Chylomicron is as the application of liver target gene therapy vector
Technical field
The present invention relates to the application of Chylomicron, particularly relate to a kind of brand-new application of Chylomicron as gene therapy vector.
Background technology
Chylomicron (chylomicron, CM) is the reprocessing lipid droplet of a kind of, diameter 80~500nm synthetic by the small intestinal mucosa epithelial cell, contains triglyceride, cholesteryl ester and some apolipoproteins such as apoA I, apoA II, apoA IV, apoB
48, apoC I, apoC II, apoC III, apoE etc., its molecular weight is greater than 50 * 10
6, density is less than 0.95g/cm
3, major function is to transport exogenous triglyceride to tissues such as skeletal muscle, cardiac muscle, fat, transports exogenous cholesterol to liver.
The apolipoprotein that lipid in the food is synthesized with rough endoplasmic reticulum behind resterification on the cell smooth endoplasmic reticulum consists of newborn Chylomicron, is secreted into the extracellular through Golgi complex, enters lymph circulation and finally enters blood.After newborn Chylomicron enters blood, accept apoC and apoE from HDL, lose simultaneously part apoA, be modified to the Chylomicron into maturation.ApoC II on the ripe molecule can activate skeletal muscle, cardiac muscle and the lipoprotein lipase (LPL) of organizing on the capillary endothelial cell outer surface such as fatty, and triglyceride hydrolysis is glycerol and fat in the catalysis Chylomicron, and phospholipid is hydrolyzed to lysophosphatide.The fatty acid that disengages is absorbed by above-mentioned tissue and utilizes, and glycerol can enter liver and be used for glyconeogenesis.By the effect of LPL, the triglyceride major part in the Chylomicron is hydrolyzed utilization, and apoA, apoC, cholesterol and phospholipid are transferred on the HDL simultaneously, and Chylomicron diminishes gradually, and becoming to contain cholesteryl ester is main Chylomicron residual particles.ApoE receptor on the liver plasma membrane can be identified the Chylomicron residual particles, and it is engulfed into hepatocyte, merges with Cytolysosome, apolipoprotein is hydrolyzed to aminoacid, cholesteryl ester is decomposed into cholesterol and fatty acid, and then can be utilized by liver or decompose, and finishes final metabolism.
So far, do not find any report of using about Chylomicron.
Hepatocarcinoma is one of very common tumor, in global sickness rate rising, the serious harm mankind's life and health.The method of Hepatoma therapy mainly is the 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 at present this art development is rapid, and gene therapy of liver cancer carrier commonly used mainly contains viral vector, liposome, plasmid, cationic polymer etc.
Viral vector efficient is high, but poor specificity, and because safety issue is used always undesirable.Liposome safety, but poor specificity.In numerous cationic polymers, polymine (PEI) has high positive charge density, can form closely complexation body 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 large cytotoxicity simultaneously, and the specificity of carrier is not strong yet.
How to realize the specificity of gene therapy, namely 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 namely the special magnetic target therapy genophore system that is oriented to hepatoma carcinoma cell becomes the from now on direction of research.
Summary of the invention
A kind of new purposes that the purpose of this invention is to provide Chylomicron, i.e. the application of Chylomicron on gene therapy medicament.
In fact, the invention provides Chylomicron as the application of the carrier of liver target gene therapy medicament.
Chylomicron is as a kind of genophore of new Hepatoma therapy, have good liver cell targeted, this is because contain apo E (apoE) in Chylomicron, and all has the apoE receptor hepatocyte and hepatoma 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 is not absorbed by other cell of health, and this has just consisted of the targeting of genophore of the present invention.
What is more important, Chylomicron are the bio-extracts of pure natural, finally can be by metabolism in hepatocyte and hepatoma carcinoma cell, and noresidue is without any cytotoxicity problem.
A kind of method of preparation Chylomicron of the present invention is: get SD strain rat, male and female are not limit, body weight 220~260g, adopt following high lipid food to feed, 15~30min blood sampling behind the feeding, 1000 rev/mins of low-speed centrifugal 15min get blood plasma, then 35000 rev/mins of ultracentrifugation 1 h get the superiors and are Chylomicron.
Described high lipid food is to take by weighing cholesterol 10g, Adeps Sus domestica 100g, egg yolk 100g, conventional animal feedstuff 2100g, Adeps Sus domestica is heated to 40 ℃ first, add the cholesterol powder after its dissolving, add again the egg yolk stirring and emulsifying, admix the conventional animal feedstuff, add that suitable quantity of water is mixed thoroughly, the group of holding, cook, by every 100g body weight 4g feedstuff every day, with naturally search for food method hello to.
Need to prove, more than be not the uniqueness preparation method of Chylomicron, can also get access to Chylomicron by other approach.The most simply approach is, many biochemical reagents sale enterprises have the Chylomicron merchandise sales, and for example, Beijing is opened the dimension benefit and become Science and Technology Ltd..
Description of drawings
Fig. 1 is the pAFP-TK-IRES2-EGFP plasmid map.
Fig. 2 is the scanning electron microscope (SEM) photograph (* 100000) of Chylomicron parcel pAFP-TK-IRES2-EGFP plasmid.
Fig. 3 is that Chylomicron is to the agarose gel electrophoresis of pAFP-TK-IRES2-EGFP plasmid protective effect.
Fig. 4 is the specifically expressing (* 300) of 1 couple of targeting vector mediation EGFP of embodiment in hepatoma cell strain MHCC97.
Fig. 5 is the pGenesil-4 plasmid map.
Fig. 6 is the scanning electron microscope (SEM) photograph (* 100000) of Chylomicron parcel pGenesil-4 plasmid.
Fig. 7 is that Chylomicron is to the agarose gel electrophoresis of pGenesil-4 plasmid protective effect.
Fig. 8 is the specifically expressing (* 300) of embodiment 5 targeting vectors mediation EGFP in liver cell line HL-7702.
The specific embodiment
Embodiment 1: two targeting genophores of preparation Chylomicron parcel pAFP-TK-IRES2-EGFP plasmid vector.
Configuration concentration is the pAFP-TK-IRES2-EGFP plasmid vector of 50 μ g/mL; Chylomicron is placed Ultrasonic Cell Disruptor ultrasonic 10 times; each 15s; ultrasonic power 150w; plasmid vector is mixed with Chylomicron with the mass volume ratio 2 μ g/mL of Chylomicron according to plasmid vector; vortex concussion 30s leaves standstill 30min, makes two targeting genophores of Chylomicron parcel pAFP-TK-IRES2-EGFP plasmid vector.
Described eukaryon expression plasmid pAFP-TK-IRES2-EGFP is 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 at the liver-cancer cell specific expression thymidine kinase after this gene is activated, this kind of enzyme can make a kind of medicine of ganciclovir that cries that enters hepatoma carcinoma cell be transformed into the medicine that can kill hepatoma carcinoma cell, simultaneously can also produce " bystander effect ", namely kill contiguous hepatoma carcinoma cell.This effect only produces in hepatoma carcinoma cell, has consisted of second targeting of the present embodiment carrier except Chylomicron.
Embodiment 2: Chylomicron is to the parcel effect of pAFP-TK-IRES2-EGFP plasmid vector.
Adopt scanning electron microscope two targeting vectors of embodiment 1 gained to be observed 100000 times of Scanning Electron Microscope photos reveal: Chylomicron is respond well to pAFP-TK-IRES2-EGFP plasmid vector parcel, and particle diameter is all between 200~300nm, such as Fig. 2.
Embodiment 3: Chylomicron is to the protective effect of pAFP-TK-IRES2-EGFP plasmid vector.
In two targeting vectors of embodiment 1 gained, add bovine pancreas DNaseⅠ; making its final concentration is 20 μ g/mL; 37 ℃ of digestion 30min; do contrast with simple pAFP-TK-IRES2-EGFP plasmid vector, agarose gel electrophoresis is observed Chylomicron to the protective effect of pAFP-TK-IRES2-EGFP plasmid vector.Observed result is seen Fig. 3, and swimming lane 1 shows simple pAFP-TK-IRES2-EGFP plasmid vector owing to there not being the protective effect of Chylomicron, is degraded by bovine pancreas DNaseⅠ; Swimming lane 2 shows that Chylomicron has very strong protective effect to the pAFP-TK-IRES2-EGFP plasmid vector in two targeting vectors, can avoid it to be degraded by bovine pancreas DNaseⅠ.The result shows: Chylomicron has very strong protective effect to the pAFP-TK-IRES2-EGFP plasmid vector in two targeting vectors, can avoid it to be degraded, and therefore two targeting vectors have higher stability.
Embodiment 4: specific expressed in hepatoma cell strain of two targeting vectors mediation EGFP.
Distinguish hepatoma cell strain MHCC97, normal liver cell strain L-02, myocardial cell strain HCMa, normal kidney cell line HK-2, colorectal cancer cell lines 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, then add the two targeting vector 1mL of embodiment 1 gained in every 10mL cell suspension, mixing is put 37 ℃ of cultivations.After the transfection 48 hours, with the expression of fluorescence inverted microscope observation of cell Green fluorescin.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.
Embodiment 5: the targeting genophore of preparation Chylomicron parcel pGenesil-4 plasmid vector.
Configuration concentration is the pGenesil-4 plasmid vector of 50 μ g/mL; Chylomicron is placed Ultrasonic Cell Disruptor ultrasonic 10 times; each 15s; ultrasonic power 150w; plasmid vector is mixed with Chylomicron with the mass volume ratio 2 μ g/mL of Chylomicron according to plasmid vector; vortex concussion 30s leaves standstill 30min, makes the targeting genophore of Chylomicron parcel pGenesil-4 plasmid vector.
Described eukaryon expression plasmid pGenesil-4 is available from the sincere industrial science and technology limited Company of Beijing Xi Er, the plasmid map of this plasmid vector such as Fig. 5, contain the CMV(cytomegalovirus on it) promoter, can start the EGFP(enhanced green fluorescence protein in its downstream) gene expression, can be at hepatocyte or hepatoma carcinoma cell expressing green fluorescent protein.
Embodiment 6: Chylomicron is to the parcel effect of pGenesil-4 plasmid vector.
Adopt scanning electron microscope the targeting vector of embodiment 5 gained to be observed 100000 times of Scanning Electron Microscope photos reveal: Chylomicron is respond well to pGenesil-4 plasmid vector parcel, and particle diameter is all between 200~300nm, such as Fig. 6.
Embodiment 7: Chylomicron is to the protective effect of pGenesil-4 plasmid vector.
In the targeting vector of embodiment 5 gained, add bovine pancreas DNaseⅠ; make its final concentration reach 20 μ g/mL; 37 ℃ of digestion 30min; do contrast with simple pGenesil-4 plasmid vector, agarose gel electrophoresis is observed Chylomicron to the protective effect of pGenesil-4 plasmid vector.Observed result is seen Fig. 7, and swimming lane 1 shows simple pGenesil-4 plasmid vector owing to there not being the protective effect of Chylomicron, is degraded by bovine pancreas DNaseⅠ; Swimming lane 2 shows that Chylomicron has very strong protective effect to the pGenesil-4 plasmid vector in the targeting vector, can avoid it to be degraded by bovine pancreas DNaseⅠ.The result shows: Chylomicron has very strong protective effect to the pGenesil-4 plasmid vector in the targeting vector, can avoid it to be degraded, so targeting vector has higher stability.
Embodiment 8: specific expressed in liver cell line of targeting vector mediation EGFP.
Distinguish normal liver cell strain HL-7702, the myocardial cell strain HCMa of transfection In vitro culture, normal kidney cell line HK-2, colorectal cancer cell lines HT-29, Skeletal Muscle Cell strain HSkMC with the targeting vector of embodiment 5 gained, in advance the cell strain of In vitro culture is mixed with the cell suspension of 10000/mL, then add embodiment 5 gained targeting vector 1mL in every 10mL cell suspension, mixing is put 37 ℃ of cultivations.After the transfection 48 hours, with the expression of fluorescence inverted microscope observation of cell Green fluorescin.The result shows: only expressing green fluorescent protein (EGFP) is (Fig. 8) in liver cell line HL-7702 for the hepatic gene treatment targeting vector of embodiment 5 gained, do not observe the expression of EGFP in other cell, prove that the hepatic gene treatment targeting vector of embodiment 5 gained has higher targeting.