Merlini Esther, Bellistri Giusi M, Tincati Camilla, d'Arminio Monforte Antonella, Marchetti Giulia
Department of Medicine, Surgery and Dentistry, Clinic of Infectious Diseases, San Paolo Hospital University of Milan, Italy.
J Vis Exp. 2011 Jun 11(52):2830. doi: 10.3791/2830.
The healthy gastrointestinal tract is physiologically colonized by a large variety of commensal microbes that influence the development of the humoral and cellular mucosal immune system. Microbiota is shielded from the immune system via a strong mucosal barrier. Infections and antibiotics are known to alter both the normal gastrointestinal tract barrier and the composition of resident bacteria, which may result in possible immune abnormalities. HIV causes a breach in the gastrointestinal barrier with progressive failure of mucosal immunity and leakage into the systemic circulation of bacterial bioproducts, such as lipopolysaccharide and bacterial DNA fragments, which contribute to systemic immune activation. Microbial translocation is implicated in HIV/AIDS immunopathogenesis and response to therapy. We aimed to characterise the composition of bacteria translocating in peripheral blood of HIV-infected patients. To pursue our aim we set up a PCR reaction for the panbacteric 16S ribosomial gene followed by a sequencing analysis. Briefly, whole blood from both HIV-infected and healthy subjects is used. Given that healthy individuals present normal intestinal homeostasis no translocation of microflora is expected in these patients. Following whole blood collection by venipuncture and plasma separation, DNA is extracted from plasma and used to perform a broad range PCR reaction for the panbacteric 16S ribosomial gene. Following PCR product purification, cloning and sequencing analyses are performed.
健康的胃肠道在生理上定植着大量共生微生物，这些微生物会影响体液和细胞黏膜免疫系统的发育。微生物群通过强大的黏膜屏障与免疫系统隔离开来。已知感染和抗生素会改变正常的胃肠道屏障以及常驻细菌的组成，这可能导致免疫异常。HIV会导致胃肠道屏障破损，黏膜免疫逐渐衰竭，细菌生物产物如脂多糖和细菌DNA片段泄漏到体循环中，从而导致全身免疫激活。微生物易位与HIV/AIDS的免疫发病机制及治疗反应有关。我们旨在表征HIV感染患者外周血中易位细菌的组成。为实现这一目标，我们针对泛细菌16S核糖体基因建立了PCR反应，随后进行测序分析。简要来说，使用HIV感染患者和健康受试者的全血。鉴于健康个体呈现正常的肠道内环境稳定，预计这些患者不会发生微生物群易位。通过静脉穿刺采集全血并分离血浆后，从血浆中提取DNA，并用于对泛细菌16S核糖体基因进行广泛的PCR反应。PCR产物纯化后，进行克隆和测序分析。
