Adriablastina antineoplastic medicine and gland correlated virus composite preparations and uses thereof
A technology of anti-tumor drugs and compound preparations, applied in the field of biomedicine, can solve the problems of low expression level of therapeutic genes, low cell infection and transduction efficiency, etc., and achieve the effects of increasing positive rate, improving curative effect, and increasing expression level
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Embodiment 1
[0079] Example 1 Adriamycin Accelerates and Improves the Transduction Efficiency and Gene Expression Level of Retinal Cells by rAAV-GFP
[0080] According to the time of adding doxorubicin, the experiment was divided into three groups: the first group was incubated with doxorubicin in advance, and rAAV was added after 24 hours 2 -GFP; the second group added doxorubicin and rAAV at the same time 2 -GFP; the third group added rAAV first 2 - For GFP infection, doxorubicin was added 24 hours later.
[0081] 2×10 per hole 5 RPE (APRE-19, ATCC) cells were seeded in 6-well cell culture plates, and after 24 hours, the cells adhered to the wall, and 1×10 9 wxya 2 -GFP with a final concentration of 0.001μg / ml (0.001μg), 0.01μg / ml (0.01μg), 0.05μg / ml (0.05μg), 0.1μg / ml (0.1μg), 1.3μg / ml (1.3μg) , 1.7 μg / ml (1.7 μg), 2.6 μg / ml (2.6 μg) doxorubicin were added to the cultured cells simultaneously or first or later. Then observe and record the brightness of GFP fluorescence, cell morp...
Embodiment 2
[0086] Example 2 Adriamycin Accelerates and Improves the Transduction Efficiency and Gene Expression Level of Retinal Cells by rAAV-Luc
[0087] 1×10 per well 5 RPE (APRE-19, ATCC) cells were seeded in 24-well cell culture plates, and after 24 hours, the cells adhered to the wall, and 1×10 6 , 1×10 7 , 1×10 8 or 1×10 9 wxya 2 -Luc alone or in combination with 0.1 μg / ml (0.1 μg) doxorubicin was added to the cultured cells at the same time. On the 3rd day after infection, after trypsinization, the cells were collected and lysed, and the corresponding substrate was added to the lysate. Detect the intensity of fluorescence in the cell lysate to analyze and compare rAAV under different conditions 2 - Transduction efficiency of RPE cells by Luc. The results are as follows: Adeno-associated virus was applied alone from 1×10 6 to 1×10 9 The intracellular Luc fluorescence intensity values (RIU) were 131, 136, 613 and 3556, respectively. for 3211, 16237, 209866 and 1637928. ...
Embodiment 3
[0088]Example 3 Doxorubicin accelerated and improved the transduction efficiency and gene expression level of rAAV-GFP and rAAV-Luc to living retinal cells, improving the therapeutic effect.
[0089] A Doxorubicin increases the expression level of AAV-mediated genes in living retinal cells
[0090] Select 10 8-week-old SD rats (one died after anesthesia), male or female, and randomly divide them into 3 groups. Group 1: Combined injection of rAAV2-GFP and doxorubicin (2.5ul) into the subretinal space of the right eye , the dosage of rAAV2-GFP was 1×10 9 For virus particles, the concentration of doxorubicin was 0.1 μg / ml (0.1 μg) and 1.3 μg / ml (1.3 μg); the left eye was the control eye, and rAAV2-GFP was injected separately in the subretinal space, 1×10 9 Virus particles (2.5ul). The second group: Adriamycin (2ul) was injected into the vitreous cavity of the right eye at a concentration of 0.1 μg / ml (0.1 μg) and 1.3 μg / ml (1.3 μg), and rAAV2-GFP was injected into the subretina...
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