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ABSTRACT: The incidence of Xuanwei lung cancer ranks first in China, and its pathogenesis requires in-depth investigation. This study aims to establish an orthotopic Xuanwei lung cancer severe combined immunodeficiency (SCID) mouse model and to provide a basic experimental platform for further study.
The Xuanwei lung cancer cell line XWLC-05 was inoculated into the lung tissue of SCID mice in high and low doses. The tumor formation rates, tumor characteristics, spontaneous metastases, and survival times of the mice were observed, taking a subcutaneously transplanted tumor as control.
The tumor formation rates of the orthotopic transplantation of lung cancer cells in high and low doses were 81% and 83%, respectively, among which mice in the high-dose group appeared cachectic on day 13. Extensive invasion and adhesion were observed in the contralateral lung and thoracic cavity, but no distant metastasis was exhibited. Mice with low-dose cells in the orthotopic transplantation group appeared cachectic and distant metastasis occurred on day 25. The tumor formation rates in the subcutaneous inoculation group by the high and low doses of cells were 100% and 94.5%, respectively, and no distant metastasis was observed. The rate of metastasis within the orthotopic transplantation group and between the orthotopic and subcutaneous inoculation groups showed a significant difference (P<0.05). A significant difference was indicated by the survival rate within and between the groups (P<0.001).
We successfully established an orthotopic XWLC SCID mouse model, which lays the foundation for a more in-depth study.
Zhongguo fei ai za zhi = Chinese journal of lung cancer 08/2012; 15(8):449-55.
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Ye Duan,
Tie-jun Gu,
Chun-lai Jiang,
Ruo-sen Yuan,
Hua-fei Zhang,
Hong-jia Hou,
Xiang-hui Yu, Yan Chen,
Yong Zhang,
Yong-ge Wu,
Wei Kong
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ABSTRACT: Rabies is a fatal infectious disease requiring efficient protection provided by post-exposure prophylaxis (PEP) with rabies immunoglobulin (RIG). The single-chain Fv fragment (scFv) is a small engineered antigen binding protein derived from antibody variable heavy (V(H)) and light (V(L)) chains. This novel antibody format may potentially replace the current application of RIG to detect and neutralize rabies virus (RV). However, the broad use of scFvs is confined by their generally low stability. In this study, a scFv (FV57) was constructed based on the monoclonal antibody, MAB57, against RV. To enhance its stability and neutralizing potency, a disulfide-stabilized scFv, ds-FV57, was also derived by introduction of cysteines at V(H)44 and V(L)100. Furthermore, the cysteine at V(L)85 of ds-FV57 was mutated to serine to construct ds-FV57(VL85Ser) in order to avoid potential mis-formed disulfide bonds which would alter the affinity of the scFv. The stability and activity of all three proteins expressed in Escherichia coli were evaluated. All of the constructed scFvs could provide efficient protection against RV infection both in vivo and in vitro. However, the stability of ds-FV57(VL85Ser) was notably improved, and its in vitro neutralizing potency against RV infection was enhanced. Our findings from these stabilization modifications support the feasibility of developing scFvs for PEP treatment of rabies.
Molecular Immunology 04/2012; 51(2):188-96. · 2.90 Impact Factor
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Yongping Yang,
Yinying Lu,
Chunping Wang,
Wenlin Bai,
Jianhui Qu, Yan Chen,
Xiujuan Chang,
Linjing An,
Lin Zhou,
Zhen Zeng,
Min Lou,
Jiyun Lv
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ABSTRACT: We assessed the safety and efficacy of sorafenib with cryotherapy (cryoRx) in advanced hepatocellular carcinoma (HCC). One hundred four HCC patients were enrolled, who met the following criteria: (i) Barcelona Clinic Liver Cancer stage C; (ii) HCC without distant metastasis; (iii) the presence of portal vein thrombosis (PVT); (iv) Child-Pugh class A or B; and (v) life expectancy of at least 12 weeks. The patients were randomly divided into sorafenib-cryoRx and sorafenib (control) groups. Primary endpoint was time to progression (TTP); secondary endpoints included overall survival (OS) and tolerability. Microvessel density (MVD) was assessed by CD34-immunostaining. After a median 10.5 (4-26) months follow-up, the data showed that median TTP was 9.5 (8.4-13.5) months in combinatorial therapy group vs. 5.3 (3.8-6.9) months in sorafenib group (P = 0.02). The median OS was 12.5 (95 % CI 10.6-16.4) months in combination therapy group vs. 8.6 (7.3-10.4) months in sorafenib group (P = 0.01). Low MVD patients in combination therapy exhibited significantly longer median TTP and OS than controls. High MVD was predictive of poor responses to sorafenib. CryoRx did not increase frequency/degree of sorafenib-related adverse events. Therefore, it was concluded that the addition of cryoRx significantly improved clinical outcomes of Sorafenib therapy in advanced HCC with acceptable tolerance and similar safety profiles as previously reported.
Cell biochemistry and biophysics 04/2012; 63(2):159-69. · 3.34 Impact Factor
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ABSTRACT: To establish a lung cancer A549 cell line stable over-expressing human integrin linked kinase (ILK) and study the effect of over-expression of ILK on biological activity of A549 cells.
Human ILK gene was amplified by RT-PCR, then cloned into pEGFP-C1 vector to construct pEGFP-ILK. After confirmed by restriction analysis and sequencing, the recombinant plasmid was transfected into A549 cells mediated with liposome, then G418-resistant clones of A549 cells (A549/pEGFP-ILK) as experimental group were obtained, and paralleled with the vector control (A549/pEGFP-C1) and A549 cell control. The expression and localization of EGFP-ILK fusion protein in A549 cells was observed by fluorescence microscopy. RT-PCR and Western blot were performed to detect the level of ILK mRNA and ILK protein of each group cells respectively. The cell proliferation was tested by methylthiazolyl tetrazolium (MTT) assay, and the cell apoptosis was measured by flow cytometry, and the morphologic changes of cells were observed by HE staining.
Both restriction analysis and sequencing proved that the pEGFP-ILK plasmid was constructed correctly. The distribution of fluorescence of stable transfected A549 cells indicated that the product of ILK gene was mainly located in cytoplasm. Compared with A549/pEGFP-C1 group and A549 group, the level of ILK mRNA and ILK protein of A549/pEGFP-ILK cells were significantly increased, which over-expression ratio was 218.18% and 245.45% respectively (P<0.05). The proliferation ability of the A549 cells over-expressing ILK was increased significantly (P<0.05). However, the apoptosis of A549/pEGFP-ILK cell was inhibited significantly by over-expression of ILK (P<0.05). After HE staining, the increased mitosis were observed only in A549/pEGFP-ILK group cells.
The lung cancer cell line stable over-expressing ILK protein was constructed successfully, and ILK over-expression could promote cell proliferation, inhibit cell apoptosis and increase mitosis.
Xi bao yu fen zi mian yi xue za zhi = Chinese journal of cellular and molecular immunology 04/2012; 28(4):340-3.
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ABSTRACT: Vascular endothelial growth factor (VEGF) has been known as a potential vasculogenic and angiogenic factor and its receptor (VEGFR2) is a major receptor to response to the angiogenic activity of VEGF. The technique that to break the immune tolerance of "self-antigens" associated with angiogenesis is an attractive approach for cancer therapy with T4 phage display system. In this experiment, mouse VEGFR2 was constructed on T4 phage nanometer-particle surface as a recombinant vaccine. T4-mVEGFR2 recombinant vaccine was identified by PCR and western blot assay. Immunotherapy with T4-mVEGFR2 was confirmed by protective immunity against Lewis lung carcinoma (LLC) in mice. The antibody against mVEGFR2 was detected by ELISPOT, ELISA and Dot ELISA. The inhibitive effects against angiogenesis were studied using CD31 and CD105 via histological analysis. VEGF-mediated endothelial cells proliferation and tube formation were inhibited in vitro by immunoglobulin induced by T4-mVEGFR2. The antitumor activity was substantiated from the adoptive transfer of the purified immunoglobulin. Antitumor activity and autoantibody production of mVEGFR2 could be neutralized by the depletion of CD4+T lymphocytes. These studies strongly suggest that T4-mVEGFR2 recombinant vaccine might be a promising antitumor approach.
Vaccine 04/2011; 29(34):5802-11. · 3.77 Impact Factor
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ABSTRACT: The mouse lung cancer orthotopic model includes spontaneous lung cancer model and endotracheal transplanted model, and etc. The spontaneous lung cancer needs longer time and does not ensure the rate of the generation of the tumor; as for endotracheal transplanted model, the position and size of the tumor are instable. In this study, the 3LL cell line was orthotopically transplanted into the lung of the C57BL/6 mice, compare to the heterotopic model, to discuss their stability and transfer-characteristics. And this study was also to optimize the method of establishing lung cancer orthotopic animal model.
Different quantity of 3LL cells were inoculated into the left oxter of C57BL/6 mice to establish the heterotopic model; or suspended with Matrigel then inoculated into the left lung of C57BL/6 mice to establish orthotopic model. The survival-time of the mice was examined. The tissue was collected for the subsequent histology assay after euthanizing the mice. Microvessels density (MVD) was observed and counted by immunohistological chemistry. CD44v was detected by flow cytometry.
TTumor-form-rate of the heterotopic group were 100%, 66.7%, 16.7%, respectively, and had no macroscopic transfer. Tumor-form-rate of the orthotopic group were 100%, 100%, 83.3%, respectively, and had widespread transfer in contralateral chest and the lung. The median survival time of the orthotopic group (38, 35, 23 days) were less than the heterotopic group (82, 72, 50 days). MVD of the orthotopic group (120.2 +/- 9.73) was higher than the heterotopic group (92.6 +/- 7.12). The expression of CD44v of orthotopic (26.46 +/- 1.56)% was higher than the heterotopic group (23.13 +/- 1.02)%.
The lung cancer orthotopic model which established by 3LL cells transplanted into the lung of the mice is simple, dependable, repeatable and has stronger transfer characteristics than the heterotopic model.
Zhongguo fei ai za zhi = Chinese journal of lung cancer 01/2010; 13(1):42-7.
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ABSTRACT: It has been known that vascular endothelial growth factor (VEGF) and its receptor (VEGFR2) play important roles in tumor angiogenesis. The aim of this study is to investigate whether an oral DNA vaccine against VEGFR2 has the inhibition effect on tumor growth and angiogenesis, and explore its mechanism in vivo.
C57BL/6 mice were respectively given the DNA vaccine encoding VEGFR2 (vaccine group), pcDNA3.1 (plasmid group) and saline (saline group). All the mice were then inoculated with Lewis lung carcinoma 3LL cells. Weight, size and microvessel density (MVD) of transplanted tumors were observed. The levels of CD3+ and CD8+ T cells in peripheral blood of mice were detected by flow cytometry.
Weight of transplanted tumors in vaccine group was significantly smaller than those in plasmid and saline groups (P < 0.05), and MVD was significantly lower in vaccine group than that in plasmid and saline groups (P < 0.05). After inoculated with 3LL cells, CD3+ and CD8+ T cell levels of vaccine group were markedly higher than those of plasmid and saline groups (P < 0.05).
The oral DNA vaccine can significantly inhibit angiogenesis and growth of transplanted tumor in mice. It may act through killing endothelial cells of tumor.
Zhongguo fei ai za zhi = Chinese journal of lung cancer 10/2007; 10(5):366-9.
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ABSTRACT: Sulfonated polybenzothiazoles with benzimidazole moieties were successfully prepared by polycondensation. For comparison purpose, sulfonated polybenzothiazoles were also synthesized. Rigid-rod sulfonated polybenzothiazoles generally show poor solubility in organic solvents, whereas these sulfonated polybenzothiazoles with high sulfonation degree are soluble, but the membranes cast from them swell excessively in water. Thus they could not be used as proton exchange membranes (PEMs). The comparison study demonstrates that the incorporation of benzimidazole moieties to sulfonated polybenzothiazoles greatly enhances the solubility of products. The resulting copolymers thus could be cast into the membranes as PEMs. The benzimidazole moieties in the resulting polymers improve thermal properties, dimensional stability, tensile strength, Young's modulus, and oxidative stability, but they decrease proton conductivity and the elongation at break, due to the ionic cross-linking and hydrogen bonds. Even so, sulfonated polybenzothiazoles with appropriate content of benzimidazole moieties (10 mol%) could still display a high proton conductivity of 0.094 S cm−1 at 80 °C and an elongation at break of 26.1%. These ionomers exhibited excellent overall properties such as excellent mechanical properties, high thermal and oxidative stability, low swelling, and high proton conductivity, thus they might be a candidate for proton exchange membranes.
Journal of Membrane Science.
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ABSTRACT: The classical Gubbio (Italy) Cretaceous-Tertiary boundary section has been resampled for both magnetostratigraphy and iridium. Paleomagnetic samples were taken over 7 m in the Maastrichtian and 6 m in the Paleocene. Previous results obtained a decade ago are confirmed. The reversal sequence is well defined and the individual reversals are somewhat more precisely located. A noticeable difference is the location of the position of the 29N/29R limit which may be lowered by 20–30 cm in the stratigraphic column. This would imply that the KTB occurs near the middle of chron 29R. Iridium measurements were made on samples from both shales and surrounding limestone beds from 2 m below to 3 m above the boundary: these measurements indicate that Ir is associated with clay minerals. Concentrations in the two types of samples are indeed compatible when reduced to a carbonate-free basis. Iridium concentrations stand above background over almost 3 m of section, corresponding to half a million years based on magnetostratigraphy. This is likely to indicate a protracted duration of the (external or internal) source of iridium, on top of which the main (short-lived) KTB anomaly proper stands.
Earth and Planetary Science Letters.
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ABSTRACT: Previous studies have demonstrated that bone morphogenetic protein-4 (BMP4) could participate in vivo endochondral ossification and is one of the main local contributing factors in the early stage of fracture healing. To investigate the effectiveness of BMP4 gene transfer, we constructed an adenoviral vector, Ad-BMP4, and evaluated its osteoinduction activity both in vitro and in vivo. In vitro study suggested that this vector could efficiently transduce mouse myoblast C2C12 cells and produce osteogenic BMP4 protein, as confirmed by immunofluorescence analysis and alkaline phosphatase activity assay. For in vivo study, Ad-BMP4 was directly injected into the hind limb muscles of male athymic nude rats. Visible new bone formation under X-ray films could be detected as early as three weeks post-injection. The bone tissue was further analyzed by histological staining and revealed a typical remodeled bone structure. In conclusion, this study is the first to establish the feasibility of adenovirus-based BMP4 gene therapy for bone regeneration.
Biochemical and Biophysical Research Communications.
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ABSTRACT: In Xenopus oocytes, Ca2+ influx through an endogenous voltage-gated Ca2+ channel activates a transient outward Cl− current (ICI(Ca)), which is potentiated by cAMP increase. The site of cAMP effect appears to be the Ca2+ channel instead of the Ca2+-activated Cl− channel, because cAMP potentiates the Ba2+ current through the Ca2+ channel in a similar way to the ICl(Ca), and cAMP does not potentiate the Ca2+-dependent CI− current in cells treated with Ca2+ ionophore. Using the catalytic subunit of protein kinase A (PKA) and PKA inhibitors, it was shown that PKA is both necessary and sufficient for the cAMP effect on ICI(Ca). Furthermore, the cAMP/PKA-mediated potentiation of IcI(Ca) was inhibited by both type 1 and type 2A protein phosphatases.
FEBS Letters.
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ABSTRACT: A study of the Neogene low-aspect-ratio Kizilkaya ignimbrite (in the calc-alkaline Central Anatolian Volcanic Province, Turkey) has been carried out using the anisotropy of magnetic susceptibility (AMS) technique on 46 sites. IRM, hysteresis loops and Curie temperature analysis indicate that magnetite is the main carrier of the magnetic signal in the ignimbrite. The shape of the AMS ellipsoids varies widely from site to site and the anisotropy is commonly low: we review the data processing used in the literature on the AMS of ignimbrites to calculate principal directions or to evaluate the quality of the results, and we develop various simple techniques (data filtering and contouring procedures) in order to more accurately define the axis or symmetry of the AMS fabric. There is no simple relationship between the shape of the AMS ellipsoids and the shape of the petrofabric ellipsoids. Similarly, there is no direct relationship between magnetic and kinematic axes. Our results show that the source of the measured magnetic signal in these rocks is a complex result of various contributions including shape anisotropy of free and fixed magnetic crystals (shape AMS), crystallization of magnetic grains on all surfaces and discontinuities during cooling of the ignimbrite (distribution anisotropy), and alteration by hydrothermal and meteoric water. Particular attention is paid to the interpretation of the AMS stereoplots departing from the “standard” fabric of sedimentary rocks formed under unidirectional flow. In order to explain these “non-standard” AMS fabrics, we discuss their possible origin: (1) in analytical and mineralogical terms (instrumental artifact, pollution by xenoclasts, mineralogical inversion of the susceptibility axes, secondary mimetic fabric); and (2) in terms of sedimentological mechanisms (rheology, depositional processes). In the present state of knowledge, we conclude that, due to the physico-chemical complexity of the magnetic source, the AMS signal should be used and interpreted with care when attempting to decipher the sedimentological or rheological processes occurring in moving pyroclastic flows. On the other hand, AMS has it greatest potential when used in conjunction with other techniques, and when a large number of sampling sites and specimens is used.
Earth and Planetary Science Letters.
