Application of alkannin and its derivative as gene therapy sensitizer
A technology of gene therapy and shikonin, which is applied in the direction of gene therapy, medical preparations containing active ingredients, drug combinations, etc., can solve the problem of undetermined target liver cancer and achieve a strong anti-tumor effect
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Embodiment 1
[0036] 1. Experimental materials and methods
[0037] 1. Compounds, Plasmids, Primers
[0038] High-performance liquid chromatography (HPLC) grade tripterine, pristimerin, shikonin, curcumin, apigenin, chrysin, luteolin, and salvianolic acid B salt were purchased from Sigma-Aldrich Co. LLC, St. Louis, USA, dissolved in DMSO at a concentration of 5 mg / ml as a stock solution. HPLC grade MG132 was purchased from Calbiochem, Merck KGaA, Darmstadt, Germany, 5 mg / ml dissolved in DMSO. HPLC grade bortezomib was purchased from Santa Cruz Biotechnology, Inc. Santa Cruz, USA, dissolved in DMSO at 5 mg / ml.
[0039] The pHelper plasmid was purchased from Agilent Technologies, Santa Clara, USA. pdsAAV-CBAp-EGFP and pAAV-CBAp-Fluc are prepared as follows: using molecular cloning technology, chicken actin promoter (CBAp) driven enhanced green fluorescent protein gene (EGFP) or luciferase gene (Fluc) Embedding into a plasmid carrying the terminal repeat (ITR) of AAV2 such that the inser...
Embodiment 2
[0078] 1. The mechanism of shikonin enhancing rAAV infection efficiency in vitro
[0079] Firstly, the coat protein of rAAV2-CB-Fluc was designed to be labeled with Cy3 dye, and then the transport of the labeled rAAV2-CB-Fluc-Cy3 virus coat in HeLa cells was observed with a Leica TCS SP5 confocal microscope. Before the official observation, the biological activity of the labeled rAAV2-CB-Fluc-Cy3 virus was detected, and HeLa cells were infected. The reporter gene expression in the high-dose group and the low-dose group could be detected, but it was higher than that of the unlabeled virus at the same dose. group had significantly lower [ Figure 5 .A]; In addition, the effect of shikonin on the infection efficiency of rAAV2-CB-Fluc-Cy3 was detected, and the results showed that shikonin could still significantly enhance its infection efficiency[ Figure 5 .B], indicating that Cy3 dye labeling has a certain negative impact on the biological activity of rAAV, but it is acceptable...
Embodiment 3
[0085] Trichosanthin (TCS) is derived from the traditional Chinese medicine Radices trichosanthis, which is a perennial climbing herb belonging to Cucurbitaceae Trichosanthis, Trichosanthis ( Trichosanthes kirilowii Maxim) or Bilateral Trichosanthes ( Trichosanthes rosthornii Herms) roots. The effects of trichosanthes are: clearing heat and purging fire, promoting body fluid and quenching thirst, reducing swelling and draining pus. It is mainly used to cure fever, thirst, thirst, jaundice, dryness of the lung and hemoptysis, carbuncle, hemorrhoids, etc. Consisting of 247 amino acid residues, TCS belongs to type I ribosome-inactivating proteins (Ribosome-inactivating proteins, RIP), and has various pharmacological activities such as inhibiting the growth of various tumor cells, anti-virus, immune regulation, and anti-AIDS virus. Shaw PC et al first confirmed the sequence of TCS gene, its length is 870bp. The present invention attempts to pack this gene into rAAV3 and use i...
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