Ping Xu

First People's Hospital Chenzhou, Chenchow, Hunan, China

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Publications (14)1.37 Total impact

  • [Show abstract] [Hide abstract]
    ABSTRACT: The expression of vascular endothelial growth factor (VEGF) is regulated by microenvironmental factors within the tumors, such as hypoxia, free radicals, pH imbalance and nutrient deficiency. The purpose of this study was to observe VEGF activity in tumor cells under different stress conditions. A plasmid was generated, consisting of green fluorescent protein (GFP) fused to a 1,217-bp sequence, which was located downstream and upstream of the transcriptional start site of VEGF, respectively. The plasmid was stably transfected into 4T1 mouse breast carcinoma cells. Cells were cultured in a medium with nitric oxide (NO) donor sodium nitroprusside (SNP), hypoxia-mimetic agent deferoxamine mesylate (DFX), H2O2, absence of serum and lowered or elevated pH, or were heat-shocked, followed by measurement of VEGF activity by reverse transcription polymerase chain reaction (RT-PCR) and ELISA. Hypoxia, SNP and H2O2 led to increments of VEGF mRNA and protein expression, as well as of GFP expression. The pH alterations, serum deprivation and heat shock reduced VEGF mRNA expression, but had little effect on GFP expression. The results demonstrated that VEGF expression may be influenced by a number of microenvironmental factors and these factors may play important roles in regulating VEGF expression during tumorigenesis.
    Biomedical reports. 07/2013; 1(4):539-544.
  • Ping Xu, Qian Huang
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    ABSTRACT: Occurrence of retinoblastoma is associated with inactivation of Rb1 gene which is located in 13q14. According to published data the inactivation of Rb1 gene occurred in 90% retinoblastoma was resulted from either deletion or point mutation or methylation of Rb1 gene. Denaturing high performance liquid chromatography (DHPLC) is capable to detect point mutation simply, rapidly and accurately. Recently developed methods are able to detect DNA methylation and analyze gene copy. DHPLC had been successfully used for gene diagnosis and genetic counseling in retinoblastoma patients and their family members.
    [Zhonghua yan ke za zhi] Chinese journal of ophthalmology 07/2007; 43(6):564-6.
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    ABSTRACT: To investigate the transduction and gene expression of the recombinant adeno-associated viruses (rAAV) of the serotypes 1 and 2 in the retinal cells. rAAV vectors of type 1 and type 2 encoding EGFP were infected into the cultured retinal pigmentary epithelium (RPE) cells of the line CRL-2302 and primarily cultured retinal neural cells from normal SD rats, and primarily cultured RPE cells from an adult cornea donor. The cultured RPE cells transduced by rAAV2-EGFP or rAAV2/1-EGFP were harvested at the 7 th and 14 th day after infection to be detected by fluorescence-activated cell sorter. The onset of EGFP gene expression and EGFP positive rate were detected by flow cytometry and fluorescence microscopy. Then, rAAV2/1-EGFP and rAAV2-EGFP were injected into the subretinal spaces of 32 SD rats to investigate the onset of EGFP fluorescence and its distribution in the fundus in vivo via fluorescence stereoscope. HE staining and immunohistochemistry were used to observe the infected cell type and immune response in the retina. The percentage of EGFP positive cells and mean intensity of EGFP fluorescence in the cells transduced by rAAV2-EGFP 7 and 14 days after transduction were 13.50% +/- 1.70% and 15.60% +/- 0.82%, and 2.75 +/- 0.12 and 3.80 +/- 0.72 respectively; and the EGFP positive cells and mean intensity of EGFP fluorescence in the cells transduced by rAAV2/1-EGFP were 1.09% +/- 0.5% and 1.98% +/- 0.45%, and 1.12 +/- 0.09 and 1.75 +/- 0.2 respectively. The EGFP fluorescence area in the retina were (5389 +/- 211) microm(2), (9832 +/- 364) microm(2), (14 454 +/- 446) microm(2), (20 528 +/- 648) microm(2), and (20 264 +/- 683) microm(2) respectively 3, 7, 14, and 75 days, and 4 month after transduction by rAAV2-EGFP in vivo; In the rat retina transduced by rAAV2/1-EGFP, the EGFP fluorescence areas were (9666 +/- 348) microm(2), (12 160 +/- 439) microm(2), (19 794 +/- 621) microm(2), (26 172 +/- 923) microm(2), and (26 022 +/- 965) microm(2) respectively 3, 7, 14, and 75 days, and 4 month after infection. rAAV2 efficiently transduces retinal cells both in vitro and in vivo. rAAV2/1 is a more effective gene-transferring vector to be used in retinal cells in vivo than rAAV2.
    Zhonghua yi xue za zhi 11/2006; 86(40):2841-6.
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    ABSTRACT: To investigate the efficacy of subretinal transplantation of CNTF gene transfected fibroblasts for preventing photoreceptor degeneration in RCS. The human fetal lung fibroblasts with high level expression of CNTF were established by liposome mediated gene transfer and MTX selection. A 5 microl of cell suspension, containing 1 x 10(5) cells, was injected through pars plana of ciliary body into the subretinal space of the right eye at postnatal 4-5 weeks, the left eye was left without injection or injected with PBS as controls. The both eyes were enucleated for histopathological examinations at 2, 4, 6, 8, 10, 12 and 15 weeks following transplantation. The level of CNTF protein (91,046.15 pg/ml) expressed in the transfected cells was determined by sandwich enzyme-linked immunosorbent assay (ELISA). The four of seven eyes examined by light microscopy and the ten of 14 eyes examined by electro microscopy showed rescue effect. The prolonged photoreceptor survival, reduction of apoptotic cells and debris were observed in transplanted eyes in comparison with untreated or sham-injected eyes. This study provides the first indication that transplanted human fibroblasts with high level expression of CNTF are able to rescue photoreceptor degeneration in RCS dystrophic rat retina.
    [Zhonghua yan ke za zhi] Chinese journal of ophthalmology 03/2006; 42(2):127-30.
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    ABSTRACT: To explore the effects and mechanisms of electroporation on the therapy of transplanted retinoblastoma tumor (RB) by transferring the expressive plasmids of sFlk-1 and ExTek. The effects and mechanisms of sFlk-1 and ExTek on RB is validated both in vitro and in vivo. In vitro, pCI-sFlk-1 was transfected into HEK293 (human embryonic kidney 293) cells with DMRIE-C Reagent, and its supernatant was collected 24 h later to mix with RPMI-1640 medium as 1:1, 2:1 and 3:1. RB cells were cultured in the mixed medium and the cell growth curve were analyzed 6 days later. The suspension of 1 x 10(7) RB cells were inoculated in 40 nude mice subcutaneously. The nude mice with RB were divided into 2 groups. The experimental group was treated with transfer of pCI-sFlk-1 (15 microg) and pCI-ExTek (15 microg) by electroporation once a week, while the control group was treated with physiological saline solution. After 3 weeks' therapy, the animals were observed for one more week. During this period the tumors were measured to draw the growth curve of the tumors and to calculate the anti-tumor rate. The vessel endothelium were tagged with smooth muscle actin (SMA) by immunohistochemical methods to inspect the micro vessel density (MVD) of the tumors. Moreover, the expression of vascular endothelial growth factor (VEGF) and its receptor Flk-1 of the tumors was confirmed by immunohistochemical staining. sFlk-1 exhibited no inhibiting effects on the growth of RB cells in vitro, while sFlk-1 combined with ExTek restrained the growth of the transplanted tumors obviously in vivo, and the anti-growth rate is 80.04%. The MVD of the experimental group is 26.69 +/- 2.95, which is evidently lower than that of the control group (44.51 +/- 6.11). The difference between these 2 groups was statistically significant (P < 0.05). As to the expression of VEGF, there was no significant difference between these 2 groups, while the expression of Flk-1 of the experimental group was much stronger than that of the control group. Transfer the expressive plasmids of sFlk-1 and ExTek by electroporation in vivo restrained the growth of the transplanted RB tumor, and the effect is due to the inhibition of angiogenesis process.
    [Zhonghua yan ke za zhi] Chinese journal of ophthalmology 10/2005; 41(10):900-4.
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    ABSTRACT: To investigate the factors regulating angiogenesis of retinoblastoma. Cultured retinoblastoma cells from HXO-Rb-44 cell lines were inoculated subcutaneously into the leg of nude-mice. Experimental retinoblastoma was studied by immunohistochemistry staining for the expression of angiogenesis factors (VEGF-A, VEGF-C, VEGF-D and bFGF), vascular endothelial cell membrane receptor tyrosine kinases (Flt-1, Flt-4, Flk-1, Tie-2 and FGFR), transcription regulated factor, Hif-1, matrix metalloproteinase (MMP-2, MMP-7 and MMP-9) and tissue inhibitor of metalloproteinase (TIMP-1 and TIMP-2). There were plenty of capillaries and small vessels in the retinoblastoma. Some retinoblastoma cells expressed Hif-1 and most cells expressed VEGF at a high level, especially VEGF-D. Flt-1, Flt-4 and FGFR were found in vascular endothelial cells in the retinoblastoma. Three matrix metalloproteinases were positive and two tissue inhibitors of metalloproteinases were negative in the retinoblastoma. Down-regulated TIMP and up-regulated MMP and VEGF signal transduction play an important role in the occurrence of angiogenesis in the retinoblastoma. This could be considered as the target of retinoblastoma treatment.
    [Zhonghua yan ke za zhi] Chinese journal of ophthalmology 06/2005; 41(5):419-22.
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    ABSTRACT: To investigate the effects of blocking VEGF expression on the retinal pigment epithelium (RPE) in vitro. RPE was transfected with plasmid encoding anti-sense of VEGF by lipofectin. RPE clones with stable expression of anti-sense of VEGF were screened by neomycin (G418). The changes of RPE and the level of VEGF secreted by RPE were observed by immunofluorescence immunohistochemical staining, Elisa and inverted-microscope. Transdifferentiation of cultured adult human retinal pigment epithelium cells to neurons was induced by blocking VEGF expression. The transdifferentiation was a direct phenotypic change from RPE cell to neurons without cell differentiation. The transdifferentiated neurons showed up-regulated expression of VEGF receptor 2 (Flk-1) and expressed several markers of retinal ganglion cell, including Thy1.1 and neurofilament. Blocking of VEGF expression and up-regulation of Flk-1 can induce transdifferetiation of adult human retinal pigment epithelium cells into ganglion cells.
    [Zhonghua yan ke za zhi] Chinese journal of ophthalmology 03/2005; 41(2):114-8.
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    ABSTRACT: To investigate the protective effects of transplantation of cell line stably expressing and secreting human ciliary neurotrophic factor (hCNTF) on the degeneration of retinal ganglion cells (RGCs) after optic nerve transection. Plasmid encoding hCNTF was transfected into human lung fibroblast (HLF) cell line, then the stably transfected clones were selected with methopterin. In adult SD rats, RGCs were labeled with retrograde axonal tracer fluorogold (FG) injected to their targets including dorsal lateral geniculate nuclei (dLGN) and superior colliculi (SC). Seven days later, the optic nerve was transected alone or in combination with transplantation of HLF cells. Five, 14, 17, 21 and 28 days after axotomy, the retinas were mounted and examined under fluorescence microscope to observe the RGCs. Compared to the controls, the density of RGC in axotomy group decreased by 67.44% and 82.73% on the 14th and 28th day, respectively. In the eyes with hCNTF-transfected HLF cells transplantation, RGC density was higher than that of the axotomy group on the 5th, 17th, 21st day after axotomy (P < 0.05). On the 5th day, the morphology of RGC in the hCNTF group remained the same as the controls, whereas the morphology of RGC in the axotomy alone group began to change. hCNTF administered at the time of optic nerve transection can protect RGC from degeneration, increasing the numbers of surviving RGCs and delaying the death of RGCs.
    [Zhonghua yan ke za zhi] Chinese journal of ophthalmology 02/2005; 41(2):119-22.
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    ABSTRACT: To investigate whether subretinal transplantation of genetically modified human retinal pigment epithelium cells can rescue photoreceptor degeneration in RCS rats. A spontaneously derived adult human retinal pigment epithelium (RPE) cell line (CRL-2302) was infected by gfp retrovirus, then transfected by liposome mediated CNTF expression plasmid transfer. Around 1 x 10(5) genetic modified cells were injected to subretinal space of the right eye at 4 - 5 weeks old, the left eye was left without injection or injected with PBS as controls. The GFP expression cells under the retina were observed from 1 to 6 weeks after transplantation. Histologic and ultrastructural assessment demonstrated substantial sparing of photoreceptors and correction of RPE phagocytosis defect. Electrophysiological assessment revealed an increased sensitivity of treated eyes to white light. The results demonstrate that potential of genetically modified RPE cells for ultimate application in therapeutic transplantation strategies for human retinal degenerative diseases.
    [Zhonghua yan ke za zhi] Chinese journal of ophthalmology 09/2004; 40(8):552-6.
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    ABSTRACT: In order to observe the growth and metastasis of the tumor directly, a new orthotopic retinoblastoma model was established with human RB cells expressing green fluorescent protein (GFP). pEGFP-N(1), the eukaryotic expressive plasmid of GFP, was transferred into human RB cell line HXO-RB(44) by liposome Dosper. Then, the cell clones expressing GFP steadily were selected by means of neomycin, fluorescence microscope, and flow cytometer. Two microliters of RB cells (density at 4.5 x 10(8) - 5.5 x 10(8) cells per ml) were injected into the subretinal space of 30 nude mice (60 eyes) under binocular operating microscope. The growth of transplanted RB was observed in vivo using fluorescence stereomicroscope. The nude mice were killed at different times post-operatively to investigate the metastasis process of the tumor to the optic nerve, brain, and other organs including lung, liver and kidney. The spread process of the tumor in the subretinal space was successfully observed under stereomicroscope. Transplanted RB developed into extra-ocular stage phase at 34 - 37 days after the operation. And metastasis to the cranium along the optic nerve was observed, with green RB cells distributing along the optic nerve sheath and long posterior ciliary artery. The histopathological characteristics of the transplanted tumor were similar to the human RB. Immunohistochemical staining showed positive expression of GFP in the tumor cells. The established orthotopic RB model expressing GFP via injection of human RB cells into the subretinal space of nude mice provides a new approach to exploring the growth and metastasis processes of RB in natural situations.
    [Zhonghua yan ke za zhi] Chinese journal of ophthalmology 05/2004; 40(4):225-8.
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    ABSTRACT: Retina is a multilayer and highly specialized tissue important in converting light into neural signals. In humans, the critical period for the formation of complex multiplayer structure takes place during embryogenesis between 12 and 28 weeks. The morphologic changes during retinal development in humans have been studied but little is known about the molecular events essential for the formation of the retina. To gain further insights into this process, cDNA microarrays containing 16361 human gene probes were used to measure the gene expression levels in retinas. Of the 16361 genes, 68.7%, 71.4% and 69.7% showed positive hybridization with cDNAs made from 12–16 week fetal, 22–26 week fetal and adult retinas. A total of 814 genes showed a minimum of 3-fold changes between the lowest and highest expression levels among three time points and among them, 106 genes had expression levels with the hybridization intensity above 100 at one or more time points. The clustering analysis suggested that the majority of differentially expressed genes were down-regulated during the retinal development. The differentially expressed genes were further classified according to functions of known genes, and were ranked in decreasing order according to frequency: development, differentiation, signal transduction, protein synthesis and translation, metabolism, DNA binding and transcription, DNA synthesis-repair-recombination, immuno-response, ion channeltransport, cell receptor, cytoskeleton, cell cycle, pro-oncogene, stress and apoptosis related genes. Among these 106 differentially expressed genes, 60 are already present in NEI retina cDNA or EST Databank but the remaining 46 genes are absent and thus identified as “function unknown”. To validate gene expression data from the microarray, real-time RT-PCR was performed for 46 “function unknown” genes and 6 known retina specific expression genes, and β-actin was used as internal control. Twenty-seven of these genes showed very similar expression profiles between the microarray and real-time RT-PCR data. In situ hybridization revealed both expression level and cellular distribution of NNAT in retina. Finally, the chromosomal locations of 106 differentially expressed genes were also searched and one of these genes is associated with autosomal dominant cone or cone-rod dystrophy. The data from present study provide insights into understanding genetic programs during human retinal development and help identify additional retinal disease genes.
    Chinese Science Bulletin 01/2004; 49(21):2277-2284. · 1.37 Impact Factor
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    ABSTRACT: To examine whether adult human retinal cells can be maintained in vitro over long time periods and to study the effects of brain-derived neurotrophic factor (BDNF),neurotrophin-4 (NT-4), epidermal growth factor (EGF), fibroblast growth factor (FGF), and retinoic acid (RA) on growth, proliferation, and apoptosis of the cultured adult human retinal cells. Adult retinal cells were isolated from donor eyes and cultured. BDNF, NT-4, EGF, FGF, and RA were added individually to the cultures. The cells were identified by morphologic criteria, growth patterns, and immunocytochemical staining. The number of neurons in each group was compared. Expression levels of c-fos, c-jun, Bcl-2, and Bax were examined. Adult human retinal cells survived for 240 days in vitro. The addition of BDNF and FGF promoted survival of adult retinal neurons, increased the number of cells positive for neuron specific enzyme (NSE), Thy1.1, c-fos, c-jun, and Bcl-2. More c-fos and c-jun positive cells were observed in cell cultures containing medium with RA. However, NT-4 and EGF treatment groups showed no significant difference from control groups in that neuron cell survival was low and fewer cells were positive for NSE, Thy1.1, c-fos, c-jun, and Bcl-2. BDNF, FGF and RA improve survival of adult human retinal neurons in vitro via a mechanism that may involve c-fos, c-jun, and Bcl-2 expression, However, EGF and NT-4 do not improve survival of the adult human retinal neurons.
    [Zhonghua yan ke za zhi] Chinese journal of ophthalmology 10/2003; 39(9):545-9.
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    ABSTRACT: The possibility of delivery genes to retina by using retinal pigment epithelium (RPE) transplantation was investigated. Cultured human adult RPE cells were transfected with retrovirus encoding green fluorescent protein (GFP), then GFP-expressing RPE cells were transplanted into the subretinal space of rabbits through pars plana vitrectomy under microscopy. The transplant sites were examined in vivo by ophthalmoscope for fluorescence. The animals were euthanatized at 1, 2, 3, 4, 6, 10, 11, 14, 18, 20, 23, 24, 25, 33, 54 weeks, respectively, and the retinas were studied histologically, including epi-fluorescent microscopy, confocal microscopy and transmission electron microscopy. The GFP-expressing transplant could be followed in the living retina up to 21 days by ophthalmoscope. The epi-fluorescent microscopy examination disclosed survival of the transplanted cells in 1 year with continual and significant GFP expression. It was observed the transplanted hRPE-gfp cells were not only present at transplant sites but spread over two to three quarters of retina. Generally, RPE xenografts integrated individually into host RPE in a regular cobblestones arrangement or formed a monolayer sheet between host RPE and neural retina. It was observed that intravitreal injection of FK-506 weekly improved the survival of RPE xenografts and reduced the infiltration of mononuclear microphages and lymphocytes in choroids from 1 through 14 weeks after transplantation. The results demonstrate a long-term survival of gene modified human RPE xenografts into the subretinal space of rabbits and support the feasibility of this approach for delivery genes to retina.
    Sheng wu hua xue yu sheng wu wu li xue bao Acta biochimica et biophysica Sinica 10/2002; 34(5):643-9.
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    ABSTRACT: The bladder transitional cell carcinoma cell line, BTT739 from the T739 mouse, was transfected with a plasmid that encoded an enhanced green fluorescence protein (GFP) and the cells stably expressing GFP were selected and subcloned. 1 x 10(3)-1 x 10(4) GFP-labeled BTT739 cells were injected under the skin of ear of T739 mice. On day 2-5 post injection, the most interesting manifestations observed were the chemotaxis-like movement of the tumor cells toward the pre-existing host vasculature, host vessel dilation and tortuosity and increased extravasation. On day 10 or later, the sprout from pre-existing host vasculature was observed. Once angiogenesis was triggered on, the tumor cells grew more rapidly and exhibited a specific growth pattern where tumor cells always associated with or surrounded the vessels. The newly formed microvessels always showed heavy extravasation. Immunohistochemistry staining revealed strong VEGF and VEGFR2 (Flk-1) expression in tumor cells. Angiography using Rhodamin-labeled dextran showed neovascularization with unprecedented clarity. However, the tumor mass, even bigger than 2 mm and being neovascularized, shrunk and then disappear in 3-5 days and left only delicated host vessels and recovered extravasation. The evidence from this observation indicated that angiogenesis induced by tumor cells after implantation into the host begins at very early stage. The micrometastases foci could not form or survive without vigorous and continuous angiogenesis. Furthermore, there was active VEGF paracrine and autocrine expression in tumor and high level VEGF secretion by tumor cells plays an important role in initiating angiogenesis and supporting micrometastases.
    Sheng wu hua xue yu sheng wu wu li xue bao Acta biochimica et biophysica Sinica 02/2002; 34(1):21-7.

Publication Stats

10 Citations
1.37 Total Impact Points

Institutions

  • 2005–2007
    • First People's Hospital Chenzhou
      Chenchow, Hunan, China
    • Shanghai Jiao Tong University
      Shanghai, Shanghai Shi, China
  • 2006
    • Shanghai University
      Shanghai, Shanghai Shi, China
  • 2004–2005
    • Hangzhou First People's Hospital
      Hang-hsien, Zhejiang Sheng, China
  • 2002
    • Shanghai Putuo District People's Hospital
      Shanghai, Shanghai Shi, China