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Quantitative measurement method of extracorporeal activity of recombinant human endostatin

A technique for quantitative determination of endostatin, applied in the application of anti-angiogenesis therapy, in the field of quantitative determination of in vitro activity, can solve the problems of poor repeatability of test results, lack of reproducibility, difficult operation control, etc., and achieve good quantitative determination. Effect, good reproducibility of results, small measurement error effect

Inactive Publication Date: 2007-10-03
江苏省基因药物工程技术研究中心
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  • Abstract
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  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, at present, angiogenesis-inhibiting drugs and biological activity assay methods have not been well resolved at home and abroad. The existing assay methods use bFGF or VEGF-stimulated in vitro endothelial cell migration test to obtain quantitative biological activity in vitro, but the test results The repeatability is poor, the operation is difficult to control, and the error of the method is as large as 50%-250%. This is mainly because the endothelial cells used need to be stimulated with bFGF or VEGF first, and then the migration inhibition test is performed. This method has a large error
The endothelial cell proliferation inhibition test also needs to use bFGF or VEGF to stimulate endothelial cells before performing the endothelial cell proliferation inhibition test. The endothelial cell culture used is poor in repeatability, and the activity stimulation effect of bFGF or VEGF itself has poor repeatability and equivalence, and the sensitivity of the experiment is poor. Therefore, there is often a lack of reproducibility, resulting in large errors in in vitro activity measurement and poor reproducibility, making it difficult to reflect the true activity level and application effect of recombinant human endostatin

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  • Quantitative measurement method of extracorporeal activity of recombinant human endostatin
  • Quantitative measurement method of extracorporeal activity of recombinant human endostatin
  • Quantitative measurement method of extracorporeal activity of recombinant human endostatin

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Example 1: Transfection of vascular endothelial cells with VEGF gene or bFGF gene

[0022] There are many kinds of vascular endothelial cells. In this embodiment, human umbilical vein endothelial cells are taken as an example. Under aseptic conditions, the umbilical cord of a newborn is taken, and put into a mixture containing penicillin-streptomycin (10 5 U / ml) umbilical cord preservation solution (containing NaCl 140mmol / L, KCl 4mmol / L, NaCl 2 HPO 4 5.2mmol / L, KH2 PO 4 1.5mmol / L, glucose 110mmol / L), rinse the umbilical vein with double-antibody-containing PBS repeatedly, perfuse with type II collagenase prepared by Hanks solution for 20min, wash the digested umbilical vein with culture medium, collect and rinse Afterwards, the culture solution containing endothelial cells was centrifuged, and then added with RPMI-1640 complete medium containing 20% ​​FBS, 2ng / ml bFGF, 1% BSA, 90μg / ml heparin sodium, 100U / ml penicillin and 100μg / ml streptomycin to cultivate. Afte...

Embodiment 2

[0024] Example 2: Endothelial cells transfected with VEGF gene or bFGF gene to inhibit proliferation in vitro activity assay

[0025] Taking hUVEC transfected with VEGF gene as an example, press 1×10 on a 96-well cell culture plate 4 Cells were seeded in RPMI-1640 complete medium with 10% FBS at 37°C in 5% CO 2 After culturing for 24 hours, recombinant human endostatin was added according to different dilution factors, and a total of 8 dilution gradients were set up, with three replicate wells for each gradient. An equal volume of sample solvent was added to the blank control group, and cultured for 72 hours. Count under a microscope after protease digestion, as shown in Figure 1, and then directly measure on a microplate reader with the MTT method or LDH method, as shown in Figure 2 and Figure 3, respectively. When using the MTT method, add 10 μl of MTT solution to each well of a 96-well cell culture plate and incubate at 37°C for 4 hours, then add 100 μl of 20% SDS, shake f...

Embodiment 3

[0028] Example 3: Endothelial cell migration inhibition activity assay in vitro transfected with VEGF gene or bFGF gene

[0029] Taking hUVEC transduced with VEGF gene as an example, use RPMI-1640 culture medium containing 10% FBS at 37°C 5% CO 2 Cultured cells, digested with trypsin to make 1.8×10 5 cells / ml cell suspension, add the same culture solution to the upper and lower tanks of the endothelial cell migration plate, 37 ° C 5% CO 2 Incubate in the incubator for more than 1 hour, dilute the sample with its own buffer solution to 8 dilution gradients, set up two duplicate wells for each gradient, add 10 μl of recombinant endostatin of different dilutions to each well of the migration plate slot, The buffer of the sample itself was added to the control group, and then 90 μl of cell suspension was added, and 540 μl of the above culture medium and 60 μl of the sample to be tested at the same dilution were added to the lower tank. Migrate plates at 37°C 5% CO 2 After incub...

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Abstract

The present invention is quantitative measurement method of extracorporeal activity of high activity soluble recombinant human endostatin produced through a genetic engineering process, and features that in the test, the vascular endothelial cell with introduced bFGF and / or VEGF gene and without need of adding additional bFGF or VEGF for excitation is added directly into the measured sample to calculate the activity quantitatively. The method is simple and accurate, and may be applied in industrial production and clinical angiogenesis antagonizing treatment.

Description

technical field [0001] The invention belongs to the field of genetic engineering biological products, and relates to a method for quantitatively measuring the in vitro activity of highly active soluble recombinant human endostatin (Endostatin) produced by genetic engineering methods, and the method is effective in the anti-angiogenic diseases. Applications in Angiogenesis Therapy. The term of the present invention relates to: recombinant human endostatin protein, recombinant human endostatin protein active fragment and PEGylated long-acting recombinant human endostatin and active fragment. Background technique [0002] Human endostatin recombinant protein can be produced and purified from Escherichia coli expression system, yeast expression system, insect expression system, eukaryotic cell expression system and virus expression system. The activities of recombinant human endostatin produced by different ways or produced by different preparation methods are often different. ...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/04C12N15/09
Inventor 徐根兴朱浩文徐家明荣志刚赵延发钟慎政
Owner 江苏省基因药物工程技术研究中心
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