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Ovarian cancer stem cell vaccine and preparation method thereof

A technology of ovarian cancer cells and stem cells, which is applied in the fields of molecular biology and genetic engineering, can solve problems such as inability to achieve effective remission and treatment effects

Inactive Publication Date: 2016-01-06
SOUTHEAST UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The current conventional treatment for ovarian cancer is surgical resection, supplemented with cisplatin / paclitaxel chemotherapy. The complete remission rate of this treatment regimen can reach 70% in patients with primary ovarian cancer. However, most patients relapse within 18 months. Cancer cells turn into resistant cells to chemotherapeutic drugs, and the existing treatment options cannot achieve effective remission.

Method used

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  • Ovarian cancer stem cell vaccine and preparation method thereof
  • Ovarian cancer stem cell vaccine and preparation method thereof
  • Ovarian cancer stem cell vaccine and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Example 1 Preparation of Ovarian Cancer Cells

[0047] a. After obtaining the tumor tissue aseptically, wash the tumor tissue with PBS containing double antibody to remove necrotic tissue, connective tissue, blood vessels, fat, etc., and cut the tumor tissue into 1mm with scissors 3 Add collagenase and DNase, digest and enzymatically digest, filter and prepare single cell suspension;

[0048] b. Use immunomagnetic beads to isolate fibroblasts;

[0049] c. After the negative selection liquid is placed in the culture flask, after the cells adhere to the wall, remove the suspended cells, replace the fresh medium, and subculture.

Embodiment 2

[0050] Example 2 Ovarian Cancer Stem Cell CD117 + CD44 + Preparation

[0051] Cell culture and immunomagnetic bead sorting

[0052] 1. Culture the human EOCSKOV3 cells with 10% fetal bovine serum RPMI1640 as a medium, and culture them at 37°C and 5% CO2 saturated humidity. After the cells grow to a certain density, trypsin digestion and blow off, then continue subculture.

[0053] 2. CD44+SKOV3 cell immunomagnetic bead sorting (magnetic activated cell sorting, MACS)

[0054] (1) Take 300μl of MACS buffer to suspend SKOV3 cells;

[0055] (2) Every 1×10 7 ~1×10 8 Cells, add 100μl FcR blocking agent, mix well and incubate at 4℃ for 30min;

[0056] (3) Every 1×10 7 ~1×10 8 Cells, add 100μl CD44+ magnetic bead labeled direct-labeled monoclonal antibody, mix well and incubate at 4°C for 30min, then add PBS to 1mL, centrifuge at 1000rpm for 8min to collect the cell pellet;

[0057] (4) Add PBS (containing EDTA) solution to 1 mL, centrifuge at 1000 rpm for 8 min, wash the cells once, discard the ...

Embodiment 3

[0071] Example 3 CD117+CD44+SKOV3 cell morphology observation and surface marker detection

[0072] Under the inverted microscope, continuously and dynamically observe the cell growth status, and take 1×10 CD117+CD44+SKOV3 cells cultured in vitro 6 Cells were labeled with CD117-PE and CD44-FITC monoclonal antibodies respectively, and incubated in a 37°C, saturated humidity, 5% CO2 incubator for 30 minutes, PBS washed to remove excess antibodies, FCM detected CD117+CD44+SKOV3 surface markers.

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Abstract

The invention relates to the field of molecular biology and discloses an ovarian cancer stem cell vaccine and a preparation method and application thereof. Cancer stem cells are insensitive to radiotherapy and chemotherapy, which is the root of cancer metastasis and recurrence. Aiming at active immunotherapy of the cancer stem cells, satisfactory therapeutic effect can be gained only on the condition that cancer 'seed' cells, namely the cancer stem cells are eliminated. The ovarian cancer stem cell CD117+CD44+ vaccine is capable of improving blood serum IFN (interferon)-gamma level, lowering TGF (transforming growth factor)-beta expression, enhancing NK cell activity, decreasing the number of ovarian cancer stem cells CD117+CD44+ remarkably and inhibiting cancer growth in animal experiments.

Description

Technical field [0001] The invention relates to the field of biotechnology, in particular to molecular biology and genetic engineering. Background technique [0002] Epithelialovarian cancer (EOC) accounts for about 90% of ovarian cancer. It is a common tumor of the female genitalia. Its incidence is second only to cervical cancer and uterine body cancer and ranks third in gynecological malignancies, but deaths However, the rate is the first of all types of gynecological tumors. Regarding the mechanism of ovarian cancer, there are reports that it is due to inflammation and / or damage repair after ovulation, and abnormal proliferation of stem cells produced during the repair process, but there is no conclusion yet. Therefore, only by discovering the cause of ovarian cancer and performing targeted treatment for the cause, especially by inhibiting the growth and metastasis of EOC, and then removing EOC and EOC cancer stem cells (CSCs), can ovarian cancer be cured fundamentally. [00...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/095A61K39/00A61P35/00
Inventor 窦骏吴迪何向锋王净蔡云郎
Owner SOUTHEAST UNIV
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