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Method for leading plasmid carrier containing gene cure segment in cell by nano particles

A nanoparticle and plasmid vector technology, used in gene therapy, introduction of foreign genetic material using vectors, pharmaceutical formulations, etc., to achieve the effects of good protection, increased transfection rate, and improved ability

Inactive Publication Date: 2008-03-26
南京凯瑞尔纳米生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] CN100335038C patent document discloses a preparation method of inorganic salt nanoparticles containing biologically active substances and traditional Chinese medicine components encapsulated, but whether this inorganic salt nanoparticles can mediate plasmid transfected cells has not been reported

Method used

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  • Method for leading plasmid carrier containing gene cure segment in cell by nano particles
  • Method for leading plasmid carrier containing gene cure segment in cell by nano particles
  • Method for leading plasmid carrier containing gene cure segment in cell by nano particles

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Preparation of Inorganic Salt Nanoparticles Encapsulated by Traditional Chinese Medicine Components

[0027] preparation before preparation

[0028] Salt solution: prepare 0.01-1.0 mole manganese chloride (or magnesium chloride, or calcium chloride solution) with deionized water.

[0029] Phosphoric acid solution: prepare 0.01-1.0 molar phosphoric acid solution with deionized water.

[0030] The active substance solution of the traditional Chinese medicine: the active substance solution of the traditional Chinese medicine astragalus polysaccharide is prepared according to the ratio of every 100ml of normal saline + 1 mg of astragalus polysaccharide.

[0031] The pH value is 7.0, and 0.01 molar phosphate buffer solution can be purchased outside.

[0032] The preparation process is as follows:

[0033] a) Preparation of inorganic salt nanoparticles:

[0034] Mix 0.01-1.0 moles of salt solution and 0.01-1.0 moles of phosphoric acid solution at a ratio of 1:1 and place ...

Embodiment 2

[0039] Method for introducing plasmid vector containing gene therapy fragment into cells by nanoparticle

[0040]preparation before preparation

[0041] 1. Cells: Human glioma cells U251 were purchased from the Cell Resource Center of Shanghai Academy of Biological Sciences.

[0042] 2. Plasmid: a plasmid vector containing gene therapy fragments, which is to insert the human telomerase reverse transcriptase (hTERT) gene into the plasmid vector pGCsi-H1 / neo / GFP, in which: the gene therapy fragments are taken from No.NM in GenBank The human telomerase reverse transcriptase (hTERT) gene of 003219 can be obtained through the human gene bank; the plasmid vector pGCsi-H1 / neo / GFP is provided by China Jikai Company.

[0043] 3. Inorganic salt nanoparticles, prepared according to the method in Example 1.

[0044] making process

[0045] a) Human brain glioma cells U251 in a good growth state were cultured overnight, digested with 0.25% trypsin, and 1×10 cells were taken after counti...

Embodiment 3

[0056] The effect of introducing plasmid vectors containing gene therapy fragments into cells by nanoparticles:

[0057] Fig. 1 shows the observation results of glioma cells U251 in good growth state taken from the control through a fluorescent inverted microscope (Nikon Eclipse TE 2000-S).

[0058] As can be seen in Figure 2, after the transfection was completed using the method of Example 2, the cell-covered coverslip was placed under a laser confocal microscope (ZEISS LSM510, 40× (magnification)) to observe the transfection of human glioma cells U251 The situation after the nanoparticles, it can be seen under the laser confocal microscope, the growth of the glioma cells after the transfection of the nanoparticles is in good condition (as indicated by the arrow), and the green part is the GFP (green fluorescent protein) after the successful transfection The green fluorescence generated by the expression (see the white place at the front of the arrow in the black and white di...

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Abstract

The present invention relates to process of leading plasmid carrier containing gene therapy segment into cell with nanometer particle. The process includes the following steps: inoculating cell in good growth condition to cell culturing plate and culturing; centrifuging nanometer inorganic salt suspension and dissolving the precipitate in deionized water in 500 microliter; dissolving 2 microliter of plasmid in 98 microliter of deionized water; compounding 1 ml of suspension with solution of nanometer particle in 5-10 mg and solution of plasmid in 4-15 microliter through shaking for best combination between the nanometer particle and the plasmid; centrifuging the suspension to obtain the precipitate, adding deionized water, adding to the cell culturing plate, and adding fresh culture liquid to culture for the nanometer particle well combined to the plasmid to transfect the cell; and finally observing the transfection effect in a microscope.

Description

technical field [0001] The invention relates to a method for introducing a plasmid vector containing gene therapy fragments into cells, in particular to a method for introducing the plasmid vector containing gene therapy fragments into cells by using nanoparticles. Background technique [0002] RNA interference (RNA interference, RNAi) is a kind of "gene silencing" induced by double-stranded RNA. During this process, the messenger RNA that has a homologous sequence to the double-stranded RNA in the disease-causing cells is degraded, thereby inhibiting the expression of the disease-causing gene. RNA interference technology has broad application prospects in probing gene functions and treating human diseases. siRNA is a short-segment double-stranded RNA molecule used for RNA interference, which can degrade specific mRNA by targeting mRNA with homologous complementary sequences, and can be used as a targeted gene therapy drug. [0003] siRNA drugs include chemically synthesize...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/63A61K48/00A61P31/12
Inventor 李瑞河清尤永平丁海霞孙申江桥张军霞石磊张勇戴如飞朱含章安欣卢娴
Owner 南京凯瑞尔纳米生物技术有限公司
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