HIV-1 latent infection activator thiostreptavidin
A technology of thiostrepton and HIV-1, applied in the field of HIV-1 latent infection activator thiostrepton, can solve the problems of incomplete reactivation, cytokine storm, life-threatening, etc., and achieve good promotion and application Value, less cytotoxic side effects
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Embodiment 1
[0032] Example 1 Construction of HIV-1 Primary Cell Latent Infection Model
[0033] 1. Transduction and amplification of Bcl-2
[0034] Firstly, CD4+ T cells in normal human PBMCs were screened out with magnetic beads. After activation of Anti-CD3 / 28 and IL-2, they were infected with lentivirus EB-FLV carrying the anti-apoptotic gene Bcl-2. In the absence of TCR stimulation and exogenous After cultured for 3-4 weeks in the presence of sex cytokines, dead cells were removed by Ficoll density gradient centrifugation to obtain primary CD4+ T cell lines that could stably express Bcl-2.
[0035] (1) Experimental method
[0036] 1. Isolate PBMC from healthy donors, and obtain CD4+ T cells by BD negative selection magnetic beads, resuspend in STCM, and the cell density is 1×10 6 / ml for activation in 6-well plates.
[0037] 2. Collect activated CD4+ T cells, take an appropriate amount of cells after centrifugation, take an appropriate amount of cells after centrifugation, and resu...
Embodiment 2
[0070] Activation of embodiment 2 thiostrepton to latent HIV-1
[0071] 1. Experimental method
[0072] The sorted GFP-negative cells were stimulated and activated by flow cytometry, and the expression of GFP in cells was detected by flow cytometry. The negative control is DMSO, the positive control is PMA / Ionomycin, and the reported latent activators such as panobinostat, bryostatin, disulfiram, JQ1 are used for control.
[0073] 2. Experimental results
[0074] Typically, reactivation of GFP-negative cells in this model induces GFP fluorescence in 1% to 6% of cells. After ensuring that the GFP positive rate of reactivated cells in the PMA / Ionomycin group reached at least 1%, the activation effect of the small molecule compound thiostrepton on latent HIV-1 was detected.
[0075] The results showed that the effect of thiostrepton was stronger than that of other known activator controls, even higher than that of the PMA / Ionomycin group ( image 3 ).
Embodiment 3
[0076] The screening of embodiment 3 thiostrepton optimum action concentration
[0077] 1. Experimental method
[0078] In order to determine the optimal concentration of thiostrepton, different drug concentrations were set up for activation experiments, including 7 groups of 100nM, 250nM, 500nM, 1μM, 2.5μM, 5μM and 10μM, and the diluent DMSO treatment group was used as Negative control, PHA as positive control. The duration of action is 3 days.
[0079] 2. Experimental results
[0080] The results showed that as the dosage of thiostrepton increased, the activation efficiency increased gradually, and the reactivation ratio reached the maximum at 1 μM, and the activation effect of donor1 was even higher than that of the positive control group at the concentration of 1 μM. Afterwards, with the increase of drug concentration, the activation rate began to decrease gradually ( Figure 4 ). This result indicates that the optimal action concentration of thiostrepton is 1 μM, and...
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