Résumé
The function of the spleen in tumor development has been investigated for years. The relationship of the spleen with hepatocellular carcinoma (HCC), a huge health burden worldwide, however, remains unknown. The present study aimed to examine the effect of splenectomy on the development of HCC and the possible mechanism. Mouse hepatic carcinoma lines H22 and Hepa1-6 as well as BALB/c and C57 mice were used to establish orthotopic and metastatic mouse models of liver cancer. Mice were divided into four groups, including control group, splenectomy control group (S group), tumor group (T group) and tumor plus splenectomy group (T+S group). Tumor growth, metastases and overall survival were assessed at determined time points. Meanwhile, myeloid-derived suppressor cells (MDSCs) were isolated from the peripheral blood (PB), the spleen and liver tumors, and then measured by flow cytometery. It was found that liver cancer led to splenomegaly, and increased the percentage of MDSCs in the PB and spleen in the mouse models. Splenectomy inhibited the growth and progression of liver cancer and prolonged the overall survival time of orthotopic and metastatic models, which was accompanied by decreased proportion of MDSCs in the PB and tumors of liver cancer-bearing mouse. It was suggested that splenectomy could be considered an adjuvant therapy to treat liver cancer.
Sujets)
Animaux , Humains , Souris , Carcinome hépatocellulaire , Chirurgie générale , Lignée cellulaire tumorale , Cytométrie en flux , Tumeurs du foie , Chirurgie générale , Cellules myéloïdes suppressives , Anatomopathologie , Tumeurs expérimentales , Chirurgie générale , Rate , Chirurgie générale , Splénectomie , MéthodesRésumé
<p><b>BACKGROUND</b>Smad4 is found mutated in many cancers. It acts as a tumor suppressor in the regulation of TGF-β signaling pathway. The objective of this work was to study the expression of Smad4 in intrahepatic cholangiocarcinoma (ICC) and its relationship with the biological behavior and prognosis of the disease.</p><p><b>METHODS</b>Forty-nine paraffin-embedded ICC specimens and nine normal liver tissues were analyzed by immunohistochemical methods using Smad4 monoclonal antibodies. The expression of Smad4 was compared with the clinical pathological characteristics of the patients.</p><p><b>RESULTS</b>The expression of Smad4 was 100% positive in normal liver tissues, which was higher than that in the ICC (44.9%). Negative labeling of the Smad4 protein was found in 26.1% (6/23) of well-differentiated ICCs and 61.5% (16/26) of poorly to moderately differentiated ICCs, and 34.3% (12/35) and 71.4% (10/14) showed negative Smad4 labeling (P = 0.018) of ICC at pathological Tumor Node Metastasis (pTNM) stage I-II and pTNM stage III-IV separately. Furthermore, 72% (8/11) of lymph node metastatic ICCs and 73.3% (11/15) of intrahepatic metastatic ICCs showed negative labeling of the Smad4 protein. The loss of Smad4 expression in those metastatic ICCs was significantly more severe compared with non-metastatic ICCs (P = 0.000).</p><p><b>CONCLUSIONS</b>The expression of Smad4 was associated with the histological grade, clinical stage, and metastasis of ICC (P < 0.05). The detection of Smad4 may be helpful in determining the degree of malignancy and prognosis of ICC.</p>
Sujets)
Adulte , Sujet âgé , Femelle , Humains , Mâle , Adulte d'âge moyen , Tumeurs des canaux biliaires , Conduits biliaires intrahépatiques , Cholangiocarcinome , Chimie , Anatomopathologie , Tumeurs du foie , Chimie , Anatomopathologie , Métastase lymphatique , Stadification tumorale , Pronostic , Transduction du signal , Physiologie , Protéine Smad-4 , Génétique , Physiologie , Facteur de croissance transformant bêta , PhysiologieRésumé
<p><b>OBJECTIVE</b>To determine whether hepatitis B virus X (HBX) protein expression affect the oval cells' response to anti-proliferative effect of transforming growth factor β1 (TGFβ1) in oval cells.</p><p><b>METHODS</b>Real-time PCR, Western blot analysis were performed to detect the expression of TGFβRII in HBX-transfected oval cells named HBX-EGFP-LE/6, and EGFP-LE/6, LE/6 control cells. In addition, exogenous TGFβ1 was added into all three oval cell lines, MTT assay was preformed to clarify different responses to the anti-proliferative effect of TGFβ1.</p><p><b>RESULTS</b>The TGFβRII mRNA levels in LE/6 and EGFP-LE/6 cells were (10.2 ± 1.8) and (8.8 ± 0.9) folds of those in HBX-EGFP-LE/6 cells, the difference was significant (P < 0.05). HBX protein expression also reduced the protein levels of TGFβRII in HBX-EGFP-LE/6 oval cells, compared to the control cells. The MTT results exhibited that, after TGFβ1 addition, proliferative inhibition rate in the HBX-EGFP-LE/6 cells was 18.1% ± 1.5% while those in control cells were 42.2% ± 2.8% and 41.9% ± 5.0%, the difference was significant (P < 0.01).</p><p><b>CONCLUSION</b>HBX protein expression affects TGFβRII transcriptional activity and protein synthesis, and insensitive oval cells to anti-proliferative effect of TGFβ1.</p>
Sujets)
Animaux , Mâle , Rats , Lignée cellulaire , Prolifération cellulaire , Foie , Biologie cellulaire , Métabolisme , ARN messager , Génétique , Transactivateurs , Génétique , Métabolisme , Transfection , Facteur de croissance transformant bêta-1 , PharmacologieRésumé
<p><b>OBJECTIVE</b>To investigate the changes of oval cell proliferation rate in the rat 2-acetylaminofluorene/partial hepatectomy (2-AAF/PH) model.</p><p><b>METHODS</b>Livers were collected from 2-AAF/PH rats at different time points after hepatectomy. Paraffin sections were investigated by double immunofluorescent staining with confocal microscopy for oval cell marker epithelial cell adhesion molecule and proliferative index proliferating cell nuclear antigen, or epithelial cell adhesion molecule and alpha-smooth muscle actin. Deposition of matrix in liver tissue was detected by sirius red staining.</p><p><b>RESULTS</b>Response of ductular oval cells could be observed in portal area at 2 days after PH, and the number of oval cells reached its peak at 9 days and then gradually declined. Oval cell proliferation rate decreased from (91.3 +/- 1.6)% at 2 days after PH to (53.6 +/- 4.4)% at 12 days (P < 0.01). In addition, oval cells infiltrating into liver parenchyma were closely associated with activated hepatic stellate cells and extracellular matrix.</p><p><b>CONCLUSIONS</b>Oval cell proliferation rate starts decreasing before its number reaches a peak in 2-AAF/PH model. Hepatic stellate cells probably tightly regulate oval cell number through secreting several factors and producing extracellular matrix.</p>
Sujets)
Animaux , Mâle , Rats , N-Fluorén-2-yl-acétamide , Pharmacologie , Division cellulaire , Prolifération cellulaire , Hépatectomie , Foie , Biologie cellulaire , Rat Sprague-Dawley , Cellules souches , Biologie cellulaireRésumé
<p><b>OBJECTIVE</b>To find out the mechanisms of HBx gene inducing oval cell malignant transformation into hepatoma carcinoma cell.</p><p><b>METHODS</b>The changes of morphology, cell cycle, differentiated markers, c-myc and TGF-alpha in pEGFP-HBx oval cells strain, which stably expressed HBx gene, were studied by inversion phase contrast microscope and transmission electron microscopy, flow cytometry, periodic acid-schiff (PAS) staining, soft agar growth assay, real-time PCR, immunocytochemistry. pEGFP-oval cells and LE/6 oval cells were used as control groups.</p><p><b>RESULTS</b>(1) The pEGFP-HBx oval cells showed bigger in size with malformed nucleus as compared with control groups. (2) Flow cytometry showed that, in contrast with the control groups, the proportion of pEGFP-HBx oval cells arrested in G0/G1 phase decreased but in S or G2/M phase rose. Moreover, the population of aneuploid cells increased obviously. (3) PAS staining showed that there were many glycogen granules in the cytoplasm of pEGFP-HBx oval cell. (4) The pEGFP-HBx oval cell formed colonies in the soft agar. (5) Compared with the control groups, the expression of HNF-4 alpha, AFP, c-myc and TGF-alpha rose obviously, whereas the expression of CK-7 and CK-19 decreased. And the expression of cps1 mRNA was not in the extent of detection.</p><p><b>CONCLUSIONS</b>The HBx gene can provoke abnormal differentiation of oval cell and induce oval cell malignant transformation.</p>
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Animaux , Rats , Lignée cellulaire , Prolifération cellulaire , Transformation cellulaire néoplasique , Vecteurs génétiques , Hépatocytes , Anatomopathologie , Transactivateurs , Génétique , TransfectionRésumé
<p><b>OBJECTIVE</b>To study the effect of Extractum trametes robiniophila murr on cardiac allograft rejection in mice.</p><p><b>METHODS</b>All abdominal heterotopic heart transplantation models were divided into three groups as follows: (A) Extractum trametes robiniophila murr group. (B) Rejection group. (C) Isograft group. In each group, mean survival times (MST) of transplanted hearts and their pathologic histological changes at postoperative fifth day were observed. With fluoroimmunoassay, granzyme B and CD8(+) expressions were examined.</p><p><b>RESULTS</b>The MST of heart allografts in group A were (6.38 +/- 0.69) d, significantly shorter than that of group B [(8.31 +/- 0.59) d] (P < 0.01). In group A, acute rejection was present in advance; transplanted hearts were seriously damaged into acute rejection pathological grade 3, and CD8(+) T lymphocytes infiltrated diffusely and the expression of granzyme B increased significantly as compared with other groups.</p><p><b>CONCLUSIONS</b>Exclusive application of Extractum trametes robiniophila murr can promote the acute rejection of graft in early phase of postoperation, and the mechanism may be the promoted proliferation and infiltration of CD8(+) T lymphocytes and the increased expression of granzyme B.</p>
Sujets)
Animaux , Femelle , Mâle , Souris , Lymphocytes T CD8+ , Allergie et immunologie , Médicaments issus de plantes chinoises , Rejet du greffon , Granzymes , Métabolisme , Transplantation cardiaque , Souris de lignée C3H , Souris de lignée C57BL , Myocarde , Allergie et immunologie , Soins postopératoiresRésumé
<p><b>OBJECTIVE</b>To investigate the role of hepatic stellate cells in the differentiation of hepatic oval cells into adult hepatocyte.</p><p><b>METHODS</b>The oval cell were cocultured with primary hepatic stellate cells (HSC) in the same well (M-coculture) or separately cultured with HSC by millIcell (S-coculture). Oval cells were cultured alone as control; the expression of adult hepatocyte marker HNF-4alpha, albumin, and oval cell marker AFP, CK-19 in each group were detected by real-time PCR and western-blot. Phenotype changes were observed by transmission electron microscope (TEM); PAS staining was used to detect the quantity of glycogen granule in oval cell. Albumin level in supernatant was detected using ELISA kit.</p><p><b>RESULT</b>(1) The relative level of HNF-4alpha and albumin mRNA expression compared with pre-coculture: M-coculture: HNF-4a: 1.9+/-0.2, 10.7+/-1.2, 12.0+/-1.3; albumin: 5.7+/-1.6, 110.7+/-13.7, 173.6+/-22.3. S-coculture: 1.4+/-0.1, 3.2+/-0.6, 8.9+/-1.4 times; albumin: 2.9+/-1.4, 22.3+/-8.5, 96.3+/-16.3. The relative level of HNF-4a and albumin mRNA expression in coculture group (M- and S-coculture) were higher than control group (LSD-t: 32.98, 10.08, 13.38, 7.96; P less than 0.01); and a higher level of HNF-4a and albumin was found in M-coculture group compared to S-coculture group (LSD-t: 32.98, 25.65; P less than 0.01). The relative level of AFP and CK-19 mRNA expression compared with pre-coculture: M-coculture: 1.1+/-0.2, 0.2+/-0.0, 0.0+/-0.0; S-coculture group: AFP: 1.0+/-0.2, 0.2+/-0.1, 0.1+/-0.0; CK-19: 0.6+/-0.1, 0.1+/-0.0, 0.0+/-0.0; control group: AFP: 1.0+/-0.1, 1.0+/-0.1, 1.1+/-0.1, CK-19: 1.0+/-0.1, 1.1+/-0.1, 1.0+/-0.1. The relative level of AFP and CK-19 mRNA expression in coculture group (M- and S-coculture) were lower than that in control group (LSD-t: 37.99, 34.50, 13.59, 22.46; P less than 0.01). (2) The albumin secretion was detected in M-coculture: 14 day: (15.30+/-0.09) ng/ml, 21: (20.98+/-0.12) ng/ml; S-coculture: 14 day: (11.41+/-0.13) ng/ml, 21 day:(15.12+/-0.17) ng/ml. (3) It showed more organelles such as endoplasmic reticulum, mitochondrion and Golgi apparatus in oval cells cocultured with HSC. And cholangiole-like structure appeared between oval cells cocultured with HSC. (4) PAS staining showed glycogen granules could be observed in coculture groups.</p><p><b>CONCLUSION</b>HSC can induce differentiation of oval cell into mature hepatocyte.</p>
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Animaux , Mâle , Rats , Albumines , Génétique , Différenciation cellulaire , Cellules cultivées , Techniques de coculture , Cellules étoilées du foie , Hépatocytes , Biologie cellulaire , Métabolisme , Foie , Biologie cellulaire , Microscopie confocale , Réaction de polymérisation en chaîne , Méthodes , ARN messager , Génétique , Rat Sprague-Dawley , Cellules souches , Biologie cellulaire , Métabolisme , Alphafoetoprotéines , GénétiqueRésumé
<p><b>OBJECTIVE</b>To construct pEGFP-HBx eukaryotic expression plasmid and establish stable and effective transfected rat oval cell (LE/6) strain expressing EGFP-HBx fusion protein to explore the roles of HBx gene and oval cell in carcinogenesis of hepatocellular carcinoma (HCC).</p><p><b>METHODS</b>HBx gene with EcoRI and Hind III endo-enzyme sites was obtained by using PCR from plasmid pcDNA3.1-HBx. The purified HBx gene fragment was inserted into pEGFP-N1 expression vector, and the recombinant plasmid pEGFP-HBx was identified by restriction endonuclease and DNA sequencing analysis. LE/6 cells were transfected with recombinant pEGFP-HBx by lipofectamine reagent. Resistant to G418 clones were selected, and expression of EGFP-HBx fusion protein in clones were examined directly with fluorescence microscope, and these clones were isolated and proliferated. The expression of HBx was detected by RT-PCR analysis and immunocytochemistry.</p><p><b>RESULTS</b>Plasmid pEGFP-HBx has whole HBx gene base and correct reading frame as indicated by restriction endonuclease and DNA sequencing analysis. After transfecting with pEGFP-HBx plasmid, LE/6 cell clones expressing EGFP-HBx fusion protein were obtained. RT-PCR analysis and immunocytochemistry showed that HBx gene was only expression in transfected pEGFP-HBx cells.</p><p><b>CONCLUSIONS</b>The pEGFP-HBx recombinant expression vector was successfully constructed, and the stable transfected LE/6 strain expressing EGFP-HBx fusion protein was successfully established. It will be helpful in the further study on the roles of HBx and liver oval cell in carcinogenesis of HCC.</p>
Sujets)
Animaux , Rats , Lignée cellulaire , Vecteurs génétiques , Hépatocytes , Biologie cellulaire , Métabolisme , Plasmides , Génétique , Cellules souches , Biologie cellulaire , Métabolisme , Transactivateurs , Génétique , TransfectionRésumé
<p><b>OBJECTIVE</b>To study the function of nuclear factor-kappaB (NF-kappaB) signaling pathway in regulating vascular endothelial growth factor (VEGF) by hepatitis B virus X protein (HBx).</p><p><b>METHODS</b>After the establishment of L02-HBx cell line with stable transfected HBx gene, NF-kappaB signaling pathway blocker PDTC was introduced to cut off its signal transduction. Double immunofluorescent staining and laser scanning confocal microscopy were applied to study the activation and deactivation of NF-kappaB signaling pathway. Real-time PCR and Western blot were used to observe the expression of VEGF gene before and after the HBx transfection, as well as the treatment with PDTC.</p><p><b>RESULTS</b>The NF-kappaB signaling pathway of L02-HBx cells was activated after transfection with HBx gene as compared to the control L02 cells without treatment. The mRNA and protein levels of VEGF in L02-HBx cells increased 4.07 +/- 0.31 and 4.34 +/- 0.64 times respectively. The difference was of statistical significance (P < 0.05) in comparison with the control cells. The mRNA levels of VEGF decreased to 2.33 +/- 0.22 and 1.86 +/- 0.18(P < 0.05) and at the same time the expression of VEGF also reduced to 2.52 +/- 0.29 and 2.17 +/- 0.34 (P < 0.05), after treatment with 25.0 micromol/L and 50.0 micromol/L PDTC for 24 h respectively when the NF-kappaB signaling pathway was blocked. There was no significant difference in VEGF mRNA and protein levels when treated with 12.5 micromol/L PDTC for 24 h.</p><p><b>CONCLUSION</b>NF-kappaB signaling pathway maybe one of the routes through which HBx up-regulate the expression of VEGF to promote angiogenesis in hepatocellular carcinoma.</p>