 4 As shown, the mitochondria of macrophages in SD rats in the body-to-lung shunt + hypoxia group were swollen, the ridges disappeared, and the number of vacuoles increased. The arrows point to the mitochondria.

[0071] like Figure 5 The figure shows the cardiac color Doppler ultrasound examination of three groups of SD rats;

[0072] like Fig. 9 and Fig.10 As shown, α-SMA is a smooth muscle cell marker. WB experiments verified that the expression of smooth muscle marker α-SMA increased, indicating that both the SH group and the MH group had hyperplasia of the middle mold layer and vascular remodeling (* indicates comparison with the CK group, # indicates comparison with the shunt + hypoxia group).

[0073] 3. Right ventricular pressure measurement

[0074] 3.1、Operation method:

[0075] After anesthesia takes effect, disinfect and lay the drape;

[0076] Cut the skin on the right side of the neck (from the thyroid cartilage to the clavicle, about 2.5 cm);

[0077] The sternocleidomastoid muscle was separated by blunt dissection with a cotton swab to expose the right internal jugular vein (1 mm in diameter);

[0078] The distal end of the internal jugular vein was ligated with 4# thread and lifted;

[0079] The needle punctures the vein, and a 21G blunt needle is inserted into the blood vessel;

[0080] Then connect the 21G needle to the pressure measuring tube;

[0081] Slowly along the vein - right atrium - tricuspid valve - right ventricle;

[0082] Read the data when the value is the highest.

[0083] 3.2、The pressure test results are as follows Figure 8 As shown, the mean right ventricular pressure (RVmVP) in the S+H group was significantly higher than that in the control group, but there was no difference with that in the M+H group. Group name annotation: control group (CK), systemic lung-shunt + hypoxia group (S+H), monocrotaline + hypoxia (M+H).

[0084] 4. Experimental results:

[0085] The changes in pulmonary artery blood flow acceleration time (PAAT) and pulmonary artery diameter in rats were used to indirectly reflect the resistance of the pulmonary artery. We measured the PAAT of three groups of rat models and compared them two by two. The results are as follows:

[0086] 4.1 PAAT

[0087] a. Control group vs. monocrotaline + hypoxia group: P of PAAT = 0.005;

[0088] b. Control group vs. shunt + hypoxia group: P for PAAT = 0.048;

[0089] c. Shunt + hypoxia group vs. monocrotaline + hypoxia group: PAAT P = 0.775

[0090] 4.2 Pulmonary artery diameter

[0091] a. Control group vs. monocrotaline + hypoxia group: P = 0.009 for pulmonary artery diameter;

[0092] b. Control group vs. shunt + hypoxia group: P = 0.003 for pulmonary artery diameter;

[0093] c. Shunt + hypoxia group vs. monocrotaline + hypoxia group: P = 0.196 for pulmonary artery diameter;

[0094] like Figure 6 and Figure 7 As shown, the PAD of the S+H group was significantly thicker than that of the control group, and there was no difference between it and the M+H group; the PAAT of the S+H group was significantly faster than that of the control group, and there was no difference between it and the M+H group.

[0095] 4.3 Tricuspid valve regurgitation: Only 3 cases of tricuspid valve regurgitation occurred in the shunt + hypoxia group, while no tricuspid valve regurgitation was found in the other control groups and the monocrotaline group.

[0096] 5. Conclusion

[0097] 5.1. In the shunt + hypoxia group and the monocrotaline + hypoxia group, the hearts of SD rats showed changes of pulmonary hypertension. Among them, four cases in the shunt + hypoxia group had tricuspid regurgitation, while the remaining experimental rats, including the normal control group, did not have tricuspid regurgitation.

[0098] 5.2. When comparing the control group with the monocrotaline + hypoxia group, and the control group with the shunt + hypoxia group, we found that the P values ​​of AT and pulmonary artery diameter were less than 0.05, indicating that there were differences between the two groups, indicating that the monocrotaline + hypoxia group and the shunt + hypoxia group had changes in pulmonary resistance, and they were statistically significant.

[0099] 5.3. When comparing the shunt + hypoxia group and the monocrotaline + hypoxia group, the P values ​​of PAAT and pulmonary artery diameter were both greater than 0.05, indicating that there was no difference between the two groups. Combined with the previous "5.2", it shows that both groups had an increase in pulmonary resistance.

[0100] Note: The model of SD rats caused by the method of monocrotaline + hypoxia has been very mature. In the experiment, the monocrotaline + hypoxia group was used as a positive control of the shunt + hypoxia group to verify that the shunt + hypoxia group could also successfully cause pulmonary hypertension in SD rats.

[0101] This specific embodiment is merely an explanation of the present invention and is not a limitation of the present invention. After reading this specification, those skilled in the art may make non-creative modifications to the present embodiment as needed. However, as long as they are within the scope of the claims of the present invention, they are protected by the patent law.

Claims

1. A method for constructing a high-altitude congenital heart disease-pulmonary hypertension model, characterized in that: The following steps are involved: S1, SD rats were adaptively raised for 3 days after arrival; S2. Preoperative preparation: fasting for 6 hours before surgery, but no drinking water; S3, anesthesia induction: air pump flow rate maintained at 250-300 ml / min, anesthetic concentration 5% for about 1-2 min, observe respiratory rate, preferably down to 20 times / min; S4. After the hair removal treatment, lie on your back and maintain the room temperature at 26 degrees Celsius. S5. Anesthesia maintenance: air pump flow rate is maintained at 250-300 ml / min, anesthetic concentration is 1.5-2%, and respiratory rate is observed at 20 times / min; S6. After anesthesia takes effect, disinfect and lay the drape; Cut the skin in the middle of the abdomen; Cut the rectus abdominis muscle; Wrap the abdominal organs with moist gauze and place them outside the right side of the rat; The retroperitoneal fat was bluntly dissected with a cotton swab to expose the abdominal aorta and the parallel inferior vena cava. The pug clamps were used to occlude the aorta and the inferior vena cava 0.8 cm above and below the proposed shunt, respectively; Puncture the inner side of the aorta and the inner side of the inferior vena cava, and trim the shunt to an appropriate size; One side of the inferior vena cava is suspended with one stitch of 8-0 Prolene; One stitch of 8-0 Prolene suture was followed by side-to-side anastomosis; After the anastomosis is completed, the pug clamp is opened and the blood can be seen to be shunted from the arterial side to the venous side; After the anastomosis is completed, check carefully for bleeding, count the instruments, and use 5-0 Pro l ene suture to close the rectus abdominis and skin layer by layer. Turn off anesthesia and wait for the animal to wake up naturally. Do not leave the animal before it wakes up. Observe the animal's condition for 30 minutes, and feed it in a separate feeding box for 3 days after confirming that the animal has recovered well. S7. After three days, the fish enter the hypoxia chamber, where the conditions are 10% oxygen concentration, 0.2% carbon dioxide, 22-26°C temperature, 60-70% humidity, 12 hours a day, and 4 weeks of breeding.

2. The method for constructing a high-altitude congenital heart disease-pulmonary hypertension model according to claim 1, characterized in that: The specific steps of S6 are as follows: cut the skin in the middle of the abdomen from the xiphoid process to 1 cm above the bladder.

3. The method for constructing a high-altitude congenital heart disease-pulmonary hypertension model according to claim 2, characterized in that: The S6 is specifically as follows: puncturing the inner side of the aorta and the inner side of the inferior vena cava, and trimming the shunt opening to a size of 2 mm.

4. Application of the high altitude congenital heart disease-pulmonary hypertension animal model prepared by the method for constructing the high altitude congenital heart disease-pulmonary hypertension animal model as described in any one of claims 1 to 3 in basic research on pulmonary hypertension.

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