Chronic Liver Injury Induces Conversion of Biliary Epithelial Cells into Hepatocytes.

Chronic liver injury can cause cirrhosis and impaired liver regeneration, impairing organ function. Adult livers can regenerate in response to parenchymal insults, and multiple cellular sources have been reported to contribute to this response. In this study, we modeled human chronic liver injuries, in which such responses are blunted, without genetic manipulations, and assessed potential contributions of non-parenchymal cells (NPCs) to hepatocyte regeneration. We show that NPC-derived hepatocytes replenish a large fraction of the liver parenchyma following severe injuries induced by long-term thioacetamide (TAA) or 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) treatment. Through lineage tracing of biliary epithelial cells (BECs), we show that BECs are a source of new hepatocytes and gain an Hnf4α+CK19+ bi-phenotypic state in periportal regions and fibrotic septa. Bi-phenotypic cells were also detected in cirrhotic human livers. Together, these data provide further support for hepatocyte regeneration from BECs without genetic interventions and show their cellular plasticity during severe liver injury.

Impact of Preoperative Hepatitis B Virus Levels on Prognosis After Primary and Repeat Hepatectomies for Hepatocellular Carcinoma Patients-a Retrospective Study.

BACKGROUND: Chronic HBV plays an important role in hepatocellular carcinoma pathogenesis. Previously, most studies have been focusing on HBV DNA levels before the primary curative hepatectomy. However, the association of virus level before repeat hepatectomy with the degrees of inflammation and fibrosis on histopathology and prognosis has not been surveyed. METHODS: From January 2002 to December 2009, all patients who were seropositive for hepatitis B surface antigen (HBsAg) were enrolled and assigned into four groups based on their HBV DNA levels before the primary and repeat hepatectomies. The cancer prognoses of these four groups of patients after the first and second operations were assessed and compared. The disease-free survival and overall survival were estimated by the Kaplan-Meier method. Univariate and multivariate analyses were performed to identify risk factors for the primary and repeat hepatectomies. RESULTS: For the 385 patients in this study, a low level of serum HBV DNA before repeat hepatectomy, but not primary hepatectomy, was significantly associated with improvement in prognosis, in terms of tumor recurrence, liver fibrosis, and liver-related mortality. CONCLUSION: The levels of HBV DNA before hepatectomies were crucial prognostic risk factors of HBV-related hepatocellular carcinoma patients. Surveillance of serum HBV DNA levels at multiple time points, rather than at a single time point, and antiviral therapy to suppress the virus to a low level had beneficial effects for these patients.

Co-culture of hepatoma cells with hepatocytic precursor (stem-like) cells inhibits tumor cell growth and invasion by downregulating Akt/NF-κB expression.

Hepatocytic stem cells (HSCs) have inhibitory effects on hepatocarcinoma cells. The present study investigated the effects of HSC activity in hepatocarcinoma cells in vitro. A Transwell co-culture system of hepatocytic precursor (stem-like) WB-F344 cells and hepatoma CBRH-7919 cells was used to assess HSC activity in metastasized hepatoma cells in vitro. Nude mouse xenografts were used to assess HSC activity in vivo. Co-culture of hepatoma CBRH-7919 cells with WB-F344 cells suppressed the growth and colony formation, tumor cell migration and invasion capacity of CBRH-7919 cells. The nude mouse xenograft assay demonstrated that the xenograft size of CBRH-7919 cells following co-culture with WB-F344 cells was significantly smaller compared with that of control cells. Furthermore, the expression levels of the epithelial markers E-cadherin and ß-catenin were downregulated, while the mesenchymal markers α-SMA and vimentin were upregulated. Co-culture of CBRH-7919 cells with WB-F344 cells downregulated NF-κB and phospho-Akt expression. In conclusion, hepatocytic precursor (stem-like) WB-F344 cells inhibited the growth, colony formation and invasion capacity of metastasized hepatoma CBRH-7919 cells in vitro and in vivo by downregulating Akt/NF-κB signaling.

miR­27a suppresses the clonogenic growth and migration of human glioblastoma multiforme cells by targeting BTG2.

miR-27a and BTG2 are implicated in gliomagenesis and glioma progression. However, hitherto, a link between miR-27a and BTG2 in glioma has not been reported. In the present study, we investigated the effects of miR-27a on the proliferation and invasiveness of glioblastoma cells in vitro and in a mouse xenograft model and further studied the relation between miR­27a expression and its target gene BTG2, which was identified by computation prediction algorithms. Our MTT and clonogenic assays showed that miR-27a overexpression significantly increased the clonogenic growth of glioblastoma U87MG and U251MG cells. The Transwell assays further revealed that miR-27a overexpression markedly increased the number of migrated U87MG and U251MG cells. TargetScan and other prediction algorithms identified BTG2 as a target gene of miR-27a, which was confirmed by EGFP reporter and immunoblotting assays showing an inverse relation between miR-27a expression and endogenous BTG2 expression. BTG2 overexpression also increased the proliferation and invasiveness of glioblastoma cells and BTG2 functioned downstream of miR-27a in modulating the proliferation and migration of glioblastoma cells. In conclusion, miR-27a modulates human glioblastoma growth and invasion by targeting BTG2.

Higher LRRFIP1 expression in glioblastoma multiforme is associated with better response to teniposide, a type II topoisomerase inhibitor.

Previous studies from this laboratory indicated that microRNA-21 (miR-21) contributes to chemoresistance of glioblastoma multiforme (GBM) cells to teniposide, a type II topoisomerase inhibitor. We also showed that LRRFIP1 is a target of miR-21. In this study, we found that higher baseline LRRFIP1 expression in human GBM tissue (n=60) is associated with better prognosis upon later treatment with teniposide. Experiments in cultured U373MG cells showed enhanced toxicity of teniposide against U373MG cells transfected with a vector that resulted in LRRFIP1 overexpression (vs. cells transfected with control vector). Experiments in nude mice demonstrated better response of LRRFIP1 overexpressing xenografts to teniposide. These findings indicate that high baseline LRRFIP1 expression in GBM is associated with better response to teniposide, and encourage exploring LRRFIP1 as a target for GBM treatment.

Alveolar adenoma combined with multifocal cysts: case report and literature review.

Alveolar adenoma is an extremely rare and benign pulmonary neoplasm; it is always asymptomatic and is usually detected incidentally on routine chest X-radiography. Typically on imaging examinations, alveolar adenoma exhibits as a peripheral, solitary, cystic nodule in the lung, which may easily imitate other lung lesions, consequently leading to difficulties in the differential diagnosis of this condition. Surgical resection is the primary treatment option. The diagnosis of alveolar adenoma is mainly based on postoperative histopathology, with features of proliferative type 2 alveolar epithelial cells and septal mesenchyme. The present case was a 60-year-old woman with alveolar adenoma, combined with systemic mutifocal cystic lesions. She underwent surgery following the obvious enlargement of this mass and a cystic nodule 7 cm in maximum diameter was resected. Postoperative histopathology confirmed a diagnosis of alveolar adenoma; her prognosis was favourable. In addition to reporting a rare case of alveolar adenoma coexisting with multifocal cysts, the English-language literature was reviewed for similar cases of alveolar adenoma.

[The derivation and validation of a prediction rule for differential diagnosis of thyroid nodules].

OBJECTIVE: To set up a prediction rule for the pro-operative differential diagnosis of thyroid nodules and evaluate its clinical value. METHODS: All patients of thyroid nodules underwent thyroid operations in Changzheng hospital from June, 1997 to July, 2012 were included in this study. They were randomly divided into the derivation cohort (2/3) and the validation cohort (1/3). A prediction rule was developed based on the logistic regression model and the scoring system was established in accordance with assigning of the value of each variable ß in the model. The prediction consistency, discriminatory power and diagnostic accuracy were conducted to evaluate the clinical value of the scoring system. RESULTS: A total of 13 980 patients were enrolled in the study with 9195 in the derivation cohort and 4785 in the validation cohort. The prediction rule consisted of 18 variables, which were gender, clinical manifestations including fever, neck sore, neck mass, palpitation and sweating, serum level of thyroid stimulating hormone (TSH) , free triiodothyronine (FT3) , thyroid peroxidase antibody (TPOAb) , thyroglobulin antibody (TgAb) , thyroglobulin (Tg) , calcitonin and carcinoembryonic antigen (CEA) , ultrasonography features including nodules number, location, size, boundaries and ethological patterns and the presence and patterns of lymph nodes. The model showed good calibration consistency (P = 0.437) and discrimination power (area under the receiver operating characteristic curve was 0.928) in the derivation cohort. The sensitivity, specificity, accuracy, positive predictive value, negative predictive value, positive likelihood ratio and negative likelihood ratio of the model were 89.3%, 81.5%, 83.2%, 56.8%, 96.6%, 4.83 and 0.13, respectively. CONCLUSION: The prediction rule and its scoring system established in the study are efficacious for the calibration and discrimination of thyroid nodules in Chinese population, which could be a useful tool for the pro-operative risk stratification.

Synchronous and metachronous multiple gastrointestinal stromal tumors.

BACKGROUND & AIMS: Sporadic multiple gastrointestinal stromal tumors (GISTs) are rare events especially those developed metachronously. This study aimed to investigate the clinico-pathologic and genetic features defining multiple GISTs. METHODS: 624 cases of GISTs were retrieved for retrospective review. 15 cases were identified as multiple GISTs including 13 synchronous and 2 metachronous ones. 32 tumors and 15 normal tissues were obtained from these cases each containing 2-3 tumor nodules and the genomic DNA was extracted for mutational analysis of KIT and PDGFRA genes. The associated patients were recruited for clinical follow-up studies, including 5 males and 10 females at 49 to 84 years of age. RESULTS: Multiple GISTs comprised of 2.4% of GIST cases in our consecutive series. Twenty-six tumors showed mutations at KIT gene in exon 11 and one at PDGFRA gene in exon 18. In seven synchronous cases, different tumors from the same patients displayed different genotypes of KIT or PDGFRA, suggesting their polyclonal origin. In the two multiple GISTs occurring metachronously, the tumors from each patient showed different KIT mutations, suggesting that the second tumors were not the relapse or metastasis of the primary GISTs. CONCLUSIONS: Based on types of KIT or PDGFRA mutations and other pathological features, multiple primary GISTs can be differentiated from multiple GISTs resulting from recurrence or metastasis of a single primary tumor. Unlike recurrence or metastasis of GISTs that are malignant, most multiple GISTs are mostly benign and do not require aggressive adjuvant therapy. Therefore, correct diagnosis is critical for proper treatment.

[Instillation of diesel exhaust particles on the posterior wall of pharynx on reproductive function in female mice].

OBJECTIVE: The present work aims to investigate the effects of subacute exposure to diesel exhaust particles (DEP) on reproductive function in female mice. METHODS: A total of 168 ICR (Institute of Cancer Research) mice were randomly divided into four groups by numeration table method, including the low (B), middle (C), high (D) dose DEP exposure groups and the control group (A). Each group consisted of 42 mice. Mice were inoculated with 30 µl DEP suspension at 0.8 (B), 3.0 (C), 12.0 (D) µg/µl, respectively, or the same volume of phosphate-buffered saline (A) on pharynx posterior wall per triduum for 4 times. The body weight and ovary weight were tested and ovary weight/body weight ratios were calculated. Rates of survival, germinal vesicle breakdown, extrusion of the first polar body, in-vitro fertilization and quantity of mitochondrial DNA for the oocytes were investigated. Ultrastructural changes of the oocytes were observed. RESULTS: The ovary weight/body weight ratios were (15.4 ± 7.3) × 10(-5), (14.1 ± 6.8) × 10(-5), (8.2 ± 0.7) × 10(-5) and (7.2 ± 2.5) × 10(-5) in groups A, B, C and D (F = 3.841, P < 0.05). In groups A, B, C and D at 48 h post-insemination, rates of oocyte survival were 64.3%, 56.8%, 39.5% and 32.9% (χ(2) = 21.575, P < 0.05), rates of extrusion of the first polar body were 75.5%, 65.3%, 37.0% and 27.1% (χ(2) = 52.772, P < 0.05), rates of 2-cell embryos were 27.9%, 39.1%, 17.6% and 12.5% (χ(2) = 20.148, P < 0.05), and rates of embryos over 2 cells were 45.3%, 32.2%, 12.5% and 13.9% (χ(2) = 32.135, P < 0.05), respectively, and were significantly lower in groups C and D compared with group A (P < 0.05). Logarithmic values of mitochondrial DNA copy numbers were 6.54 ± 0.13, 6.48 ± 0.09, 5.57 ± 0.15 and 5.41 ± 0.07 in groups A, B, C and D, respectively, and were significantly lower in groups C and D compared with group A or B (F = 89.241, P < 0.05). A number of mitochondria of the oocytes exhibited tremendous tumescence and vacuolization in groups C and D, which was contrast to a roughly normal appearance in groups A and B. CONCLUSIONS: DEP is noxious to murine female reproduction. Subacute exposure to DEP injures the ovary and oocyte resulting in compromised ovarian function and fertilizability of the oocyte.

Downregulation of ABCG2 expression in glioblastoma cancer stem cells with miRNA-328 may decrease their chemoresistance.

ABCG2, which encodes an ATP-binding cassette transporter protein, is associated with the phenotype of cancer stem cells and is used to define the pluripotential side population cells by flow cytometry and slide-cytometry. MicroRNAs control a wide array of biological processes (e.g., cell differentiation, proliferation and apoptosis) whose dysregulation is a hallmark of cancer. MicroRNA-328 (miR-328) is underexpressed in many cancers including glioblastoma multiforme and contributes to tumor resistance to chemotherapy. ABCG2 is associated with multi-drug resistance and is also highly expressed in glioblastoma. Some preliminary studies have shown that ABCG2 is the target gene for miRNA-328. Thus, we hypothesize that modulating ABCG2 expression by targeting miRNA-328 in glioblastoma cancer stem cells could represent a promising strategy for therapeutic manipulation to increase the efficacy of chemotherapeutic agents for glioblastoma, a highly lethal type of cancer.

Hepatocytic precursor (stem-like) WB-F344 cells reduce tumorigenicity of hepatoma CBRH-7919 cells via TGF-beta/Smad pathway.

Hepatic stem cells (HSCs) are involved in repair of liver injury. Stem cells may have inhibitory effects on tumor cell growth and apoptosis. However, it is unknown whether HSCs regulate the biological functions of hepatocarcinoma cells, especially tumor cell growth and apoptosis. The present study was designed to determine the effects of hepatocytic precursor (stem-like) WB-F344 cells on the growth and apoptosis of hepatoma CBRH-7919 cells. Using a Transwell chamber culture system, we co-cultured WB-F344 cells and CBRH-7919 cells in serum-free conditioned medium at 3 different ratios: 1:1 (2 x 10(5): 2 x 10(5) cells/well), 1:5 (4 x 10(4): 2 x 10(5) cells/well), and 5:1 (2 x 10(5): 4 x 10(4) cells/well). We determined the effects of stem cells on tumor cells using in vivo xenograft assay in nude mice and determining gene expression by RT-PCR and Western blot analyses. With the increment proportion of the WB-F344 cells in the co-culture system, tumor formation was inhibited in nude mice. Moreover, down-regulation of bone morphogenetic protein 4 (BMP4), Bcl-2, and c-Myc and upregulation of PTEN also occurred along with the inhibitory effects. Western blotting showed that the TGF-beta/Smad pathway played a prominent role in tumor inhibition, which may have been mediated by the cytokines released from the stem cells. In conclusion, hepatocytic precursor (stem-like) WB-F344 cells inhibit the tumorigenicity of hepatoma CBRH-7919 cells, and the effect is mediated by TGF-beta/Smad signaling pathway.

Repression of the miR-17-92 cluster by p53 has an important function in hypoxia-induced apoptosis.

We here report that miR-17-92 cluster is a novel target for p53-mediated transcriptional repression under hypoxia. We found the expression levels of miR-17-92 cluster were reduced in hypoxia-treated cells containing wild-type p53, but were unchanged in hypoxia-treated p53-deficient cells. The repression of miR-17-92 cluster under hypoxia is independent of c-Myc. Luciferase reporter assays mapped the region responding to p53-mediated repression to a p53-binding site in the proximal region of the miR-17-92 promoter. Chromatin immunoprecipitation (ChIP), Re-ChIP and gel retardation assays revealed that the binding sites for p53- and the TATA-binding protein (TBP) overlap within the miR-17-92 promoter; these proteins were found to compete for binding. Finally, we show that pri-miR-17-92 expression correlated well with p53 status in colorectal carcinomas. Over-express miR-17-92 cluster markedly inhibits hypoxia-induced apoptosis, whereas blocked miR-17-5p and miR-20a sensitize the cells to hypoxia-induced apoptosis. These data indicated that p53-mediated repression of miR-17-92 expression likely has an important function in hypoxia-induced apoptosis, and thus further our understanding of the tumour suppressive function of p53.

Epithelioid hemangioendothelioma of thoracic vertebra: a case report (2008: 11b).

Epithelioid hemangioendothelioma is a rare, low-grade malignant hemangioendothelioma first reported in 1982. The disease is more likely to occur between 20 and 30 years with a 2:1 male-to-female ratio. Epithelioid hemangioendothelioma may occur in multiple organs and tissues, but it rarely involves the skeleton. We present a case in a thoracic vertebra.

Isolation and characterization of bipotent liver progenitor cells from adult mouse.

Liver progenitor cells have drawn a great deal of attention both for their therapeutic potential and for their usefulness in exploring the molecular events surrounding liver development and regeneration. Despite the intensive studies on liver progenitors from rats, equivalent progenitor cells derived from mice are relatively rare. We used retrosine treatment followed by partial hepatectomy to elicit liver progenitors in mice. From these animals showing prominent ductular reactions, mouse-derived liver progenitor cell lines (LEPCs) were isolated by single-cell cloning. Phenotypic and lineage profiling of the LEPC clones were performed using immunochemistry, reverse transcription-polymerase chain reaction, and a dual-color system comprising the reporter EGFP under the control of the cytokeratin 19 promoter and the DsRed reporter under the control of the albumin promoter. LEPCs expressed liver progenitor cell markers. LEPCs also expressed some markers shared by bone marrow-derived hematopoietic stem cells c-Kit and Thy-1 but not CD34 and CD45. When cultured as aggregates in Matrigel, LEPCs differentiated into hepatocyte upon treatment with 50 ng/ml epithelial growth factor or differentiated into biliary lineage cells upon treatment with 20 ng/ml hepatocyte growth factor. In the presence of 2% dimethyl sulfoxide and 2% Matrigel, LEPCs acquired predominantly bile lineage phenotypes, with occasional patches of cells exhibiting hepatocyte phenotypes. Upon transplantation into CCl4-injured-liver, LEPCs engrafted into liver parenchyma and differentiated into hepatocytes. Considering the amenability of the mouse to genetic manipulation, these mouse-derived LEPCs may be useful tools as in vitro models to study molecular events in liver development and regeneration and can shed light in studying the therapy potential of liver stem cells.

[The relationship between infestation of Demodex folliculorum and epidermal neoplasm on face].

OBJECTIVE: To discuss the relationship between infestation of Demodex folliculorum and facial epidermal neoplasm. METHODS: A retrospective analysis was made with the pathological data of 153 cases collected in the recent four years on facial basal cell carcinoma, squamous cell carcinoma, seborrheic keratosis and trichilemmoma. The infection rate of Demodex folliculorum in the four types of neoplasm was evaluated and the relationship between the infection rate and the location of neoplasm and age was analyzed by V2 test. RESULTS: There was a significant difference in the infestation rate of Demodex folliculorum in the four types of epidermal neoplasm(P < 0.05), with the highest rate in basal cell carcinoma(56%), compared with seborrheic keratosis (21%), trichilemmoma (20%), and squamous cell carcinoma (14%). The infestation rate of Demodex folliculorum was significantly different in variant locations of epidermal neoplasm (P < 0.05). The highest infestation rate was in cases of nasal neoplasm (71%), compared with other parts. In addition, among twelve cases of Demodex folliculorum positive nasal neoplasm, nine were basal cell carcinoma; ten of thirty-six basal cell carcinoma occurred on nose. CONCLUSION: The highest infestation rate of Demodex folliculorum was in cases of nasal epidermal neoplasm compared with other locations, and the cases of basal cell carcinoma showed the highest infestation rate among the four types of neoplasm.

Antigenic and immunogenic changes due to mutation of s gene of HBV.

AIM: To investigate the change of immunological characteristics of HBsAg caused by the mutation at codon 145 of HBsAg using DNA-based immunization. METHODS: Plasmids expressing mutant and wild type envelope antigens were transfected into human hepatocellular carcinoma cells via electrotransformation. The antigenicity of HBsAg was studied with EIA and immunocytochemical staining. Then plasmids were used to immunize 5 C57BL/6 mice. Sera of mice were detected for anti-HBs and anti-preS2 with ELISA. RESULTS: The mutant HBsAg could be detected by native antibody in EIA and immunocytochemical study. But the A((450 nm)) value of the mutant HBsAg in the supernatant was apparently lower than that of the wild-type. Both mutant and native HBsAg expression plasmid could stimulate a strong humoral immune response to HBsAg and preS2 antigen in mice. Protective antibodies against HBsAg elicited by the native HBsAg occurred earlier than that elicited by the mutant HBsAg about one to two weeks. The occurrence of protective antibodies against preS2 antigen was one to two weeks earlier than that of anti-HBs. CONCLUSION: The amino acid substitution causes changes of the antigenicity and immunogenicity of HBsAg, but mutant HBsAg can still induce a protective humoral immune response in mice.

Expression of hepatitis B virus X protein in transgenic mice.

AIM: To establish a mice model harboring hepatitis B virus x gene (adr subtype) for studying the function of hepatitis B virus X protein, a transactivator of viral and cellular promoter/enhancer elements. METHODS: Expression vector pcDNA3-HBx, containing CMV promoter and hepatitis B virus x gene open reading fragment, was constructed by recombination DNA technique. Hela cells were cultured in DMEM and transfected with pcDNA3-HBx or control pcDNA3 plasmids using FuGENE6 Transfection Reagent. Expression of pcDNA3-HBx vectors in the transfected Hela cells was confirmed by Western blotting. After restriction endonuclease digestion, the coding elements were microinjected into male pronuclei of mice zygotes. The pups were evaluated by multiplex polymerase chain reaction (PCR) at genomic DNA level. The x gene transgenic mice founders were confirmed at protein level by Western blotting, immunohistochemistry and immunogold transmission electron microscopy. RESULTS: Expression vector pcDNA3-HBx was constructed by recombination DNA technique and identified right by restriction endonuclease digestion and DNA direct sequencing. With Western blotting, hepatitis X protein was detected in Hela cells transfected with pcDNA3-HBx plasmids, suggesting pcDNA3-HBx plasmids could express in eukaryotic cells. Following microinjection of coding sequence of pcDNA3-HBx, the embryos were transferred to oviducts of pseudopregnant females. Four pups were born and survived. Two of them were verified to have the HBx gene integrated in their genomic DNA by multiplex PCR assay, and named C57-TgN(HBx)SMMU1 and C57-TgN(HBx)SMMU3 respectively. They expressed 17KD X protein in liver tissue by Western blotting assay. With the immunohistochemistry, X protein was detected mainly in hepatocytes cytoplasm of transgenic mice, which was furthermore confirmed by immunogold transmission electon microscopy. CONCLUSION: We have constructed the expression vector pcDNA3-HBx that can be used to study the function of HBx gene in eukaryotic cells in vitro. We also established HBx gene (adr subtype) transgenic mice named C57-TgN (HBx)SMMU harboring HBx gene in their genome and express X protein in hepatocytes, Which might be a valuable animal system for studying the roles of HBx gene in hepatitis B virus life cycle and development of hepatocellular carcinoma in vivo.

[Induction of specific humoral immune response in mice by recombinant mutant HBV s gene vaccine].

AIM: To construct the recombinant eukaryotic expression vector pCMV-S2. S + 145R(PR) containing mutant HBV s gene and detect the specific humoral immune response to the PR in mice. METHODS: The PR was constructed by positional cloning of restriction endonuclease, and then it was transfected into human hepatocellular carcinoma cell line Hep G2 through electrotransformation. The antigenicity of PR was examined by EIA, ELISA and immunocytochemical staining. The PR and empty vector pcDNA3.0 were then used respectively to immunize intramuscularly 5 C57BL/6 mice, dosage being 100 pg purified plasmid each mouse. The titers of serum anti-HBs and anti-HBs2 antibodies were detected by ELISA. RESULTS: In vitro experiment showed that mutant HBsAg could bind to anti-HBs antibody. The PR could induce anti-HBs and anti-HBs2 antibody production in immunized mice. But the appearance time of serum anti-HBs2 antibody one or two weeks earlier than that of serum anti-HBs antibody. CONCLUSION: The expression product of PR had a good antigenicity, which can induce specific hu-moral immune response in C57BL/6 mice.