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| Isolation and Cultivation of Guinea Pig (Cavia porcellus) Peritoneal Macrophage and Its Immunological Function in Response to Tuberculosis |
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Abstract Abstract Macrophages are key cells associated with innate immunity, pathogen containment and modulation of the immune response and also the key regulators of tissue repair, regeneration and fibrosis. Commonly used model systems for studying macrophage biology have centered on macrophage-like leukemic cell lines, primary macrophages derived from model organisms and primary macrophages differentiated from blood monocytes. Although these cells have provided important insights into macrophage-associated biology, there are issues that need consideration. The primary drawback of understanding macrophage biology is that reliable and scalable macrophage models for cellular and genetic studies is scarce, limiting their utility in genetic studies. Based on these, in order to elucidate the role and mechanism of guinea pig macrophages in tuberculosis immunization, in the present study, guinea pig peritoneal macrophages were isolated and characterized by using the following methods: Morphological observation was by phase contrast microscope, cell viability analysis was performed using Trypan blue staining method, phagocytosis assay was carried out using the method of swallowing ink particles, the expression of intracellular enzyme was detected using α-naphthol acetate esterace (α-NAE) staining method, and the expression of cell surface specific antigen analysis was detected by flow cytometry. Furthermore, in order to study the effect of Bacillus Calmette-Guérin (BCG) on guinea pig macrophage apoptosis and its production of nitric oxide (NO), guinea pig macrophage was infected by BCG in vitro. The results showed that the guinea pig macrophage performed multiple cell morphology and maintained the characteristic of macrophage-specific morphology, which resembled round shape, oval shape, spindle shape and irregular shape under the microscope. Pseudopodium and bulge were observed in some of the cells. The cells had a large amount of cytoplasm and large nuclei. The result of Giemsa staining showed that the nuclei of macrophage was darker and horseshoe shaped, round or irregular shape, locating at one side of the cells. The macrophage had the ability of favourable phagocytosis, and the phagocytic rate was (94.3±1.04)%. The results of nonspecific esterase staining showed that the rate of positive staining cells was (94.3±1.44)%, which indicated that the purity of the isolated macrophages was higher. Meanwhile, flow-cytometric surface phenotyping of guinea pig macrophage revealed expression of classical macrophage markers such as CD14, CD40 and CD68, and the percentages of positive staining of CD14, CD40 and CD68 were 97.6%, 96.9% and 92.1% respectively. Moreover, the results showed that the production of NO in guinea pig macrophage which were infected by BCG was significantly increased (P＜0.01) compared with the control group. Meanwhile, the results of apoptosis analysis by FITC Annexin V Apoptosis Detection Kit assay combined with fluorescence activated cell sorter (FACS) showed that the ratio of apoptosis of the BCG infected group was (14.4±2.86)% which was significantly higher than that of the control group (4.26±1.06)% (P＜0.01). These results initially confirmed that pre-stimulation of starch broth solution could extract a large number of guinea pig peritoneal macrophages, and confirmed that the guinea pig macrophage had the function in tuberculosis immunity. And the present results may provide new experimental material and data support for the research on interaction between macrophage and Mycobacterium tuberculosis.
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Received: 04 February 2017
Published: 20 July 2017
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| [1] Weavers H, Evans IR, Martin P, et al. 2016. Corpse Engulfment Generates a Molecular Memory that Primes the Macrophage Inflammatory Response[J]. cell, 165(7):1658-1671.[2] Li Y, Wang J, He HY , et al. 2011. Immunohistochemical demonstration of airway epithelial cell markers of guinea pig[J]. Tissue & cell, 43: 283-290.[3] Kaufmann E, Spohr C, Battenfeld S, et al. 2016. BCG Vaccination Induces Robust CD4+ T Cell Responses to Mycobacterium tuberculosis Complex-Specific Lipopeptides in Guinea Pigs[J]. J Immunol, 196(6):2723-2732.[4] Sakthi S, Palaniyandi K, Gupta UD, et al. 2016. Lipoprotein LpqS deficient M. tuberculosis mutant is attenuated for virulence in vivo and shows protective efficacy better than BCG in guinea pigs[J]. Vaccine ,34(6):735-743.[5] Sander P, Clark S, Petrera A, et al. 2015. Deletion of zmp1 improves Mycobacterium bovis BCG-mediated protection in a guinea pig model of tuberculosis[J]. Vaccine, 33(11):1353-1359.[6] Tiwari P, Arora G, Singh M, et al. 2015. MazF ribonucleases promote Mycobacterium tuberculosis drug tolerance and virulence in guinea pigs. [J]. Nat Commun, 6:6059.[7] Clark S, Hall Y, Williams A. 2014. Animal models of tuberculosis: Guinea pigs[J]. Cold Spring Harb Perspect Med, 5(5):a018572.[8] Vrettou C, Traeger-Synodinos J, Tzetis M, et al. 2004. Real-time PCR for single-cell genotyping in sickle cell and thalassemia syndromes as a rapid, accurate, reliable, and widely applicable protocol for preimplantation genetic diagnosis. Hum Mutat, 23(5):513-521.[9] Chan E D, Morris K R, Belisle J T, et al. 2001. Induction of inducible nitric oxide synthase-NO* by lipoarabinomannan of Mycobacterium tuberculosis is mediated by MEK1-ERK, MKK7-JNK, and NF-kappaB signaling pathways [J]. Infection and Immunit, 69(4): 2001-2010.[10] Pathania R, Navani N K, Gardner A M, et al. 2002. Nitric oxide scavenging and detoxification by the Mycobacterium tuberculosis haemoglobin, HbN in Escherichia coli[J]. Molecular Microbiology, 45(5): 1303-1314.[11] Lee J, Remold H G, Ieong M H, et al. 2006. Macrophage apoptosis in response to high intracellular burden of Mycobacterium tuberculosis is mediated by a novel caspaseindependent pathway[J]. The Journal of Immunology, 176(7): 4267-4274.[12] 刘云霞, 张万江. 2012. 结核分枝杆菌与巨噬细胞相互作用的研究进展[J]. 中国细胞生物学学报, 34(6): 617-622.(Liu Y X, Zhang W J. 2012. Progress of interactions between Mycobacterium tuberculosis and macrophages[J]. Chinese Journal of Cell Biology, 34(6): 617-622.)[13] Patel, N. R., Swan K., et al. 2009. Impaired M. tuberculosis-mediated apoptosis in alveolar macrophages from HIV+ persons: potential role of IL-10 and BCL-3[J]. J Leukoc Biol, 86(1): 53-60.[14] Tang J, Zhan L, Qin C. 2016. Inhibition of TLR8 mediated signaling promotes BCG induced apoptosis in THP-1 cells[J]. Microb Pathog. 2016 Apr;93:78-82. doi: 10.1016/j.micpath.2015.11.028.[15] Riendeau C J, Kornfeld H. 2003. THP-1 cell apoptosis in response to Mycobacterial infection[J]. Infect Immun. 71(1):254-259.[16] 何玉龙, 张文清, 何承文, 等. 2015. 结核分枝杆菌BCG和H37Ra对巨噬细胞凋亡的影响及其机制研究[J]. 农业生物技术学报, 2015, 23(5):571-578.(He Y L, Zhang W Q, He C W, et al. 2015. Effect and Its Mechanism of Mycobacterium tuberculosis BCG and H37Ra on Macrophage Apoptosis[J]. Journal of Agricultural Biotechnology, 23(5): 571~578. ) |
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