Background To suppress vascular endothelial growth factor (VEGF) is a researching hot topic for the treatment and prevention of retinal neovascularization.Some detectable efficacy of VEGF small interference RNA (VEGF siRNA) in anti-tumor neovascularization has been well-known.But relevant study on VEGF siRNA on retinal neovascularization is seldom.Objective Present study was to investigate the inhibiting effect of VEGF siRNA on retinal neovascularization.Methods The 48 clean C57BL/6J mice aged 7-day-old were randomly divided into normoxia group,hypoxia control group,vector group and VEGF siRNA group and 12 mice for each.Hypoxia models were established by raising the pups with mother mice in the airtight oxygen-cabin for 5 days.The lipofectamineTM 2000 (LF2000)-mediated vector plasmids or VEGF siRNA recombinant plasmids were then injected intravitreally in 12 12-day-old pup mice respectively.The animals were sacrificed in 1 week after intravitreal injection,and the numbers of vascular endothelial cell nuclei extending beyond the internal limiting membrane (ILM) were counted by hematoxylin-eosin stain.The expressions of VEGF protein and mRNA in retina were assayed by immunoinfluorescence technique and RT-PCR.Results The numbers of vascular endothelial cell nuclei extending beyond the ILM were 0.19±0.09,24.89±2.03,23.65±2.15 and 8.83±1.12 in normoxia group,model control group,vector group and VEGF siRNA group separately,showing significant decrease in VEGF siRNA group compared with model control group or vector group (q=5.67,q=4.97,P＜0.01).RT-PCR revealed that VEGF mRNA was faintly expressed in mouse retina in normoxia group.However,in model control group and vector group,the level of VEGF mRNA was 52.3 times and 36.7 times more than that of normoxia group respectively and only 3.5 times in VEGF siRNA group,presenting a inhibitory rate of 43.39% of VEGF siRNA on VEGF.Immunofluorescence showed that the expression of VEGF was weaker in normoxia group and strong positive response in model control group and vector group,but the expression intensity of VEGF protein was significantly weaker in VEGF siRNA group.Conclusion VEGF siRNA recombinant plasmids can efficiently inhibit retinal neovascularization in oxygen-induced retinopathy mouse model through intravitreal injection.

OBJECTIVETo investigate the expression variation and significance of Skp2 and p27(kip1) during the proliferation of lymphoma cell line Jurkat cells.

METHODSThe binding of p27(kip1) and Skp2 in Jurkat cells were detected by immunoprecipitation. Jurkat cells were treated with serum starvation and release synchronization. The expression variation and subcellular localization of p27(kip1) and Skp2 were detected by subcellular fractionation, Western blot and double immunofluorescence labelling.

RESULTSThe results of immunoprecipitation suggested that p27(kip1) and Skp2 could bind each other in Jurkat cells. During the proliferation of Jurkat cells, the protein expression of p27(kip1) decreased and intranuclear p27(kip1) decreased significantly, while the Skp2 protein increased and cytoplasmic Skp2 increased significantly.

CONCLUSIONDuring the proliferation of Jurkat cells, the increased cytoplasmic synthesis of Skp2 may speed up p27(kip1) degradation via the ubiquitin-proteasome pathway, then intranuclear p27(kip1) decreases significantly, leading to an increased cell cycling activity.

Cell Nucleus ; metabolism ; Cell Proliferation ; Cyclin-Dependent Kinase Inhibitor p27 ; metabolism ; Cytoplasm ; metabolism ; Humans ; Jurkat Cells ; Lymphoma, B-Cell ; metabolism ; pathology ; Protein Binding ; S-Phase Kinase-Associated Proteins ; metabolism

OBJECTIVETo study the expressions of p35 and p25 and Cdk5 kinase activity in cultured rats hippocampal neurons following X-ray exposure to provide experimental evidence for prevention and treatment of radiation encephalopathy.

METHODSThe hippocampal neurons cultured for 12 days were subjected to a single-dose X-ray exposure of 30 Gy. Western blotting was used to detect the p35 and p25 protein levels, and the effect of pretreatment with roscovitine, a Cdk5 inhibitor, on the apoptosis of the hippocampal neurons following the exposure was examined with 4',6-diamidino-2-phenylindole (DAPI) staining.

RESULTSThe protein level of p35 increased significantly 3.5 and 4 h after the irradiation by 1.51-/+0.13 and 1.45-/+0.14 folds in comparison with the control level, respectively (P<0.01), and the p25 level increased significantly 6 h after irradiation by 1.62-/+0.28 folds (P<0.05). Nuclear condensation occurred in (24.8-/+3.97)% of the neurons 24 h after 30 Gy X-ray exposure, a rate significantly higher than that in the nonexposed cells [(1.82-/+1.08)%, P<0.01) and that in roscovitine-pretreated neurons [(7.74-/+2.27)%, P<0.01).

CONCLUSIONX-ray exposure activates Cdk5 by increasing the p35 and p25 expressions in rat hippocampal neurons, and inhibition of Cdk5 activity with roscovitine can significantly protect the neurons from apoptosis.

Animals ; Animals, Newborn ; Cells, Cultured ; Cyclin-Dependent Kinase 5 ; genetics ; metabolism ; Female ; Hippocampus ; cytology ; metabolism ; radiation effects ; Male ; Neurons ; cytology ; metabolism ; radiation effects ; Phosphotransferases ; genetics ; metabolism ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo silence annexin II gene expression by using small interference RNA (siRNA) in prostate cancer cell line PC3.

METHODSFor in vitro transcription, four sequences of 29-nucleotide DNA template oligonucleotides were designed, and one pair of the sequences were complementary to annexin II gene. The other pair was negative control. The 8 nucleotides at the 3' end of each oligonucleotide were complementary to the T7 Promoter Primer. The sense and antisense siRNA templates were transcribed by T7 RNA polymerase and the resulting RNA transcripts were hybridized to create dsRNA. The siRNA was transfected into prostate cancer cell PC3. For assaying the efficiency of siRNA, confocal microscopy, Northern blotting, and Western blotting were employed to examine the expression of annexin II protein and its mRNA. 3H thymidine was used to measure DNA synthesis.

RESULTSThe siRNA sequence specific to annexin II gene was capable of inhibiting the expression of annexin II protein and its mRNA. And cellular DNA synthesis was significantly reduced in siRNA transfected cells.

CONCLUSIONSThe protocol for the synthesis of siRNA by T7 RNA polymerase is feasible. Annexin II might be involved in DNA synthesis.

Annexin A2 ; genetics ; Cell Line, Tumor ; DNA Replication ; DNA, Neoplasm ; genetics ; Humans ; Male ; Promoter Regions, Genetic ; genetics ; Prostatic Neoplasms ; genetics ; RNA Interference ; RNA, Neoplasm ; genetics ; RNA, Small Interfering ; genetics ; Transcription, Genetic

OBJECTIVETo investigate the effect of Hyper-IL-6 on the proliferation of L-02 cells.

METHODSThe recombinant lentiviral vectors encoding Hyper-IL-6 (FIV-Hyper-IL-6), IL-6 (FIV-IL-6) and the lentiviral (FIV) were prepared using the FIV-based lentiviral vectors and the packaging system. The titer of FIV-Hyper-IL-6, FIV-IL-6 and FIV was tested with puromycin. L-02 cells were infected with FIV-Hyper-IL-6, FIV-IL-6, FIV, or mock-infected. The growth rate of L02 cells was analyzed with MTT at different time points after infection. The changes of Haptoglobin in L-02 cells were analyzed with RT-PCR.

RESULTSFIV-Hyper-IL-6, FIV-IL-6 and FIV were successfully constructed, and the titer was 107 pfu/ml. 48 hours after infection, the absorbance of the cells infected with FIV-Hyper-IL-6 was 0.6267+/-0.0256, and the absorbances of FIV-IL-6 infected cells, FIV infected cells and mock-infected cells were 0.5563+/-0.0112, 0.5040+/-0.0078 and 0.4790+/-0.0201, respectively. There were significant differences between the FIV-Hyper-IL-6 group and the other groups (F = 41.09, P less than 0.01). The ratio of the absorbance between haptoglobin mRNA and beta-actin was 0.7030+/-0.0106, 0.3355+/-0.0093, 0.1145+/-0.0076 and 0.1143+/-0.0153, respectively, in FIV-Hyper-IL-6 infected cells, FIV-IL-6 infected cells, FIV infected cells and mock-infected cells. There were significant differences between the FIV-Hyper-IL-6 group and the others (q = 57.5007, P less than 0.01).

CONCLUSIONCompared with IL-6, Hyper-IL-6 is more potent to stimulate proliferation and induce the expression of Haptoglobin in L-02 cells.

Cell Proliferation ; Genetic Vectors ; Haptoglobins ; genetics ; metabolism ; Hep G2 Cells ; Hepatocytes ; cytology ; metabolism ; Humans ; Interleukin-6 ; genetics ; metabolism ; Lentivirus ; genetics ; RNA, Messenger ; genetics ; metabolism ; Recombinant Fusion Proteins ; genetics ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Time Factors ; Transfection

OBJECTIVETo observe the changes of the cytokines following stereotactic irradiation for hepatocarcinoma with cirrhosis in rabbits.

METHODSSixteen rabbits with liver cirrhosis and hepatocarcinoma (experimental group) were randomized into two equal groups to receive stereotactic irradiation at single dose of 20 and 30 Gy, respectively. Eight rabbits with hepatocarcinoma (control group) were divided into two equal groups and treated in identical manner. All the rabbits were killed 3 weeks after irradiation, and EV two-step method was used to observe the cytokine changes of proliferating cell nuclear antigen (PCNA) and glutathione S-transferase pi (GST-pi) after irradiation.

RESULTSAfter irradiation, PCNA and GST-pi expression showed significant difference in the adjacent liver tissue between the experimental and control rabbits with irradiation at 20 Gy (P=0.010), but not with the irradiation dose of 30 Gy (P=1.000). Irradiation at different doses resulted in significant difference in the cytokine expression in the experimental rabbits (P=0.010). In the liver tissue exposed to irradiation, different irradiation doses resulted in significant difference in PCNA and GST-pi protein expression (P=0.010).

CONCLUSIONSFor hepatocarcinoma with cirrhosis in rabbits, radiation at the single dose of 30 Gy produces better response than 20 Gy, and PCNA and GST-pi may serve as good indexes for evaluating the therapeutic effect.

Animals ; Glutathione S-Transferase pi ; biosynthesis ; Immunohistochemistry ; Liver Cirrhosis, Experimental ; metabolism ; radiotherapy ; Liver Neoplasms, Experimental ; metabolism ; radiotherapy ; Male ; Proliferating Cell Nuclear Antigen ; biosynthesis ; Rabbits ; Radiotherapy Planning, Computer-Assisted ; methods ; Radiotherapy, Conformal ; Random Allocation

OBJECTIVE: To determine the influence of different registration scopes and methods on kilo-voltage cone beam CT (kVCBCT) image and CT image and to estimate the appropriate clinical target volume (CTV)-to-planned target volume (PTV) margin for central-type non-small cell lung cancer in image-guided radiation therapy (IGRT). METHODS: Twenty-six patients with central-type non-small cell lung cancer (NSCLC) who received IGRT were enrolled in this study and totally 104 flames of kVCBCT scanning acquired before radiotherapy were analyzed. First, registrations were performed by gray plus manual match and were compared among different registration scopes of tumor and tumor plus vertebra. Then, the results of registration as well as time cost using tumor plus vertebra as registration scope were compared among different registration methods of gray plus manual match and bone plus manual match. At last, 200 registrations using tumor plus vertebra as the registration scope performed by bone plus manual match were recorded and the CTV-to-PTV margin was calculated. RESULTS: There was no significant difference in X, Y and Z translational and rotational movements between the registration scopes of tumor and tumor plus vertebra when gray plus manual match was used as the registration method (P>0.05). The registration results of gray plus manual match and bone plus manual match were equal when tumor plus vertebra was used as the registration scope (P>0.05), but the time cost of bone plus manual match [(1.9±0.3) min] was shorter than that of gray plus manual match [(3.1±0.2) min]. The CTV-to-PTV margins were 5.3, 4.9 and 5.7 mm in X, Y, and Z directions respectively. CONCLUSION For central-type NSCLC, tumor and vertebra can be used as registration scope and the bone plus manual match is suggested in IGRT. To avoid errors, we suggest a CTV-to-PTV margin of 6 mm.
Aged ; Carcinoma, Non-Small-Cell Lung ; radiotherapy ; Cone-Beam Computed Tomography ; Female ; Humans ; Lung Neoplasms ; radiotherapy ; Male ; Middle Aged ; Patient Positioning ; Radiotherapy Planning, Computer-Assisted ; methods ; Radiotherapy, Image-Guided ; methods

OBJECTIVETo investigate the effect of adiponectin on hepatocyte steatosis.

METHODSL02 cells were transfected with pEGFP-N1-AdipoQ, a plasmid encoding pEGFP-adiponectin fusion protein, or pEGFP-N1. Lipid droplets in the hepatocytes were observed by oil red staining at 72 h. The contents of TG, FFA and glycerol in hepatocytes were measured.

RESULTSCompared to cells transfected with pEGFP-N1-AdipoQ plasmid, much more lipid droplets were observed in cells transfected with pEGFP-N1 plasmid. TG, FFA and glycerol contents in L02 cells and L02/pEGFP-N1 cells were significantly higher than those in L02/pEGFP-N1-AdipoQ cells.

CONCLUSIONSOverexpression of adiponectin prevent hepatocyte steatosis.

Adiponectin ; genetics ; Cell Line ; Fatty Acids, Nonesterified ; analysis ; Fatty Liver ; metabolism ; Genetic Vectors ; Glycerol ; analysis ; Hepatocytes ; cytology ; metabolism ; Humans ; Plasmids ; Recombinant Fusion Proteins ; genetics ; Transfection ; Triglycerides ; analysis

OBJECTIVETo study the protective effect of ginsenoside Rg1 on cultured rat hippocampal neurons against radiation injury and explore new therapies for preventing radiation encephalopathy.

METHODSRat hippocampal neurons cultured for 12 days were subjected to a single-dose X-ray exposure of 30 Gy. 4',6-diamidino-2-phenylindole (DAPI) staining was used to detect the neuronal apoptosis and NOS activity kit utilized to evaluate NOS activity in the cells after the exposure.

RESULTSNuclear condensation was detected in (25.3-/+3.57)% of the neurons 24 h after 30 Gy X-ray exposure, a rate significantly higher than that in the control cells [(1.95-/+0.78)%, P<0.01]. In the neurons pretreated with ginsenoside Rg1, only (7.43-/+1.51)% of the cells presented with nuclear condensation after the exposure, significantly different from the rates in the control cultures and the exposed cultures (P<0.01). The NOS activity of exposed cultures was 6.46-/+0.95 U/ml, significantly higher than that in the control cultures (3.20-/+0.70 U/ml, P<0.01). The NOS activity of the neurons pretreated with ginsenoside Rg1 was 3.85-/+0.69 U/ml, significantly different from that in the control cultures (P<0.05) and the exposed cultures (P<0.01).

CONCLUSIONGinsenoside Rg1 offers significant protective effect on rat hippocampal neurons from radiation-induced apoptosis by reducing the activity of NOS.

Animals ; Cells, Cultured ; Ginsenosides ; pharmacology ; Hippocampus ; cytology ; drug effects ; radiation effects ; Neurons ; drug effects ; radiation effects ; Neuroprotective Agents ; pharmacology ; Nitric Oxide ; metabolism ; Nitric Oxide Synthase ; metabolism ; Radiation Injuries ; metabolism ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo investigate the therapeutic effect of recombinant human parathyroid hormone(1-34) [rhPTH(1-34)] on osteoporosis of ovariectomized rats.

METHODSThe model of osteoporosis was formed after 3 months of ovariectomy with 6-month age of 80 rats. Another 20 rats was control of sham operation. rhPTH(1-34) was subcutaneously injected once daily with 5, 10, 20, 40 micrograms/kg for 3 months. There were 10 rats in each group. The control of therapy included Salmon Calcitonin to 10 rats and Alendronate sodium to 10 rats. The bone weight of dry and ash, bone mineral density, bone biomechanical property, trabecular area, bone mineral deposition and serum alkaline phosphatase, Ca, P and urinary Pyridinoline/creatin (Pyd/Cr) were measured after the end of therapy.

RESULTSWhen administered to animals as a single subcutaneous injection once daily, rhPTH(1-34) increased obviously bone mass, bone biomechanical property and trabecular area, as well as bone deposition compared with the animals of control group. The bone architecture was ultimately improved by rhPTH(1-34) therapy.

CONCLUSIONSRats of ovariectomized-induced osteoporosis possess obvious effect of treatment with low dose of rhPTH(1-34) administered once daily.

Animals ; Female ; Osteoporosis ; drug therapy ; etiology ; Ovariectomy ; Rats ; Recombinant Proteins ; therapeutic use ; Teriparatide ; therapeutic use