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Method for mediated gene transfection

A gene transfection and mediation technology, which is applied in the fields of biomedicine and bioengineering, can solve the problems of transient transfection effect and low transfection efficiency, so as to reduce virus dosage, improve transfection efficiency, and increase targeting sexual effect

Inactive Publication Date: 2009-07-08
SHANGHAI FIRST PEOPLES HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] However, UTMD has its application defects. One is that the transfection efficiency is still low, and the other is that the transfection effect is transient.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1 Determining the implementation of UTMD combined with AAV-mediated gene transfection method

[0030] 1) UTMD pre-irradiated rAAV2

[0031] The mechanism of UTMD-mediated gene transfection is mainly related to the instantaneous cavitation effect, that is, under the irradiation of high ultrasonic sound pressure, the microbubbles rapidly expand and contract, and even collapse, and then generate strong shock waves and acoustic waves around the microbubbles. Jet flow and a large number of free radicals, etc. These effects can be increased to a certain extent to affect the performance of proteins and genes.

[0032] In this example, rAAV2-EGFP was administered with a certain dose of UTMD before being added to the cell well: ultrasonic intensity 2 , irradiation time<120seconds and microbubble<60μl, frequency 1MHz, duty cycle 50%, pulse repetition frequency 100Hz, pre-irradiation, the results showed that the efficiency of infecting human renal cancer cells (786-0) was...

Embodiment 2

[0037] Example 2 Optimizing the effect of UTMD combined with AAV-mediated gene transfection method

[0038] A. Irradiation with different sound intensities

[0039] Since the cavitation effect caused by UTMD is positively correlated with the cell transfection rate, but negatively correlated with the cell viability, it is necessary to optimize its influencing factors in the in vivo and in vitro experiments to achieve the highest transfection efficiency while ensuring the cell survival rate. Minimal damage. The main factors of UTMD-mediated gene transfection include ultrasonic irradiation conditions and microbubble dosage. The magnitude of the ultrasonic alternating sound pressure is proportional to the amplitude of the microbubble vibration, that is, when the irradiation sound intensity increases, the nonlinear vibration of the microbubble intensifies, so that the microbubble is more likely to rupture, resulting in stronger shock waves, microjet flow and shear stress, etc., in...

Embodiment 3

[0049] In vivo and in vitro research application of UTMD combined with AAV-mediated gene transfection method

[0050] A. In vitro experiments

[0051] A). Human kidney cancer cells

[0052] Renal cancer cells are rAAV2 non-tropic cells. Human kidney cancer cells were purchased from the Cell Resource Center, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. In the present invention, rAAV2 (1×10 4 ~5×10 6 MOI) was used to infect human renal carcinoma (786-0) cells, and the transfection rate of EGFP was detected after 48 hours. The results showed that the infection efficiency of rAAV2 to 786-0 cells was low, with an average of (14.31±2.50)%. With the optimal UTMD irradiation conditions (sound intensity 1W / cm 2 , microbubble 30μl, irradiation time 60s, frequency 1MHz, duty cycle 50%, pulse repetition frequency 100Hz) combined with rAAV2, the infection efficiency increased by 200%-300% on average.

[0053] B). Human liver cancer cells

[0054] Human l...

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Abstract

The present invention, pertaining to the field of biomedicine and bioengineering, relates to the gene delivery system technology related to the gene therapy, specifically to a mediated gene conversion method. The invention combines the ultrasonic targeted damage microbubble technology and gland relevant viral vector technology at the appropriate time and mode to realize advantage complementary, so that the target gene can be more safely and efficiently entered in the histiocytes to realize the long-term stable expression of the target gene. The method described in this invention can be used in the vivo experimental investigation of AAV carrying of different target genes for gene therapy, specifically in the non-AAV direction cells or tissues; the invention can safely improve the AAV carrying gene transfection efficiency, speed up gene body expression, increase the targeting of the transfection, reduce the virus dosage and diminish the toxic side effects.

Description

technical field [0001] The invention relates to the fields of biomedicine and bioengineering, and relates to the gene delivery system technology related to gene therapy. In particular, it relates to a method for mediating gene transfection. Background technique [0002] With the completion of the Human Genome Project, the selection of therapeutic genes is no longer a problem. Gene therapy has become a very potential treatment method for malignant tumors and some congenital diseases or chronic diseases. Relevant studies have shown that the whole process of gene therapy mainly involves three aspects: target gene, gene transfer technology and recipient cells. In the case of certain recipient cells, gene transfer technology is the key to determine the effect of gene therapy. The pros and cons of gene transfer technology platforms depend on whether exogenous genes can efficiently and safely enter tissue cells and be stably and persistently expressed in tissue cells. [0003] ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/87C12N15/861C12N15/864
Inventor 杜联芳王慧萍李凡李红丽郑孝志伍瑛史秋生
Owner SHANGHAI FIRST PEOPLES HOSPITAL
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