CN104013635A - Application of substance for inhibiting LSECtin in preparing medicine for treating and/or preventing viral hepatitis B - Google Patents
Application of substance for inhibiting LSECtin in preparing medicine for treating and/or preventing viral hepatitis B Download PDFInfo
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Abstract
The invention discloses application of a substance for inhibiting LSECtin in preparing a medicine for treating and/or preventing viral hepatitis B. The invention provides application of the substance for inhibiting LSECtin in preparing a product with the following purposes: (a) treating and/or preventing viral hepatitis B; (b) promoting elimination of hepatitis B virus antigen in an infected person of hepatitis B virus; (c) treating and/or preventing infection of HBV (Hepatitis B Virus); (d) inhibiting duplication of hepatitis b viral hepatitis virus; (e) promoting increase of proportion of CD25 positive cells in liver lymphocyte; (f) promoting decrease of CD62 positive cells in liver lymphocyte; (g) promoting decrease of proportion of CD127 positive cells in liver lymphocyte; (h) promoting increase of proportion of cells which secrete interferon-gamma in liver lymphocyte. The substance disclosed by the invention has huge social meaning and economic value.
Description
Technical field
The present invention relates to material for suppressing LSECtin treats and/or prevents the medicine of hepatitis B application in preparation.
Background technology
Hepatitis B is called for short hepatitis B, is to infect by hepatitis B virus (HBV) a kind of common transmittable disease causing.Approximately there are 2,000,000,000 HBV the infecteds in the whole world at present, and China belongs to the high Endemic Area that HBV infects.Although preventative vaccine for clinical, still has the chronic carrier that exceedes 3.5 hundred million in nineteen eighty-two every year, wherein 75% is distributed in the Asian-Pacific area.Chronic carrier is faced with the risk of the decompensation of development liver function, liver cirrhosis or hepatocarcinoma, estimates that the annual death toll in the whole world has 100 ten thousand left and right.
For chronic carrier, how thoroughly to remove HBV and remain very stubborn problem.So far, to comprise interferon-ALPHA, Polyethylene Glycol interferon-ALPHA and nucleoside analog (nucleoside analog is according to different points of 3 classes of structure) for the drug main of the treatment of chronic viral hepatitis B.Interferon is only effective to the patient of 20-40%, but also can bring many side effect.The side effect of nucleoside analog is less, but the drug resistance that long-term prescription brings sudden change is had a greatly reduced quality curative effect, causes new round virus replication to increase the weight of hepatic disease thereby discontinue medication.
Treating hepatitis B will pass through following three " mountains ": virus continues to exist in vivo; Hepatopathy abnormal (inflammation, necrosis, fibrosis etc.) of science; The immunoreation disorder of body anti-hepatitis virus.Only suppress virus replication and cannot recover impaired immune system; must further improve body disease-resistant poison immunologic function by external force; on antiviral and liver protecting therapy basis, combine effective immune modulating treatment; help patient that the Virus mutation of HBeAg and HBsAg occurs; fully recover patient's Immune responses of the antivirus, finally reach the lasting object of removing virus, the immunity of recovery body protective.In " the guideline " issued in China, explicitly point out " immune modulating treatment is one of important means for the treatment of of chronic ".Therefore by the effective activating immune system of immune modulating treatment, break immunity of organism tolerance, strengthen specificity antivirus immunologic function, for control HBV continue to copy, obtain continue virological response and final virus sweep extremely important.
Developing immunoregulation medicament, strengthening anti-HBV immunne response is a kind of inexorable trend.Existing immunoregulation medicament is as follows: (1) DC vaccine: quantity and the equal defectiveness of function of DC in Chronic Hepatitis B body, this has directly affected effective CTL antiviral response, therefore utilize the external sensitization DC of HBsAg and/or HBcAg to feed back again to patient, can improve the antigen presentation function of DC, thereby strengthen the reaction of HBV specific CTL; (2) CIK cell therapy: patient self PERIPHERAL BLOOD MONONUCLEAR CELL is obtained to the powerful immunologically competent cell of a group after cytokine profiles stimulation in vitro, feed back to again in patient body, can not damage under the prerequisite of body immune system 26S Proteasome Structure and Function, direct killing hepatopathy cell, and the immunologic function of adjusting and enhancing body, for treating chronic hepatitis B provides new approach; (3) seal immune negative regulator molecule therapy: the special t cell responses of HBV is impaired is that body cannot be removed one of key reason of pathogen, and immune negative regulator molecule is the key factor of impelling T cell function to exhaust, therefore taking T cellular immunization negative regulator molecule as target, cause in the world scientific research widely thereby seal its inhibit feature reverse immunity of organism inhibitory state, corresponding clinical practice is within sight.
LSECtin(Liver Sinusoidal Endothelial Cells lectin) be II type transmembrane glycoprotein, be positioned at mankind 19p13.3, be the newcomer of C type lectin family.
Summary of the invention
The object of this invention is to provide material for suppressing LSECtin treats and/or prevents the medicine of hepatitis B application in preparation.
The invention provides and suppress the application in preparing product of the material of LSECtin; The purposes of described product be following (a) and (b), (c), (d), (e), (f), (g) and (h) at least one:
(a) treat and/or prevent hepatitis B;
(b) removing of the hepatitis B virus antigen in promotion Hepatitis B Virus Infection body;
(c) treat and/or prevent the infection of HBV hepatitis B virus;
(d) suppress hepatitis B virus replication;
(e) impel the shared ratio of CD25 positive cell in liver lymphocyte to raise;
(f) impel the shared ratio of CD62L positive cell in liver lymphocyte to reduce;
(g) impel the shared ratio of CD127 positive cell in liver lymphocyte to reduce;
(h) impel the shared ratio of cell of secreting interferon-γ in liver lymphocyte to raise.
Described " suppressing the material of LSECtin " specifically can be the function of the described LSECtin of blocking-up or suppresses the material of its expression.
In described (b), described hepatitis B virus antigen can be HBeAg(HBeAg), HBcAg(hepatitis B virus core antigen) and HBsAg(hepatitis B surface antigen) at least one.
Described LSECtin can be (1) or (2) as follows:
(1) protein being formed by the aminoacid sequence shown in sequence in sequence table 1;
(2) by the aminoacid sequence of sequence 1 through replacement and/or disappearance and/or the interpolation of one or several amino acid residue and there is the protein being derived by sequence 1 of lectins function.
Describedly specifically can be GlcNAc β 1-2Man for suppressing the material of LSECtin.
GlcNAc β 1-2Man molecular formula is C
14h
25nO
11, claiming again b1-2N-Acetylglucosamine-mannose or β 1-2N-Acetylglucosamine-mannose, molecular weight is that 383.35, CAS registration number is 34621-73-3, can buy and obtain from commercial channels.
The present invention also protects a kind of product, and its active component is the material for suppressing LSECtin; The purposes of described product be following (a) and (b), (c), (d), (e), (f), (g) and (h) at least one:
(a) treat and/or prevent hepatitis B;
(b) removing of the hepatitis B virus antigen in promotion Hepatitis B Virus Infection body;
(c) treat and/or prevent the infection of HBV hepatitis B virus;
(d) suppress hepatitis B virus replication;
(e) impel the shared ratio of CD25 positive cell in liver lymphocyte to raise;
(f) impel the shared ratio of CD62L positive cell in liver lymphocyte to reduce;
(g) impel the shared ratio of CD127 positive cell in liver lymphocyte to reduce;
(h) impel the shared ratio of cell of secreting interferon-γ in liver lymphocyte to raise.
Described " suppressing the material of LSECtin " specifically can be the function of the described LSECtin of blocking-up or suppresses the material of its expression.
In described (b), described hepatitis B virus antigen can be HBeAg(HBeAg), HBcAg(hepatitis B virus core antigen) and HBsAg(hepatitis B surface antigen) at least one.
Described LSECtin can be (1) or (2) as follows:
(1) protein being formed by the aminoacid sequence shown in sequence in sequence table 1;
(2) by the aminoacid sequence of sequence 1 through replacement and/or disappearance and/or the interpolation of one or several amino acid residue and there is the protein being derived by sequence 1 of lectins function.
Describedly specifically can be GlcNAc β 1-2Man for suppressing the material of LSECtin.
GlcNAc β 1-2Man molecular formula is C
14h
25nO
11, claiming again b1-2N-Acetylglucosamine-mannose or β 1-2N-Acetylglucosamine-mannose, molecular weight is that 383.35, CAS registration number is 34621-73-3, can buy and obtain from commercial channels.
The present invention also protects the application of described LSECtin as the target of inhibition hepatitis B virus replication.
The present invention also protects described LSECtin application in auxiliary development or deisgn product as target spot;
The purposes of described product be following (a) and (b), (c), (d), (e), (f), (g) and (h) at least one:
(a) treat and/or prevent hepatitis B;
(b) removing of the hepatitis B virus antigen in promotion Hepatitis B Virus Infection body;
(c) treat and/or prevent the infection of HBV hepatitis B virus;
(d) suppress hepatitis B virus replication;
(e) impel the shared ratio of CD25 positive cell in liver lymphocyte to raise;
(f) impel the shared ratio of CD62L positive cell in liver lymphocyte to reduce;
(g) impel the shared ratio of CD127 positive cell in liver lymphocyte to reduce;
(h) impel the shared ratio of cell of secreting interferon-γ in liver lymphocyte to raise.
In described application, described " as target spot " specifically can be the function of the described LSECtin of blocking-up or suppresses its expression.
The present invention proves by experiment: (1) suppresses after the function of LSECtin, and HBV infecting mouse body inner virus antigen is removed and accelerated, and shows as S antigen, and the level of E antigen and C antigen declines, and the positive rate of HBsAg reduces; (2) use the depressant of functions GlcNAc β 1-2Man of LSECtin to block the function of LSECtin, HBV infecting mouse body inner virus antigen is removed and is accelerated; (3) LSECtin suppresses the function of liver inner virus specificity T, and the ability of its secretion interferon-γ is reduced, thereby delays the removing of liver virus; Taking T cellular immunization negative regulator molecule L SECtin as target, seal its inhibit feature, strengthen the function of HBV specific T-cells, can accelerate liver inner virus and remove.
The present invention has huge social meaning and economic worth.
Brief description of the drawings
Fig. 1 is the HBsAg concentration in the 20 times of diluents of serum of first group of mice in embodiment 1.
Fig. 2 is the HBeAg concentration in the 20 times of diluents of 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 the 20 times of diluents of serum of second group of mice in embodiment 1.
Fig. 5 is the HBeAg concentration in the 20 times of diluents of 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 the 20 times of diluents of serum of first group of mice in embodiment 2.
Fig. 9 is HBsAg and the HBeAg concentration in the 20 times of diluents of serum of second group of mice in embodiment 2.
Figure 10 is the HBsAg positive rate dynamic change of second group of mice in embodiment 2.
Figure 11 is the SABC photo of second group of mice in embodiment 2.
Figure 12 is HBsAg and the HBeAg concentration in the 20 times of diluents of serum of the 3rd group of mice in embodiment 2.
Figure 13 is the HBsAg positive rate dynamic change of the 3rd group of mice in embodiment 2.
Figure 14 is HBsAg and the HBeAg concentration in 20 times of diluents of serum of embodiment 3 small mouses.
Figure 15 is the HBsAg positive rate dynamic change of embodiment 3 small mouses.
Figure 16 is the flow cytometer testing result in embodiment 4.
Figure 17 is the situation of the liver lymphocyte secretion interferon-γ in embodiment 4.
Detailed description of the invention
Following embodiment is convenient to understand better the present invention, but does not limit the present invention.Experimental technique in following embodiment, if no special instructions, is conventional method.Test material used in following embodiment, if no special instructions, is and purchases available from routine biochemistry reagent shop.Quantitative test in following examples, all arranges and repeats experiment, results averaged for three times.HBeAg represents HBeAg.HBcAg represents hepatitis B virus core antigen.HBsAg represents hepatitis B surface antigen.
C57BL/6 mice: 20 ± 2 grams of body weight, 6-8 week, purchased from magnificent Fukang company.BalB/c mice: 20 ± 2 grams of body weight, 6-8 week, purchased from magnificent Fukang company.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 CD62L antibody of PE labelling: purchased from eBioscience company, article No. is 12-0621-82.The Mus source CD127 antibody of PE labelling: purchased from eBioscience company, article No. is 12-1271-82.Mouse interferon-γ detects ELISPOT test kit: purchased from Mabtech company, article No. is 3321-2AW-Plus.HBcAg antibody: purchased from company of Zhong Shan Golden Bridge, article No. is ZM-0421.GlcNAc β 1-2Man: purchased from Dextra company, article No. is M292, and molecular formula is C
14h
25nO
11, molecular weight is 383.35.
Balb/c LSECtin knock-out mice (is " 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.
C57BL/6LSECtin knock-out mice (is " the LSECtin KO Mice " in document, " the Generation of LSECtin KO Mice " part seeing reference in document): 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 and the Preliminary Applications of Chronic Hepatitis B Virus infecting mouse model ", doctor: Li Lei, tutor: Yang Dongliang.Plasmid pBS-HBV1.3 imports after Balb/c mice, expresses HBV virus, impels 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..Plasmid pAAV/HBV1.2 imports after Balb/c mice, expresses HBV virus, impels Balb/c mice generation B virus acute hepatitis.Plasmid pAAV/HBV1.2 imports after C57BL/6 mice, expresses HBV virus, impels C57BL/6 mice generation B virus chronic hepatitis.
GlcNAc β 1-2Man solution used in embodiment all obtains with PBS buffer dilution GlcNAc β 1-2Man.
PBS buffer is by solute and solvent composition, and solvent is water, and solute and concentration thereof are as follows: 135mM NaCl, 2.7mM KCl, 1.5mM KH
2pO
4with 8mM K
2hPO
4; PH7.2.
Embodiment 1, set up HBV infecting mouse model
One, set up the animal model of HBV infection Balb/c mice
First group: the concentration of adjusting plasmid pBS-HBV1.3 with normal saline, then in tail vein 5sec, inject Balb/c mice and (adopt the injection system of tail vein injection, plasmid enters liver substantially entirely), every injected in mice 10 microgram plasmids, the mass ratio of injection and mice is 8%;
Second group: adjust the concentration of plasmid pAAV/HBV1.2 with normal saline, then in tail vein 5sec, inject Balb/c mice, every injected in mice 10 microgram plasmids, the mass ratio of injection and mice is 8%.
Start timing from injection, every 24 hours is 1 day, within the 1st day, (test and inject after 24 hours for the 1st day respectively at experiment, the like), within the 4th day, the 5th day, the 6th day, the 7th day and the 10th day, get serum, be diluted to and deliver to 307 hospitals of PLA after 20 times of volumes and detect the concentration of HBsAg and HBeAg (being all to adopt Roche Cobas automatic tester to detect) with PBS buffer.The concentration of HBsAg and HBeAg is all with light emission intensity (COI) numerical representation method.HBsAg concentration in 20 times of diluents of the serum of first group of mice is shown in Fig. 1 (meansigma methodss of 10 mices), and the HBeAg concentration in 20 times of diluents of serum is shown in Fig. 2 (meansigma methodss of 10 mices).
Start timing from injection, every 24 hours is 1 day, within the 2nd day, (test and inject after 48 hours for the 2nd day respectively at experiment, the like), the 4th day, the 7th day and the 10th day put to death mice, get liver, after fixing with formalin, make paraffin section, adopt HBcAg antibody to carry out SABC.The photo of the SABC of first group of mice is shown in that photo is shown in Fig. 3.
Start timing from injection, every 24 hours is 1 day, within the 1st day, (test and inject after 24 hours for the 1st day respectively at experiment, 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, get serum, be diluted to and deliver to 307 hospitals of PLA after 20 times of volumes and detect the concentration of HBsAg (adopting Roche Cobas automatic tester to detect) with PBS buffer.The concentration of HBsAg is with light emission intensity (COI) numerical representation method.HBsAg concentration in 20 times of diluents of the serum of second group of mice is shown in Fig. 4 (meansigma methodss of 10 mices).
Start timing from injection, every 24 hours is 1 day, respectively at testing the 0th day (before injection), within the 1st day, (testing and inject after 24 hours for the 1st day, the like), within the 3rd day, the 5th day, the 7th day, the 10th day and the 15th day, get serum, deliver to 307 hospitals of PLA and detect the concentration of HBeAg (being all to adopt Roche Cobas automatic tester to detect).The concentration of HBeAg is with light emission intensity (COI) numerical representation method.HBeAg concentration in 20 times of diluents of the serum of second group of mice is shown in Fig. 5 (meansigma methodss of 10 mices).
Start timing from injection, every 24 hours is 1 day, within the 1st day, (test and inject after 24 hours for the 1st day respectively at experiment, the like), the 3rd day, the 7th day and the 14th day are each puts to death mice, get liver, after fixing with formalin, make paraffin section, adopt HBcAg antibody to carry out SABC.The photo of the SABC of second group of mice is shown in that photo is shown in Fig. 6.
Two, set up the animal model of HBV infection C57BL/6 mice
Adjust the concentration of plasmid pAAV/HBV1.2 with normal saline, then in tail vein 5sec, inject C57BL/6 mice, every injected in mice 10 microgram plasmids, the mass ratio of injection and mice is 8%.Inject altogether 7 C57BL/6 mices.
Start timing from injection, every 24 hours is 1 day, within the 28th day, (test and inject after 24 × 28 hours for the 28th day respectively at experiment, the like), within the 49th day, the 63rd day, the 91st day, the 112nd day, the 140th day, the 168th day, the 196th day, the 224th day, the 252nd day, the 280th day, the 308th day, the 364th day and the 448th day, get serum, be diluted to and deliver to 307 hospitals of PLA after 20 times of volumes and detect the concentration of HBsAg (adopting Roche Cobas automatic tester to detect) with PBS buffer.The concentration of HBsAg is with light emission intensity (COI) numerical representation method.If the COI numerical value of the sign HBsAg concentration in 20 times of diluents of the serum of mice is greater than 1, 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 dynamic change of HBsAg positive rate.
The result of step 1 and step 2 shows: after plasmid pBS-HBV1.3 or plasmid pAAV/HBV1.2 injection Balb/c mice, all can successfully set up the mouse model of B virus acute hepatitis; After plasmid pAAV/HBV1.2 injection C57BL/6 mice, can successfully set up the mouse model of B virus chronic hepatitis.
After the function of embodiment 2, inhibition LSECtin, HBV infecting mouse body inner virus antigen is removed and is accelerated
First group: adjust the concentration of plasmid pBS-HBV1.3 with normal saline, then in tail vein 5sec, inject Balb/c mice or Balb/c LSECtin knock-out mice, every injected in mice 10 microgram plasmids, the mass ratio of injection and mice is 8%;
Second group: adjust the concentration of plasmid pAAV/HBV1.2 with normal saline, then in tail vein 5sec, inject Balb/c mice or Balb/c LSECtin knock-out mice, every injected in mice 10 microgram plasmids, the mass ratio of injection and mice is 8%;
The 3rd group: adjust the concentration of plasmid pAAV/HBV1.2 with normal saline, then in tail vein 5sec, inject C57BL/6 mice or C57BL/6LSECtin knock-out mice, every injected in mice 10 microgram plasmids, the mass ratio of injection and mice is 8%.
First group of mice, start timing from injection, every 24 hours is 1 day, within the 1st day, (test and inject after 24 hours for the 1st day respectively at experiment, the like), within the 4th day, the 5th day, the 6th day and the 7th day, get serum, be diluted to the concentration of delivering to 307 hospitals of PLA after 20 times of volumes and detect HBsAg and HBeAg with PBS buffer.The concentration of HBsAg and HBeAg is all with light emission intensity (COI) numerical representation method.HBsAg in 20 times of diluents of the serum of mice and HBeAg testing result are shown in Fig. 8 (each black circle represents a Balb/c LSECtin knock-out mice, and each soft dot represents a Balb/c mice).
Second group of mice, start timing from injection, every 24 hours is 1 day, within the 7th day, (test and inject after 24 × 7 hours for the 7th day respectively at experiment, the like), within the 10th day, the 15th day, the 21st day and the 28th day, get serum, be diluted to the concentration of delivering to 307 hospitals of PLA after 20 times of volumes and detect HBsAg and HBeAg with PBS buffer.The concentration of HBsAg and HBeAg is all with light emission intensity (COI) numerical representation method.HBsAg in 20 times of diluents of the serum of mice and HBeAg testing result are shown in Fig. 9 (pillar of light grey fill color represents the meansigma methods of 10 Balb/c mices, and the pillar of black fill color represents the meansigma methods of 6 Balb/c LSECtin knock-out mices).Respectively at experiment the 1st day, the 5th day, the 7th day, the 10th day, the 15th day, the 21st day and the 27th day statistics HBsAg positive rate, Figure 10 is shown in the HBsAg positive rate dynamic change (soft dot) of 10 Balb/c mices and the HBsAg positive rate dynamic change (black circle) of 6 Balb/c LSECtin knock-out mices.
Second group of mice: start timing from injection, every 24 hours is 1 day, within the 3rd day, (test and inject after 24 × 3 hours for the 3rd day respectively at experiment, the like), the 7th day and the 14th day put to death mice, get liver, after fixing with formalin, make paraffin section, adopt HBcAg antibody to carry out SABC.The photo of SABC is shown in that photo is shown in Figure 11.
The 3rd group of mice, start timing from injection, every 24 hours is 1 day, within the 7th day, (test and inject after 24 × 7 hours for the 7th day respectively at experiment, the like), within the 14th day, the 21st day, the 28th day, the 35th day, the 42nd day, the 49th day, the 56th day, the 63rd day, the 70th day, the 77th day and the 84th day, get serum, be diluted to the concentration of delivering to 307 hospitals of PLA after 20 times of volumes and detect HBsAg with PBS buffer.The concentration of HBsAg is with light emission intensity (COI) numerical representation method.The concentration of HBsAg in 20 times of diluents of the serum of mice is shown in Figure 12 (soft dot represents the meansigma methods of 8 C57BL/6 mices, and black circle represents the meansigma methods of 9 C57BL/6LSECtin knock-out mices).Respectively at experiment the 1st day, the 14th day, the 21st day, the 28th day, the 35th day, the 42nd day, the 49th day, the 56th day, the 63rd day, the 70th day, the 77th day and the 84th day statistics HBsAg positive rate, Figure 13 is shown in the HBsAg positive rate dynamic change (soft dot) of 8 C57BL/6 mices and the HBsAg positive rate dynamic change (black circle) of 9 C57BL/6LSECtin knock-out mices.
Above result shows, in LSECtin knock-out mice body, HBV antigen is removed significantly and accelerated, and suppresses after the function of LSECtin HBV infecting mouse body inner virus antigen and removes and accelerate.
Embodiment 3, GlcNAc β 1-2Man impel HBV infecting mouse body inner virus antigen to remove by the function of blocking-up LSECtin and accelerate
1, experiment the 1st day by the concentration of normal saline adjustment plasmid pAAV/HBV1.2, is then injected C57BL/6 mice in tail vein 5sec, every injected in mice 10 microgram plasmids, and the mass ratio of injection and mice is 8%.
2, experiment the 4th day, is divided into two groups by the mice of step 1, carries out respectively following parallel processing:
Experimental group (10): every mice lumbar injection every day 0.2ml13 μ M GlcNAc β 1-2Man solution, is injected to experiment the 42nd day continuously;
Matched group (9): every mice lumbar injection every day 0.2ml PBS buffer, is injected to experiment the 42nd day continuously.
Start timing from injection, respectively within the 7th day, the 14th day, the 21st day, the 28th day, the 35th day, the 49th day, the 56th day, the 63rd day, the 70th day, the 84th day and the 98th day, getting serum, be diluted to the concentration of delivering to 307 hospitals of PLA after 20 times of volumes and detect HBsAg and HBeAg with PBS buffer.The concentration of HBsAg and HBeAg is all with light emission intensity (COI) numerical representation method.HBsAg in 20 times of diluents of the serum of mice and HBeAg testing result are shown in Figure 14 (black circle represents the meansigma methods of 10 experimental mice, and 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 day, the 49th day, the 56th day, the 63rd day, the 70th day, the 84th day and the 98th day statistics HBsAg positive rate, Figure 15 was shown in the HBsAg positive rate dynamic change (soft dot) of 9 control group mice and the HBsAg positive rate dynamic change (black circle) of 10 experimental mice.
Above result shows, gives to have blocked after GlcNAc β 1-2Man processes the function of LSECtin, and HBV infecting mouse body inner virus antigen is removed and 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, then in tail vein 5sec, inject each 6 of Balb/c mice or Balb/c LSECtin knock-out mice, every injected in mice 10 microgram plasmids, the mass ratio of injection and mice is 8%.
After 10 days, separate liver lymphocyte by density gradient centrifugation.
Detect the expression of liver lymphocyte (CD8+T cell) middle CD25, CD62L and CD127 with flow cytometer.The results are shown in Figure 16.
Liver lymphocyte is seeded to Tissue Culture Plate, 3 × 10
5after stimulating, the cAg peptide (87-95:SYVNTNMGL) that the surface antigen peptide (28 – 39:IPQSLDSWWTSL) that individual cells/well is 10ug/ml with final concentration and final concentration are 10ug/ml adopt mouse interferon-γ detection ELISPOT test kit also to detect the situation of liver lymphocyte secretion interferon-γ by test kit description.
Figure 17 A is shown in by photo.In every hole, secrete the number of the cell of interferon-γ and see Figure 17 B.Every cubic millimeter of average spot size is shown in Figure 17 C.
Above result shows: suppress after the function of LSECtin, in liver lymphocyte (CD8+T cell) more, the CD62L positive cell of CD25 positive cell still less, CD127 positive cell still less, the cell of secretion interferon-γ is more.
Claims (10)
1. the application of the material of inhibition LSECtin in preparing product; The purposes of described product be following (a) and (b), (c), (d), (e), (f), (g) and (h) at least one:
(a) treat and/or prevent hepatitis B;
(b) removing of the hepatitis B virus antigen in promotion Hepatitis B Virus Infection body;
(c) treat and/or prevent the infection of HBV hepatitis B virus;
(d) suppress hepatitis B virus replication;
(e) impel the shared ratio of CD25 positive cell in liver lymphocyte to raise;
(f) impel the shared ratio of CD62L positive cell in liver lymphocyte to reduce;
(g) impel the shared ratio of CD127 positive cell in liver lymphocyte to reduce;
(h) impel the shared ratio of cell of secreting interferon-γ in liver lymphocyte to raise.
2. the method for claim 1, is characterized in that: in described (b), described hepatitis B virus antigen is at least one in HBeAg, HBcAg and HBsAg.
3. application as claimed in claim 1 or 2, is characterized in that: described LSECtin is following (1) or (2):
(1) protein being formed by the aminoacid sequence shown in sequence in sequence table 1;
(2) by the aminoacid sequence of sequence 1 through replacement and/or disappearance and/or the interpolation of one or several amino acid residue and there is the protein being derived by sequence 1 of lectins function.
4. the application as described in claim 1 or 2 or 3, is characterized in that: described is GlcNAc β 1-2Man for suppressing the material of LSECtin; The described molecular formula for the material that suppresses LSECtin is C
14h
25nO
11.
5. a product, its active component is the material for suppressing LSECtin; The purposes of described product be following (a) and (b), (c), (d), (e), (f), (g) and (h) at least one:
(a) treat and/or prevent hepatitis B;
(b) removing of the hepatitis B virus antigen in promotion Hepatitis B Virus Infection body;
(c) treat and/or prevent the infection of HBV hepatitis B virus;
(d) suppress hepatitis B virus replication;
(e) impel the shared ratio of CD25 positive cell in liver lymphocyte to raise;
(f) impel the shared ratio of CD62L positive cell in liver lymphocyte to reduce;
(g) impel the shared ratio of CD127 positive cell in liver lymphocyte to reduce;
(h) impel the shared ratio of cell of secreting interferon-γ in liver lymphocyte to raise.
6. product as claimed in claim 5, is characterized in that: in described (b), described hepatitis B virus antigen is at least one in HBeAg, HBcAg and HBsAg.
7. the product as described in claim 5 or 6, is characterized in that: described LSECtin is following (1) or (2):
(1) protein being formed by the aminoacid sequence shown in sequence in sequence table 1;
(2) by the aminoacid sequence of sequence 1 through replacement and/or disappearance and/or the interpolation of one or several amino acid residue and there is the protein being derived by sequence 1 of lectins function.
8. the product as described in claim 5 or 6 or 7, is characterized in that: described is GlcNAc β 1-2Man for suppressing the material of LSECtin; The described molecular formula for the material that suppresses LSECtin is C
14h
25nO
11.
9.LSECtin is as the application of the target of inhibition hepatitis B virus replication.
10.LSECtin is the application in auxiliary development or deisgn product as target spot; The purposes of described product be following (a) and (b), (c), (d), (e), (f), (g) and (h) at least one:
(a) treat and/or prevent hepatitis B;
(b) removing of the hepatitis B virus antigen in promotion Hepatitis B Virus Infection body;
(c) treat and/or prevent the infection of HBV hepatitis B virus;
(d) suppress hepatitis B virus replication;
(e) impel the shared ratio of CD25 positive cell in liver lymphocyte to raise;
(f) impel the shared ratio of CD62L positive cell in liver lymphocyte to reduce;
(g) impel the shared ratio of CD127 positive cell in liver lymphocyte to reduce;
(h) impel the shared ratio of cell of secreting interferon-γ in liver lymphocyte to raise.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201310064236.7A CN104013635B (en) | 2013-02-28 | 2013-02-28 | Application of substance for inhibiting LSECtin in preparation of medicine for treating and/or preventing viral hepatitis B |
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| Application Number | Priority Date | Filing Date | Title |
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| CN201310064236.7A CN104013635B (en) | 2013-02-28 | 2013-02-28 | Application of substance for inhibiting LSECtin in preparation of medicine for treating and/or preventing viral hepatitis B |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110475873A (en) * | 2017-03-16 | 2019-11-19 | 中央研究院 | The method of judging viral infection |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101152558A (en) * | 2007-09-30 | 2008-04-02 | 中国人民解放军军事医学科学院放射与辐射医学研究所 | New use of LSECtin |
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2013
- 2013-02-28 CN CN201310064236.7A patent/CN104013635B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101152558A (en) * | 2007-09-30 | 2008-04-02 | 中国人民解放军军事医学科学院放射与辐射医学研究所 | New use of LSECtin |
Non-Patent Citations (2)
| Title |
|---|
| BIAO LIU: "Liver Sinusoidal Endothelial Cell LectinInhibits CTL-Dependent Virus Clearance inMouse Models of Viral Hepatitis", 《THE JOURNAL OF IMMUNOLOGY》, vol. 190, no. 8, 15 April 2013 (2013-04-15) * |
| 曹红梅: "《西南大学硕士学位论文》", 17 May 2009, article "一种新的乳腺癌细胞粘附分子LSECtin的功能研究" * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110475873A (en) * | 2017-03-16 | 2019-11-19 | 中央研究院 | The method of judging viral infection |
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