动物造模,灭活H37Rv全菌体,致敏模型 z-bio 2021-12-15 615

　　Tuberculosis is a chronic infectious disease that is zoonotic. It is highly pathogenic and lethal worldwide and is caused by Mycobacterium tuberculosis. Sputum smears and sputum culture from patients' sputum can be used to detect tuberculosis. Although smears are simple and easy to perform, the positive rate is low. Although the culture is the gold standard, the cycle is too long to meet clinical needs. Through conventional intradermal allergy

　　(TST) is still the best and fastest clinical tuberculosis screening and diagnosis method. The development of diagnostic reagents for tuberculosis intradermal allergy is still the current difficulty. One is that there are fewer animal models for research and development. Guinea pigs are highly sensitive to Mycobacterium tuberculosis, and their pathological changes after infection are similar to those of humans. They are easy to model and have a high survival rate. They are recognized as experimental animal models of tuberculosis. Guinea pigs have a very strong delayed-type hypersensitivity reaction, which has been widely used in the detection of anti-tuberculosis drugs and related biological agents, and has become the gold standard for the efficacy of human tuberculin skin tests and the identification of tuberculosis biological products. This study aims to explore different concentrations of inactivated H37Rv whole cells sensitized guinea pigs, pass standard PPD skin test, observe delayed sensitization (DTH), and obtain the best concentration of inactivated H37Rv whole cells when sensitized guinea pigs, and Observe the effect of sensitization on the welfare of guinea pigs, such as the effect of body weight and injection site.

　　1 Materials and methods

　　1.1 Preparation of injection bacterial solution

　　H37Rv strain was cultured in Middlebrook 7H11 medium for 21 days, the cells were collected, washed 3 times with PBS, inactivated at 115°C, 10lb for 30 minutes, dried, and placed in a refrigerator at 4°C for later use. Weigh the whole inactivated H37Rv cells, add normal saline for injection and the same amount of incomplete Freund's adjuvant, grind and emulsify evenly until dripping into clear water without dispersing.

　　1.3 Experimental animals

　　SPF Hartley guinea pigs, 35 to 45 days old. The animals are kept in the barrier animal laboratory, and the experimental animal use license number: SYXK (Guangdong) 2012-0122.

　　1.4 Sensitization and excitation

　　After 7 days of quarantine adaptation for guinea pigs, each guinea pig was injected subcutaneously with the corresponding concentration of inactivated H37Rv in the groin on the inner thigh of each guinea pig, and injected into 3 sites for sensitization, with a total injection of 0.5 mL.

　　After sensitization, it was challenged at different time periods. The sensitized guinea pigs were taken, the back was shaved, and 0.1mL PPD (5IU) was injected intracutaneously. Each guinea pig was injected into the same part.

　　1.5 Observation index

　　After sensitization, the guinea pigs were weighed continuously 5 times, once a week, to make a body weight growth curve; observe the ulceration of the subcutaneous injection site in the groin. The guinea pigs were observed once at 24h and 48h after the challenge. The longitudinal and transverse diameters of the skin test erythema induration were measured and recorded, and the mean value was calculated.

　　1.6 Statistical analysis

　　Perform statistical processing on the sensitization and challenge results of guinea pigs and list them, express them as mean plus or minus standard deviation (), and use t-test and least significant range method for statistical comparison. P≤0.05 indicates that the difference is statistically significant.

　　2 Results

　　2.1 Body weight growth curve of sensitized guinea pigs

　　After sensitizing the guinea pigs with different concentrations of bacteria liquid, no abnormal condition was found. The diet and drinking water were normal, and the weight continued to increase. Although the initial weights of guinea pigs in each group were not consistent, their weights tended to be the same in the 5th week after sensitization.

　　2.2 Changes of injection site in guinea pigs after sensitization

　　After guinea pigs are injected with different concentrations of inactivated H37Rv whole cells, ulcers will appear at the injection site. No ulcers were observed in the injection part of guinea pigs within 3 weeks after the injection of the bacterial solution. In the 4th week, 18.9% of the guinea pig injection sites had ulcers, and 33.3% of the guinea pig injection sites had ulcers in the 5th week. All guinea pig injection sites had ulcers. Section.

　　2.3 Comparison of erythema size after inoculation with various sensitizing doses

　　In the sensitized 0.2mg/mL group, there was a significant difference in the mean value of erythema length and breadth between males and females 48h after the standard challenge. There was no significant difference between males and females in other groups; 0.2mg/mL, 0.3mg/mL, 0.5mg/mL There was a significant difference in the mean value of the longitudinal and longitudinal erythema 24h after the standard was challenged between the groups, with 0.2mg/mL the mean value of the longitudinal and longitudinal erythema being the largest; between the 0.2mg/mL and 0.5mg/mL groups, the longitudinal and lateral erythema was 48h after the standard was challenged. There is no significant difference in the mean diameter.

　　The mean values of longitudinal and transverse diameters of erythema in three sensitization and single sensitization guinea pig skin tests were 24h and 48h, and there was no significant difference in the measurement results.

　　Repeated skin test results showed that 0.3mg/ml H37Rv whole cell sensitized guinea pig skin test erythema average value, the first skin test is better than the skin test repeated many times later.

　　3 Discussion

　　Guinea pigs are highly sensitive to Mycobacterium tuberculosis and are recognized as an experimental animal model of tuberculosis in the world. Guinea pig model is the first choice animal for BCG test and evaluation. In addition, the guinea pig tuberculosis model is often used to develop new antibiotics for the treatment of tuberculosis or to evaluate the effects of combination drugs. Since the tuberculin reaction was discovered by Robert Kirk a century ago, guinea pigs have become an animal model for the study of delayed sensitization (DTH). The DTH response of guinea pigs is very similar to the PPD skin test performed by humans. Finally, the pathological changes of granulomatous gangrene in the guinea pig model are very similar to those of humans.

　　Through this study, it can be concluded that the concentration of 0.2mg/mL, 0.3mg/mL, 0.5mg/mL inactivated H37Rv whole bacteria sensitized guinea pigs, guinea pigs can produce DTH response well, of which 0.2mg/mL inactivated H37Rv The average value of the erythema longitudinal and transverse diameter is the largest after the challenge of the whole-cell sensitized guinea pig standard. The average longitudinal and longitudinal diameter of the erythema in multiple sensitized skin tests is not significantly different from that of a single sensitization; the first skin test erythema is better than the subsequent repeats. The results of the skin test. Our research results show that the average value of the erythema in the 24-hour and 48-hour skin test is significantly greater than that of the model prepared by Zhang Lingxia et al. [11]. She used 0.1ml (5mg) to inactivate the whole H37Rv cells to sensitize guinea pigs 4 times, 1 week per week. The average values of longitudinal and transverse diameters of erythema at 24 hours and 48 hours were 8.25±1.73mm and 7.41±1.22mm, respectively. Suely S. Kashino [8] The use of 18b Mycobacterium tuberculosis to prepare a guinea pig model is stronger than the use of inactivated H37Rv whole-cell sensitized guinea pig model. DTH reaction is stronger, and will cause animal weight loss and death. 18b Mycobacterium tuberculosis is 5 weeks after sensitization of guinea pigs The PPD skin test was performed at 14 weeks and 28 weeks. After 24 hours, the average longitudinal and longitudinal diameters of the erythema in the DTH skin test were 18 ± 3 mm, 19 ± 0.7 mm, and 15.5 ± 1.9 mm, respectively. According to our research results and known literature, the intensity of the DTH reaction in the guinea pig model is related to the death or life of Mycobacterium tuberculosis and the dose, but is less related to the number of inoculations. Live Mycobacterium tuberculosis can cause a strong DTH reaction in guinea pigs, but it may also cause death in guinea pigs; using a lower dose to inactivate the whole H37Rv cell, the DTH reaction is strong. In short, the specific mechanism of the DTH reaction in guinea pigs caused by H37Rv whole cells needs further study.

　　After human infection with Mycobacterium tuberculosis, it can cause drowsiness, malaise, weight loss, and death. In this study, inactivated H37Rv whole cells were used to sensitize guinea pigs. The weight of guinea pigs continued to grow. No obvious uncomfortable reaction was observed in guinea pigs. The ulceration in the injection part is mainly caused by the incomplete Freund's adjuvant. The use of multiple injections can appropriately reduce the incidence of ulceration.

　　In conclusion, this study improved the method of preparing inactivated H37Rv whole-cell sensitized guinea pig models, making it simpler and more reliable to prepare guinea pig models using inactivated H37Rv whole-cell sensitization, and can be widely used in the detection of anti-tuberculosis drugs and related biological agents. . Refer to 0.2mg/mL inactivated H37Rv whole cell subcutaneous injection into the groin of the inner thigh of the guinea pig to prepare the sensitized guinea pig model.