CN104911212A

CN104911212A - Efficient HaCaT cell transfection method

Info

Publication number: CN104911212A
Application number: CN201510279140.1A
Authority: CN
Inventors: 李志远; 李彩月; 马文波
Original assignee: Guangzhou Institute of Biomedicine and Health of CAS
Current assignee: Guangzhou Institute of Biomedicine and Health of CAS
Priority date: 2015-05-27
Filing date: 2015-05-27
Publication date: 2015-09-16

Abstract

The invention discloses an efficient HaCaT cell transfection method. In the process that transfection plasmids enter a HaCaT cell through a liposome method, TX-10 is added in a transfection system of the HaCaT cell. As the TX-10 is added in the transfection process, permeability of a eukaryotic cell membrane is improved, and a liposome-DNA compound is promoted to effectively enter the cell. The method is very effective for HaCaT cells thick in cell membrane and difficult to transfect through a traditional liposome method and improves the transfection efficiency by several times. The method also has the applicability for other cells thick in cell membrane and difficult to transfect; as polyethylene glycol octylphenol ether has toxicity to cells when the use amount of polyethylene glycol octylphenol ether is high and the effect is not obvious when the use amount is low, the use amount of the polyethylene glycol octylphenol ether needs to be considered. For HaCaT cells, the best dosage of the TX-10 is 0.005%; under the best dosage, the death rate of HaCaT cells is lowest and the transfection efficiency is highest.

Description

A kind of method of high-efficiency transfection HaCaT cell

Technical field:

The invention belongs to biological field, be specifically related to a kind of method of high-efficiency transfection HaCaT cell.

Background technology:

HaCaT cell is the human skin immortalized keratinocytes strain gone out by people's normal skin Keratinocyte derived, in view of the advantage that it is easily cultivated and can repeatedly go down to posterity, it is used as a kind of vehicles cells and replaces research people normal keratinocyte, studies the cell proliferation of human keratinocytes, cell cycle and dermal pathology mechanism etc. by the impact on HaCaT cell such as various medicine, compound, chemical factors.

Cell transfecting refers to eukaryotic cell obtains new genetic marker process because foreign DNA mixes.Foreign gene transfection enters cell and mainly contains four kinds of methods: electric shocking method, calcium phosphate method, virus-mediated methods and liposome mediated-method.Experiment condition due to electric shocking method and calcium phosphate method control comparatively tight, difficulty is larger; The early-stage preparations of virus method are more complicated and may have considerable influence for cell; So now for a lot of ordinary cells system, general transfection method many employings liposome method.Cationic-liposome surface band positive charge, in DNA molecular being wrapped up by electrostatic interaction with the phosphate radical of nucleic acid, form DNA mono-fat complex body, also can be adsorbed by the cytolemma of surface band negative charge, again by the fusion of film or the endocytosis of cell, also by the direct osmosis of aperture on cytolemma, DNA transmission is entered cell once in a while.

The cytolemma of HaCaT cell is thicker, is difficult to digestion, more difficult transfection, and foreign DNA be entered HaCaT cell by liposome transfection is a very difficult thing, and transfection efficiency is very low, does not usually reach requirement of experiment.The efficiency of being carried out transfection by electric shocking method and viral method is not high yet, and experimental implementation process is cumbersome, how to utilize simple lipofection to enter HaCaT cell become a technical barrier to improve foreign DNA transfection.

Triton X-100 (TX-10) is a kind of nonionic surface active agent.

Summary of the invention:

The object of this invention is to provide a kind of method that greatly can improve the high-efficiency transfection HaCaT cell of the transfection efficiency of HaCaT cell.

The method of high-efficiency transfection HaCaT cell of the present invention, is characterized in that, it is entering in the process of HaCaT cell, adds Triton X-100 (TX-10) in the rotaring redyeing system of HaCaT cell.

Preferably, the described content of Triton X-100 (TX-10) in the rotaring redyeing system of HaCaT cell is 0.005g/100ml.

The present invention and traditional liposome transfection are unlike adding Triton X-100 (TX-10) in transfection process, it can improve the permeability of eukaryotic cell membrane, thus promote that liposome-DNA complex effectively enters cell, cell membrane is thicker, be difficult to the HaCaT cell carrying out transfection by traditional liposomal method, this method is very effective, transfection efficiency is made to improve several times, it also has suitability to the cell of the difficult transfection of some other cell thickness, just to note the consumption of Triton X-100, because its consumption is high toxic to cell, consumption is low DeGrain.For HaCaT cell, the optimum dose of TX-10 is 0.005% (0.005g/100ml), and under this consumption, the cell mortality of HaCaT cell is minimum, and transfection efficiency is the highest.

Accompanying drawing illustrates:

Fig. 1 is the fluorescent microscope shooting figure of control group and experimental group, wherein contrast represents control group, + 0.005%TX-10 represents experimental group, employ light field and details in a play not acted out on stage, but told through dialogues two kinds of modes, light field can see HaCaT cellular form clearly, as seen from the figure, no matter be control group or+0.005%TX-10 group, under light field, cell adherent all fine, dead cell is little; And details in a play not acted out on stage, but told through dialogues can only see the cell with GFP green fluorescent protein of transfection success, as seen from the figure, under details in a play not acted out on stage, but told through dialogues, the cell fluorescence number of+0.005%TX-10 group is more than control group, fluorescence intensity is also stronger, illustrates that the TX-10 adding 0.005% when transfection can increase substantially the transfection efficiency of HaCaT cell.

Fig. 2 is that the HaCaT cell of the green fluorescent protein GFP to transfection carries total protein, and carry out immunoblot experiment figure with the antibody of anti-GFP, wherein contrast represents control group, + 0.005%TX-10 represents experimental group, can find from figure, the expression amount ratio of GFP albumen in control group is much lower at the expression amount of the experimental group adding 0.005%TX-10 (to show that cell quantity is roughly the same) when internal reference is consistent, the transfection that the TX-10 proving to add when transfection 0.005% can effectively promote GFP plasmid in HaCaT cell and expression.

Embodiment:

Following examples further illustrate of the present invention, instead of limitation of the present invention.

Embodiment 1:

1, be inoculated in 12 orifice plates by HaCaT cell, plating density is about 40-50%, cultivates 12-24h in 37 DEG C of incubators;

2, cell transfecting grouping: GFP plasmid transfection group (GFP is green fluorescent protein, can judge transfection efficiency according to fluorescence intensity), 0.005%TX-10+GFP plasmid transfection group.Wherein front group is set to control group, and rear group is experimental group.

3, cell transfecting step: a, with perfect medium (DMEM+10%FBS), Triton X-100 (TX-10) is diluted to 0.1% (0.1g/100ml, namely the Triton X-100 of 0.1g is added in every 100ml perfect medium, miscible); B, get a 1.5ml EP pipe, add 100 μ l DNA respectively and concentrate damping fluid (EC buffer), 3.2 μ l Enhancer and 600ng GFP plasmid, leave standstill 5min; C, add 10 μ l Effectene, piping and druming evenly gently, leaves standstill 10min; D, add the perfect medium of 290 μ l again, be made into the plasmid transfection liquid that cumulative volume is 400 μ l; E, take out and completed 12 orifice plates of HaCaT cell, go stoste, add the perfect medium of 800 μ l in the hole slot of control group, experimental group then adds the substratum of 750 μ l; F, respectively get the plasmid transfection liquid of 200 μ l, be slowly added dropwise in corresponding hole, jiggle evenly; G, in the hole slot of experimental group, add the TX-10 diluent of the 50 μ l 0.1% of step a, make its final concentration be 0.005% (0.005g/100ml), jiggle evenly; H, continuation cultivation in cell culture incubator, after 4-6h, by stoste sucking-off, and wash twice cell with PBS, then adds 1ml perfect medium and continue to cultivate.

4, after 24h, the transfection efficiency of fluorescence microscope experimental group and cellular control unit can be utilized, and utilize digital image collection system pictures taken; Also the method for using immunoblots detects the expression amount of GFP albumen in cell.

No matter result as depicted in figs. 1 and 2, as can be seen from Figure 1, is control group or+0.005%TX-10 group (experimental group), under light field, and cell adherent all fine, dead cell is little; And details in a play not acted out on stage, but told through dialogues can only see the cell with GFP green fluorescent protein of transfection success, as seen from the figure, under details in a play not acted out on stage, but told through dialogues, the cell fluorescence number of+0.005%TX-10 group is more than control group, fluorescence intensity is also stronger, illustrates that the TX-10 adding 0.005% when transfection can increase substantially the transfection efficiency of HaCaT cell.As seen from Figure 2, to transfection, the HaCaT cell of green fluorescent protein GFP carries total protein, and carry out immunoblot experiment with the antibody of anti-GFP, the expression amount ratio of GFP albumen in control group is much lower at the expression amount of the experimental group adding 0.005%TX-10 to find (to show that cell quantity is roughly the same) when internal reference is consistent, the transfection that the TX-10 proving to add when transfection 0.005% can effectively promote GFP plasmid in HaCaT cell and expression.

Claims

1. a method for high-efficiency transfection HaCaT cell, is characterized in that, it is entering with liposome transfection plasmid in the process of HaCaT cell, in the rotaring redyeing system of HaCaT cell, is adding Triton X-100.

2. the method for high-efficiency transfection HaCaT cell according to claim 1, is characterized in that, the content of described Triton X-100 in the rotaring redyeing system of HaCaT cell is 0.005g/100ml.