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Engineering-transformed macrophages and application of macrophages to septicopyemia

A technology of macrophages and inflammation, applied in the field of biomedicine, can solve the problems of difficult anti-inflammatory effects, short effective time of about 2-10 minutes, etc.

Active Publication Date: 2019-03-29
THE FIFTH MEDICAL CENT OF CHINESE PLA GENERAL HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, macrophages do not secrete IL-4 themselves, and the half-life of IL-4 in the body is very short, the effective time is about 2-10 minutes, and it is easy to be cleared by the body, which makes it difficult for direct injection of IL-4 to play a role in the body anti-inflammatory effect

Method used

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  • Engineering-transformed macrophages and application of macrophages to septicopyemia
  • Engineering-transformed macrophages and application of macrophages to septicopyemia
  • Engineering-transformed macrophages and application of macrophages to septicopyemia

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Example 1. Acquisition of a macrophage cell line stably expressing mIL4 protein (i.e. RAW264.7 cells or IL-4-RAW cells infected with lentivirus pCDH-mIL4) and detection of its secreted mIL4 protein

[0052]1. Construction of recombinant plasmid pCDH-mIL4

[0053] Insert the mIL4 gene between the recognition site of the restriction endonuclease EcoRI and the BamHI recognition site of the vector pCDH-MSCV-MCS-EF1α-copGFP-T2A-Puro (the nucleotide sequence is shown in sequence 1 in the sequence listing) , to obtain the recombinant plasmid pCDH-mIL4.

[0054] The recombinant plasmid pCDH-mIL4 expresses mIL4 protein, and its amino acid sequence is shown in sequence 2 in the sequence listing.

[0055] 2. Obtaining the lentivirus pCDH-mIL4 concentrate

[0056] 1. Inoculate the 293T cells grown to the logarithmic growth phase in a petri dish (60 mm in diameter) filled with 10 mL of conventional medium, at 37 °C, 5% CO 2 nourish.

[0057] 2. Gently mix 1 μg pVSVg, 3 μg psPAX2...

Embodiment 2

[0089] Embodiment 2, the application of IL-4-RAW cell

[0090] C57BL / 6 wild-type mice are products of Beijing Huafukang Biotechnology Co., Ltd.

[0091] PBS buffer is pH7.2-7.4, 67mM PO 4 (Ca and Mg free) PBS buffer.

[0092]1. Take 24 C57BL / 6 wild-type mice at 6-8 weeks, and inject LPS intraperitoneally (the mouse sepsis model is prepared by intraperitoneally injecting LPS), and the injection dose is 20 mg / kg.

[0093] 2. One hour after completing step 1, 24 C57BL / 6 wild-type mice were randomly divided into three groups (8 mice in each group) of PBS group, CON-RAW group and IL-4-RAW group. Then proceed as follows:

[0094] PBS group (negative control group): each mouse was injected with 200 μL PBS buffer solution into the tail vein.

[0095] CON-RAW group (negative control group): Each mouse was injected with 200 μL of CON-RAW cell suspension into the tail vein.

[0096] The preparation method of CON-RAW cell suspension is as follows: collect CON-RAW cells, and then resu...

Embodiment 3

[0103] Example 3, IL-4-RAW cells release interleukin-4 for a long time

[0104] Female healthy C57BL / 6J mice are products of Beijing Huafukang Biotechnology Co., Ltd.

[0105] PBS buffer is pH7.2-7.4, 67mM PO 4 (Ca and Mg free) PBS buffer.

[0106] 1. Take 12 female healthy C57BL / 6J mice of 7-8 weeks, inject ketamine (for anesthesia) into each mouse intraperitoneally, and the injection dose is 220 mg / kg; On the stage, the neck hair was cut off, disinfected, and the skin of the neck was cut through a longitudinal incision to fully expose the trachea. A syringe was inserted into the trachea between the two tracheal cartilage rings, and LPS solution was instilled at a dose of 15 mg / kg.

[0107] 2. After completing the step 10.5 hours, 12 female healthy C57BL / 6J mice were randomly divided into three groups (4 mice in each group) of PBS group, IL-4 group and IL-4-RAW group. Then proceed as follows:

[0108] PBS group (negative control group): 25 μL of PBS buffer was injected in...

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Abstract

The present invention discloses a method of preparing macrophages expressing mIL4 protein. The method comprises the steps: enabling macrophages to express mIL4 protein so as to obtain macrophages expressing the mIL4 protein. According to the steps of the method, the inventor prepares RAW264.7 cells infected with lentivirus pCDH-mIL4. Experiments show that the survival rate of mice is significantlyincreased after the injection of the RAW264.7 cells infected with the lentivirus pCDH-mIL4 into the caudal vein of mouse septicopyemia model; and the survival rate of the mice is also significantly increased after the injection of the RAW264.7 cells infected with the lentivirus pCDH-mIL4 into the trachea of mice with acute lung injury. Therefore, the RAW264.7 cells infected with the lentivirus pCDH-mIL4 can treat septicopyemia and acute lung injury. The RAW264.7 cells have important application value.

Description

technical field [0001] The invention belongs to the field of biomedicine, and in particular relates to engineered macrophages and their application in sepsis. Background technique [0002] Sepsis is a common clinical emergency and severe disease with a high mortality rate. So far, there is still a lack of drugs and treatments with high efficacy and few side effects. Bacterial infection is the most common cause of sepsis. Bacterial products, such as LPS and exotoxins, which are components of the bacterial outer wall, can directly or indirectly stimulate monocytes, macrophages, polymorphonuclear neutrophils, etc. to initiate the inflammatory process and produce a large amount of TNF-α, IL-6, Excessive secretion of pro-inflammatory cytokines such as IL-1β leads to severe inflammatory reactions, resulting in septic shock and even the death of the body. Inhibiting the inflammatory response of macrophages has become an important direction for the prevention and treatment of seps...

Claims

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Application Information

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IPC IPC(8): C12N5/10C12N15/867C12N15/24
CPCC12N5/0645C12N15/86C07K14/5406C12N2510/02C12N2740/15043Y02A50/30
Inventor 张硌夏夏陆琤刘慧莹柏长青何园张鹏飞周晨辰张鹏李巍
Owner THE FIFTH MEDICAL CENT OF CHINESE PLA GENERAL HOSPITAL
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