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Application of polycation carrier coated recombinant adenovirus to preparation of anti-tumor gene drug
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A recombinant adenovirus and polycation technology, which can be used in anti-tumor drugs, gene therapy, drug combinations, etc., and can solve the problems of limited clinical application, toxic side effects and high cost.
Inactive Publication Date: 2018-04-20
KUNMING MEDICAL UNIVERSITY
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Due to the rapid renal clearance, it is still necessary to repeatedly inject large doses of IL-15 cytokines to achieve an effective therapeutic dose. The toxic side effects and high cost caused by this shock therapy also limit its wide clinical application.
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Embodiment 1
[0063] Packaging and expression of Adv-IL5:
[0064] The titer was measured by the TCID50 method:
[0065] Adv-IL5: The titer of the virus is (0.7×5×10 10.5 )PFU=1.106×10 11 PFU / mL
[0066] Adv–Luciferase: The titer of the virus is (0.7×5×10 10.41 )PFU=9.0×10 10 PFU / mL
[0067] Production and expression detection of H1 / Adv-IL15 In order to determine the optimal packaging ratio, we first used different ratios of H1 to package Adv-Luc. Such as image 3 Shown, H1:Adv-Luc=70nmol:2×10 6 pfu showed the highest transfection efficiency, therefore, this ratio was determined to be the optimal packaging ratio and used in future experiments.
[0068] Such as Figure 4 As shown, B16 cells were transduced with H1 / Adv-IL15, and IL-15 cells were highly expressed in the culture medium.
[0069] Such as Figure 5 Shown: Compared with the control group, Adv-IL15 and HI / Adv-IL15 treatment, the growth rate of the tumor volume was significantly reduced, indicating that the above treatmen...
Embodiment 2
[0073] HI / Adv-IL15 reduces pulmonary metastatic nodules of malignant melanoma and prolongs the survival time of tumor-bearing mice:
[0074] Such as Figure 7 Shown: Adv-Luc, H1 / Adv-Luc, Adv-IL15 and HI / Adv-IL15 treatment groups reduced the number of pulmonary nodules in mice, and the reduction in HI / Adv-IL15 treatment group was obvious and had statistical significance ( student t-t test, n=6, p<0.05).
[0075] Such as Figure 8 Shown: Adv-IL15 and HI / Adv-IL15 treatment groups prolong the survival time of metastatic tumor model mice.
Embodiment 3
[0077] HI / Adv-IL15 treatment increases the number of peripheral blood CD4+ T cells:
[0078] We used flow cytometry to detect the number of CD4+ T cells in peripheral blood lymphocytes. Representative flow cytometry histograms are shown in Figure 10 , the statistics show that Figure 9 , Compared with the control group HI / Adv-Luc, Adv-Luc, 5% Glu, HI / Adv-IL15, Adv-IL15 treatment significantly increased the number of peripheral blood CD4+ T cells (P<0.05).
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Abstract
The invention discloses application of polycation carrier coated recombinant adenovirus to preparation of an anti-tumor genedrug. A polycation carrier is polycation formed by polymerizing cyclodextrin and polyethyleneimine folic acid; the anti-tumor genedrug is a genedrug obtained by transducing human-derived interleukin 15 on the recombinant adenovirus. H1 / rAdv-15 can be used for effectively inhibiting the growth of tumors on a subcutaneous and metastatic malignant melanoma mouse model, the survival time of a tumor-bearing mouse is prolonged and the curative effect is better than that of independent Adv-IL15. An anti-tumor mechanism of the H1 / rAdv-15 mainly has the effects of activating multiplication of CD4+ and CD8+T cells and NK (Natural Killer) cells, stimulating the killing activeof the cells and inhibiting an immunosuppressioncell Treg to express the anti-tumor activity, so that a new thought is provided for immunization therapy of malignant melanoma.
Description
technical field [0001] The invention belongs to the technical field of application of anti-tumor drugs, and in particular relates to the application of a polycation carrier-coated recombinant adenovirus in the preparation of anti-tumor gene drugs. Background technique [0002] Cutaneous malignant melanoma (melanoma) is the most malignant tumor in skincancer. Occur in Caucasians more often, in our country, the sickness rate of evil black is relatively low, but the sickness rate increases rapidly. With the advent of an aging society, some scholars have even suggested that malignant melanoma will become an important issue affecting public health in the next decade. Due to geographical and living habits and other factors, photodermatosis and chronic skin damage are more common in Yunnan Province, and the occurrence rate of dark spots is relatively high. [0003] Based on the immunogenic properties of malignant melanoma, immunotherapy has become an important adjuvant therapy f...
Claims
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