Inhibitor of stem cell proliferation and uses thereof
A technology of sequence, cysteine residue, applied in application, genetic engineering, plant genetic improvement, etc., can solve the problems of increased infection rate, low stem cell frequency and high susceptibility
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Embodiment 1
[0155] Example 1: In vivo stem cell proliferation inhibition assay
[0156] To measure the proliferation of stem cells, by 3 The H-TdR "suicide method" measures the number of CFU-S in the S phase of the cell cycle (Becker et al., Blood 26:296-308, 1965).
[0157] Immature hematopoietic progenitors - colony-forming units of the spleen (CFU-S) can be detected in vivo by macroscopic colonies formed in the spleen of lethally irradiated mice 8-12 days after intravenous injection of hematopoietic cells (Till & McCulloch 1961).
[0158] For standard CFU-S proliferation assays, typically 3 H-TdR "Suicide Method" (Becker et al., 1965). The method is based on radiolabeled thymine ( 3 H-TdR) is incorporated into cells as a synthetic DNA precursor. CFU-S, which was in the S phase of the cell cycle at the time of detection, was killed by high radioactivity and thus failed to form colonies in the spleen. Then, by injecting with 3 H-TdR co-cultured cell samples and no 3 The differe...
Embodiment 2
[0177] Example 2 In vitro stem cell proliferation inhibition analysis
[0178] The direct effect of INPROL was shown using the following detection system (Lord et al., The Inhibitors of Hematopoiesis, pp. 227-239, 1987). Multilineage factor (IL-3) dependent cell line FDCP mix A 4 (A 4 ), maintained in IMDM medium containing 20% horse serum and 10% WEHI-3 conditioned medium as a source of colony-stimulating IL-3.
[0179] use 3 Proliferation detected by H-TdR incorporation assay: A 4 cells (5×10 4 in 100 μl medium containing 20% horse serum and 50% WEHI-3 conditioned culture supernatant) at 37°C, 5% CO 2 cultured for 16 hours.
[0180] Begin adding INPROL or crude BME (Bone Marrow Extract) (Component IV), then add to each group 3 H-TdR (3.7KBq in 50μl at 740GBq / mmol) was incubated for an additional 3 hours. Proliferation rates were determined by harvesting cells and calculating percent inhibition using the following formula.
[0181]
[0182] FDCPmix-A grown i...
Embodiment 3
[0183] Example 3 In vivo injection of INPROL results in inhibition of CFU-S proliferation: dose and effect duration
[0184] The effects of INPROL injection in vivo showed that INPROL could effectively block the recruitment of CFU-S into the cell cycle. Thus, these cells are protected from cytotoxic effects upon further treatment. It shows its potential clinical application value.
[0185] This protocol has two objectives: to examine the effect of INPROL on CFU-S when injected in vivo and to determine the effective duration of INPROL activity involving cell cycle stem cells.
[0186] To stimulate CFU-S proliferation, according to the effects mentioned in Example 1, testosterone propionate (TSP) was injected.
[0187] BDF in 0 days 1 Mice were injected with TSP (10mg / 100g), and 24 hours later, mice in each experimental group (4 mice / group) were intraperitoneally injected with pINPROL 0μg, 5μg, 10μg and 15μg / mouse.
[0188] Twenty-four hours after pINPROL injection, mice...
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