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Method for acquiring solid tumor cell with successful in-vitro transfection

A technology for solid tumors and cells, which is applied to cells modified by introducing foreign genetic material, biochemical equipment and methods, and the determination/inspection of microorganisms. It can solve the problems of low transfection efficiency and unstable experimental results. Improved therapeutic efficacy, simple operation, and high transfection efficiency

Inactive Publication Date: 2010-06-09
SHANDONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the aggregation of three-dimensional growth solid tumor cells into clusters, it is difficult for the cells inside the clusters to come into contact with the transfection reagent during the in vitro transfection process, resulting in low transfection efficiency and unstable experimental results

Method used

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  • Method for acquiring solid tumor cell with successful in-vitro transfection
  • Method for acquiring solid tumor cell with successful in-vitro transfection
  • Method for acquiring solid tumor cell with successful in-vitro transfection

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1 Establishment of a three-dimensional growth solid tumor cell model

[0032] Coated Poly-HEMA culture plate

[0033] Dissolve 2.4g of Poly-HEMA powder in 20mL of 95% ethanol, shake and mix at 65°C for more than 8h, prepare Poly-HEMA storage solution, and store in a sealed container at 4°C. Dilute the working solution with 95% ethanol at a ratio of 1:2.3. Under sterile conditions, gently add the working solution to the bottom of the well of the culture plate along the well wall to cover the whole bottom of the well, and then irradiate with ultraviolet light in the ultra-clean bench overnight. , that is, the Poly-HEMA culture plate was prepared, covered and stored at 4°C for one week, irradiated with ultraviolet light for 0.5h before use, and washed 3 times with sterile PBS.

[0034] Establishment of 3D growing solid tumor cell models

[0035] 1) The BEL7402 cell line was cultured with RPMI1640 plus 10% fetal bovine serum at 37°C and 5% CO 2 Cultured under condi...

Embodiment 2

[0043] Example 2 Direct transfection of antisense oligonucleotides into two-dimensional and three-dimensional growing cells

[0044] 1) BEL7402 cells were treated with 3×10 per well 5 The concentration of cells was inoculated on a 24-well plate, wherein the bottom of the hole was covered with Poly-HEMA (i.e. "Poly-HEMA culture plate" in Example 1) was a three-dimensional growth group, and the group without Poly-HEMA was a two-dimensional growth group. Three wells were repeated in each group, and the cells were routinely cultured for 20 hours.

[0045] 2) Add PS-asODNs / ANGPTL4 or PS-rODNs (final concentration: 10 μmol / L) to each well, 37°C, 5% CO 2 After culturing for 24 hours, the cells were collected in 0.5mL TRIZOL.

[0046] 3) Extracting total cellular RNA, and obtaining cDNA through reverse transcription reaction.

[0047] 4) Using the obtained cDNA as a template, PCR amplifies ANGPTL4 and the internal reference gene β-actin.

[0048] 5) 10 μL of the PCR reaction produ...

Embodiment 3

[0050] Example 3 Liposome-mediated transfection of antisense oligonucleotides into two-dimensional and three-dimensional growing cells

[0051] 1) with embodiment 2.

[0052] 2) Mix the antisense oligonucleotide mixture, take 6 μL of PS-asODNs / ANGPTL4 or PS-rODNs antisense oligonucleotide and add 145 μL of Optim MEM and mix well.

[0053] 3) To dilute liposomes, take 29 μL lipofectamine 2000 and add 461 μL Optim MEM, mix well and place at room temperature for 5 minutes.

[0054] 4) Mix 151 μL (2) and 151 μL (3) evenly, let stand at room temperature for 20 minutes, then add 50 μL / well to each well of PS-asODNs / ANGPTL4 group or PS-rODN group, and add 151 μL (3) to each well of Optim MEM to mix Add 50 μL / well to each well of the liposome control group, and set up a blank control group, add the same volume of Optim MEM, mix well, 37 ° C, 5% CO 2 Continue to culture for 24h, collect the cells in 0.5mL TRIZOL.

[0055] 5) All the other steps are the same as in Example 2.

[0056...

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Abstract

The invention discloses a method for acquiring a solid tumor cell with successful in-vitro transfection, comprising the following operation steps: 1) taking single-cell suspension of an exponential phase, culturing for 20h at the temperature of 37 DEG C by serum culture solution; 2) mixing antisense oligonucleotide in a sterile EP tube; 3) mixing liposome in another sterile EP tube, placing at room temperature for 5min after mixing; 4) taking the above antisense oligonucleotide mixture and lipofectamine 2000 mixture; evenly mixing, placing at room temperature for 20min for later use; and 5) transfecting. The invention effectively solves the problem that an in-vitro transfection technology can not stabilize the cells in transfection three-dimensional auxocyte mass; the cells can be evenly transfected into the three-dimensional auxocyte to be transfected, thus achieving the highest transfection efficiency, and simulating the functions of the related molecules in vivo to the maximum.

Description

technical field [0001] The invention relates to a method for obtaining solid tumor cells successfully transfected in vitro. Background technique [0002] Since the first establishment of in vitro culture cell technology in 1885 by isolating cells from chicken embryos, monolayer adherent cell culture technology has been widely used in scientific research. However, scientific research in the field of life sciences is not only about whether they can divide Growth, and more importantly, whether the grown cells can represent the real situation in the body. At present, most of the studies on the screening and therapeutic effects of anti-tumor drugs in vitro are based on the two-dimensional growth model of monolayer adherent growth, and this growth mode is very different from the real state of tissue cells in vivo. This difference has caused the experimental conclusions drawn by the two-dimensional growth model to be inconsistent with animal experiments and clinical experiments. ...

Claims

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Application Information

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IPC IPC(8): C12N5/10C12Q1/68C12Q1/06G01N21/31
Inventor 孙汶生韩丽辉曹莉莉梁晓红杜娟张之勇曲忠花刘玉刚刘素侠石永玉高立芬
Owner SHANDONG UNIV
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