| 其他摘要 | Trichosanthin (TCS) is a type I ribosome inactivating protein (REP) isolated from the root tubers of the Chinese medicinal herb Trickosanthes kirilowii Maxim. TCS possesses a wide spectrum of biological and pharmacological properties, including anti-tumor, immunosuppressive, abortifacient and antiviral activity. TCS have been found preferentially inhibited replication of human immunodeficiency virus type 1 (HIV-1) in both acutely infected T-lymphoblastoid cells and chronically infected macrophages in vitro. The mechanism is still unknown, but generally believed that the antiviral activity of TCS is related to the ribosome inactivating (RI) activity. The side effects of TCS have hindered its further clinical trial as anti-HIV/AIDS reagent. It is necessary to modify TCS molecule in order to decrease its neurotoxicity and anaphylactic side effects. Therefore the study of structure-functional relationship of TCS anti-HIV-1 will be helpful and favorable for the foundational and applied investigations of RIPs, and will widen the clinical trial of TCS. The anti-HIV-1 activity, the potential mechanism and the structure-functional relationship were studied in this dissertation. Firstly, cytotoxicity of TCS on C8166 T lymphocyte cell line, HIV-1 chronically infected H9 cell line (H9/HIV-lmB), and peripheral blood mononuclear cell (PBMC) were tested by using MTT colorimetric assay or trypan blue exclusive assay; The inhibition of TCS on HIV-1 induced cytopathic effect was quantitated by the syncytium formation; and the replication inhibition of TCS on ffiV-lmB in C8166, drug-resisted strain HIV-I74V in C8166 and clinically primary isolated strain HIV-1 KMOH in PBMC were assayed by using capture p24 antigen ELISA. Secondly, the inhibition effect of TCS on HIV attachment and fusion steps were tested by using fluoresence-based real-time quantify RT-PCR; The depurination effect of TCS on naked HIV-1 RNA was assayed by using HPLC system; What is more, the direct killing effect of TCS on HIV-1 particle, the inhibition on HIV-1 recombination reverse transcriptase (RT) activity and the inhibition on fusion of HIV infected cells with uninfected cells were tested. Finally, the stracture-fimctional relationship of TCS anti-HTV-l was investigaed by using 14 TCS mutants, including TCS acitive site mutants (TCSM(12W23> TCSEI C-tenninus mutants ( TCSQ, TC3C*" TCSCM, TCSKDEL , TCScww), and antigen determinant site mutants (Q219C , K173C " S7C) and their PEGylation forms(PEG20K-Q219C, PEG2GK-K173C, PEGIOK-STC). The results showed that TCS efficiently inhibited laboratory strain HIV-1ms replication in C8166 cells and the cytopathic effect of host cells, also TCS displayed inhibition effect in HTV-IKMOI S replication in PBMC and HIV-174V replication in C8166 cells. Though TCS exhibited a direct killing effect on HIV-1 chronically infected H9 ceEs, there was no direct inhibition on HIV-1 replication in such cells. Another, all of the inhibition effects of TGS on entry of HIV into host cells, the fusion of infected and uninfected cells, and rRT activity, were not observed. What is more, TCS could not directly kill HIV-1 particle. TCS could depurinate naked HIV-1 RNA. Maybe TCS could recognize other structures located in HIV-1 RNA besides of the classical stem-loop-like structure which always was recognized for the depurination of 28S rRNA. The enrichment of depurinated adenine by TCS in cytoplasm of infected cells would contribute to the drop of of mitochondria A^Pm, the release of cytochrome C, the addition of reactive oxygen species and the decrease of anti-apoptosis factors, which would lead to apoptosis. The results also showed that the active site mutants TCSM < 120-123 > and TCSEI6OA/EI89 with greatest decrease in RI activity, almost lost all of the anti-HIV-1 activity, another active site mutant TCSR1220, which exhibited a 160-fold drop in RI activity, retained some anti-HIV-1 activity. The RI activity of the C-terminal deletion mutants (TCSQ, TCS04 and TCSCM) decreased by 1.2-3.3 fold with parallel downshifting of its anti-HIV-1 activity (1.4-4.8 fold). These results from these studies suggested that RI activity of TCS might have significant contribution to its anti-HIV-1 property. While the anti-HIV-1 activity of TCS was not entirely dependent on the RI activity, because there were two TCSCi9aa and TCSKDEL, having 19 amino acids extension and a KDEL signal sequence; added.to the C-terminus, retained all RI activity but subsequently lost most of the anti-HIV-1 activity. Maybe there were another mechanisms involved in TCS anti-HIV-1 action. The mutant of TCS antigen determinant sites had no significant effect on its anti-HIV-1 activity, while the site-directed PEGylation of TCS had led to a substantial drop in its anti-HIV-1 activity. |
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