CN102198277A - Dual-targeting gene treatment vector for liver cancer and preparation method thereof - Google Patents

Abstract

The invention relates to a dual-targeting gene treatment vector for liver cancer, which is a complex consisting of chylomicron and an eukaryotic expression plasmid vector containing an alpha-fetoprotein (AFP) promoter and a suicide gene. Apolipoprotein E (apo E) is contained in the chylomicron, and apo E receptors present on membrane surfaces of liver cells and liver cancer cells, and can identity and combine the apo E specifically, so the plasmid vector can be carried by the chylomicron specifically to be oriented to the liver cells and the liver cancer cells without being absorbed by other cells of bodies; and the eukaryotic expression plasmid vector contains the AFP promoter for promoting gene expression, and the promoter can be promoted only under the specific AFP promoting environment to promote the gene expression at the downstream of the eukaryotic expression plasmid vector, so the dual-targeting gene treatment vector has high and obvious liver cell targeting, and can be promoted and expressed only in the liver cancer cells.

Description

Two targeting vectors of a kind of gene therapy hepatocarcinoma and preparation method thereof

Technical field

The present invention relates to a kind of genophore, relate in particular to a kind of genophore that is used to carry treatment hepatocarcinoma genes of interest, and the preparation method of this genophore.

Background technology

Hepatocarcinoma is one of very common tumor, rises the serious harm mankind's life and health day by day at global sickness rate.The method of treatment hepatocarcinoma mainly is means such as chemotherapy, operation, intervention at present, but effect is all not ideal enough.Growing along with genetic science and technology, gene therapy hepatocarcinoma becomes possibility.The key issue of gene therapy is the carrier problem of carrying gene, and this art development at present is rapid, and gene therapy of liver cancer carrier commonly used mainly contains viral vector, liposome, plasmid, cationic polymer etc.

Viral vector efficient height, but poor specificity, and because safety issue is used undesirable always.Liposome safety, but poor specificity.In numerous cationic polymers, polymine (PEI) has high positive charge density, can form complexation body closely with DNA, be in recent years in the cell transfecting of inside and outside research the widest, the macromolecule carrier that transfection efficiency is the highest.But high positive charge density makes PEI show very big cytotoxicity simultaneously, and the specificity of carrier is not strong yet.

How to realize the specificity of gene therapy, promptly targeting is the key issue of gene therapy of liver cancer.Consider from biocompatibility and biological degradability, the design low cytotoxicity, the carrier of high targeting has become problem demanding prompt solution.Selection can the direct killing hepatoma carcinoma cell and do not damage normal liver cell, and the just special magnetic target therapy genophore system that is oriented to hepatoma carcinoma cell becomes the direction of research from now on.

Summary of the invention

The purpose of this invention is to provide a kind of genophore for the treatment of hepatocarcinoma, it is good liver cell targeted that it is had, and hepatoma carcinoma cell had great lethal effect, but can not kill and wound normal hepatocyte, and simultaneously, the present invention also provides the preparation method of this genophore.

Below be to realize technical scheme of the present invention:

A kind of two targeting vectors of gene therapy hepatocarcinoma, the complex of forming by Chylomicron and the eukaryon expression plasmid that contains AFP promoter and suicide gene, and the proportioning of eukaryon expression plasmid and Chylomicron is adding 2 μ g plasmid vectors in every mL Chylomicron.

The present invention also provides the preparation method of two targeting vectors of said gene treatment hepatocarcinoma, specifically may further comprise the steps:

1) configuration concentration is the eukaryon expression plasmid that contains AFP promoter and suicide gene of 50 μ g/mL;

2) Chylomicron is placed Ultrasonic Cell Disruptor ultrasonic 10 times, each 15s, ultrasonic power 150w;

3) according to the ratio that adds 2 μ g plasmid vectors in every mL Chylomicron, with step 1) and step 2) product mix, vortex concussion 30s leaves standstill 30min, obtains two targeting vectors of gene therapy hepatocarcinoma of the present invention.

Contain apo E (apoE) in the Chylomicron, and all there is the apoE receptor in hepatocyte regulating liver-QI cancer cell membrane surface, identification that can be special and in conjunction with apoE, therefore, Chylomicron can specificly be carried plasmid vector and is oriented to hepatocyte and hepatoma carcinoma cell, and do not absorbed by other cell of health, this has just constituted first targeting of carrier of the present invention.And finally meeting metabolism in hepatocyte and hepatoma carcinoma cell of Chylomicron, the no cytotoxicity problem.The Chylomicron that the present invention uses opens the dimension benefit available from Beijing and becomes Science and Technology Ltd..

Wherein, described eukaryon expression plasmid is pAFP-TK-IRES2-EGFP, available from the sincere industrial science and technology limited Company of Beijing Xi Er, the plasmid map of this plasmid vector such as Fig. 1, contain alpha-fetoprotein (AFP) promoter that promotor gene is expressed on it, the AFP promoter can only just can be activated under specific AFP startup environment, and then start the gene expression in its downstream, also carry suicide gene TK(thymidine kinase gene on this plasmid vector), can be after this gene is activated at the liver-cancer cell specific expression thymidine kinase, a kind of medicine of ganciclovir that cries that this kind of enzyme enables to enter hepatoma carcinoma cell is transformed into the medicine that can kill hepatoma carcinoma cell, simultaneously can also produce " bystander effect ", promptly kill contiguous hepatoma carcinoma cell.This effect only produces in hepatoma carcinoma cell, and this has just constituted second targeting of carrier of the present invention.

The present invention has designed the gene therapy vector of two targeting, one, and Chylomicron is the bio-extract of pure natural, contains apolipoprotein apoE, and Chylomicron definitely has no side effect noresidue finally by metabolism.They are two years old, according to the characteristics of having only hepatoma carcinoma cell high expressed alpha-fetoprotein (AFP), the interior existence of hepatoma carcinoma cell just can start the environment of AFP gene expression, contain the AFP promoter on the plasmid vector that adopts, that is to say, this plasmid vector only just can be activated expression in hepatoma carcinoma cell, express suicide gene hepatoma carcinoma cell is committed suiside, and has significant liver cell targeted and to the significant lethality of hepatoma carcinoma cell.

Description of drawings

Fig. 1 is the plasmid map of eukaryon expression plasmid pAFP-TK-IRES2-EGFP.

Fig. 2 is the sem photograph (* 100000) of Chylomicron parcel plasmid vector pAFP-TK-IRES2-EGFP.

Fig. 3 is the agarose gel electrophoresis figure of Chylomicron to the plasmid vector protective effect.

Fig. 4 is specific expressed (* 300) of the two targeting vector mediation EGFP of the present invention in hepatoma cell strain MHCC97.

The specific embodiment

Embodiment 1: the preparation of two targeting vectors.

1. configuration concentration is the pAFP-TK-IRES2-EGFP plasmid vector of 50 μ g/mL.

2. Chylomicron is placed Ultrasonic Cell Disruptor ultrasonic 10 times, each 15s, ultrasonic power 150w.

3. the ratio (mass/volume) according to plasmid vector and Chylomicron is 2 μ g/mL, and with the product mixing of step 1 and step 2, vortex concussion 30s leaves standstill 30min, makes two targeting vectors.

Embodiment 2: Chylomicron is to the parcel effect of plasmid vector.

Adopt scanning electron microscope that two targeting vectors of embodiment 1 gained are observed, the result shows: when the ratio (mass/volume) of plasmid vector and Chylomicron is 2 μ g/mL, Chylomicron is best to the parcel effect of plasmid vector, and particle diameter is all between 200～300nm, as Fig. 2.

Embodiment 3: Chylomicron is to the protective effect of plasmid vector.

In two targeting vectors of embodiment 1 gained, add the Pancreas Bovis seu Bubali deoxyribonuclease; making its final concentration is 20 μ g/mL; 37 ℃ of digestion 30min do contrast with simple plasmid vector, and agarose gel electrophoresis is observed the protective effect of Chylomicron to plasmid vector.Observed result is seen Fig. 3, and swimming lane 1 shows simple plasmid vector owing to there is not the protective effect of Chylomicron, is degraded by the Pancreas Bovis seu Bubali deoxyribonuclease; Swimming lane 2 shows that Chylomicron has very strong protective effect to plasmid vector in two targeting vectors, can avoid it to be degraded by the Pancreas Bovis seu Bubali deoxyribonuclease.The result shows: Chylomicron has very strong protective effect to plasmid vector in two targeting vectors, can avoid it to be degraded, and therefore two targeting vectors have advantages of higher stability.

Embodiment 4: two targeting vectors mediation EGFP specific expressed in hepatoma cell strain.

Distinguish hepatoma cell strain MHCC97, normal liver cell strain L-02, myocardial cell strain HCMa, normal kidney cell line HK-2, colorectal cancer cells strain HT-29, the Skeletal Muscle Cell strain HSkMC of transfection In vitro culture with two targeting vectors of embodiment 1 gained, in advance the cell strain of In vitro culture is mixed with the cell suspension of 10000/mL, add the two targeting vector 1mL of embodiment 1 gained in every then 10mL cell suspension, mixing is put 37 ℃ of cultivations.After the transfection 48 hours, with the expression of green fluorescent protein in the fluorescence inverted microscope observation of cell.The result shows: only expressing green fluorescent protein (EGFP) is (Fig. 4) in hepatoma cell strain MHCC97 for the two targeting vectors of the gene therapy of liver cancer of embodiment 1 gained, do not observe the expression of EGFP in other cell, prove that the two targeting vectors of gene therapy of liver cancer of embodiment 1 gained have higher targeting.

Claims (3)

1. two targeting vectors of a gene therapy hepatocarcinoma, the complex of forming by Chylomicron and the eukaryon expression plasmid that contains AFP promoter and suicide gene, and the proportioning of eukaryon expression plasmid and Chylomicron is adding 2 μ g plasmid vectors in every mL Chylomicron.

2. two targeting vectors of gene therapy hepatocarcinoma according to claim 1 is characterized in that described eukaryon expression plasmid is pAFP-TK-IRES2-EGFP.

3. prepare the method for two targeting vectors of claim 1 gene therapy hepatocarcinoma, may further comprise the steps:

1) configuration concentration is the eukaryon expression plasmid that contains AFP promoter and suicide gene of 50 μ g/mL;

2) Chylomicron is placed Ultrasonic Cell Disruptor ultrasonic 10 times, each 15s, ultrasonic power 150w;

3) according to the ratio that adds 2 μ g plasmid vectors in every mL Chylomicron, with step 1) and step 2) product mix, vortex concussion 30s leaves standstill 30min, obtains two targeting vectors.

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