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Application of substance for inhibiting CLEC5A in preparation of medicine for treating and/or preventing viral hepatitis B

A viral hepatitis and drug technology, applied in the field of biomedicine, can solve the problems of unclear correlation of infection, etc., and achieve the effect of reduced replication level and high clearance efficiency

Pending Publication Date: 2021-11-30
ACADEMY OF MILITARY MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the correlation between CLEC5A and HBV viral infection remains unclear

Method used

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  • Application of substance for inhibiting CLEC5A in preparation of medicine for treating and/or preventing viral hepatitis B
  • Application of substance for inhibiting CLEC5A in preparation of medicine for treating and/or preventing viral hepatitis B
  • Application of substance for inhibiting CLEC5A in preparation of medicine for treating and/or preventing viral hepatitis B

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1 CLEC5A in THP1 cells promotes the replication of HBV in HepG2.2.15 cells

[0039] To study the function of CLEC5A in macrophages in HBV chronic infection, such as figure 1 As shown, we constructed a non-contact co-culture system of THP-1-induced macrophages and HepG2.2.15 cells. First, THP-1 or control THP-1 cells that interfere with the expression of CLEC5A were induced into M2 macrophages: THP-1 cells were collected and counted, and 2×10 5 THP-1 cells were seeded in a 12-well plate and induced by adding PMA (final concentration: 100 ng / mL) for 24 hours. Then add IL4 (final concentration 20ng / mL) and IL13 (final concentration 20ng / mL) to continue induction for 48h. Second, the 1×10 5 A HepG2.2.15 cell was inoculated into the upper chamber of the co-culture system, and 48-72 cells were co-cultured without contact. Finally, the cell culture supernatant in the co-culture system was collected, centrifuged at 1000 g, and the supernatant was taken to quantitati...

Embodiment 2

[0045] Example 2 CLEC5A in PBMC cells promotes the replication of HBV in HepG2.2.15 cells

[0046] To further confirm the function of CLEC5A in promoting HBV replication, we repeated the experiment in human peripheral blood mononuclear cells (PBMC). Such as Figure 4 As shown, we constructed a non-contact co-culture system of PBMC-induced macrophages and HepG2.2.15 cells. First, fresh human peripheral blood was collected and PBMCs were isolated, and 2×10 5 PBMC cells were inoculated in a 12-well plate, and hrM-CSF (final concentration: 20 ng / mL) was added to induce macrophages for 4 days. The lentivirus that stably knocked down CLEC5A or the lentivirus that stably overexpressed CLEC5A and its control lentivirus were used. The macrophages induced by virus infection of PBMC were sorted by flow cytometry after 48 hours, and the cells positive for green fluorescence were sorted, and the RNA and protein were extracted to detect the knockdown and overexpression effects of CLEC5A. ...

Embodiment 3

[0049] Example 3 CLEC5A in macrophages inhibits the secretion of inflammatory cytokines such as IL-1β during HBV infection

[0050] Further, we used ELISA technology to detect the levels of IL6, IL1β and TNFα in the cell culture supernatant in the co-culture system. First, after knockdown of CLEC5A in THP-1-induced macrophages, the levels of IL6, IL1β and TNFα in the supernatant of the co-culture system were significantly increased ( Figure 7 a-c); After overexpressing CLEC5A in THP-1-induced macrophages, the levels of IL6, IL1β and TNFα in the supernatant of the co-culture system were significantly reduced ( Figure 7 e-f).

[0051] Second, after knockdown of CLEC5A in PBMC-induced macrophages, the levels of IL6, IL1β and TNFα in the supernatant of the co-culture system were significantly increased ( Figure 8a-c); After overexpressing CLEC5A in PBMC-induced macrophages, the levels of IL6, IL1β and TNFα in the supernatant of the co-culture system were significantly reduced...

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Abstract

The invention relates to the field of biological medicine, in particular to application of a substance for inhibiting CLEC5A in preparation of a medicine for treating and / or preventing viral hepatitis B. Experimental results show that after CLEC5A is knocked down in macrophages induced by THP-1 and PBMC, the levels of HBV DNA, HBV RNA, HBsAg and HBeAg in HepG2.2. 15 cells are all remarkably reduced, and the levels of inflammatory cytokines such as IL-1[beta] and the like are remarkably increased; and meanwhile, the removal efficiency of the HBV in the mouse of which the Clec5a gene is knocked out is higher, and the HBV replication level in HepG2.2. 15 cells after Clec5a is knocked out in the sorted mouse macrophages is remarkably reduced.

Description

technical field [0001] The invention relates to the field of biomedicine, in particular to the application of substances for inhibiting CLEC5A in the preparation of medicines for treating and / or preventing hepatitis B virus. Background technique [0002] HBV Infection and Persistence HBV is a heterosexual virus that can cause liver diseases, including acute hepatitis, cirrhosis, liver cancer, and hepatocellular carcinoma (HCC). There are about 250 million people in the world suffering from chronic hepatitis B virus infection, causing nearly 1 million deaths every year, and the HBV infection rate in my country is 60%. Therefore, HBV infection is still a public health problem that endangers people's healthy life for a long time in our country. [0003] Most normal adults newly infected with HBV can effectively clear HBV, but about 5% of adults and most newborns cannot effectively clear the invading HBV after infection, resulting in persistent HBV infection. About 25% of peop...

Claims

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Application Information

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IPC IPC(8): A61K45/00A61P1/16A61P31/20
CPCA61K45/00A61P1/16A61P31/20
Inventor 周钢桥杨爱清曹鹏博
Owner ACADEMY OF MILITARY MEDICAL SCI
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