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Pancreatic cancer stably transfected cell line as well as construction method and application thereof

A technology for pancreatic cancer cells and construction methods, applied in the field of pancreatic cancer stable cell lines and their construction and application, can solve the problems of unsatisfactory treatment effects and achieve good tumorigenic effects

Pending Publication Date: 2021-12-03
TIANJIN TUMOR HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The disease is mainly treated by methods such as surgery, radiotherapy and chemotherapy, but the therapeutic effect is not satisfactory.

Method used

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  • Pancreatic cancer stably transfected cell line as well as construction method and application thereof
  • Pancreatic cancer stably transfected cell line as well as construction method and application thereof
  • Pancreatic cancer stably transfected cell line as well as construction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1 Transformation

[0033] A plasmid vector pHS-AVC-0700 for overexpressing OVAL-Gallus gallus and other genes was constructed, and its element sequence was pLV-EF1a-hluc-P2A-mNeongreen-CMV-OVAL-3Xflag-P2A-puro.

Embodiment 2

[0034] Embodiment 2 plasmid amplification

[0035] 1. Experimental materials

[0036] tryptone

[0037] Yeast extract

[0038] Sodium chloride

[0039] agar

[0040] beaker

[0041] glass rod

[0042] volumetric flask

[0043] Erlenmeyer flask

[0044] Parafilm

[0045] kraft paper

[0046] rubber band

[0047] Sterilizer

[0048] Clean bench

[0049] pH meter

[0050] HCl

[0051] NaOH

[0052] Ampicillin

[0053] 10cm petri dish

[0054]Water bath

[0055] Competent Escherichia coli stbl3

[0056] Target plasmid pHS-AVC-0700

[0057] Packaging plasmids psPAX2, pMD2G

[0058] pipette

[0059] Float

[0060] EP pipe rack

[0061] shaker

[0062] coated glass rod

[0063] Centrifuge tube

[0064] long tweezers

[0065] Absorbent paper

[0066] TIANGEN endotoxin-free plasmid mini-prep kit

[0067] 2. Experimental steps

[0068] 2.1 LB medium preparation

[0069] S1. Use a balance to weigh 10g of tryptone, 5g of yeast extract, and 10g of sodium chlo...

Embodiment 3

[0087] Embodiment 3 virus packaging

[0088] 1. Experimental materials

[0089] Clean Bench

[0090] 293T cells passaged in a 6-well plate were revived in advance (the confluence is 70%-90%)

[0091] Thermo Fisher liposome lipofectamine3000 kit

[0092] Target plasmid pHS-AVC-0700

[0093] Packaging plasmids psPAX2, pMD2G

[0094] Pipettes and Tips

[0095] 1.5ml clean centrifuge tube

[0096] Cell culture 6-well plate

[0097] cell culture incubator

[0098] DMEM basal medium

[0099] DMEM complete medium

[0100] 2. Experimental steps (operation in the ultra-clean bench between cells)

[0101] S1, transfection tool cell 293T:

[0102] per system:

[0103] Prepare in a 1.5ml centrifuge tube:

[0104] Tube 1: 125 μL DMEM basal medium + 7.5 μL lipofectamine3000

[0105] Tube 2: 125 μL of DMEM basal medium + 2.5 μg of DNA + 5 μL of P3000

[0106] Among them, the mass ratio of the target plasmid pHS-AVC-0700:packaging plasmid psPAX2:packaging plasmid pMD2G in the D...

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Abstract

The invention discloses a pancreatic cancer stably transfected cell line as well as a construction method and application thereof. The construction method comprises the following steps: (1) constructing a plasmid vector overexpressing OVA gene; (2) amplifying the plasmid vector overexpressing OVA gene, transfecting the plasmid vector into a tool cell, and carrying out virus packaging to obtain a virus supernatant; and (3) transfecting the virus supernatant into target cells, and screening to obtain the stable pancreatic cancer cell line. The pancreatic cancer cell line established by the invention has characteristics of stable growth speed, stable passage, good mouse tumorigenicity and the like, can be used for establishing a pancreatic cancer cell model and an animal model so as to provide a good tumor model for research of pancreatic cancer, and can be applied to the aspects of occurrence, development and metastasis mechanisms of pancreatic cancer, immunotherapy and related combined therapy thereof, drug resistance principles, new drug screening and the like.

Description

technical field [0001] The present invention relates to the construction of tumor cell lines, in particular to a pancreatic cancer stable cell line and its construction method. The present invention further relates to the use of the pancreatic cancer stable cell line as a tool cell for the development and metastasis of pancreatic cancer, immunotherapy and Its application in combination therapy, drug resistance principle and new drug screening belongs to the field of pancreatic cancer stable cell line and its application. Background technique [0002] Pancreatic cancer is a malignant tumor that occurs in the exocrine gland of the pancreas. Pancreatic malignant tumors can come from pancreatic exocrine glands, endocrine glands, or non-epithelial tissues, and 95% of them are pancreatic cancers, which have the worst prognosis, high morbidity and mortality, and a 5-year survival rate of less than 8%. Mainly use methods such as surgery, radiotherapy and chemotherapy to treat the d...

Claims

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Application Information

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IPC IPC(8): C12N5/10C12N15/12C12N15/867C12Q1/02A01K67/027
CPCC12N5/0693C12N5/0676C12N15/86C07K14/77G01N33/5011A01K67/0271C12N2740/15043C12N2503/02G01N2500/10A01K2207/12A01K2227/106A01K2267/0331
Inventor 郝继辉高松周博栋袁帅陈志强朱文博
Owner TIANJIN TUMOR HOSPITAL
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