动物造模,eNOS基因敲除 z-bio 2021-05-07 874

　　Hypertension is a complex polygenic disease characterized by elevated arterial blood pressure. It is one of the main risk factors for coronary heart disease, myocardial infarction and stroke. The morbidity and mortality are increasing year by year, posing a serious threat to humans. ..health. The development of hypertension is associated with increased secretion or activity of vasoconstrictors and decreased secretion or activity of vasodilators. Endothelial nitric oxide synthase (eNOS) plays an important role in regulating blood pressure. Vascular endothelial cells produce nitric oxide (N0), which acts on vascular smooth muscle cells, causing vasodilation and ultimately lowering blood pressure. The establishment of the eNOS gene knockout rat model can lay the foundation for the research on the etiology and treatment of hypertension.

　　1 Materials and methods

　　1.1 Animal

　　SPF grade SD rats, purchased from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd. [SCXK (Kyo) 2012.0001]. All experimental mice were raised in the Department of Experimental Animal Science of China Medical University [SYXK(?)2013.0001] at a temperature of 24±2°C and a relative humidity of 50%±10%. Automatic dimming (bright for 12 hours, dark for 12 hours) [SYXK (辽)2013-0007].

　　Preparation of

　　1.2eOS gene knockout rat

　　1.2.1 Purification of injectable genes Use zinc finger nuclease (ZFN) technology to construct knockout vectors, digest them with restriction enzymes, and use them together with Ambion's gate SSAGEm MACHINE. T7ULTRA kit was transcribed in vitro and purified by Qiagennesay Mini kit.

　　The purified fragment was dissolved in water and stored at -80°C. Dilute before injection, the injection concentration is 10ng/? TL. 1.2.2 In the rat PMSG25IU/mouse intraperitoneal injection, ovulation and receptor preparation are too much, 46"-? 48 hours later, hCG25IU/mouse intraperitoneal injection, and by normal SD male rats in the same Day 1 in 5-week-old SD rats: Mate in a cage, check the vaginal suppository the next morning, and then check the pregnancy suppository as a donor mouse, wild type, meanwhile over-ovulate the cage, select SD female mice And mate with the ligated SD male mice. The next morning, move the vagina to the vagina, and see Shuan’s pregnant mice.

　　1.2.3 The fertilized eggs were collected by cervical dislocation and the donor female rats were sacrificed. The Faropius test tube was collected and placed in the preheated M2 medium, and the swollen area was torn apart. After treatment with hyaluronidase at 1 mg/mL, one fertilized egg was obtained. After washing with M2 for 3-5 times, the amount of fertilized egg was recorded and transferred to overnight culture.

　　1.2.4 Microinjection The in vitro transcription and purification of mRNA encoding eNOS-specific zinc finger enzymes were injected into fertilized rat eggs, and the eggs were transferred to a COz incubator and incubated. 30 minutes before ovulation. Transfer.

　　1.2.5 Anesthetize the pseudopregnant rats with 0.1 g/mL containing 0.31 mL/100 g SD via intraperitoneal injection embryo transfer. Perform alcohol disinfection and then amputation. Cut it open. For the kidney, cut the mouth near the small corner of the small canal of Faropius, insert the fallopian tube from the incision into the canal of Farobian, blow the fertilized egg into the ampoule, let it stand for a while and then pull it

　　1.2. Detect the tail tissues of 6 rats aged 10 to 15 days and digest them in a water bath shaker at 55°C. Under the action of proteinase K, the rats were extracted by the phenol-chloroform method. 5'-TGTGTTCTCTTCTGGCTCAGG-3', reverse primer: 5'-CAAAGATCcTrCCACGAAC. The conditions are as follows: 94°C pre-denaturation for 5 minutes, 94°C 45S, 60°C 45S, 72°C 45S, 34 cycles, and finally extended at 72°C for 10 minutes. The PCR product was sequenced and cloned by TA to confirm the gene deletion. 1.2.7 Homozygous screening The primary positive mice and wild SD rats are bred to produce F1 heterozygous rats, F1 heterozygous rats are bred with each other and homozygous rats are selected.

　　1.3 Western blot detection

　　ENOS took gene knockout rat heart, quickly placed it in SD wild-type rat, crushed kidney tissue, liquid nitrogen, and tissue, lysed with protein lysate, centrifuged, and collected the supernatant, which is a kind of protein. After protein quantification, denaturation, SDS PAGE gel electrophoresis, transfer membrane, place skim milk and incubate with antibody, the primary antibody is Immunoway Company antibody, and the secondary antibody is goat anti-rabbit antibody. After imaging with ECL chemiluminescence reagent, the expression of eNOS was detected. At the same time, GAPDH is used as internal control.

　　1.4 4 Observation of appearance and phenotype of homozygous rats knocked out with eNOS gene Visually inspect the head, trunk, limbs, tail, etc. of homozygous rats and littermate wild-type rats to observe whether there are abnormalities. .

　　1.5 Histological and Morphological Observations

　　After the rats were sacrificed, the vascular tissues of the heart, liver, spleen, lung, kidney, muscle and abdominal aorta were collected and fixed with 0.25 g/mL paraformaldehyde. It was embedded in paraffin, sectioned and stained. Hematoxylin-eosin (HE) is used to observe and photograph changes in tissue structure and morphology through an optical microscope. 1.6 Body weight comparison 5 males and 5 males of wild-type SD rats, and 5 males and 5 males of eNOS gene knockout rats were selected. The weight is compared at 8 to 16 weeks of age. 1.7 Measure heart rate, blood pressure, systolic blood pressure, and diastolic blood pressure. 5 males and 5 male wild SD rats (8-9 weeks old) and 5 males and 5 male IVD, select one knockout rat. Using Softron BP-2010A equipment, the rat heart rate, blood pressure, systolic blood pressure and diastolic blood pressure were measured by the tail cuff method. Measure each rat 3 times, and record the average value as the rat's heart rate. Blood pressure, systolic and diastolic blood pressure.

　　1.8 Statistical analysis

　　The data are expressed as mean±standard deviation±. The difference between the t-test groups, and P\u003c0.05 is considered to be statistically significant. Results 2 2.1 genotype test results of homozygous rats with eNOS gene knockout The eNOS gene knockout vector was transcribed and purified in vitro, and injected into fertilized SD rat eggs. A total of 441 fertilized eggs were injected, of which 365 survived. Twelve SD rats were transplanted to produce a total of 23 FO generation rats. Sequencing and analysis of PCR products produced a total of 4 positive rats, of which rat No. 8 was selected for subculture and homozygous selection. In knockout-positive rats, the fourth exon has a 22-base deletion, resulting in a frameshift mutation. 2.2 Western blot detection uses GAPDH as an internal reference. Wild-type SD rats express eNOS protein in the heart and kidney, while eNOS knockout rats express eNOS protein. 2.3 Appearance and phenotype observation Homozygous eNOS gene knockout rats showed varying degrees of limb deficiency.

　　2.4 Pathological observation

　　Wild-type SD rat arterial intima endothelial cell monolayer with smooth surface, but eNOS gene knockout rat arterial endothelial cells swell, the surface is uneven, and the adventitia is damaged. 4) .. There is no obvious change in the morphology and structure of other organizations. Laboratory Animal and Comparative Medicine 2015, 35 (4)

　　Note: Compared with wild-type rats of the same sex, P\u003c0.05, "P\u003c0.01

　　3 is the study of physiology, pharmacology and behavior, especially discussing the behavior and neurological research of certain cardiovascular diseases in rats. An animal model that is more meaningful and ideal than mice. Rats are evolutionarily closer to humans than mice. Complicated with inflammation, used for research and neurological diseases. As an animal model, its phenotype is similar to human clinical manifestations. For example, in a large mouse model of Alzheimer's disease with the same genes, the phenotype of transgenic rats is not exactly the same as that of mice. This model effectively reproduces the brain pathological changes of Alzheimer's disease. Its phenotype is similar to the development of human diseases. It is more suitable as an animal model of Alzheimer's disease. The role of rats is positive in life science research. Becoming more and more important. Due to physiological, behavioral and metabolic reasons, certain human diseases (such as hypertension) are very good. You cannot have a new mouse model and must rely on the establishment of a rat model. Rat genetic engineering Technology is very important for the development of large animals. The large-scale mouse model is limited by the culture conditions of rat embryonic stem cells (ES cells). The traditional gene targeting technology based on rat ES cells has been greatly affected, and the modification technology independent of ES cell genes is more important. outstanding. New genome editing methods such as ZFNs technology and TALENs technology [1O, CRISPCas9 technology] have been introduced, and various effective research tools have been developed. These research tools provide new ways to study gene function. The application of these technologies eliminates this demand. This study used ZFN technology to create an eNOS knockout rat. The fourth exon of the rat lacked 22 bases, resulting in a frame shift. The rat protein expression test showed that the gene was not expressed in the kidney and heart, and the immunohistochemistry experiment showed the same result (not shown), achieving the goal of gene knockout

　　Observation of

　　eNOS Knockout rats have varying degrees of limb defects in homozygotes. Knockout rats. However, heterozygous rats were found to have a normal appearance. The size of knockout rats is smaller than that of wild-type rats. The body weight of 16-week-old rats was compared and analyzed. The body weight of knockout rats is much lighter than that of wild-type rats. This is consistent with the results of previous studies on eNOS knockout mice. Female mice showed a reduced number of embryos and a high static birth rate. Knockout rats have varying degrees of limb defects, which also affect some eating and affect the body. quality improvement. eNOS is the key to regulating vascular function and also plays an important role in regulating endothelial cell angiogenesis [14,15]. In this study, HE staining of rat arteries showed that the appearance of the arteries was markedly damaged and had a constant effect on vascular function. The eNOS gene for limb development was found in knockout mice, but no phenotype was found, and the phenotype was obvious in knockout rats. The effect of this gene on limb development needs further research. In eNOS knockout rats, blood pressure increased significantly, and systolic and diastolic blood pressure also increased significantly. Under normal physiological conditions of the human body, vascular tension is mainly regulated by vasoconstrictors and relaxation factors. As an important vasodilator in the body, NO, together with other vasodilators, can mediate the vasodilation reaction and resist the vasoconstriction effect of substances such as angiotensin II and renin in the body. In most blood vessels, the endothelium-dependent relaxation function is mainly attributed to NO products derived from eNOS. Down-regulation of eNOS can lead to a large reduction in NO and weaken the vasodilator function. Research on eNOS knockout mice showed that the blood pressure of eNOS knockout mice was significantly higher than that of wild-type mice, indicating that eNOS affects the blood pressure level of mice. Savard et al. ENOS gene transfer can enhance the release of NO and prevent the deterioration of hypertension. In addition to the increase in blood pressure, the study also observed that the heart rate of the knockout female mice was significantly reduced, while the male mice did not. This may be the response of the baroreceptor reflex mechanism to increased blood pressure. In summary, this study successfully established a rat that can knock out the eNOS gene as an ideal animal model for studying the etiology of hypertension, and laid the foundation for the study of eNOS gene function.