CN107335054B - Therapeutic DC vaccine for chronic hepatitis B - Google Patents
Therapeutic DC vaccine for chronic hepatitis B Download PDFInfo
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- CN107335054B CN107335054B CN201710524296.0A CN201710524296A CN107335054B CN 107335054 B CN107335054 B CN 107335054B CN 201710524296 A CN201710524296 A CN 201710524296A CN 107335054 B CN107335054 B CN 107335054B
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- C12N2730/00—Reverse transcribing DNA viruses
- C12N2730/00011—Details
- C12N2730/10011—Hepadnaviridae
- C12N2730/10111—Orthohepadnavirus, e.g. hepatitis B virus
- C12N2730/10134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
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- C12N2740/00—Reverse transcribing RNA viruses
- C12N2740/00011—Details
- C12N2740/10011—Retroviridae
- C12N2740/15011—Lentivirus, not HIV, e.g. FIV, SIV
- C12N2740/15041—Use of virus, viral particle or viral elements as a vector
- C12N2740/15043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
Abstract
The invention discloses a chronic hepatitis B therapeutic DC vaccine, which comprises a gene modified dendritic cell, wherein the gene modified dendritic cell contains granulocyte-macrophage cluster factor receptor signal peptide for guiding fusion gene segments of a preS2 region and an S region of an encoding M-HBsAg.
Description
Technical Field
The invention relates to the technical field of biological medical treatment, in particular to a therapeutic DC vaccine for chronic hepatitis B.
Background
Hepatitis B Virus (HBV), the most prevalent hepatotropic virus worldwide, can cause acute, chronic and severe Hepatitis after infection, and is closely related to the onset of cirrhosis and liver cancer. In China, about 9300 million HBV carriers, wherein the number of chronic hepatitis B patients is as high as 2500 million, which is one of the three most serious infectious diseases in China. At present, the main clinical treatment medicines for chronic hepatitis B patients comprise nucleoside (acid) analogues, interferon and immunomodulators, the medicines cannot completely eliminate hepatitis B virus, and the recurrence rate is high. Therefore, the development of effective treatment strategies is very urgent.
How to induce powerful specific cellular immune response and break the in vivo HBV specific T cell immune tolerance has become the main research direction at present. Because of its unique advantages in inducing specific cellular immunity, DC vaccines have been extensively studied as potential immunotherapeutic approaches against chronic hepatitis b. The current focus of chronic hepatitis b DC vaccine research: (1) how to obtain an effective HBV antigen (the antigen must be intolerant in chronic hepatitis b). (2) How to further enhance the immune effect of DC vaccines.
The HBV surface protein or antigen (HbsAg) is three envelope proteins (L protein, M protein and S protein) encoded by the alternate translation of the genes of the preS (preS1 and preS2) region and the S region through each of the three initiation codons. The S-HbsAg consists of 226 amino acids, only comprises an S region, is a main component of pseudovirions in serum of a chronic hepatitis B patient, and is also a component of a current clinical preventive hepatitis B vaccine; M-HBsAg is composed of 281 amino acids, including S and preS2 regions, and is also an important component of pseudovirion; L-HBsAg is composed of about 400 amino acids (389 ay-type and 400 ad-type depending on the subtype of hepatitis B) and includes a preS1 domain, a preS2 domain and an S domain. The current research shows that: during chronic HBV infection, different viral antigens present different tolerance states. The S region is strongly immune-tolerant and directly used as a vaccine, and cannot produce therapeutic effects in HBV-resistant mice.
Disclosure of Invention
To compensate for the above deficiencies, the preS1 region as a vaccine could elicit a cellular immune response in an HBV-resistant model. And combined with the preS1 vaccine and the HBsAg vaccine, the serum conversion of the HBsAg-HBsAb can be induced, and the hepatitis B virus infection in the liver can be reduced. It is thus clear that vaccines containing both preS1 antigen and S antigen are more immunogenic than vaccines containing only S antigen. Therefore, the invention provides a DC vaccine for treating chronic hepatitis B
The scheme of the invention is as follows:
a chronic hepatitis B therapeutic DC vaccine comprising genetically modified dendritic cells containing a granulocyte-macrophage cluster factor receptor signal peptide targeting a fusion gene segment encoding the preS2 region and the S region of M-HBsAg.
As a preferred technical scheme, the granulocyte-macrophage cluster factor receptor signal peptide is a nucleotide sequence shown in a sequence table SEQ ID NO. 1.
As a preferred technical scheme, the M-HBsAg is a nucleotide sequence shown in a sequence table SEQ ID NO. 2.
The invention also provides a method for loading the dendritic cells with the fusion gene segments, which comprises the following steps: and (2) slowly inserting gene fragments of a preS2 region and an S region of the granulocyte-macrophage cluster factor receptor signal peptide guide coding M-HBsAg into a lentivirus expression vector pLent-C-GFP, packaging to obtain lentivirus carrying GM-CSFR SP-M-HBsAg coding genes, inducing peripheral blood mononuclear cells of chronic hepatitis B patients to form imDCs, infecting the lentivirus carrying GM-CSFR SP-M-HBsAg coding genes into the imDCs, and inducing the infected imDCs by maturation factors to obtain mature DC vaccines.
The invention also provides a method for inducing the peripheral blood mononuclear cells of the chronic hepatitis B patient into imDCs, which comprises the following steps: collecting peripheral venous blood 50ml of chronic hepatitis B patient, separating mononuclear cells with lymphocyte separating medium, culturing at 37 deg.C with 5% CO2And after culturing for 2-3 hours under the condition, pouring off the suspended T cells, reserving adherent cells, adding cell factors rhIL-4 and rhGM-GSF to induce the mononuclear cells to differentiate into DCs, replacing a new culture medium every 48 hours, and culturing for 5 days to obtain the imDCs.
As a preferred technical scheme, the maturation factor is a maturation factor TNF-alpha.
The invention also provides a slow-release agent packaged to carry GM-CSFR SP-M-HBsAg coding geneThe virus method comprises the following steps: the lentivirus packaging cell line 293T was seeded in 10cm petri dishes containing DMEM + 10% FBS at 37 ℃ in 5% CO2Culturing under the condition, and performing transfection after the anchorage rate is 70-80%; co-transfecting 293T cells by using a calcium phosphate transfection method through the lentivirus packaging plasmid and the target expression plasmid; after 24 hours of transfection, cells are obviously enlarged and spherical, cell nucleuses are enlarged and become round, and the wall-attaching capability is reduced and the cells are easy to fall off; after 48 hours, the expression of green fluorescent protein in the cells is observed under an inverted fluorescence microscope; and after 72h, collecting the supernatant, filtering and sterilizing to obtain the recombinant GM-CSFR SP-M-HBsAg lentivirus, wherein the recombinant GM-CSFR SP-M-HBsAg lentivirus is stored in a low-temperature refrigerator at the temperature of-80 ℃ for later use.
Due to the adoption of the technical scheme, the chronic hepatitis B therapeutic DC vaccine comprises the genetically modified dendritic cells, wherein the genetically modified dendritic cells contain a fusion gene segment of a granulocyte-macrophage cluster factor receptor signal peptide guide and code a preS2 region and an S region of M-HBsAg.
Compared with the prior art, the invention has the following advantages and beneficial effects:
(1) the preS2 region and the S region are used as antigen genes for preparing the chronic hepatitis B DC vaccine for the first time, and the traditional hepatitis B vaccine, namely hepatitis B surface antigen small protein, has a strong immune tolerance state and cannot produce a therapeutic effect in chronic hepatitis B patients. M-HBsAg containing preS2 antigen plays an important role in the adhesion of HBV to corresponding receptors of liver cells, and when a body is immunized, characteristic antibodies aiming at preS2 antigen can be generated, and the antibodies have good effect of neutralizing HBV virus, so that a host is protected from being infected by the HBV virus.
(2) The granulocyte-macrophage cluster factor receptor signal peptide is adopted to guide the expression of the M-HBsAg antigen, so that the expression efficiency of the antigen can be improved, the immune effect of the antigen can be enhanced, and a stronger anti-HBV specific immune response can be generated.
(3) The lentivirus is used as an expression vector, the DC cell infection rate is high, the M-HBsAg antigen gene can be stably integrated into a DC cell genome and expressed for a long time, and a large amount of M-HBsAg antigens continuously stimulate and activate the DC, so that the DC antigen presentation efficiency is improved.
Drawings
FIG. 1 is a graph comparing HBsAg-specific T cell responses of wild type mice in a control group, an M-HBsAg vaccine group and a GM-CSFR SP-M-HBsAg group;
FIG. 2 is a graph comparing HBsAg-specific T cell responses in mice with tolerance in the control group, the M-HBsAg vaccine group, and the GM-CSFR SP-M-HBsAg group;
FIG. 3 is a graph showing the comparison of HBsAg antigen levels in serum of mice resistant to the control group, M-HBsAg vaccine group and GM-CSFR SP-M-HBsAg group;
FIG. 4 is a graph comparing the serum HBsAg antibody levels in the resistant mice of the control group, the M-HBsAg vaccine group and the GM-CSFR SP-M-HBsAg group;
Detailed Description
The specific implementation is as follows:
1. the gene fragment GM-CSFR SP-M-HBsAg is inserted into a lentivirus expression vector pLent-C-GFP.
(1) GM-CSFR SP nucleic acid artificial sequence (SEQ ID NO.1)
(2) M-HBsAg nucleic acid artificial sequence (SEQ ID NO.2)
NotI and AsiSI site restriction sites (SEQ ID NO.4) are respectively added at two ends of a nucleic acid artificial sequence of GM-CSFR SP-M-HBsAg, two complete expression frames are synthesized by committee Biotechnology Limited company, a lentivirus pLent-C-GFP vector (Invitrogen) NotI-AsiSI site is inserted after restriction enzyme treatment, the vector is transformed to E.coli (DH5 alpha), and after nucleic acid sequencing identification is correct, a plasmid is extracted and purified by using a plasmid purification kit of Qiagen company, so that a high-quality plasmid of a recombinant expression vector is obtained.
2. Recombinant lentivirus packaging and titer detection
The lentivirus packaging cell line 293T was seeded in 10cm petri dishes containing DMEM + 10% FBS at 37 ℃ in 5% CO2And (5) culturing under the condition, and performing transfection after the anchorage rate is 70-80%. The slow virus packaging plasmid and the target expression plasmid are cotransfected with 293T cells by a calcium phosphate transfection method, and are cloned by a reference molecule. After 24h after transfection, cells are obviously enlarged and spherical, cell nucleuses are enlarged and become round, and the wall-attaching capability is reduced and the cells are easy to fall off. Observed under an inverted fluorescence microscope after 48hGreen fluorescent protein is expressed in cells. After 72h, the supernatant was collected, filtered to remove bacteria, and stored in a low temperature freezer at-80 ℃ for further use. According to Lenti-XTM Go StixTMThe kit (product of Beijing Huaxia ocean technology Co., Ltd.) determines the virus titer, and the result shows that the titer of the recombinant GM-CSFR SP-M-HBsAg lentivirus is 2.74 multiplied by 106pfu/ml。
3. Induction of immature DC (imDC)
Collecting peripheral venous blood 50ml of chronic hepatitis B patient, separating mononuclear cells with lymphocyte separation medium, culturing at 37 deg.C with 5% CO in culture medium (purchased from TaKaRa, GT-T551)2After culturing for 2-3 hours under the condition, pouring off the suspended T cells, reserving adherent cells, adding cell factors rhIL-4 (final concentration: 50ng/ml) and rhGM-GSF (final concentration: 100ng/ml), inducing the mononuclear cells to differentiate into DCs, replacing a new culture medium every 48 hours, and culturing for 5 days to obtain the imDCs.
4. Infection of imDC by recombinant lentivirus and maturation of DC cells
Mix imDC at 1 × 106Inoculating the amount of each cell/hole in a 12-hole culture plate, infecting the imDC by recombinant lentivirus with the MOI of 10, after infecting for 8-12h, washing the cells for 2-3 times by PBS, adding a maturation factor TNF-alpha, continuing culturing and inducing to obtain mature DC cells (mDC), observing the cell morphology after the DC is matured by an inverted microscope, and detecting the expression condition of M-HBsAg genes by a probe; PCR analysis of chromosome integration of the lentiviral vector; and analyzing the expression of the DC cell maturation marker by a flow cytometer. After the DC precursor cells are infected by the recombinant lentiviruses, the M-HBsAg positive cell rate is more than 92.3%, and flow cytometry analysis results show that the average value of maturation markers CD11c + CD86+ of the DC cells in a virus infection group is 74.04%, the average value of CD11c + CD83+ is 88.05%, and the high-expression CD83 and CD86 are obtained. The mDC can be directly injected as a vaccine for preventing HBV.
Preparation of M-HBsAg vaccine:
1. the gene fragment M-HBsAg is respectively inserted into a lentivirus expression vector pLent-C-GFP.
(1) M-HBsAg nucleic acid artificial sequence (SEQ ID NO.2)
NotI and AsiSI site restriction sites (SEQ ID NO.3) are respectively added at two ends of a nucleic acid artificial sequence of the M-HBsAg, two complete expression frames are synthesized by a committee biotechnology limited company, a lentivirus pLent-C-GFP vector (Invitrogen) NotI-AsiSI site is inserted after restriction enzyme treatment, the lentivirus pLent-C-GFP vector is transformed to E.coli (DH5 alpha), and after correct nucleic acid sequencing identification, a plasmid is extracted and purified by using a plasmid purification kit of a Qiagen company to obtain a high-quality plasmid of a recombinant expression vector.
2. Recombinant lentivirus packaging and titer detection
The lentivirus packaging cell line 293T was seeded in 10cm petri dishes containing DMEM + 10% FBS at 37 ℃ in 5% CO2And (5) culturing under the condition, and performing transfection after the anchorage rate is 70-80%. The slow virus packaging plasmid and the target expression plasmid are cotransfected with 293T cells by a calcium phosphate transfection method, and are cloned by a reference molecule. After 24h after transfection, cells are obviously enlarged and spherical, cell nucleuses are enlarged and become round, and the wall-attaching capability is reduced and the cells are easy to fall off. After 48h, the expression of green fluorescent protein in the cells was observed under an inverted fluorescence microscope. After 72h, the supernatant was collected, filtered to remove bacteria, and stored in a low temperature freezer at-80 ℃ for further use. According to Lenti-XTM Go StixTMThe kit (product of Beijing Huaxia ocean technology Co., Ltd.) determines the virus titer, and the result shows that the titer of the recombinant M-HBsAg lentivirus is 2.8 multiplied by 106pfu/ml。
3. Induction of immature DC (imDC)
Collecting peripheral venous blood 50ml of chronic hepatitis B patient, separating mononuclear cells with lymphocyte separation medium, culturing at 37 deg.C with 5% CO in culture medium (purchased from TaKaRa, GT-T551)2After culturing for 2-3 hours under the condition, pouring off the suspended T cells, reserving adherent cells, adding cell factors rhIL-4 (final concentration: 50ng/ml) and rhGM-GSF (final concentration: 100ng/ml), inducing the mononuclear cells to differentiate into DCs, replacing a new culture medium every 48 hours, and culturing for 5 days to obtain the imDCs.
4. Infection of imDC by recombinant lentivirus and maturation of DC cells
Mix imDC at 1 × 106The amount of each cell/well was inoculated in 12-well culture plates, the imDCs were infected with the recombinant lentivirus with MOI 10, after 8-12h, the cells were washed 2-3 times with PBS, and addedContinuously culturing and inducing by using a maturation factor TNF-alpha to obtain mature DC cells (mDC), observing the cell morphology after the DC is mature by using an inverted microscope, and detecting the expression condition of the M-HBsAg gene by using a probe; PCR analysis of chromosome integration of the lentiviral vector; and analyzing the expression of the DC cell maturation marker by a flow cytometer. After the DC precursor cells are infected by the recombinant lentiviruses, the M-HBsAg positive cell rate is more than 92.3%, and flow cytometry analysis results show that the average value of maturation markers CD11c + CD86+ of the DC cells in a virus infection group is 75.64%, the average value of CD11c + CD83+ is 88.34%, and the high-expression CD83 and CD86 are obtained.
And (3) comparison test:
effects of HBV-specific DC vaccine in prevention and treatment of chronic hepatitis B
Establishment of a chronic HBV infection model, 6-8 week old C57BL/6 mice (purchased from the university of traditional Chinese medicine, Guangzhou, female, SPF (III) -level animals), 10. mu.g of pAAV/HBV1.2 plasmid (purchased from Acanthopanax beijing and molecular medical research institute, Inc.) was injected by tail vein high pressure injection. Stable HBV tolerance and chronic HBV infection models were established 4 weeks after infection. By the model, the chronic hepatitis B therapeutic DC vaccine is verified, three groups are prepared for comparison, namely a control group, an M-HBsAg vaccine group and a chronic hepatitis B therapeutic DC vaccine containing GM-CSFR SP-M-HBsAg group, wherein the control group is a blank control group.
The blank control group was not subjected to any treatment;
the M-HBsAg vaccine group; at 1 × 106The immunization was performed with a total of two injections of the M-HBsAg vaccine per tail, with the first and second immunizations being separated by 7 days.
The invention relates to a DC vaccine group for treating chronic hepatitis B, which comprises the following components in part by weight: at 1 × 106The DC-based HBV viral vaccine of the present invention was injected tail-in a single cell/tail for immunotherapy for a total of two immunizations, with 7 days between the first and second immunizations. It was found by comparison of ELISPOT (ex eBioscience) and ELISA (ex Thermo Fisher Scientific) assays that it was able to induce a robust T cell immune response specific for HBsAg in both wild and tolerant mice. And immune response generated in HBV-resistant miceCan completely clear the HBsAg in serum and simultaneously induces partial serology HBsAb transformation, which is clinically regarded as a key index for curing HBV, so the vaccine prepared by the invention has better treatment effect.
Although specific embodiments of the invention have been described in detail, those skilled in the art will appreciate. Various modifications and substitutions of those details may be made in light of the overall teachings of the disclosure, and such changes are intended to be within the scope of the present invention. The full scope of the invention is given by the appended claims and any equivalents thereof.
Sequence listing
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Claims (4)
1. A therapeutic DC vaccine for chronic hepatitis B, characterized by: comprises a genetically modified dendritic cell which contains a granulocyte-macrophage cluster factor receptor signal peptide to guide a fusion gene segment GM-CSFR SP-M-HBsAg which codes the M-HBsAg;
the nucleotide sequence of the fusion gene fragment is shown as SEQ ID NO. 4;
the nucleotide sequence of the granulocyte-macrophage cluster factor receptor signal peptide is shown in a sequence table SEQ ID NO. 1;
the preparation method of the DC vaccine comprises a method for loading the dendritic cells with the fusion gene segments: slowly inserting the fusion gene fragment into a lentivirus expression vector pLent-C-GFP, packaging to obtain lentivirus carrying GM-CSFR SP-M-HBsAg encoding genes, simultaneously inducing peripheral blood mononuclear cells of chronic hepatitis B patients to form imDC, infecting the imDC with the lentivirus carrying GM-CSFR SP-M-HBsAg encoding genes, and inducing the infected imDC by a maturation factor to obtain a mature DC vaccine; the nucleotide sequence of the M-HBsAg is shown in a sequence table SEQ ID NO. 2.
2. The chronic hepatitis B therapeutic DC vaccine of claim 1The preparation method of the DC vaccine is characterized by further comprising a method for inducing peripheral blood mononuclear cells of the chronic hepatitis B patient into imDC: collecting peripheral venous blood 50ml of chronic hepatitis B patient, separating mononuclear cells with lymphocyte separating medium, culturing in culture medium at 37 deg.C with 5% CO2And after culturing for 2-3 hours under the condition, pouring off the suspended T cells, reserving adherent cells, adding cell factors rhIL-4 and rhGM-GSF to induce the mononuclear cells to differentiate into DCs, replacing a new culture medium every 48 hours, and culturing for 5 days to obtain the imDCs.
3. The therapeutic DC vaccine of claim 1, wherein: the maturation factor is maturation factor TNF-alpha.
4. The chronic hepatitis b therapeutic DC vaccine of claim 1, further comprising a method of packaging said fusion gene fragment into a lentivirus carrying the GM-CSFR SP-M-HBsAg encoding gene: the lentivirus packaging cell line 293T was seeded in 10cm dishes containing DMEM + 10% FBS at 37 ℃ with 5% CO2Culturing under the condition, and performing transfection after the anchorage rate is 70-80%; co-transfecting 293T cells by using a calcium phosphate transfection method through a lentivirus packaging plasmid and a lentivirus expression vector pLent-C-GFP containing a fusion gene segment; after 24h of transfection, the cells are obviously enlarged and spherical, the cell nucleuses are enlarged and become round, the wall attaching capability is reduced and the cells are easy to fall off; after 48 hours, the expression of green fluorescent protein in the cells is observed under an inverted fluorescence microscope; and after 72h, collecting the supernatant, filtering and sterilizing to obtain the recombinant GM-CSFR SP-M-HBsAg lentivirus, wherein the recombinant GM-CSFR SP-M-HBsAg lentivirus is stored in a low-temperature refrigerator at the temperature of-80 ℃ for later use.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1981866A (en) * | 2006-04-30 | 2007-06-20 | 解放军三○二医院生物治疗研究中心 | DC vaccine for treating chronic hepatitis B |
| CN101897965A (en) * | 2010-06-29 | 2010-12-01 | 中国人民解放军第三军医大学 | Hepatitis B virus dendritic cell therapeutic vaccine and preparation method thereof |
| CN104189897A (en) * | 2014-05-21 | 2014-12-10 | 深圳先进技术研究院 | Preparation method for dendritic cells capable of loading antigens efficiently |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102787097B (en) * | 2012-05-30 | 2014-11-05 | 深圳市博泰生物医学科技发展有限公司 | Modified dendritic cell and vaccine containing the same |
| CA2945305C (en) * | 2014-04-10 | 2023-10-17 | Seattle Children's Hospital (dba Seattle Children's Research Institute) | Method and compositions for cellular immunotherapy |
-
2017
- 2017-06-30 CN CN201710524296.0A patent/CN107335054B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1981866A (en) * | 2006-04-30 | 2007-06-20 | 解放军三○二医院生物治疗研究中心 | DC vaccine for treating chronic hepatitis B |
| CN101897965A (en) * | 2010-06-29 | 2010-12-01 | 中国人民解放军第三军医大学 | Hepatitis B virus dendritic cell therapeutic vaccine and preparation method thereof |
| CN104189897A (en) * | 2014-05-21 | 2014-12-10 | 深圳先进技术研究院 | Preparation method for dendritic cells capable of loading antigens efficiently |
Non-Patent Citations (12)
| Title |
|---|
| AF477491.3;GenBank;《https://blast.ncbi.nlm.nih.gov/Blast.cgi#alnHdr_22711891》;20030129;第1-2页 * |
| HBsAg基因修饰DC瘤苗不同免疫途径对肝癌治疗的实验研究;幸思忠;《万方中国学位论文全文数据库》;20080924;第1-51页 * |
| HBsAg重组腺病毒载体的构建及在树突状细胞的表达;黄茵等;《浙江预防医学》;20060630;第18卷(第6期);第1-11页 * |
| HBV preS2S-rhGM-CSF融合基因表达质粒的构建和表达;郭晓兰等;《细胞与分子免疫学杂志》;20041013;第20卷(第5期);第548-551页 * |
| HBVPreS2S-rhGM-CSF融合基因表达质粒的构建及其体外表达研究;郭晓兰;《中国优秀博硕士学位论文全文数据库 (硕士)医药卫生科技辑》;20030315(第01期);第E059-65页，摘要 * |
| KX000906.1;GenBank;《https://www.ncbi.nlm.nih.gov/nucleotide/KX000906.1?report=genbank&log$=nuclalign&blast_rank=1&RID=T10DMAKW015》;20160424;第1-3页 * |
| 慢性乙型肝炎合并肝癌患者树突状细胞负载HBV抗原的研究;钱莘;《中国优秀博硕士学位论文全文数据库(硕士)医药卫生科技辑》;20051115(第07期);第9页第4段，第一部分第二节 * |
| 慢性乙型肝炎合并肝癌患者树突状细胞负载乙型肝炎病毒抗原后表型和功能的研究;钱莘等;《中华医学杂志》;20050126;第85卷(第4期);第248-252页 * |
| 治疗型双质粒 HBV DNA疫苗的构建及其鉴定;何晓嫱等;《解放军医学杂志》;20030630;第28卷(第6期);摘要，第1.2.2节，第2.1节 * |
| 编码hGM-CSF基因慢病毒载体的构建及其介导的肿瘤活性的研究;林阿丽;《中国优秀硕士学位论文全文数据库医药卫生科技辑》;20070915(第03期);第E072-17页，摘要、前言 * |
| 自体树突状细胞疫苗治疗乙型肝炎病毒感染者的免疫机制研究;石庆凤;《万方中国学位论文全文数据库》;20091224;第1-66页 * |
| 自体树突状细胞疫苗治疗慢性乙型肝炎的临床研究;李用国等;《中华肝脏病杂志》;20030430;第11卷(第4期);第206-208页 * |
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