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Novel application of intestinal alkaline phosphatase and product activity quality control method of preparation of intestinal alkaline phosphatase

A phosphatase, a new application technology, applied in the direction of medical preparations containing active ingredients, non-central analgesics, biochemical equipment and methods, etc., can solve the research on the biological activity, physiological function and clinical application of alkaline phosphatase Limited and other problems, to achieve the effect of inhibiting inflammation and inhibiting removal

Pending Publication Date: 2020-04-28
李鑫荣
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0029] It can be seen that the above-mentioned prior art is relatively limited to the research on the biological activity, physiological function and clinical application of alkaline phosphatase, and there is still room for further development.

Method used

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  • Novel application of intestinal alkaline phosphatase and product activity quality control method of preparation of intestinal alkaline phosphatase
  • Novel application of intestinal alkaline phosphatase and product activity quality control method of preparation of intestinal alkaline phosphatase
  • Novel application of intestinal alkaline phosphatase and product activity quality control method of preparation of intestinal alkaline phosphatase

Examples

Experimental program
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Effect test

Embodiment 1

[0057] Purpose: Preparation of recombinant human intestinal alkaline phosphatase

[0058] Methods: A chimeric recombinant human intestinal alkaline phosphatase (recIAP) cDNA (references 24, 25) was used to construct the expression vector pMH3-recIAP (reference 26), and then the constructed pMH3-recIAP was transformed into CHO-S Cells (CVCL_7183, Life Technologies). Use DMEM / F12 culture medium containing 10% fetal bovine serum (FBS) to obtain stable and high-expression cell clones through G418 pressurized screening as seed cell lines for harvesting proteins, expand culture in animal cell bioreactors, and obtain harvest liquid for purification For treatment, the harvest liquid was centrifugally concentrated and added to an anion column (Q XK50, GE Healthcare), and the eluate containing high-concentration recIAP was collected and depyrogenated through a hollow fiber column (Borgeron Biotechnology Co., Ltd.) for later use. The purity of the solution containing high concentration ...

Embodiment 2

[0065] Objective: Intestinal alkaline phosphatase inhibits human neutrophil emigration

[0066] method:

[0067] 1. Isolation of Neutrophils and Mononuclear Cells from Human Venous Blood

[0068] A total of 6 healthy volunteers, aged 26±5 years old, blood collection was approved by the Medical Ethics Committee of Changchun Jiahe Surgical Hospital and I agreed. The present invention adopts the sugar density gradient centrifugation method and the human venous blood leukocyte separation kit (endotoxin<0.1EU) (Tianjin Haoyang Huake Biotechnology Co., Ltd.) to separate venous blood. Collect human venous blood at room temperature and centrifuge at 1800rpm for 25 minutes, absorb the mononuclear cell layer (lymphocytes and a small number of monocytes) and the multinuclear cell layer (mainly neutrophils) to mix, fully lyse the red blood cells, wash twice, and use 3ml Resuspended in 1640 medium containing 10% FBS for use. The cell morphology was observed by staining with leukocyte di...

Embodiment 3

[0074] Objective: Study on Phagocytosis of Fluorescent Particles by Human Neutrophils

[0075] Methods: Human neutrophils were adjusted to 2×106 cells / ml in RPMI-1640 medium containing 10% FBS. Add 200ul cells per well, inoculate in a 24-well plate, add 5U / ml recIAP and 1ng / ml LPS separately or simultaneously to stimulate neutrophils. Add 3.5ul of carboxylate-modified polystyrene (L3030 Sigma-Aldrich) to each well of latex beads with a diameter of 2um, adjust the density of fluorescent particles to 2×107 / ml, and establish the best phagocytosis model of neutrophils and fluorescent particles After phagocytosis culture at 37°C for 1 h, flow cytometer (FACSCalibur US PE Company) 488nm wavelength laser collects red fluorescence with a wavelength of 575nm to obtain the phagocytosis rate of neutrophils, so as to study the phagocytosis ability of neutrophils. After the experiment is completed, freshly extract another human venous blood leukocytes to repeat the experiment to ensure th...

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Abstract

The invention discloses application of intestinal alkaline phosphatase as a human neutrophile granulocyte removal inhibitor, a human mononuclear cell removal inhibitor, a humanneutrophile granulocytephagocytosis inhibitor and a mononuclear cell apoptosis accelerant in the presence or absence of endotoxin, and application of the intestinal alkaline phosphatase as a reversion preparation for treating diseases that human neutrophile granulocyte apoptosis is inhibited by the endotoxin. The invention further discloses application of the intestinal alkaline phosphatase in preparing medicines for treating related diseases and in treatment on related diseases. The invention further discloses a product activity quality control method of an industrial or commercial alkaline phosphatase preparation.The application disclosed by the invention is independent from known anti-inflammation mechanisms and application that the endotoxin is inactivated by the intestinal alkaline phosphatase to inhibit secretion of human leukocyte TNF-alpha and IL-6, and application of the alkaline phosphatase is greatly widened.

Description

technical field [0001] The invention relates to the field of intestinal alkaline phosphatase, in particular to a new application of intestinal alkaline phosphatase and a product activity quality control method for its preparation. Background technique [0002] Alkaline phosphatase is divided into multiple subtypes (TNAP, IAP, PLAP), which are widely distributed in different tissues of the human body, including heart, brain, epithelial cells of placental blood vessels, intestinal mucosa, liver cells, etc. (references 1, 2, 3, 4, 5). However, the physiological function of alkaline phosphatase has not been clarified for half a century. The earliest widely accepted function of alkaline phosphatase was its involvement in bone calcification (Ref. 6). In the early 1990s, the inventors used gene transfer technology to introduce alkaline phosphatase gene into different cells, and found that alkaline phosphatase was related to pathological vascular calcification (references 7 and 8)...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K38/46A61P7/00A61P29/00C12Q1/42C12Q1/02
CPCA61K38/465C12Y301/03001A61P7/00A61P29/00C12Q1/42G01N33/5029G01N2333/916
Inventor 姜媛媛贾潇潇郭田田王家麒李鑫荣崔加友吴书音赵方圆惠鑫瑶惠觅宙
Owner 李鑫荣
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