CN104013635B - Application of substance for inhibiting LSECtin in preparation of medicine for treating and/or preventing viral hepatitis B - Google Patents
Application of substance for inhibiting LSECtin in preparation of medicine for treating and/or preventing viral hepatitis B Download PDFInfo
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Abstract
The invention discloses an application of a substance for inhibiting LSECtin in preparing a medicament for treating and/or preventing virus B hepatitis. The invention provides an application of a substance for inhibiting LSECtin in preparing a product with the following purposes: (a) treating and/or preventing viral hepatitis B; (b) promoting the elimination of hepatitis B virus antigen in the body of the hepatitis B virus infected person; (c) treatment and/or prevention of infection by HBV hepatitis b virus; (d) inhibiting viral hepatitis b virus replication; (e) promoting the proportion of CD25 positive cells in liver lymphocytes to be increased; (f) promoting the proportion of CD62L positive cells in liver lymphocytes to be reduced; (g) promoting the reduction of the proportion of CD127 positive cells in liver lymphocytes; (h) promoting the proportion of cells secreting interferon-gamma in liver lymphocytes to be increased. The invention has great social significance and economic value.
Description
Technical field
The present invention relates to the material for suppressing LSECtin at preparation treatment and/or the medicine of prevention hepatitis B
Application in thing.
Background technology
Hepatitis B is called for short hepatitis B, is to be infected a kind of common transmittable disease caused by hepatitis B virus (HBV).
The whole world there are about 2,000,000,000 HBV infection persons at present, and China belongs to the high Endemic Area of HBV infection.Although preventative vaccine was used in nineteen eighty-two
Clinic, but still have more than the chronic carriers of 3.5 hundred million every year, wherein 75% it is distributed in the Asian-Pacific area.Chronic carriers is faced with
Development liver function decompensation, liver cirrhosis or the risk of hepatocarcinoma, estimate that the annual death toll in the whole world has about 1,000,000.
For chronic carriers, the most thoroughly remove HBV and remain very stubborn problem.So far, for chronic viral hepatitis B
Drug main interferon-ALPHA to be included, Peg-IFN alpha-2b α and the nucleoside analog for the treatment of (nucleoside analog is according to structure not
With point 3 classes).Interferon is only effective to the patient of 20-40%, but also can bring many side effect.The side effect of nucleoside analog
Less, but the drug resistance sudden change that long-term prescription brings makes curative effect have a greatly reduced quality, and discontinues medication and causes new round virus replication
Thus increase the weight of hepatic disease.
Treating hepatitis B will be through following three " mountains ": viral persistence exists in vivo；Liver pathology is abnormal, and (liver is scorching
Disease, necrosis, fibrosis etc.)；The immunoreation of body anti-hepatitis virus is disorderly.Only suppression virus replication cannot recover impaired exempting from
Epidemic disease system, must improve body disease-resistant poison immunologic function further, i.e. combine on the basis of antiviral and liver protecting therapy by external force
Effective immune modulating treatment, helps patient that the Virus mutation of HBeAg and HBsAg occurs, is thus capable of sufficiently recovering the antiviral of patient
Immunne response, the purpose be finally reached and persistently remove virus, recovered body protective immunity.At " the chronic type b that China issues
Hepatitis guideline of prevention and treatment " in explicitly point out " one of the important means that immune modulating treatment is treatment of chronic ".Therefore lead to
Cross the effective activating immune system of immune modulating treatment, break immunity of organism tolerance, strengthen specificity antivirus immunologic function, for
Control HBV persistently to replicate, obtain continued viral response and final virus sweep is extremely important.
Development immunoregulation medicament, enhancing Anti-HBV activity immunne response are a kind of inexorable trends.Existing immunoregulation medicament is such as
Under: (1) DC vaccine: in Chronic Hepatitis B body, quantity and the function of DC are the most defective, and it is disease-resistant that this directly affects effective CTL
Poison reaction, feeds back to patient hence with external sensitization DC of HBsAg and/or HBcAg again, can improve the antigen presentation function of DC,
Thus strengthen the reaction of HBV specific CTL；(2) CIK cell treatment: by the most multiple for patient self PERIPHERAL BLOOD MONONUCLEAR CELL
Cytokine obtains, after stimulating, the immunologically competent cell that a group is powerful, then feeds back in the patient, can not damage machine
On the premise of body immune system 26S Proteasome Structure and Function, direct killing hepatopathy cell, and regulate and the immunologic function of enhancing body, for
Treating chronic hepatitis B provides new approach；(3) immunity negative regulatory molecule therapy is closed: the reaction of HBV specific T cell is impaired is machine
Body cannot one of the key reason that remove pathogen, and immunity negative regulatory molecule be the key that promotes T cell function to exhaust because of
Element, therefore with T cell immunity negative regulatory molecule as target, closes its suppression function thus reverses immunity of organism inhibitory state and exist
Causing scientific research widely in the world, corresponding clinical practice is pointed the day and await for it.
LSECtin(Liver Sinusoidal Endothelial Cells lectin) it is II type transmembrane glycoprotein, position
In mankind 19p13.3, it is the newcomer of c-type lectin family.
Summary of the invention
It is an object of the invention to provide the material for suppressing LSECtin at preparation treatment and/or prevention B virus
Application in the medicine of hepatitis.
The invention provides the material suppressing LSECtin application in preparing product；The purposes of described product is as follows
At least one in (a) and (b), (c), (d), (e), (f), (g) and (h):
A () treats and/or prevents hepatitis B；
B () promotes the removing of the hepatitis B virus antigen in Hepatitis B Virus Infection body；
C () treats and/or prevents the infection of HBV hepatitis B virus；
(d) suppression hepatitis B virus replication；
E () promotes the ratio rising in liver lymphocyte shared by CD25 positive cell；
F () promotes the ratio reduction in liver lymphocyte shared by CD62L positive cell；
G () promotes the ratio reduction in liver lymphocyte shared by CD127 positive cell；
H () promotes to secrete the ratio shared by the cell of interferon-γ in liver lymphocyte and raises.
Function that described material of LSECtin " suppression " concretely blocks described LSECtin or the thing suppressing it to express
Matter.
In described (b), described hepatitis B virus antigen can be HBeAg(HBeAg), HBcAg(hepatitis B core
Antigen) and HBsAg(hepatitis B surface antigen) at least one.
Described LSECtin can be following (1) or (2):
(1) protein being made up of the aminoacid sequence shown in sequence in sequence table 1；
(2) aminoacid sequence of sequence 1 through replacement and/or the disappearance of one or several amino acid residue and/or is added
Add and have the protein derived by sequence 1 of lectins function.
Described for suppressing the material concretely GlcNAc β 1-2Man of LSECtin.
GlcNAc β 1-2Man molecular formula is C14H25NO11, also known as b1-2N-Acetylglucosamine-mannose or β
1-2N-Acetylglucosamine-mannose, molecular weight is 383.35, and CAS registration number is 34621-73-3, can be from business
Approach is commercially available.
The present invention also protects a kind of product, and its active component is the material for suppressing LSECtin；The purposes of described product
For at least one in following (a) and (b), (c), (d), (e), (f), (g) and (h):
A () treats and/or prevents hepatitis B；
B () promotes the removing of the hepatitis B virus antigen in Hepatitis B Virus Infection body；
C () treats and/or prevents the infection of HBV hepatitis B virus；
(d) suppression hepatitis B virus replication；
E () promotes the ratio rising in liver lymphocyte shared by CD25 positive cell；
F () promotes the ratio reduction in liver lymphocyte shared by CD62L positive cell；
G () promotes the ratio reduction in liver lymphocyte shared by CD127 positive cell；
H () promotes to secrete the ratio shared by the cell of interferon-γ in liver lymphocyte and raises.
Function that described material of LSECtin " suppression " concretely blocks described LSECtin or the thing suppressing it to express
Matter.
In described (b), described hepatitis B virus antigen can be HBeAg(HBeAg), HBcAg(hepatitis B core
Antigen) and HBsAg(hepatitis B surface antigen) at least one.
Described LSECtin can be following (1) or (2):
(1) protein being made up of the aminoacid sequence shown in sequence in sequence table 1；
(2) aminoacid sequence of sequence 1 through replacement and/or the disappearance of one or several amino acid residue and/or is added
Add and have the protein derived by sequence 1 of lectins function.
Described for suppressing the material concretely GlcNAc β 1-2Man of LSECtin.
GlcNAc β 1-2Man molecular formula is C14H25NO11, also known as b1-2N-Acetylglucosamine-mannose or β
1-2N-Acetylglucosamine-mannose, molecular weight is 383.35, and CAS registration number is 34621-73-3, can be from business
Approach is commercially available.
The present invention also protects described LSECtin as the application of the target of suppression hepatitis B virus replication.
The present invention also protects described LSECtin as target spot application in auxiliary development or deisgn product；
The purposes of described product is at least one in following (a) and (b), (c), (d), (e), (f), (g) and (h):
A () treats and/or prevents hepatitis B；
B () promotes the removing of the hepatitis B virus antigen in Hepatitis B Virus Infection body；
C () treats and/or prevents the infection of HBV hepatitis B virus；
(d) suppression hepatitis B virus replication；
E () promotes the ratio rising in liver lymphocyte shared by CD25 positive cell；
F () promotes the ratio reduction in liver lymphocyte shared by CD62L positive cell；
G () promotes the ratio reduction in liver lymphocyte shared by CD127 positive cell；
H () promotes to secrete the ratio shared by the cell of interferon-γ in liver lymphocyte and raises.
In described application, described " as target spot " concretely blocks the function of described LSECtin or suppresses it to express.
The present invention is experimentally confirmed: after the function of (1) suppression LSECtin, HBV infection Mice Body inner virus antigen is removed
Accelerating, show as S antigen, the level of E antigen and C antigen declines, and the positive rate of HBsAg reduces；(2) by the function of LSECtin
Inhibitor GlcNAc β 1-2Man blocks the function of LSECtin, and HBV infection Mice Body inner virus antigen is removed and accelerated；(3)
The function of LSECtin suppression liver inner virus specificity T so that it is the ability of secretion interferon-γ reduces, thus delay liver virus
Removing；With T cell immunity negative regulatory molecule LSECtin as target, close its suppression function, strengthen HBV specific T-cells
Function, can accelerate liver inner virus and remove.
The present invention has huge social meaning and economic worth.
Accompanying drawing explanation
Fig. 1 is the HBsAg concentration in 20 times of diluents of the serum of first group of mice in embodiment 1.
Fig. 2 is the HBeAg concentration in 20 times of diluents of the serum of first group of mice in embodiment 1.
Fig. 3 is the photo of the SABC of first group of mice in embodiment 1.
Fig. 4 is the HBsAg concentration in 20 times of diluents of the serum of second group of mice in embodiment 1.
Fig. 5 is the HBeAg concentration in 20 times of diluents of the serum of second group of mice in embodiment 1.
Fig. 6 is the photo of the SABC of second group of mice in embodiment 1.
Fig. 7 is the dynamic change of HBsAg positive rate in embodiment 1.
Fig. 8 is HBsAg and the HBeAg concentration in 20 times of diluents of the serum of first group of mice in embodiment 2.
Fig. 9 is HBsAg and the HBeAg concentration in 20 times of diluents of the serum of second group of mice in embodiment 2.
Figure 10 is that the HBsAg positive rate of second group of mice in embodiment 2 dynamically changes.
Figure 11 is the SABC photo of second group of mice in embodiment 2.
Figure 12 is HBsAg and the HBeAg concentration in 20 times of diluents of the serum of the 3rd group of mice in embodiment 2.
Figure 13 is that in embodiment 2, the HBsAg positive rate of the 3rd group of mice dynamically changes.
Figure 14 is HBsAg and the HBeAg concentration in 20 times of diluents of the serum of embodiment 3 small mouse.
Figure 15 is that the HBsAg positive rate of embodiment 3 small mouse dynamically changes.
Figure 16 is the flow cytomery result in embodiment 4.
Figure 17 is the situation of the liver lymphocyte secretion interferon-γ in embodiment 4.
Detailed description of the invention
Below example facilitates a better understanding of the present invention, but does not limit the present invention.Experiment in following embodiment
Method, if no special instructions, is conventional method.Test material used in following embodiment, if no special instructions, is certainly
Routine biochemistry reagent shop is commercially available.Quantitative test in following example, is respectively provided with three times and repeats experiment, and result is made even
Average.HBeAg represents HBeAg.HBcAg represents hepatitis B virus core antigen.HBsAg represents hepatitis B surface antigen.
C57BL/6 mice: body weight 20 ± 2 grams, 6-8 week, purchased from Fukang company of China.BalB/c mice: body weight 20 ± 2 grams,
6-8 week, purchased from Fukang company of China.The Mus source CD8 antibody of APC labelling: purchased from eBioscience company, article No. is 17-0081-
82.The Mus source CD25 antibody of FITC labelling: purchased from eBioscience company, article No. is 12-0251-82.The Mus source of PE labelling
CD62L antibody: purchased from eBioscience company, article No. is 12-0621-82.The Mus source CD127 antibody of PE labelling: be purchased from
EBioscience company, article No. is 12-1271-82.Mouse interferon-γ detects ELISPOT test kit: public purchased from Mabtech
Department, article No. is 3321-2AW-Plus.HBcAg antibody: purchased from company of Zhong Shan Golden Bridge, article No. is ZM-0421.GlcNAcβ1-
2Man: purchased from Dextra company, article No. is M292, and molecular formula is C14H25NO11, molecular weight is 383.35.
Balb/c LSECtin knock-out mice (i.e. " LSECtin in document-/-Mice "): list of references: Yunfei
Zuoetc, Novel roles of liver sinusoidal endothelial cell lectin in colon
Carcinoma cell adhesion, migration and in-vivo metastasis to the liver,
Published by group.bmj.com, published on May25,2012as10.1136/gutjnl-2011-
300593。
(i.e. " the LSECtin KO Mice " in document, sees reference in document C57BL/6LSECtin knock-out mice
" Generation of LSECtin KO Mice " part): list of references: Liver sinusoidal endothelial
cell lectin,LSECtin,negatively regulates hepatic T-cell immune
response.Gastroenterology,2009,137.4:1498-1508。
Plasmid pBS-HBV1.3: list of references: thesis for the doctorate " foundation of Chronic Hepatitis B Virus infecting mouse model and
Preliminary Applications ", doctor: Li Lei, tutor: Yang Dongliang.After plasmid pBS-HBV1.3 imports Balb/c mice, express HBV virus,
Promote Balb/c mice generation B virus acute hepatitis.
Plasmid pAAV/HBV1.2: list of references: An immunocompetent mouse model for the
tolerance of human chronic hepatitis B virus infection.PNAS,2006,103(47):
17862–17867..After plasmid pAAV/HBV1.2 imports Balb/c mice, express HBV virus, promote Balb/c mice generation second
The viral acute hepatitis of type.After plasmid pAAV/HBV1.2 imports C57BL/6 mice, express HBV virus, promote C57BL/6 mice
There is B virus chronic hepatitis.
GlcNAc β 1-2Man solution used in embodiment is all to obtain with PBS dilution GlcNAc β 1-2Man
's.
PBS is made up of solute and solvent, and solvent is that water, solute and concentration thereof are as follows: 135mM NaCl, 2.7mM
KCl、1.5mM KH2PO4With 8mM K2HPO4；pH7.2.
Embodiment 1, set up HBV infection mouse model
One, the animal model of HBV infection Balb/c mice is set up
First group: adjust the concentration of plasmid pBS-HBV1.3 with normal saline, in tail vein 5sec, then inject Balb/
C mice (uses the injection system of tail vein injection, plasmid enters liver the most entirely), every injected in mice 10 g plasmid,
Injection is 8% with the mass ratio of mice；
Second group: adjust the concentration of plasmid pAAV/HBV1.2 with normal saline, then inject in tail vein 5sec
Balb/c mice, every injected in mice 10 g plasmid, injection is 8% with the mass ratio of mice.
Starting timing from injection, every 24 hours is 1 day, respectively at experiment the 1st day (test the 1st day and i.e. injects after 24 hours,
The like), within the 4th day, the 5th day, the 6th day, the 7th day and the 10th day, take serum, send after being diluted to 20 times of volumes with PBS
Concentration (being all to use Roche Cobas automatic tester to detect) to hospital of PLA 307 detection HBsAg and HBeAg.
The concentration of HBsAg and HBeAg is all with light emission intensity (COI) numerical representation method.In 20 times of diluents of the serum of first group of mice
HBsAg concentration see Fig. 1 (meansigma methodss of 10 mices), the HBeAg concentration in 20 times of diluents of serum is shown in Fig. 2, and (10 are little
The meansigma methods of Mus).
Starting timing from injection, every 24 hours is 1 day, respectively at experiment the 2nd day (test the 2nd day and i.e. injects after 48 hours,
The like), the 4th day, the 7th day and the 10th day put to death mice, take liver, with formalin fixing after make paraffin section, adopt
SABC is carried out with HBcAg antibody.The photo of the SABC of first group of mice is shown in that Fig. 3 is shown in by photo.
Starting timing from injection, every 24 hours is 1 day, respectively at experiment the 1st day (test the 1st day and i.e. injects after 24 hours,
The like), within the 3rd day, the 5th day, the 6th day, the 10th day, the 15th day, the 21st day and the 28th day, take serum, dilute with PBS
Release to 20 times of volumes deliver to hospital of PLA 307 detection HBsAg concentration (use Roche Cobas automatic tester examine
Survey).The concentration of HBsAg is with light emission intensity (COI) numerical representation method.In 20 times of diluents of the serum of second group of mice
HBsAg concentration is shown in Fig. 4 (meansigma methodss of 10 mices).
Starting timing from injection, every 24 hours is 1 day, respectively at experiment the 0th day (i.e. before injection), (experiment the 1st in the 1st day
After it i.e. injects 24 hours, the like), within the 3rd day, the 5th day, the 7th day, the 10th day and the 15th day, take serum, deliver to PLA
The concentration (being all to use Roche Cobas automatic tester to detect) of 307 hospital detection HBeAg.The concentration of HBeAg is put with light
Penetrate intensity (COI) numerical representation method.HBeAg concentration in 20 times of diluents of the serum of second group of mice see Fig. 5 (10 mices
Meansigma methods).
Starting timing from injection, every 24 hours is 1 day, respectively at experiment the 1st day (test the 1st day and i.e. injects after 24 hours,
The like), within the 3rd day, the 7th day and the 14th day, respectively put to death mice, take liver, with formalin fixing after make paraffin section,
HBcAg antibody is used to carry out SABC.The photo of the SABC of second group of mice is shown in that Fig. 6 is shown in by photo.
Two, the animal model of HBV infection C57BL/6 mice is set up
Adjust the concentration of plasmid pAAV/HBV1.2 with normal saline, in tail vein 5sec, then inject C57BL/6 little
Mus, every injected in mice 10 g plasmid, injection is 8% with the mass ratio of mice.7 C57BL/6 mices of injection altogether.
Starting timing from injection, every 24 hours is 1 day, the 28th day respectively at experiment (tests and i.e. injects 24 × 28 on the 28th day
After hour, the like), the 49th day, the 63rd day, the 91st day, the 112nd day, the 140th day, the 168th day, the 196th day, the 224th
My god, within the 252nd day, the 280th day, the 308th day, the 364th day and the 448th day, take serum, after being diluted to 20 times of volumes with PBS
Deliver to the concentration (using Roche Cobas automatic tester to detect) of hospital of PLA 307 detection HBsAg.The concentration of HBsAg
With light emission intensity (COI) numerical representation method.If the COI number of the sign HBsAg concentration in the 20 of the serum of mice times of diluents
Value is more than 1, and testing result is positive.The number of elements ÷ 7 × 100% of the mice of test positive in the mice of HBsAg positive rate=7.
Fig. 7 is shown in the dynamically change of HBsAg positive rate.
The result of step one and step 2 shows: plasmid pBS-HBV1.3 or plasmid pAAV/HBV1.2 injection Balb/c is little
After Mus, all can be successfully established the mouse model of B virus acute hepatitis；Plasmid pAAV/HBV1.2 injection C57BL/6 is little
After Mus, the mouse model of B virus chronic hepatitis can be successfully established.
Embodiment 2, suppression LSECtin function after, HBV infection Mice Body inner virus antigen remove accelerate
First group: adjust the concentration of plasmid pBS-HBV1.3 with normal saline, in tail vein 5sec, then inject Balb/
C mice or Balb/c LSECtin knock-out mice, every injected in mice 10 g plasmid, injection with the mass ratio of mice is
8%；
Second group: adjust the concentration of plasmid pAAV/HBV1.2 with normal saline, then inject in tail vein 5sec
The quality of Balb/c mice or Balb/c LSECtin knock-out mice, every injected in mice 10 g plasmid, injection and mice
Ratio is 8%；
3rd group: adjust the concentration of plasmid pAAV/HBV1.2 with normal saline, then inject in tail vein 5sec
The quality of C57BL/6 mice or C57BL/6LSECtin knock-out mice, every injected in mice 10 g plasmid, injection and mice
Ratio is 8%.
First group of mice, starts timing from injection, and every 24 hours is 1 day, the 1st day respectively at experiment (tests and i.e. notes for the 1st day
After penetrating 24 hours, the like), within the 4th day, the 5th day, the 6th day and the 7th day, take serum, be diluted to 20 times of volumes with PBS
After deliver to hospital of PLA 307 detection HBsAg and HBeAg concentration.The concentration of HBsAg and HBeAg is all with light emission intensity
(COI) numerical representation method.HBsAg and HBeAg testing result in 20 times of diluents of the serum of mice is shown in Fig. 8 (each filled circles
Point represents a Balb/c LSECtin knock-out mice, and each soft dot represents a Balb/c mice).
Second group of mice, starts timing from injection, and every 24 hours is 1 day, the 7th day respectively at experiment (tests and i.e. notes for the 7th day
After penetrating 24 × 7 hours, the like), within the 10th day, the 15th day, the 21st day and the 28th day, take serum, be diluted to PBS
The concentration of hospital of PLA 307 detection HBsAg and HBeAg is delivered to after 20 times of volumes.The concentration of HBsAg and HBeAg is all put with light
Penetrate intensity (COI) numerical representation method.HBsAg and HBeAg testing result in 20 times of diluents of the serum of mice is shown in Fig. 9 (light gray
The pillar of color fill color represents the meansigma methods of 10 Balb/c mices, and the pillar of filled black color represents 6 Balb/c
The meansigma methods of LSECtin knock-out mice).Respectively at experiment the 1st day, the 5th day, the 7th day, the 10th day, the 15th day, the 21st day and the
27 days statistics HBsAg positive rates, the HBsAg positive rate of 10 Balb/c mices dynamically changes (soft dot) and 6 Balb/c
The HBsAg positive rate of LSECtin knock-out mice dynamically changes (black circle) and sees Figure 10.
Second group of mice: starting timing from injection, every 24 hours is 1 day, the 3rd day respectively at experiment (tests and i.e. notes for the 3rd day
After penetrating 24 × 3 hours, the like), the 7th day and the 14th day put to death mice, take liver, with formalin fixing after make paraffin
Section, uses HBcAg antibody to carry out SABC.The photo of SABC is shown in that Figure 11 is shown in by photo.
3rd group of mice, starts timing from injection, and every 24 hours is 1 day, the 7th day respectively at experiment (tests and i.e. notes for the 7th day
After penetrating 24 × 7 hours, the like), the 14th day, the 21st day, the 28th day, the 35th day, the 42nd day, the 49th day, the 56th day, the 63rd
My god, within the 70th day, the 77th day and the 84th day, take serum, deliver to after being diluted to 20 times of volumes with PBS hospital of PLA 307 inspection
Survey the concentration of HBsAg.The concentration of HBsAg is with light emission intensity (COI) numerical representation method.In 20 times of diluents of the serum of mice
The concentration of HBsAg is shown in that (soft dot represents the meansigma methods of 8 C57BL/6 mices to Figure 12, and black circle represents 9 C57BL/
The meansigma methods of 6LSECtin knock-out mice).Respectively at experiment the 1st day, the 14th day, the 21st day, the 28th day, the 35th day, the 42nd day,
49th day, the 56th day, the 63rd day, the 70th day, the 77th day and the 84th day statistics HBsAg positive rate, 8 C57BL/6 mices
HBsAg positive rate dynamically changes the HBsAg positive rate of (soft dot) and 9 C57BL/6LSECtin knock-out mices and dynamically changes
(black circle) is shown in Figure 13.
Result above shows, in LSECtin knock-out mice body, HBV antigen is removed and dramatically speeded up, and i.e. suppresses the merit of LSECtin
After energy, HBV infection Mice Body inner virus antigen is removed and is accelerated.
Embodiment 3, GlcNAc β 1-2Man promote HBV infection Mice Body inner virus antigen by the function blocking LSECtin
Remove and accelerate
1, experiment the 1st day, adjusts the concentration of plasmid pAAV/HBV1.2, then notes in tail vein 5sec with normal saline
Penetrating C57BL/6 mice, every injected in mice 10 g plasmid, injection is 8% with the mass ratio of mice.
2, experiment the 4th day, is divided into two groups by the mice of step 1, carries out following parallel processing respectively:
Experimental group (10): 0.2ml13 μM of GlcNAc β 1-2Man solution of every mice lumbar injection every day, injects continuously
To experiment the 42nd day；
Matched group (9): every mice lumbar injection every day 0.2ml PBS, is injected to test the 42nd day continuously.
From injection start timing, respectively at the 7th day, the 14th day, the 21st day, the 28th day, the 35th day, the 49th day, the 56th day,
63rd day, the 70th day, the 84th day and the 98th day take serum, deliver to PLA 307 and cure after being diluted to 20 times of volumes with PBS
The concentration of institute detection HBsAg and HBeAg.The concentration of HBsAg and HBeAg is all with light emission intensity (COI) numerical representation method.Mice
HBsAg and HBeAg testing result in 20 times of diluents of serum is shown in that (black circle represents the average of 10 experimental mice to Figure 14
Value, soft dot represents the meansigma methods of 9 control group mice).Respectively at the 7th day, the 14th day, the 21st day, the 28th day, the 35th
My god, the 49th day, the 56th day, the 63rd day, the 70th day, the 84th day and the 98th day statistics HBsAg positive rate, 9 control group mice
HBsAg positive rate dynamically changes the HBsAg positive rate of (soft dot) and 10 experimental mice and dynamically changes (black circle)
See Figure 15.
Result above shows, gives to have blocked after GlcNAc β 1-2Man processes the function of LSECtin, HBV infection Mice Body
Inner virus antigen is removed and is accelerated, and HBsAg positive rate reduces.
Embodiment 4, LSECtin delay the mechanism that virus antigen is removed
Adjust the concentration of plasmid pAAV/HBV1.2 with normal saline, in tail vein 5sec, then inject Balb/c mice
Or each 6 of Balb/c LSECtin knock-out mice, every injected in mice 10 g plasmid, injection with the mass ratio of mice is
8%。
After 10 days, by density gradient centrifugation separation liver lymphocyte.
With the expression of CD25, CD62L and CD127 in flow cytomery liver lymphocyte (CD8+T cell).
Result is shown in Figure 16.
Liver lymphocyte is seeded to Tissue Culture Plate, 3 × 105Individual cells/well, with the table of final concentration of 10ug/ml
CAg peptide (87-95:SYVNTNMGL) thorn of face antigenic peptides (28 39:IPQSLDSWWTSL) and final concentration of 10ug/ml
After Jiing use mouse interferon-γ detection ELISPOT test kit and press test kit description detection liver lymphocyte secrete do
Disturb the situation of element-γ.
Figure 17 A is shown in by photo.The number of the cell secreting interferon-γ in every hole is shown in Figure 17 B.Every cubic millimeter of average blob
Size is shown in Figure 17 C.
Result above shows: after the function of suppression LSECtin, and in liver lymphocyte (CD8+T cell), CD25 is positive thin
Born of the same parents are more, CD62L positive cell less, CD127 positive cell is less, and the cell of secretion interferon-γ is more.
Claims (5)
1. the material of suppression LSECtin application in preparing product；The purposes of described product be following (a), (b), (c),
At least one in (d), (e), (f), (g) and (h):
A () treats and/or prevents hepatitis B；
B () promotes the removing of the hepatitis B virus antigen in Hepatitis B Virus Infection body；
C () treats and/or prevents the infection of HBV hepatitis B virus；
(d) suppression hepatitis B virus replication；
E () promotes the ratio rising in liver lymphocyte shared by CD25 positive cell；
F () promotes the ratio reduction in liver lymphocyte shared by CD62L positive cell；
G () promotes the ratio reduction in liver lymphocyte shared by CD127 positive cell；
H () promotes to secrete the ratio shared by the cell of interferon-γ in liver lymphocyte and raises.
2. the method for claim 1, it is characterised in that: in described (b), described hepatitis B virus antigen be HBeAg,
At least one in HBcAg and HBsAg.
Apply the most as claimed in claim 1 or 2, it is characterised in that: described LSECtin is following (1) or (2):
(1) protein being made up of the aminoacid sequence shown in sequence in sequence table 1；
(2) by the aminoacid sequence of sequence 1 through the replacement of one or several amino acid residue and/or disappearance and/or interpolation and
There is the protein derived by sequence 1 of lectins function.
4. the application as described in claim 1 or 2 or 3, it is characterised in that: described for suppressing the material of LSECtin to be
GlcNAc β 1-2Man, its molecular formula is C14H25NO11, CAS registration number is 34621-73-3.
5.LSECtin is as target spot application in auxiliary development or deisgn product；The purposes of described product be following (a),
At least one in (b), (c), (d), (e), (f), (g) and (h):
A () treats and/or prevents hepatitis B；
B () promotes the removing of the hepatitis B virus antigen in Hepatitis B Virus Infection body；
C () treats and/or prevents the infection of HBV hepatitis B virus；
(d) suppression hepatitis B virus replication；
E () promotes the ratio rising in liver lymphocyte shared by CD25 positive cell；
F () promotes the ratio reduction in liver lymphocyte shared by CD62L positive cell；
G () promotes the ratio reduction in liver lymphocyte shared by CD127 positive cell；
H () promotes to secrete the ratio shared by the cell of interferon-γ in liver lymphocyte and raises.
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