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Anti-liver cancer whole cell vaccine modified by hbx and its preparation method and application

A whole cell vaccine, liver cancer cell technology, applied in the field of anti liver cancer whole cell vaccine, can solve the problems of weak immunogenicity and difficulty in causing immune response, etc.

Active Publication Date: 2015-07-29
SICHUAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although tumor cells carry all the antigens of the tumor, it is difficult to elicit a strong immune response due to the weak immunogenicity of tumor cell vaccines

Method used

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  • Anti-liver cancer whole cell vaccine modified by hbx and its preparation method and application
  • Anti-liver cancer whole cell vaccine modified by hbx and its preparation method and application
  • Anti-liver cancer whole cell vaccine modified by hbx and its preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Preparation of the anti-hepatoma whole cell vaccine of embodiment 1 HBx gene modification

[0051] Hepa1-6 (mouse hepatoma cells, purchased from ATCC) were inoculated in a culture dish with a density of 70-80% and in the logarithmic growth phase, using a non-replicating recombinant adenovirus vector carrying the HBx gene (structure and construction process) Refer to reference 1 and reference 2 for the construction process of AdHBx, the nucleotide sequence of its expression cassette is shown in Seq ID NO.3) to infect cells with a multiplicity of infection (MOI) of 20, discard the virus after 2 hours of virus infection, Change to fresh DMEM medium containing 10% fetal bovine serum and continue culturing for 24 hours, digest with 0.25% trypsin, collect cells by centrifugation, wash 3 times in serum-free DMEM medium, resuspend the cell pellet with an appropriate amount of cell culture medium, adjust the cell density into 1×10 7 individual / mL. The treated cells were irradi...

Embodiment 2

[0052] Example 2 In vivo anti-tumor experiment of the HBx gene-modified anti-hepatoma whole cell vaccine of the present invention

[0053] Will 2.5×10 6 Hepa1-6 / HBx mouse liver cancer cells were inoculated subcutaneously on the right flank of 6-8 week-old C57 mice (purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.). When the tumor diameter reached 3 mm, the mice were randomly Divide into 4 groups, 6 mice in each group, perform 1×10 on the left side of the back of the mice 6 Cells / vaccine injection (recorded as day 0), boosted once on day 14 and day 21 respectively. After immunization, the long diameter (a) and wide diameter (b) of the tumor were measured every two days with a vernier caliper, with (a×b 2 )×0.52 to calculate the tumor volume and draw the tumor growth curve. see results figure 1 .

[0054] figure 1 Show: There are significant differences in the tumor volumes of mice in each group ( figure 1 , P3 , the tumors of the remaining fi...

Embodiment 3

[0055] Example 3 The anti-tumor mechanism of the anti-hepatoma whole cell vaccine with HBx gene modification of the present invention

[0056] 1. T lymphocyte typing

[0057] One week after the last booster immunization in Example 2, the mice were sacrificed by cervical dislocation, the spleen was aseptically removed, ground and filtered on a 200-mesh metal sieve, the suspension was collected, lysed with erythrocyte lysate, and washed three times with PBS. Take 10 for each sample 5 Add fluorescent dye-labeled anti-CD4 (purchased from Biolegend, H129.19) and anti-CD8 antibody (purchased from Biolegend, 53-6.7) to each cell, label at 4°C for 30 min, wash three times with PBS, and use a flow cytometer (purchased From BD Biosciences, FACSVerse). see results figure 2 .

[0058] figure 2 In the middle, lymphocytes were isolated after three times of immunotherapy, and CD4 and CD8 were labeled with fluorescent dyes, and flow cytometric detection was performed. The results show...

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Abstract

The invention belongs to the technical field of cellular immunotherapy, and specifically relates to a hepatitis B virus X protein (HBx) gene modified liver-cancer-inhibiting whole-cell vaccine. The invention aims at solving a technical problem of providing a novel gene-modified liver-cancer-inhibiting whole-cell vaccine which can be used for treating HBV-related liver cancer. The invention provides an HBx-modified liver-cancer-inhibiting whole-cell vaccine. The active components of the vaccine comprise liver cancer cells modified by liver cancer antigen gene, wherein the liver cancer antigen gene is HBx. The invention also provides a preparation method of the liver-cancer-inhibiting whole-cell vaccine. The liver-cancer-inhibiting whole-cell vaccine provided by the invention can be used in targeted killing of HBx-positive liver cancer cells, and shows good liver-cancer-inhibiting effect.

Description

technical field [0001] The invention belongs to the technical field of cellular immunotherapy, and in particular relates to a whole-cell vaccine against liver cancer genetically modified by hepatitis B virus X protein (HBx). Background technique [0002] There are 626,000 new liver cancer patients every year in the world, and its death rate ranks third in the death rate of malignant tumors; my country is a high-incidence area of ​​liver cancer, and its incidence has been increasing year by year in recent years. The incidence of liver cancer in my country accounts for 45% of the world's total. It ranks second in the incidence of cancer in my country. Liver cancer has a high degree of malignancy. The average 6-month survival rate of patients after diagnosis is less than 50%, the 1-year survival rate is 24%, and the 5-year survival rate is about 10%. [0003] Liver cancer is not sensitive to radiotherapy and chemotherapy. Surgical resection is the preferred treatment method for...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K48/00A61K39/00A61P1/16A61P35/00
Inventor 程平魏于全杨莉李玉华文艳君
Owner SICHUAN UNIV
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