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Background and Purpose: Gene therapy is one of the hot research at home and abroad, with rapid progress in recent years as a research based viral vectors. Adenoviral vector transduction efficiency of target gene high, low pathogenicity, high titer and in the body does not integrate into the host cell chromosome and other advantages, has been considered one of the most efficient gene transfer vector, and are widely used in human gene therapy. However, the host immune response is still a major obstacle to obtaining transgenic long-term expression and repeated use of adenoviral vectors. So far, adenoviral vectors from three generations carrier. A first generation adenovirus vector, E1/E3 gene expression cassette has been to addition, but the lack of expression of the E1 region is not conducive to the transcriptional expression of the vector, is mainly due to the immune response against virus package shell and transgenic expression product of the The gene expression was inhibited. Addition to some or all of the E2 gene in order to suppress the occurrence of immune responses, and improve the efficiency of gene transduction, built after the adenoviral vector, resulting in the deletion or mutation of the viral genes, weakening the expression of viral proteins, and joined to regulate gene expression temperature-sensitive factor, eliminating the possibility of replicate DNA and produce replicable adenovirus (vector) (Replication Competent Adenovirus, RCA), which is the so-called second-generation adenovirus vector. More \to the terminal repeat sequences (inverted terminal repeats, ITR) and packaging signals, and generating the helper virus and appropriate complementary packaging cell, and more necessary to further purification, often referred to as a helper virus-dependent adenovirus type or shell adenovirus or having a high packaging capacity adenovirus (high-capacity or gutted or helper-dependent Adenovirus vectors, HD-AdV). Adenoviral vectors caused by the body's immune response: innate and acquired immunity. Adenoviral vectors into the body first start of the innate immune system, the innate immune response mainly neutrophils, monocyte-macrophage cells, NK cells activation and swallowed into the body of the virus vector transduction products. The activation of these cells secrete cytokines, including IL-2, IL-6 and TNF-a by mononuclear macrophages and other cells to produce acute inflammatory response and immune-enhancing, has a very important role. The Acquired Immune divided into cellular and humoral immune cell immune response in the body to produce specific cytotoxic T lymphocytes (cytotoxic Tlymphocytes, CTL) destruction and elimination of transduction of target cells. Humoral immunity through the body to produce specific antibody to the elimination of the adenovirus vectors and transductants. IL-2, IL-6 and TNF-a cytokines is a key factor of the innate immune and acute inflammatory response, also played an important role in adaptive immunity. In addition, the cellular immune currently known T cells are activated and proliferating would need at least two signals, the first signal is a necessary condition of the activated T cells, but to make the proliferation of the virus or the product was transduced antigen-specific CTL and obtain all effector function, still need a second signal that costimulatory interactions between the molecules and the corresponding T cell surface receptors. Be able to pass the second signal costimulatory molecules include the B7 family of molecules, cell adhesion molecules, and LFA-3, etc., wherein the B7-1 (CD80) in combination with the T cell surface CD28 can induce the secretion of IL-2 and other lymphokines, is determining whether the first signal can generate the activation of the T cell function key factors. This topic is intended to be used SPF grade feeding SD rats as experimental animals through the subarachnoid space and peritoneal two injection routes at different time points after injection of comparing the first-generation adenoviral vectors, and third-generation adenoviral vectors related indicators of immune response changes. At different time points serum IL-2, IL-6, and the content of cytokines such as TNF-α and serum immunoglobulin IgG content; the flow cytometry virus injection after different time points spleen cells were detected by ELISA costimulatory molecules CD80 expression changes; another virus intraperitoneal injection of inflammatory infiltration of the lung tissue was observed with HE staining method. By the above method to analyze the immune response of the two adenovirus vector difference. Detection methods: 1, IL-2, IL-6, TNF-a and other cytokines: animal experiments grouping SD rats were divided into two major groups: the subarachnoid injection group and intraperitoneal injection group, in which each group subdivided as a first generation adenovirus vector injection group, the third-generation adenovirus vector injection group, the control group (saline injected group). At different time points, respectively (the day before the injection of T0, 1 day after injection, T1, 3 days after injection T2, seven days after injection, T3, 14 days after injection in T4) postorbital picking venous 1000r/min centrifuged 20min supernatant - The 20 ℃ saved. IL-2, IL-6, TNF-a and other content detected by ELISA. Costimulatory molecules B7-1 (CD80) detection: animal experiments grouping, SD rats were divided into two groups: subarachnoid injection group and intraperitoneal injection group, where each group of first-generation adenovirus vector injection group, the third-generation adenovirus vector injection group, the control group (saline injected group). At different time points, respectively (the day before the injection of T0, one day after injection, T1, three days after injection, T2, 7 days after injection of T3, after injection 14 days T4), anesthetized SD rats were sacrificed, ground into cells from rat spleen suspension of spleen cells detected by flow cytometry CD80 molecule expression, CD80 molecule primary antibody labeled with PE. 3 Determination of rat IgG Ibid: Animal experiments packet, respectively, at different time points (day before injection T0, 1 day after injection, T1, 3 days after injection, T2, T3, seven days after injection, 14 days after injection, T4), taken retro-orbital venous 1000r/min centrifugal 20min and the supernatant was stored at -20 ℃. The serum IgG levels were measured by ELISA. HE staining of lung tissue inflammation infiltration: Virus intraperitoneal injection, the first-generation adenoviral vector injection group, the third-generation adenovirus vector injection group and the control group (saline injection group). At different time points, respectively (the day before the injection of T0, one day after injection, T1, three days after injection, T2, 7 days after injection of T3, after injection 14 days T4), anesthetized SD rats were sacrificed, the lung tissue, paraformaldehyde fixed saved detecting. Results: 1. Before injection (T0), each set of indicators no significant difference. Test results of the innate immune response: viral vectors after subarachnoid and intraperitoneal injection of SD rats, the same point in time serum IL-6 cytokine secretion levels of TNF-a and other first-generation adenoviral vector injection group third-generation adenoviral vector injection group and two groups were higher. The test results of the cellular immune response: viral vectors after subarachnoid and intraperitoneal injection of SD rats, 3 days and 7 days spleen costimulatory molecules CD80 expression of first-generation adenoviral vector injection group were higher than the third generation adenovirus viral vector injection group, but the two groups were higher; secretion levels of IL-2 in the serum of the same point in time, the first generation of injection group than in the third generation of injection, but the two groups were higher. 4 the detection result of the humoral immune response: viral vector by subarachnoid and intraperitoneal injection SD rats, the serum IgG levels in the same time point, the first generation adenovirus vector injection group is higher than the third-generation adenovirus vector injection group, and two groups were higher. By intraperitoneal injection of 5. Generations viral vectors SD rats HE staining of lung tissue showed no significant difference in groups. Conclusion: The successful use of animal experiments compared the immune response of the two adenovirus vector. Innate immune response indicators to detect the virus in serum after injection of IL-6, TNF-a level of cytokine secretion; cellular immune response indicators to detect the virus after injection of serum IL-2 secretion levels and spleen cells CD80 expression; humoral immune reaction, detection of a virus after injection, the content of serum IgG. Conclusion: the immune response of the third-generation adenovirus vector is less than a first generation adenovirus vector, indicating the third generation adenoviral vector, more suitable for gene therapy because of its weaker immune response through the detection of the different immune reaction stage also open up broader prospects for gene therapy. From the experimental results can be seen that the third-generation adenoviral vectors can cause certain immune response raised new problems for our research work, which requires us to seek ways to further improve the adenovirus vector in future research work strive to minimize the immune response.
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