BackgroundEndostatin (ES) is currently the strongest endogenous angiognesis inhibitor,and it can inhibit the occurrence of neovascularization.Various studies demonstrated that the poly RGD sequence can enhance the function of the ES gene.ObjectiveThis study was to evaluate the use of gene therapy of modified ES for alkaline burn-induced corneal neovascularization (CNV).MethodsOne hundred and two clean SD rats were randomly divided into the normal control group,the pCI empty vector group,the pCI-ES group,and the pCI-RGDRGDES group.Corneal neovascularization models were established by placing a piece of 3 mm filter paper with 1 mol/L NaOH at the central cornea for 40 seconds.3 μg of the pCI blank vector,ES-tranfected pCI blank vector,or RGDRGD-ES-transfected pCI vector was injected into the superior bulbar conjunctiva after the alkali burn twice at 1-week intervals.Area of CNV and edema of the cornea in the various groups of rats were examined daily under the slit lamp biomicroscope.1,4,7 and 14 days after operation,the rats were sacrificed by the excessive anesthesia method and corneal tissues were obtained to evaluate pathological changes.The expression of CD34 in vascular endothelial cells was detected by immunochemistry to calculate the corneal neovascular density.The expressions of VEGF mRNA and Flk-1 protein in the corneas were detected by reverse transcription polymerase chain reaction (RT-PCR) and Western blot analysis.The use and maintenance of animals followed the Statement of ARVO.Results Seven to fourteen days after corneal alkali-burning,the corneal neovascular area was smaller in the pCI-ES group and pCI-RGDRGD-ES group compared with the normal control group and pCI blank vector group (P＜0.05,P＜0.01 ),and nevascular area in the pCI-RGDRGD-ES group was smaller than that in the pCI-ES group (P＜0.05).The expression level of CD34 was significantly lower in the pCI-ES group and pCI-RGDRGD-ES group than that in the normal control group and pCI blank vector group (P＜0.05,P＜0.01 ),and the expression level of CD34 was further declined in the pCI-RGDRGD-ES group compared with the pCI-ES group (P＜0.05 ).Compared with the normal control group and pCI vector group,the expressions of the Flk-1 protein and VEGF mRNA were decreased in the pCI-ES group and pCI-RGDRGD-ES group on the fourth day after corneal alkali-burning (P＜0.01,P＜0.05 ),and those in the pCI-RGDRGD-ES group were less than the pCI-ES group (P＜ 0.05,P＜ 0.05 ).Conclusions Subconjunctival injection of both ES and modified RGDRGD-ES genes result in significant suppression of CNV in vivo,and modified RGDRGD-ES appears to be more effective than native ES.The main mechanism of ES in inhibiting neovascularization is to downregulate the expression of VEGF and Flk-1.

Objective To investigate the influence of Ethyl pyruvate(EP) on the level of high mobility group box 1 (HMGB1) in brain injury of infant rats induced by lipopolysaccharide (LPS) and its significance.Methods Two hundred and forty normal healthy 1-month-old Spragne-Dawley (SD) rats were randomly divided into 3 groups:9 g/L sodium chloride (NS) group(n=80),LPS group (n=80),and EP group (n=80).LPS (1 mg/kg) was injected via internal carotid.In EP group,after injecting LPS,each rat was immediately administrated 4 mL EP(40 mg/kg) intraperitoneally; in control group,4 mL Ringer's solution was given instead of EP.Rats were decapitated at 6,12,24,48 and 72 h following drug injection.The evan's blue (EB) content of brain tissues was meteraged by the formamide methods.Immunohistochemistry technology was used to detect the expression of HMGB1,neuron specific enolase(NSE) and glial fibrillary acidic protein(GFAP) protein,and reverse transcribe polymerase chain reaction technology was applied to study the expression level of HMGB1 mRNA in brain tissue.Meanwhile,the pathological changes of brain tissues were observed under the light microscope.Results Six hours after LPS was given,brain EB content,the levels of NSE,GFAP protein started to rise,reaching the peak at 24 h,and still higher than those in control group at 48 h and 72 h.The expression pattern of HMGB1 protein and mRNA in brain tissue was consistent with the severity of brain injury,increased at 6 h after LPS was given and reached the peak at 24 h,still higher than those in control group at 48 h and 72 h.Positive correlation was found among HMGB1 protein,HMGB1 mRNA and EB content,NSE protein,GFAP protein,respectively.In EP group,the levels of HMGB1 protein and mRNA,the levels of brain EB content,NSE,GFAP protein were found,positive correlation was still gotten between HMGB1 protein,EB content and NSE,GFAP protein,HMGB1 mRNA in LPS group and EP group.Conclusion EP has neuroprotective effect on brain injury induced by LPS,which may be relevant to decreasing of the expression of HMGB1 and suppressing inflammation action.

Adult ; Aged ; Autoimmune Diseases ; diagnosis ; metabolism ; Female ; Humans ; Insulins ; metabolism ; Male ; Middle Aged

Adult ; Female ; Humans ; Lymphoma, T-Cell ; pathology ; physiopathology ; Panniculitis ; physiopathology ; Subcutaneous Tissue ; pathology ; physiopathology

Background Corneal epithelial abrasion results in corneal ulcer and stroma cloudy evenb irreversible visual impairment.Previous drugs for corneal epithelial injury can only alleviate the inflammatory irritation.So it is very important to seek a drug which regulate the growth of corneal epithelium.Objective This study was to investigate the effects of recombinant human BIGH3 protein eye drops on corneal epithelial abrasion.Methods Fifty right eyes of 50 clean adult New Zealand white rabbits were collected.Two rabbits were sacrificed right away following establishment of corneal epithelial abrasion models (0 hour group).The other 48 rabbits were randomly divided into recombinant human epidermal growth factor (EGF) derivative group (positive control group),normal saline solution group (negative control group),0.25％ or 0.5％ recombinant human BIGH3 protein eye drops group.Corneal abrasion models were created with alcohol corrosion method with a defect area of 7 mm2.The corresponding eye drops were used separately in 4 groups for four times per day after operation.Experimental eyes were examined by the slit lamp microscope,and fluorescein vital staining were performed 12,24,36,48,72 hours after operation.Planimetry was performed and the corneal photographs were analyzed with computer software.The rabbits were sacrificed 12,24,36,48 and 72 hours after operation,respectively,and the histopathological examination of corneal tissue was carried out.Results No obvious irritation response was seen after administered of eye drops in the recombinant human EGF derivative group,normal saline solution group,0.25％ and 0.5％ recombinant human BIGH3 protein eye drops groups.Histopathological examination revealed a full-thickness defect of corneal epithelium after modeling.The defect area was gradually smaller with time lapse,and corneal epithelium migrated from periphery toward the center zone.Corneal epithelial cells increased with time lapse.Compared with normal saline solution group,the defect area of corneal epithelium lessened 12,24,36,48 hours after operation in the 0.25％,0.5％ recombinant human BIGH3 protein eye drops groups and recombinant human EGF derivative group (all at P =0.000),but at 12and 24,36 hours after operation,no significant differences were found between the recombinant human EGF derivative group and normal saline solution group (P =0.321,0.057,0.126).The defect area was smaller in the 0.5％recombinant human BIGH3 protein eye drops group than that of the recombinant human EGF derivative group at various time points (P=0.042,0.039,0.025,0.008).However,significant smaller defect area was exhibited only at 12 hours and 24 hours after operation in the 0.25％ recombinant human BIGH3 protein eye drops group (P=0.047,0.042).No significant differences were seen in corneal defect area at various time points between 0.25％ and 0.5％recombinant human BIGH3 protein eye drops groups (P =0.358,0.259,0.108,0.062).In addition,the corneal defect area was (0.51 ±0.42)mm2 72 hours after operation in the normal saline group;while that in the recombinant human EGF derivative group and recombinant human BIGH3 protein eye drops groups was disappeared.The repairing curves in the recombinant human BIGH3 protein eye drops groups were superior to those of the recombinant human EGF derivative group and normal saline solution group.Conclusions 0.25％ and 0.5％ recombinant human BIGH3 protein eye drops have facilitation effect on the growth of corneal epithelial cells and the healing of corneal injury.

Objective To investigate the effect of long noncoding RNA nuclear paraspeckle assembly transcript 1(lncRNA NEAT1)on autophagy in membranous nephropathy(MN)and its mechanism.Methods HK-2 cells were induced by albumin to construct MN model in vitro.HK-2 cells were divided into 5 groups:control group,model group,LY294002(PI3K inhibitor)group,OV-NEAT1(NEAT1 overexpression)group and OV-NEAT1+LY294002 group.Cell proliferation was detected by CCK-8.Cell apoptosis was detected by flow cytometry.Expressions of autophagy and PI3K/Akt/mTOR pathway-related mRNA and protein were detected by qRT-PCR and Western blotting.Results Compared with control group,the cell proliferation ability,LC3-Ⅱ and Beclin 1 mRNA levels as well as LC3-Ⅱ/Ⅰ and Beclin 1 protein expression levels in the model group were signifi-cantly decreased(all P＜0.01).The apoptosis rate,LC3-Ⅰ,PI3K,Akt,mTOR mRNA and p-PI3K,p-Akt,p-mTOR protein ex-pression levels were significantly increased(all P＜0.01).Compared with model group,the cell proliferation ability,LC3-Ⅱ and Beclin 1 mRNA and LC3-Ⅱ/Ⅰ and Beclin 1 protein expression levels in OV-NEAT1 group were significantly decreased(all P＜0.05).The apoptosis rate,LC3-Ⅰ,PI3K,Akt,mTOR mRNA and p-PI3K,p-Akt,p-mTOR protein expression levels were sig-nificantly increased(all P＜0.01).LY294002 group witnessed the opposite trend.Compared with LY294002 group,cell prolifer-ation ability,LC3-Ⅱ and Beclin 1 mRNA and LC3-Ⅱ/Ⅰ and Beclin 1 protein expression levels in OV-NEAT1+LY294002 group were significantly decreased(all P＜0.05).The apoptosis rate,LC3-Ⅰ,PI3K,Akt,mTOR mRNA and p-PI3K,p-Akt,p-mTOR protein expression levels were significantly increased(all P＜0.05).Conclusion lncRNA NEAT1 can inhibit albumin-induced autophagy in HK-2 cells,and the mechanism may be related to the activation of PI3K/Akt/mTOR signaling pathway.

Objective: To investigate the anticancer effect and its mechanism of SN-38 combined with sorafenib on hepatocellular cancer cell lines HepG-2 and BEL-7402 . Methods: SRB colorimetry was employed to measure the viability of HepG-2 and BEL-7402 cells after the treatment of SN-38 with sorafenib .Propidium iodide flow cytometric assay and DAPI staining were used to evaluate the apoptosis of HCC cells. Western blotting was conducted to detect the expression level of apoptosis-related and DNA damage-related proteins .Results: SRB colorimetry showed the synergistic anticancer activities of SN-38 combined with sorafenib , with a combination index of <0.9.The apoptotic rates of HepG-2 cells in control, 60 nmol/L SN-38, 2.5 μmol/L sorafenib and combination groups were 4.25%±2.45%, 28.95%±10.75%, 3.49%±2.49% and 53.19%±11.21%, respectively ( P <0 .05 ) .Western blotting showed that the combination of these two drugs increased the enzymolysis of PARP , Caspase-8 and Caspase-3 , and promoted the expression levels of p53 , p21 andγ-H2 AX significantly .Conclusion:SN-38 and sorafenib have synergistic anticancer activity on hepatocellular carcinoma cells in vitro with the augmentation of apoptosis .

Multiple displacement amplification (MDA) is a new technology for whole genome amplification (WGA), which can generate large amount of high-quality DNA and features high amplification efficiency and fidelity. MDA combined with conventional PCR techniques has been successfully applied for preimplantation genetic diagnosis, which has broaden latter's clinical applications.
Genome, Human ; Humans ; Nucleic Acid Amplification Techniques ; methods ; Polymerase Chain Reaction ; methods ; Preimplantation Diagnosis ; methods

OBJECTIVETo study the function and clinicopathological significance of RNA-29c (miR-29c) in the carcinogenesis and development of gastric cancer.

METHODSMicroRNA microarray was applied to assess the miRNAs expression profile of gastric cancer. Quantitative real-time PCR was used to detect the expression of miR-29c in 64 cases of gastric cancer tissues and corresponding normal gastric epithelium, as well as cell lines GES-1, BGC-823 and SGC-7901 cells. MTT assay and flow cytometry were applied to detect the effects of forced expression of miR-29c in gastric cancer BGC-823 cells including cell proliferation, apoptosis, cell cycle and drug sensitivity. Quantitative real-time PCR, Western blot and luciferase reporter assay were used to explore the targeted relationship between miR-29c and myeloid cell leukemia-1 (Mcl-1).

RESULTSCompared with normal gastric epithelium, seven microRNAs (miR-374b*, miRPlus-E1212, miR-338-5p, miR-297, miR-21, miR-135b, miR-18a) were significantly up-regulated more than 2-folds, and nine microRNAs (miR-29b-2*, miR-1260, miRPlus-E1241, miR-S1-5p, miR-148a, miR-29c, miR-647, miR-196b*, ebv-miR-BART5) were significantly down-reguated in gastric cancer tissues. The average expression level of miR-29c in gastric cancer tissues was 0.70 ± 0.34 and in corresponding normal epithelium was 1.00 ± 0.06 (P < 0.05). miR-29c expression was related to tumor size, lymph node metastasis, clinical stage, Laurén classification, Borrmann classification and Ming classification (P < 0.05). The poorer differentiation degree of gastric cell lines, the lower was miR-29c expression level (P < 0.05). Overexpression of miR-29c in gastric cancer BGC-823 cells suppressed cell proliferation, stimulated cell apoptosis, induced cell cycle arrest in S phase and increased the chemotherapy sensitivity to drug docetaxel (all were P < 0.05). The average expression level of Mcl-1 mRNA in gastric cancer tissues was 3.47 ± 1.34 and corresponding epithelialium was 1.00 ± 0.20 (P < 0.05). The expression level of miR-29c was negatively related with that of Mcl-1 mRNA in gastric cancer tissues. miR-29c directly targeted to regulation of Mcl-1 expression.

CONCLUSIONSThere are special miRNA expression profile in gastric cancer. The expression of miR-29c is closely related to biological behavior of human gastric cancer. miR-29c is involved in targeted regulation of Mcl-1, and may be one of mechanisms of the carcinogenesis of gastric cancer.

Antineoplastic Agents ; therapeutic use ; Apoptosis ; Cell Cycle Checkpoints ; Cell Differentiation ; Cell Line, Tumor ; Cell Proliferation ; Drug Resistance, Neoplasm ; Gene Expression Regulation, Neoplastic ; Humans ; Lymphatic Metastasis ; MicroRNAs ; genetics ; metabolism ; Microarray Analysis ; Myeloid Cell Leukemia Sequence 1 Protein ; genetics ; metabolism ; Neoplasm Staging ; RNA, Messenger ; metabolism ; Stomach Neoplasms ; drug therapy ; genetics ; metabolism ; pathology ; Taxoids ; therapeutic use ; Transcriptome ; Tumor Burden

Objective To evaluate the clinical characteristics,outcome and prognostic factom of adolescent nasopharyngeal carcinoma.Methods Between Jan 1990 and Dec 2009,totally 148 pathological confirmed nasopharyngeal carcinoma(NPC)patients with age≤20 years were treated in our hospital,including stage Ⅱ 8,stage Ⅲ 58,stage Ⅳ 81,and unknown 1 when restaged by TNM system(UICC 2002),ninty-four(63.5%)patients were treated with radiotherapy alone,54(36.5%)patients were treated with radiotherapy combined with cisplatin-based chemotherapy.Results The median follow-up time for all patients was 44.5 months.The 5-year overall survival(OS),local-regional control(LRC)and distant metastasis-free survival(DMFS)rateswere 82.9%,85.1%and 78.6%.There were 42 patients(28.4%)failed with 16 regional recurrence and 29 distant metastasis,and 3 with both;bone metastasis was the most common site of distant metastasis(22/29).In univariate analysis,the adverse prognostic factors for OS were stage T4(X2=5.61,P=0.018),radiation dose<70 Gy(X2=5.30,P=0.021),for LRC was radiation dose<70 Gy and for distant metastasis-free survival was the disease history≥6month(X2=4.24,P=0.039).In multivariate analysis,radiation dose<70 Gy and stage T4 were the independent prognostic factors for OS(X2=5.73、5.56,P=0.017、0.018),for LRC was radiation dose<70 Gy(X2=5.81,P=0.016).Conclusions The outcome of the present series was excellent,total nagopharyngeal radiation dose less than 70 Gy is inappropriate.Reduce the distant metastasis and late toxicities were the future direction for the treatment of adolescent nasopharyngeal carcinoma.