动物造模,艾滋病 z-bo 2022-08-09 500

　　1. animal models created by different routes

　　 The three methods of AIDS infection are through blood, sex and mother-to-child transmission. There are two types of sexually transmitted infections, that gay and straight. In preparation for SIV animal model, we must first consider it value. The best animal model for human disease can be maximized, including the use of similar or similar natural infection method. SIVmac251 by the above method were seeded onto primary rhesus monkey naturally infected, you can create an animal model of infection in four different ways: a blood vein model of infection, the virus in the blood of CD4, + T direct infection If the lymphocytes 1m12 x 100000 TCID50 of SIVmac251 slowly pushed into monkeys by intravenous, China will increase the chance of infection in rhesus monkeys.

　　2. The heterosexual model is transmitted through the vaginal mucosal epithelium, and the virus can directly infect Langerhans cells and epithelial cells. Studies in primate models of AIDS have shown that injecting progesterone into the body can promote the transmission of SIV, while estrogen can inhibit the transmission of SIV. These hormones may affect the thickness of the vaginal wall, and affect the expression of CCR5 and CXCR4 receptor by IL-2. relationship. For an appropriate menstrual cycle, or when a specific amount of progesterone is used, 1 ml of 2×10 7 TCID50 SIVmac251 is slowly catheterized into the monkey’s vagina to establish an animal model of heterosexual infection. Do it. 3. The same model of infection spread through the epithelial mucosa, direct infection of M cells and epithelial cells. Physiological and rectal epithelial cells very thin thickness than rectal infection vaginal infection is more likely. In order to establish an animal model of infection is gay, will be 1 ml 2x10 7 TCID50 SIVmac251 slowly into the rectum of the monkey. Small amount a plurality of times after infection (usually 5 to 8 times of infection) over a period of time, can achieve 100% infection.

4. The child transmission model Recently, an animal model of neonatal AIDS neonatal macaques with SIV vaccination established. Symptoms similar to those of infants infected with HIV have been observed. Through the highly virulent SIV strain, the neonatal disease of rhesus monkeys progresses very rapidly. This model has practical value in studying the characteristics and treatment of HIV-infected infants. Some scholars have found that mothers infected with HIV has little monkey born with AIDS in monkeys. This is the most ideal animal model for mother-to-child transmission, but its availability limits its application. Note that by intravenous, vaginal or rectal infected effector cells are different. Intravenous injection directly infect CD4 + T cells, Langerhans cells and infected vaginal epithelial cells, M cells and is a rectal epithelial cells. Different structures rectal and vaginal mucosa, the vaginal squamous cell mainly, rectal mainly columnar epithelial cells, vaginal epithelial cells, and 10 times a thickness of rectal epithelial cells. Because the virus infection by a variety of methods are exposed to the body's immune pressure, so a variety of infection methods with a particular impact on areas such as clinical and virological indicators.

　　二. Preparation of animal model SAIDS SIV / (1) Preparation of SIV / SAIDS 1. Experimental animal model SIVmac251 virus strains or SIVmac239, TCID50 of virus stock solution of 2 × 10. 7 power/ml 2. Experimental animals are SPF rhesus monkeys weighing 4 to 6 kg, as well as simian immunodeficiency virus (SIV), simian retrovirus type D (SRV-1,2,5) and simian T lymphocyte I Viruses other than STLV. -1) infection. If possible, perform animal genetic background analysis. Normal physical examination before the experiment. Animal infection experiments should be carried out in ABSL-3 rooms.

3. SIVmac251 virus in rhesus monkeys inoculated into three ways: intravenous, vaginal and rectal. Infective dose and method as follows: (1) the intravenous route. Virus solution in 1 ml of a solution of 1: 100 dilution, then slowly injected into the body via the vaginal route. Using 1 ml of virus stock solution, place the animal in the prone position, slowly inject the virus solution into the edge of the vagina, and maintain the prone position for 30 minutes ③rectal route. Use 1 ml of the stock virus solution in the prone position of the animal, and slowly inject the virus solution into the anal sinus for 30 minutes in the prone position. 4. Whole blood virus isolation 4d, 7d, 10d, 14d, 21d, 28d, and other meaningful time points after inoculation of the virus, the collected RIV SIV infected monkey heparin anticoagulated whole blood to obtain whole blood. Isolate the virus. Specific steps are as follows: Take a 24-well plate, and serially diluted whole blood sample in a 5-fold in the 1640 medium with 10% calf serum. After dilution, the final volume of the whole blood sample were in each well 200μl, 40μl, 8μl, 1.6μl, 0.32μl and 0.064μl. To each well was added 0.4 ml CEMx174 cells, at a concentration of 2x100000 / ml, and finally with 10% bovine serum was prepared 1.6 ml of 1640 medium per well, every 3-4 days disposed 37 ° C, 5% CO2 incubator in. trading. Once the liquid. Per day for 4 weeks to observe CPE. The highest CPE dilution that can occur is the virus titer of the sample.

　　5. PBMC are separated virus virus inoculation, rhesus heparin 4,7,10,14,21,28 and other meaningful time points after normal monkey whole blood and SIV infection. Collect (without SIV, STLV)-1, SRV and B virus infection) whole blood is anticoagulated with heparin, PBMC are separated by density gradient centrifugation, counted and adjusted to a cell concentration of 1 x 1000000/ml. Normal monkey PBMC was stimulated with PHA (final concentration 5μg/ml), SIV-infected monkey PBMC was stimulated with IL-2 (final concentration 100 U/ml) and PHA (final concentration 5μg/ml), placed at 37°C, 5% In the CO2 incubator. I was taken away. 3d. Three days later, the PBMCs of normal monkeys and infected monkeys were mixed 1ml in a 24-well plate, and exchanged with 1640 medium containing IL-2 (25U/ml) and 10% bovine serum. Observe changes in cell morphology (CPE) and growth every day. When the number of cells is small and the condition is not good, fresh, well-growing normal monkey PBMC can be appropriately supplemented. Every 3-4 days the culture supernatant was aspirated, P27 antigen measured by ELISA, add fresh 1640 medium to a final volume of 1.6ml, and cultured at 37 ℃ at in 5% CO 2 incubator. .. Culture the cells for up to 4 weeks.

 6. Viral load detection SYBR Green? Real-time quantitative RT-PCR with fluorescent dyes is used to determine viral load. The specific steps are as follows: use EDTA anticoagulated whole blood, collect the supernatant by centrifugation, extract viral RNA by TRIzol method, and use Qiagen's QuantiTect SYBRGreenT-PCR kit and Roche LightCycler 3.5 real-time PCR instrument for measurement. Primers: 2f (5'-GTAACTATGTCCACCTGCCATTA-3') and 2r (5'-CAGCCTCCTCGTTTATGATGT-3'), the length of the amplified fragment is 209bp. The reaction system as follows: 2xQuantiTectSYBRGreenIRT-PCRMasterMix 10μl; primer 2f and 2r (10μmol / L) of each 1μl, a final concentration of 0.5μmol / L; QuantiTectRTMix 0.2μl; template 1μl; PCR grade water 6.8μl. The reaction conditions are 50°C 20 minutes (×1), 95°C 10 minutes (×1), 94°C 15 seconds (×45), 56°C 20 seconds (×45), 72°C 30 seconds (× 45), 75 determines the absorbance value at 3°C (×45)°C.

7. Measure CD4 +/CD8 + T lymphocytes to take 50μl EDTA anticoagulated whole blood, add 10μl PerCP-CD3/FITC-CD4/PE-CD8 fluorescently labeled antibody, mix gently, and store at room temperature for 15 minutes at room temperature; with 1 ml of 10-fold diluted hemolysate was dissolved FACSLysingSouLution 10 minutes. Wash twice with PBS for flow cytometry, add 0.5 ml of 1% paraformaldehyde, store in a refrigerator at 4°C, machine test within 48 hours, CD4 + and CD8 + T in lymphocytes to determine the cell percentage And calculate the CD4 +/CD8 + ratio. 8. Plasma antibody determination Immunofluorescence method (IFA) detects the level of antibodies in the plasma of infected monkeys. CEMx174 was infected with SIVmac251 to prepare antigen slides. First, dilute the sample to be tested at a ratio of 1:20, gradually dilute it to 1:1280, drop it on the antigen sheet, and then react for 30 minutes in a humid chamber at 37°C to completely remove the antigen and antibody. Allowed to bind, and then washed to remove unbound serum components. Add FITC fluorescently labeled anti-monkey IgG antibody, incubate in a humidified box at 37°C for 30 minutes, wash, and observe the result on a fluoroscope. 9. The cellular immunoassay (Elispot) was pre-coated with anti-monkey IFN-γ antibody on a 96-well plate, placed overnight at 4°C, and then washed with PBS (PBST) containing 0.05% Tween-20, and 1% BSA was added . 200μlPBS allowed to stand at 37 ° C 1 hour. Separate monkey PBMC by Ficoll density gradient centrifugation, calculate the cell concentration in a cell-free medium to adjust it to 3x1000000/ml, add PBMC to the wells of a closed 96-well plate, and add 100μl of cell solution to each well . In addition, incubate at 37°C for 24 hours in a 5% CO2 incubator, and add 5 g to each well. After incubation, pour out the cell solution, treat it with 200μl of ice-cold deionized water, wash the plate 10 times with PBST, add biotinylated detection antibody and GABA-conjugated anti-biotin antibody, and then incubate at 37°C. Incubate for several hours. After incubation, the plates were washed 5 times with PBST. The substrate was then added for 15-20 minutes at room temperature. After the color was developed, the plate was washed twice with distilled water, dried and stored in the dark. Use an inverted microscope and ELISPOT plate reader to count the completed ELISPOT plates. 10. The method for determining neutralizing antibodies is to sequentially dilute the test serum, incubate the diluted serum with a fixed amount of virus solution at 37°C for 1 hour, and then use the neutralizing solution to induce TZM. - bl infected cells and infection was observed. To determine the serum neutralization concentration.

　　 (2) AIDS SIV/SAIDS animal model evaluation criteria

　　 1. Clinical evaluation criteria Experimental monkeys developed anorexia from 7 to 14 days after infection, lasting for more than 2 weeks, weight loss, and groin area decreased. Obvious swelling remained in the superficial lymph nodes. Six months after infection, the infected monkeys showed great loss of appetite, weight loss, diarrhea, decreased inguinal lymph nodes, multiple surface infections, and the animals died due to fatigue, especially intravenous infections. Due to individual differences in animals, the severity of the disease, the clinical course and manifestations may be different, and the symptoms of AIDS may not be typical. 2. Standard etiology virus isolation results showed that the virus is usually isolated from whole blood monkeys after infection three days, 10-40 days after infection the highest separation rate. Occurs, and then measure the virus titer in the whole blood. It drops rapidly and remains low. By detecting the plasma viral load, the change trend of the three ways after infection is basically the same, and it rises rapidly after infection, reaching 10 to 7 copies/ml in about 14 days, and maintains for about 40 days and then rapidly decreases. In general, the earlier discovered vein virus infection in monkeys and rectal infection in monkeys, and finally vaginal infection in monkeys. After the acute phase, the monkey's viral load fluctuates to a certain extent. The viral GagP27 antigen can be easily detected in the acute phase.

　　 3. Immunological standards

　　 (1) Cytological changes: The CD4 +/CD8 + ratio of experimental monkeys began to decline 7 days after infection, and reversed. This phenomenon can continue for a long time. During the entire post-infection period, the CD4 +/CD8 + ratio fluctuates less than 1, (2) Changes in humoral immunity: in infected monkeys, the antibody test was found to be positive on day 10-14, and the antibody titer was on day 40 -24 weeks gradually reached its peak. It rises, then falls, and continues to exist. (3) Changes in cellular immunity: IFN-γ levels in infected monkeys repeatedly increased and decreased. In the early stages of infection, IFN-γ monkeys PBMC were contained in a small number of secreting cells (SFC), and a small peak appeared at 30 days post infection in monkeys and stabilization of the SFC number of stable Around 100. Another peak appeared on the 126th day, followed by a period of fluctuation, with the average SFC fluctuating between (100-200)/1,000,000 PBMC. (4) Pathological changes: After SIV establishes a long-term chronic infection in the human body, with the development of the disease, it will affect the functions and morphology of various human systems, and eventually lead to complex and diverse diseases. It may show physical changes. On the other hand, the formation of AIDS also involves damage to various system organs, such as the destruction of lymphatic and hematopoietic tissues. Observation and examination of pathological changes in monkeys infected with SIV model, will help to understand the causes of the model and to further evaluate its usefulness.

　　3. Preparation of AIDS SIV/SAIDS rhesus monkey model

　　SHIV virus is an artificial chimeric virus with SIV virus as the backbone and genetically recombined with HIV-1 virus. We will use the SIV genome as the background to express the inherent genetic components of HIV. This overcomes the HIV envelope and main structure and antigenic differences of SIV, and to overcome the trouble of using SIV infection assess HIV vaccines and drugs in animal models. Differences in virus types limit the accuracy of model use and evaluation. Therefore, the SIV infection model is the main advancement of the SIV infection model. In theory, the monkey SIV infection model should be the best animal model for AIDS at present, but most of these synthetic SIVs cannot cause typical monkey AIDS like SIV. At present, it has been reported internationally that SIV is contained in strains that have successfully established animal models of SIV infection. -Strains such as SF162, SIV-89.6P, SIV-HXBc2 These SIV strains replicate at high levels after passage in pig-tailed monkeys, causing loss of CD4 + T cells and causing opportunistic infections. Eventually you will die. At present, most SIVs are constructed based on HIV-1B subtype strains. For example, SIV-89.6 is a chimeric virus mainly composed of SIVmac239 and having the env gene of HIV-1B subtype. HIV-189.6 is the parent strain of SIV-89.6, a trophic strain with T lymphocyte and macrophage tropism. SIV-89.6, which expresses HIV-189.6 tat, rev, vpu and env genes, can infect rhesus monkeys, but it does not cause AIDS-like symptoms in monkeys. 140 base deletion occurs, changing the 12 amino acid extracellular gp120 and gp41 regions, resulting in a strong pathogenic strain SIV-89.6P. This pathogenic strain quickly depleted CD4 + T lymphocytes, and rhesus monkeys in India caused AIDS-like symptoms. SHIV-KB9 is a clone of SHIV-89.6P. Foreign animal studies have shown that SIV-KB9 is a mature SIV virus that is strongly pathogenic to macaques and cynomolgus monkeys in India. In recent years, it has been widely used abroad to study the pathogenesis of AIDS and evaluate HIV vaccines. Chinese scholars have also constructed an SIV virus strain that integrates the HIV-1B'/C strain circulating in China and conducted infection experiments in Chinese rhesus monkeys. The results showed that viremia and lymphocyte changes were observed. It has been shown to be effective infection. To establish an SIV rhesus monkey animal model, the SIV infection dose must first be determined. If the infection rate is too low, the rhesus monkey will not be infected with the SIV virus; if the infection rate is too high, the rhesus monkey may die soon. Usually, the virus concentration in experimental monkeys needs to be titrated to determine the infectious dose. eimann, who succeeded to 400 TCID 50 dose of SIV-HXBc2 and rhesus monkeys infected with SIV-89.6. The SIV-KB9 virus solution with a concentration of 4.8 x 100000 copies/ml or higher can successfully infect Chinese rhesus monkeys. SHIV also passes through three main routes of infection. Venous infections, mucosal infections (including vaginal and rectal routes) and mother-to-child transmission, respectively, simulate the blood, sexual contact and mother-to-child transmission of human HIV infection. Currently, most research focuses on venous and mucosal routes. Intravenous infection is usually used to study the action and mechanism of HIV vaccines and anti-HIV drugs. SHIV strains commonly SIV-89.6P and SIV-KB9. Infection is often used to study the mechanism of mucosal immunity and the effects of vaccines and drugs. The commonly used strains are SIV-sfl62P3 and SIV-1157i.

　　1. Materials and methods for establishing the SIV/SAIDS animal model. Currently, there is no SIV strain in the experimental stage in China. The SHIV-89.6P virus is relatively mature and may be introduced in the United States by NIH. The animal rhesus monkey and other indicators are the same as the above SIV introduction.

　　2. SIV strains with different evaluation criteria in the SHIV/SAIDS animal model have very different clinical manifestations, viremia, lymphocyte changes and disease levels in the infected experimental monkeys. Animals infected with SIV-89.6P virus will show symptoms and signs, such as anorexia nervosa, restlessness, watery diarrhea, weight loss and weight loss, and even death. ELISA method for the determination of plasma monkeys P27 antigen. In seven to ten days after 10-14 days and virus isolation after infection, it becomes positive. The plasma viral load usually becomes positive from the 7th day, and the viral load becomes positive from the 10th day to the 21st day, reaching a peak (1.0 x 10 copies/ml higher than the 7th power), and then continuing to decrease. It may be tested positive for a long time, or it may be difficult to detect. Monkeys before infection of CD4 + / CD8 + ratios are greater than 1, and is gradually reduced and reversed within 10 to 21 days after infection. Cycle fluctuations, sometimes reversals and normal rotations. Before virus infection, the absolute number of CD4 + T cells in the experimental monkeys exceeded 1000/μl. After infection, the number of CD4 + cells in the experimental monkeys decreased sharply, usually reaching the lowest point between 14 and 21 days, after which it remained low and fluctuated unstable.