CDC20 and PTTG1 are Important Biomarkers and Potential Therapeutic Targets for Metastatic Prostate Cancer.

INTRODUCTION: Metastatic prostate cancer (mPCa) is responsible for most prostate cancer (PCa) deaths worldwide. The present study aims to explore the molecular differences between mPCa and PCa. METHODS: The authors downloaded GSE6752, GSE6919, and GSE32269 from the Gene Expression Omnibus and employed integrated analysis to identify differentially expressed genes (DEGs) between mPCa and PCa. Functional and pathway-enrichment analyses were performed, and a protein-protein interaction (PPI) network and modules were constructed. Clinical mPCa specimens were collected to verify the results by performing RT-qPCR. The Cancer Genome Atlas database was used to conduct a survival analysis, and an immunohistochemical assay was performed. The invasion ability of PCa cells was verified by Transwell assay. RESULTS: One-hundred six consistently DEGs were found in mPCa compared with PCa. DEGs significantly enriched the positive regulation of cell proliferation, cell division, and cell adhesion in small cell lung cancer and PCa. Cell division, nucleoplasm, and cell cycle were selected from the PPI network, and the top 10 hub genes were selected. CDC20 and PTTG1 with genetic alterations were significantly associated with poorer disease-free survival. Immunohistochemical assay results showed that the expression levels of CDC20 and PTTG1 in mPCa were higher than those in PCa. The results of the migration assay indicated that CDC20 and PTTG1 could enhance the migration ability of PCa cells. CONCLUSION: The present study revealed that CDC20 and PTTG1 contribute more to migration, progression, and poorer prognoses in mPCa compared with PCa. CDC20 and PTTG1 could represent therapeutic targets in mPCa medical research and clinical studies.

Nanotoxicity of different sizes of graphene (G) and graphene oxide (GO) in vitro and in vivo.

Graphene family nanomaterials (GFNs) have attracted significant attention due to their unique characteristics and applications in the fields of biomedicine and nanotechnology. However, previous studies highlighted the in vitro and in vivo toxicity of GFNs with size and oxidation state differences are still elusive. Therefore, we prepared graphene (G) and graphene oxide (GO) of three different sizes (S-small, M-medium, and L-large), and characterized them using multiple surface-sensitive analytical techniques. In vitro assays using HEK 293T cells revealed that the small and large sizes of G and GO significantly reduced the cell viability and increased DNA damage, accompanying with activated reactive oxygen species (ROS) generation and induced various expressions of associated critical genetic markers. Moreover, the bacterial assays highlighted that G and GO caused strong acute toxicity on Tox2 bacteria. Effects of G were higher than GO and showed size dependent effect: L > M > S, while the medium size of GO induced mild genetic toxicity on RecA bacteria. In vivo assays revealed that exposure to G and GO caused the developmental toxicity, induced ROS generation, and activated related pathways (specifically GO) in zebrafish. Taken together, G showed stronger ability to decrease the survival rate and induce the acute toxicity, while GO showed obvious toxicity in terms of DNA damages, ROS generation, and abnormal gene expressions. Our findings highlighted that G and GO differentially induced toxicity based on their varying physical characteristics, especially sizes and oxidation state, and exposure concentrations and sensitivity of the employed in vitro and in vivo models. In short, this study provided deep insights on the negative effects of GFNs exposure.

[The Influence of New Medium with RGD on Cell Growth,Cell Fusion and Expression of Exogenous Gene].

OBJECTIVE: To investigate the influence of a new culture medium added with RGD on cell growth,cell fusion and expression of exogenous gene. METHODS: A new medium was prepared by adding different concentrations of RGD to ordinary culture medium. The optimum concentration of RGD was determined by observation of the growth of human pancreatic epithelial cell line HPDE6-C7. After determining the optimum concentration of RGD,different concentrations of cells HPDE6-C7 (5×104,105,5×105 mL－1) were inoculated in the two mediums. The morphology,adherence,growth and density of the cells were observed by inverted microscope; The ratio of clone formation and the positive rate of cloning were compared between the two cultures after fusion; The fluorescence intensity after the transfection of plasmid with green fluorescent protein (GFP) and the protein expression after transfection of plasmid with KRAS were observed to campare the expression of exogenous genes between the new medium with ordinary medium. RESULTS: Firstly,the optimal concentration of RGD was 10 ng/mL. Compared with the normal medium,the cultured cells with RGD had better morphology,adhesion and faster proliferation. In addition,both of the number and positive rate of clones formed in the new medium were significantly higher than that in the ordinary medium (P<0.05);The fluorescence intensity after transfection of exogenous gene GFP in the new medium was significantly higher than that in normal medium (P<0.05); Expression level of exogenous gene KRAS of the new medium was also significantly higher than that in normal medium. CONCLUSION: The new culture medium has highlighted advantages in cell growth,cell fusion and expression of exogenous genes. RGD peptide has widely prospect and potential value in the cell culture.

Mechanism of fatty acid synthase in drug tolerance related to epithelial-mesenchymal transition of breast cancer.

OBJECTIVE: The mechanism of action of fatty acid synthase (FASN) in drug tolerance of breast cancer cells with epithelial-mesenchymal transition (EMT) features was investigated. METHODS: The breast cancer cell line MCF-7-MEK5 with stably occurring EMT and tumour necrosis factor-α (TNF-α) tolerance was used as the experimental model, whereas MCF-7 acted as the control. Tumour cells were implanted into nude mice for in vivo analysis, and cerulenin was used as a FASN inhibitor. RT-PCR, real-time quantitative PCR and Western blot were employed to detect the expression of FASN, TNFR-1, TNFR-2, Wnt-1, ß-catenin and cytC at the RNA and protein levels. RESULTS: Compared with MCF-7, TNFR-1 expression in MCF-7-MEK5 was slightly changed, TNFR-2 was decreased, and FASN, Wnt-1, ß-catenin and cytC were increased. The expression of Wnt-1 and ß-catenin in MCF-7-MEK5 decreased after cerulenin treatment, whereas cytC expression increased. CONCLUSIONS: The important function of FASN in the drug tolerance of breast cancer may be due to the following mechanisms: FASN downregulated TNFR-2 expression through lipid rafts to make the cells less sensitive to TNF-α, and simultaneously activated the Wnt-1/ß-catenin signalling pathway. Thus, cytC expression increased, which provided cells with anti-apoptotic capacity and induced drug tolerance.

[Comparision of intracellular localization and recycling route of mouse nepmucin and CD31 in endothelial cells].

AIM: To compare the localization and recycling between nepmucin and CD31 molecules on transfected endothelial cells, and attempted to clarify the recycling mechanisms of nepmucin in endothelial cells. METHODS: Recycling assay and internalization assay were employed to compare the localization and recycling pathway of nepmucin and CD31. The internalized and recycling nepmucin and CD31 molecules on transfected endothelial cells were double or single stained with specific fluorchrome-labeled monoclonal antibodies against nepmucin (Alexa Fluor 488-ZAQ5) and/or CD31 (Alexa Fluor 488-anti-CD31 or Alexa Fluor 594-anti-CD31), then observed under confocal microscopy. RESULTS: Mouse nepmucin underwent intracellular recycling like CD31, but which recycling rate was significantly lower. The CD31 and nepmucin molecules showed largely distinct localization in endothelial cells. CD31 was found mainly on the cell surface, while nepmucin was found predominantly in the deep area of cytoplasm and partly on the cell membrane. CONCLUSION: The distribution of mouse nepmucin in endothelial cells are different from CD31. Nepmucin underwent intracellular recycling like CD31 but employed different mechanisms.

[Preparation and identification of monoclonal antibody against hMOF].

OBJECTIVE: To prepare and identify the monoclonal antibody (mAb) against hMOF. METHODS: BALB/C mice were immunized with protein from the spleen cells isolated and fused with SP2/0 cells. After several rounds of screening and cloning, the hybridoma cell strain secreting anti-hMOF mAb was obtained. Its specificity was evaluated with ELISA and Western blot, and the titer, immunoglobulin subtype and affinity of the mAb were measured. RESULTS: One cell strains of hybridoma were obtained and named as 4C1C8. The anti-hMOF mAb secreted by the hybridoma cell strain was identified as IgG1 subtype. The mAb titers in ascitic fluid were 1 409600, as determined with ELISA with an affinity reaching to 7.65 x 10(6) L/mol. Western blot demonstrated that the antibodies could specifically recognize the immunogen. The cell immunohistochemistry proved that the antibody could recognize the hMOF antigens expressed on the normal cells HL7702. CONCLUSION: The success in anti-hMOF mAb preparation provides the basis for further study of hMOF.

Effect of anaphylatoxin C3a, C5a on the tubular epithelial-myofibroblast transdifferentiation in vitro.

BACKGROUND: Tubulointerstitial renal fibrosis is the common end point of progressive kidney diseases, and tubular epithelial-myofibroblast transdifferentiation (TEMT) plays a key role in the progress of tubulointerstitial renal fibrosis. Anaphylatoxin C3a and C5a are identified as novel profibrotic factors in renal disease and as potential new therapeutic targets. The aim of this study was to investigate whether C3a, C5a can regulate TEMT by transforming growth factor-ß1 (TGF-ß)/connective tissue growth factor (CTGF) signaling pathway and the effects of C3a and C5a receptor antagonists (C3aRA and C5aRA) on C3a- and C5a-induced TEMT. METHODS: HK-2 cells were divided into C3a and C5a groups which were subdivided into four subgroups: control group, 10 ng/ml TGF-ß1 group, 50 nmol/L C3a group, 50 nmol/L C3a plus 1 µmol/L C3aRA group; control group, 10 ng/ml TGF-ß1 group, 50 nmol/L C5a group, 50 nmol/L C5a plus 2.5 µmol/L C5aRA group. TGF-ß1 receptor antagonist (TGF-ß1RA) 10 µg/ml was used to investigate the mechanism of C3a- and C5a-induced TEMT. Electron microscopy was used to observe the morphological changes. Immunocytochemistry staining, real-time PCR and Western blotting were used to detect the expressions of a smooth muscle actin (α-SMA), E-cadherin, Col-I, C3a receptor (C3aR), C5aR, CTGF and TGF-ß1. RESULTS: HK-2 cells cultured with C3a and C5a for 72 hours exhibited strong staining of α-SMA, lost the positive staining of E-cadherin, and showed a slightly spindle-like shape and loss of microvilli on the cell surface. The expressions of α-SMA, E-cadherin, Col-I, C3aR, C5aR, TGF-ß1 and CTGF in C3a- and C5a-treated groups were higher than normal control group (P < 0.05). C3aRA and C5aRA inhibited the expressions of α-SMA, Col-I, C3aR, C5aR, and up-regulated the expression of E-cadherin (P < 0.05). TGF-ß1 and CTGF mRNA expressions induced by C3a and C5a were partly blocked by TGF-ß1RA (P < 0.05). CONCLUSION: C3a and C5a can induce TEMT via the up-regulations of C3aR and C5aR mRNA and the activation of TGF-ß1/CTGF signaling pathway in vitro.

[Preparation and identification of a monoclonal antibody against recombinant human L-type pyruvate kinase N].

OBJECTIVE: To prepare and identify the monoclonal antibody (mAb) against pyruvate kinase N terminal (PK-N). METHODS: BALB/C mice were immunized with immunogen PK-N-GST-tag. Then the spleen cells were isolated and fused with SP2/0 cells. After several rounds of detecting and cloning, the hybridoma cell strains secreting anti-PK-N mAb were obtained. Its specificity was evaluated with ELISA and Western blot, and the titer, immunoglobulin subtype and affinity of the mAb were measured. RESULTS: Two cell strains of hybridoma, 2B2E4G and 2C6F5, were obtained. The hybridoma cell strains secreting anti-PK-N mAb belonged to IgG2b subtype, with a mAb titer in ascetic fluid of 1 : 409600 and 1 : 102400, respectively. Their affinity reached 3.54 x 10(8) L/mol and 2.72 x 10(8) L/mol, respectively, as determined by ELISA. Western blot demonstrated that the mAb could specifically recognize the immunogen and the natural cell lysis protein. The cell immunohistochemistry proved that the antibody could recognize human L type pyruvate kinase expressed in the plasma of HL-7702 cell strain and paraffin slice of hepatoma. CONCLUSION: The success in anti-PK-N mAb preparation provides a foundation for further studies into glycolysis in normal condition and metabolic diseases.

[The expression of interleukin-17 in blood and nasal tissue of patients with allergic rhinltis and nasal polyps].

OBJECTIVE: To study the expression of IL-17 in blood and nasal tissue of allergic rhinitis and nasal polyps, and to investigate the possible mechanism of IL-17 in the progress of allergic rhinitis and nasal polyps. METHODS: There were 41 patients enrolled, including the patients with allergic rhinitis, nasal polyps and deviated nasal septum. The blood and nasal mucosa tissue or nasal polyps were collected in all the patients. The expression of IL-17 was measured by the methods of ELISA and immunohistochemisty, and the number of eosinophils and IL-17 positive cells was measured. RESULTS: The IL-17 expression of nasal polyps was significantly elevated in the blood and nasal tissue, which was obviously correlated with the number of eosinophils infiltrated in nasal tissue. However, for the patients with allergic rhinitis, there was only apparent expression of IL-17 in nasal mucosa. CONCLUSION: IL-17 plays an important role in the development of nasal polyps, but for allergic rhinitis, it needs further study as an potential important aspect of the pathogenesis.

[The effects of intense pulsed light and 5-aminolevulinic acid on the cultured B16 marine melanoma cells].

OBJECTIVE: To investigate the effects of IPL and IPL plus 5-ALA on the proliferation, melanogenesis and tyrosinase activity of cultured B16 murine melanoma cells. METHODS: B16 cells were pretreated with or without ALA and irradiated with IPL (20, 30, 40 J/cm2), also the negative control group and the positive control UVA irradiation group (3, 4, 5 J/cm2) were designed. The proliferation of the treated B16 cells were evaluated with MTT method, the melanogenesis of the cells was tested with NaOH assay as well as tyrosinase activity was detected at 1 d, 2 d, 3 d, 4 d, and 5 d after irradiation. RESULTS: 5 mmol/L 5-ALA did not affect the proliferation, melanogenesis and tyrosinase activity in B16 melanoma cell. The cell yielding in UVA group increased significantly (P < 0.05) compared with that of negative control group after UVA irradiation (3, 4 J/cm2), but the proliferation of the treated B16 cells were suppressed after UVA irradiation (5 J/cm2). IPL (20, 30, 40 J/cm2) or IPL plus ALA did not have any significant effects on cell proliferation. IPL or IPL plus ALA have inhibitory effects on melanogenesis without any influence on cell tyrosinase activity in B16 cells. ALA, as the photosensitizer, combined with IPL could have stronger effect than IPL alone. CONCLUSIONS: We demonstrated that UVA could stimulate the proliferation of B16 cells, increase the melanogenesis and tyrosinase activity of the cells. IPL or IPL plus ALA have inhibitory effects on melanogenesis without any influence on cell proliferation and tyrosinase activity in B16 cells. IPL plus ALA had stronger effect than IPL alone.

[Effect of recombinant super-compound interferon (rSIFN-co) on human breast cancer cells in vitro].

OBJECTIVE: To study the anti-cancer activities of recombinant super-compound interferon (rSIFN-co) against human breast cancer cells, its cooperative anti-tumor effect with chemotherapy and the possible mechanism of these effects. METHODS: The rSIFN-co, infergen, epirubicin were applied separately or combined in cultured human breast cancer cells MCF-7 and adriamycin-resistant strains of MCF-7/ADR cells. MTT assays and flow cytometry were applied to determine the growth inhibition and cell apoptosis index (AI) of MCF-7 and MCF-7/ ADR after different treatment. Immunohistochemical was used to detect the expression of P53, Bcl-2, CerbB-2 in treated MCF-7 and MCF-7/ADR cells. RESULTS: The rSIFN-co demonstrated a efficiency of inhibiting the cell proliferation, induce apoptosis of both MCF-7 cells and MCF-7/ADR cells, which was stronger than that of infergen in the same concentration; rSIFN-co combined with epirubicin has a synergistic action in inhibiting cell proliferation, inducing apoptosis of both type of breast cancer cells; rSIFN-co can also decrease the expression level of P53 and CerbB-2, increase the expression of Bcl-2 in both cell lines. CONCLUSION: In vitro conditions, rSIFN-co can inhibit cell growth of human breast cancer cell MCF-7 and MCF-7/ADR, which may attribute to influence the proliferation-associated factor expression, and induce apoptosis of cancer cells.

[Autophosphorylation of erythrocyte insulin receptors in patients with polycystic ovary syndrome].

OBJECTIVE: To investigate the tyrosine autophosphorylation of insulin receptors in patients with polycystic ovary syndrome (PCOS). METHODS: Forty patients with PCOS and 20 age- and body mass index matched healthy women were included in the study. The patients with PCOS were classified as HI-PCOS (n=20) or non-HI-PCOS (n=20) based on the fasting insulin level (>or< or =15 mIU/L). 1) Serum gonadotropins and ovarian steroids were determined. Oral glucose tolerance tests (OGTT) and insulin releasing tests were performed to calculate the insulin resistance index (HOMA-IR). 2) Blood samples were obtained at five time-points during the OGTT (Fasting and 30 min, 60 min, 120 min, and 180 min after taking 75 g glucose orally). The erythrocytes were isolated and the autophosphorylation insulin receptors (APIR) and total insulin receptors (TIR) were measured by enzymatic immunoassay. 3) The in vivo autophosphorylation of insulin receptors was indicated by the APIR/TIR ratio. RESULTS: The HI-PCOS patients had lower APIR/TIR ratio than the non-HI-PCOS patients and healthy controls at the 60th minute after OGTT (P<0.05). No differences in other time-points were significant. CONCLUSION: The autophosphorylation of insulin receptors in HI-PCOS patients decrease, which might be a mechanism for insulin resistance in patients with PCOS.

[Preparation and application of biotinylated anti-AIB1 moniclonal antibody].

OBJECTIVE: To cross-link the McAb 1A2E1 to BNHS reagents and apply this biotinylated McAb to detect AIB1 antigen of target cells. METHODS: The McAb 1A2E1 was mixed with BNHS reagents to generate Biotin-1A2E1. The competitive inhibition ELISA and immunocytochemical method were applied to detect the biologic activity of antibody. RESULTS: The antibody kept high biological activities (with a titer of 1 : 3200) and sensitivities in detecting the AIB1 protein of breast cancer cell. CONCLUSION: The method of preparing biotinylated McAb is successful, and the prepared biotinylated McAb can be applied to detect target cells which express AIB1 antigen. This McAb provide useful tool for tumor auxiliary diagnosis.

[Effect of metastasis suppressor gene KAI1 on the proliferation and invasive ability of cervical carcinoma cells].

OBJECTIVE: To explore the effect of metastasis suppressor gene KAI1 on the proliferation and invasive ability of cervical cancer cell line CaSki. METHODS: pCMV-KAI1 cDNA plasmid was transferred into cervical carcinoma cell line CaSki by liposome, which had low level of endogenous KAI1 expression. The expressions of KAI1 protein and mRNA were determined by immunohistochemistry and real-time fluorescence quantitative PCR (RT-PCR), the proliferation of KAI1-transfected CaSki cells was investigated by MTT assay and the invasive ability of these cells was evaluated by in vitro invasion assays. RESULTS: After the transfection of pCMV-KAI1 cDNA, the level of KAI1 mRNA and protein expression in CaSki cell were increased (P < 0.05), while the cell proliferation was suppresssed, and the migrative ability of passing through the membrane filte also decreased evidently (P < 0.05). CONCLUSION: The KAI1 metastasis suppressor gene suppressed the ability of proliferation and invasion of cervical cancer cell CaSki in vitro.

[Inhibition of proliferation in MCF-7 breast cancer cells by plasmid-based siRNA targeting to oncogene c-myc].

OBJECTIVE: To investigate the effects of plasmid-based siRNA targeting to oncogene c-myc on c-myc/ c-Myc expressions and cells proliferation in MCF-7 breast cancer cells. METHODS: siRNA eukaryotic expression plasmid p-Mat01-1 targeting to the sequence 589-609 of oncogene c-myc and its mismatch plasmid p-Mis09-1 were constructed, and transiently transfected MCF-7 cells using Lipo2000. Semi-quantitative RT-PCR and Western blot were used to analyze the expressions of c-myc/c-Myc in MCF-7 cells, and cells proliferation was detected by MTT assay. RESULTS: p-Mat01-1 inhibited the expressions of c-myc mRNA (24 h: P < 0.01) and c-Myc protein (5 d. P < 0.01) in MCF-7 cells as compared with pEGFP-C1 and p-Mis09-1 controls, and suppressed the proliferation of MCF-7 cells significantly (3 d: P < 0.05, 5, 7 d: P < 0. 01). CONCLUSION: Plasmid-based siRNA targeting to oncogene c-myc could inhibit the expressions of c-myc/c-Myc in MCF-7 breast cancer cells efficiently, suggesting that the downregulation of c-myc/c-Myc could suppress the proliferation of MCF-7 cells in vitro.

[Effect of aurora A on carcinogenesis of human prostate cancer].

OBJECTIVE: To investigate whether Aurora A is involved in prostate cancer carcinogenesis. METHODS: The expressions of Aurora A mRNA and protein in prostate cancer tissue and cell line were measured using RT-PCR, immunohistochemistry and Western blot analyses. Meanwhile, A DNAzyme targeting at Aurora A mRNA was performed to detect the inhibition effect of Aurora A in regulating the growth of prostate cancer cell line PC3. RESULTS: Aurora A expression was up-regulated in 91% prostate cancer tissue as well as in prostate cancer cell lines such as PC3, LNCaP and Du145. Furthermore, Aurora A expression suppressed by cell transduction of a DNAzyme targeting at Aurora A mRNA made the PC3 cells both arrest the cell cycle in G2/M phase and increase the cell apoptosis. CONCLUSIONS: These findings suggest that Aurora A up-regulation may be involved in carcinogenesis of prostate cancer, and that aurora A gene may be a valuable target for treatment of prostate cancer.

[Preparation and characterization of a monoclonal antibody against human SOCS3].

OBJECTIVE: To prepare and characterize the monoclonal antibody (mAb) against human SOCS3. METHODS: BALB/c mice were immunized with recombinant GST-SOCS3 protein, from which the spleen cells were isolated and fused with Sp2/0 cells. After several rounds of screening and cloning, the hybridoma cell strain secreting anti-SOCS3 mAb was obtained, whose specificity was evaluated using ELISA and Western blotting, and the titer, immunoglobulin subtype and affinity of the mAb were also measured. RESULTS: The hybridoma cell strain secreting anti-SOCS3 mAb was identified to belong to IgG2a subtype. The mAb titers in cultural supernatant and acetic fluid were 1:640 and 1:25600, respectively, as determined by ELISA with affinity reaching 4.84x10(6) L/mol. CONCLUSION: The success in anti-SOCS3 mAb preparation provides the basis for further study of the negative regulation of cytokine signal transduction and the immunoregulation in microorganism infections.

Preparation, characterization and uptake by primary cultured rat hepatocytes of liposomes surface-modified with glycyrrhetinic acid.

3-succinyl-30-stearyl glycyrrhetinic acid (Suc-GLAOSt) was synthesized as a targeting molecule, and incorporated in ordinary to liposomes (LP) to prepare a liposome surface-modified with glycyrrhetinic acid (LP-GLA), which could bind to the hepatocyte through the specific binding site of glycyrrhetinic acid (GLA) on the surface of rat cellular membrane. The maximal molar ratio of Suc-GLAOSt to total lipids in LP-GLA was 1:10. Calcein loaded liposome (Cal-LP) and calcein loaded LP-GLA (Cal-LP-GLA) were prepared by an ethanol injection method. The average diameter of Cal-LP and Cal-LP-GLA was 65 nm +/- 16 nm and 68 nm +/- 21 nm, respectively. The characteristics of cellular uptake of the two types of liposome were investigated through cellular uptake and competitive inhibition experiments. The uptake of Cal-LP-GLA by rat hepatocytes was markedly higher (3.3-fold) than that of Cal-LP (P < 0.01). The uptake of Cal-LP-GLA was inhibited, but the uptake of Cal-LP was not influenced by adding extraneous GLA. LP-GLA may be internalized by hepatocytes via the specific binding site, and can be used as a novel and promising carrier for targeting drug delivery to hepatocytes.

[The preparation and identification on monoclonal antibody to platelet-derived growth factor receptor].

OBJECTIVE: Through genetic engineering to produce the fusion protein of glutathione S-transferase (GST) linked to amino-terminal end of platelet-derived growth factor receptor (PDGFR), and to prepare the bioactive monoclonal antibody. METHODS: With taking GST-PDGFR-N fusion protein as immunogen, the anti-PDGFR monoclonal antibody was produced by using the hybridoma technique, of which then the antigen binding characteristic was identified by indirect enzyme-linked immunosorbent assay (ELISA), Western blot and immunohistochemistry methods. RESULTS: Two cell strains of hybridoma were obtained and named as 3B12F5 and 3C6H7C11 which secreted the anti-PDGFR monoclonal antibody, of which the class and subtype identification demonstrated both strains to produce all type of IgG1. The indirect ELISA result showed that the titers of ascites fluid which two hybridoma induced were 1 : 102400 and 1 : 25600. Western blot demonstrated that the two antibodies could recognize specifically the immunogen on PDGFR and U251 cell line. The cell immunohistochemistry proved that the antibody could recognize the expressed PDGFR antigens of neurogliocytoma U251 and bladder carcinoma BIU87 cell lines. CONCLUSION: We prepare successfully the PDGFR monoclonal antibody and provide a useful tool for researching on the PDGFR expression and clinical detection.

[Effect of PPAR-gamma ligand RGZ on inhibiting the cell proliferation of cholangiocarcinoma].

OBJECTIVE: To investigate the effect that PPAR-gamma (peroxisome proliferator-activated receptor gamma, PPAR-gamma) ligand rosiglitazone (rosiglitazone, RGZ) inhibits the cell proliferation of cholangiocarcinoma. METHODS: The cell line QBC939 of cholangiocarcinoma was interfered with different concentration of RGZ, and then calculated for the rate of cell proliferation inhibited at different concentration. The change of cell cycle and the rate of cell apoptosis at each concentration were detected by FCM. RESULTS: RGZ showed the significant effect on inhibiting the cell proliferation of cholangiocarcinoma, especially on the 1200 mg/L concentration group. The highest inhibited rate of QBC939 cell proliferation could be up to 83.66%. After RGZ used to treat the cultured QBC939 cell for 48 h and 72 h, the inhibited rates of QBC939 cell proliferations of 1200 mg/L, 600 mg/L,300 mg/ L,150 mg/L, 75 mg/L and 37.45 mg/L groups were compared to those of control group, and with the statistics result of P < 0.001. Meanwhile the cell cycles were controlled significantly as well, 62.77% of the cells were detained in stage G0/G1. CONCLUSION: In vitro PPAR-gamma ligand rosiglitazone has the significant proliferation inhibition effect to cell lines QBC939 of cholangiocarcinoma.