Objective To investigate the expression time and the position of VEGF in the choroid of diabetic rats and compare them with the VEGF expression in the choroid and retina of normal rats.Methods Healthy male Wistar rats were selected and randomly divided into diabetic group and normal group.Type 1 diabetes rat model was induced by being injected of large-dose streptozotocinum(STZ) into abdominal cavity.The retina and choroid of rats were obtained to detect the VEGF expression by immunohistochemistry at 1~(st),2~(nd),3~(rd),4~(th),5~(th) month after diabetes induction.Results VEGF expression in retina and choroid of rats was of obvious difference between normal group and one-month diabetic group.In two-month diabetic group,VEGF expression in choroid was positive(33.3%),while VEGF expression in retina was not significantly different with that of normal group.In three-month diabetic group,VEGF expression in choroid was positive(55.6%),and VEGF expression in inner nuclear layer and ganglion cell layer of retina was positive(33.3%).In four-month diabetic group,VEGF expression in choroid was positive(66.7%),and VEGF expression in inner limiting membrane,inner nuclear layer,outer nuclear layer and ganglion cell layer of retina was positive(77.8%).In five-month diabetic group,VEGF expression in choroid was positive(88.9%),and VEGF expression in the whole retina was positive(88.9%).The expression density of VEGF in choroid and retina gradually increased((P

Background Age-related macular degeneration (AMD) is the leading cause of blindness in people over 50 years old,of which 90％ cases are caused by choroidal neovascularization (CNV).Current treatments on AMD have gained great achievements,but there are still some drawbacks.So we need to search for new targets to cure CNV.Objective This study was to construct two combined lentiviral vectors for rat Slit2 gene RNA interference (RNAi) and verify its interfering effects on Slit2 gene in rat retinal pigment epithelial (RPE) cells.Methods Two specific siRNA sequences targeting towards rat Slit2 gene were designed and were annealed to DNA sequences.The DNA sequences and GV248-enhanced green flourescent protein (EGFP) vectors were combined together as recombinant vectors and then were identified.The GV248-EGFP vector,helper 1.0 and helper 2.0 were transfected together into 293T cells and the two combined lentiviral vectors for rat Slit2 RNAi were gained from the cell supernatant after 72 hours of transfection.The titers of the combined lentiviral vectors were measured.The cells were divided into blank control group,Lv-EGFP vector group,Lv-rSlit2-siRNA1 group and Lv-rSlit2-siRNA2 group.The interference efficacy of the combined lentiviral vectors targeting to rat Slit2 gene were identified by real-time fluorescence quantitative PCR and Western blot.The sequence with higher interference efficacy was transfected to rat RPE cells again.The transfected and nontransfected rat RPE cells were treated with 0,100,200 and 400 μmol/L CoCl2 for the preparation of hypoxia models.The expression of vascular endothelial growth factor-A (VEGFA) mRNA in rat RPE cells was finally measured by real-time fluorescence quantitative PCR and the concentration of VEGFA protein in cell supernatant was assayed by ELISA.Results The recombined lentiviral vectors for rat Slit2 gene R NAi were successfully constructed.The titers of the two reconbinant sequences were 5× 108 TU/ml and 3×l08 TU/ml,with the transfected rate ≥70％.The relative expression levels of Slit2 mRNA in the Lv-rSlit2-siRNA1 group and LvrSlit2-siRNA2 group were 0.67±0.09 and 0.23±0.11,respectively,which were lower than 1.03±0.31 and 0.92± 0.07 in the blank control group and Lv-EGFP vector group (all at P＜0.01).The expression levels of Slit2 protein in the Lv-rSlit2-siRNA1 group and Lv-rSlit2-siRNA2 group were 0.62±0.07 and 0.49±0.02,respectively,which were lower than 1.00±0.10 in blank control group and 0.95±0.11 in Lv-EGFP vector group (all at P＜0.01).Significant differences were found in the expression of VEGFA mRNA and protein in RPE cells among different concentrations of CoC12 groups (mRNA:F tration =127.998,P＜0.01;Fgroup =69.663,P＜0.01.Protein:F ion =17.059,P＜ 0.01;Fgroup =91.791,P＜0.01),and the expression of VEGFA mRNA and the concentration of VEGFA protein were evidently lower in the Lv-rSlit2-siRNA2 group than those in the blank control group after being treated by 100,200 and 400 μmol/L CoCl2 (all at P＜0.01).Conclusions Recombined lentiviral vector for rat Slit2 gene RNAi is successfully constructed,which can effectively knockdown rat Slit2 gene and inhibit the expression of VEGFA in rat RPE cells.

OBJECTIVE:To observe the therapeutic effect of fleabane in the treatment of non-arteritis anterior ischemic optic neuropathy(AION).METHODS:38non-arteritis AION patients were divided into two groups at random:group A and group B were treated with fleabane and salvia miltiorrhiza respectively.RESULTS:The eyesight of both groups has been im-proved in a different degree and the improvement of visual field of group A surpassed that of group B.CONCLUSION:Flea-bane has certain amelioration and protection effects in the treatment of non-arteritis AION.

Objective To observe the therapeutic effect of ultrasonic microbubble combined with bevacizumab (Avastin) on choroidal neovascularization induced by photocoagulation in rabbits.Methods CNV was induced by photocoagulation with argon laser in 30 rabbits (60 eyes).All of the rabbits underwent fundus fluorecein angiography (FFA) 21 days after photocoagulation;6-8 hours later,3 rabbits were randomly chosen to be executed to having the immunohistochemical examination.Twenty-one days after photocoagulation,27 rabbits were divided randomly into 3 groups:bevacizumb,ultrasonic microbubble+bevacizumb,and control group;each group has 9 rabbits (18 eyes).The rabbits in control group had no interference treatment;while the rats in bevacizumb and ultrasonic microbubble+bevacizumb group underwent injection with bevacizumb or ultrasonic microbubble+bevacizumb respectively.FFA was performed on all of the rabbits 7,14,and 28 days after photocoagulation to observe the inhibition of CNV;immunofluorecence and Western blot were used to detect the expression of VEGF in retina and choroid.Twenty-eight days is the time point to determine the therapeutic efficacy.The expression of VEGF and the results of FFA were the sdandards of the judgement of therapeutic efficacy.Results Proliferaion of CNV to the retinal inner layer and the obvious leakage of fluoresein in the photocoagulation area indicated that the model of CNV was set up successfully.Twenty-eight days after injection,obvious fluorescent leakage was found in the control group,and the average fluorescent leakage in bevacizumab group differed much from the control group(t=16.2952,P<0.05);while the difference between ultrasonic microbubble+bevacizumb group and bevacizumab group was also significant (t=4.7955,P<0.05).At the same time point,the expression of VEGF in bevacizumab group detected by immunofluorecent assay and Western blot differed much from the control group (t=7.0327,9.2596;P<0.05),and the difference of VEGF between ultrasonic microbubble+bevacizumb group and bevacizumab group was significant (t=2.9724,17.1937;P<0.05).this experiment show that ultrasound combined bevacizumab intravitreal injection of the therapeutic effect of CNV superior to other groups(P<0.01).Conclusion Ultrasound microbubble combined with bevacizumab injection may improve the therapeutic effect on CNV by inhibiting the expression of VEGF.

Objective To observe the changes in cytokines, oxygen free radicals, acidulous production in serum and to investigate the effectiveness of monitoring metabolism of cells and the method for evaluating cell injury in patients with severe trauma. Method The detailed data of 117 patients rescued and managed carefully from May 2005 to February 2007 were assessed and stratified with ISS and APACHE Ⅱ , and the serum levels of arterial blood lactate(ABL) ,base deficit(BD) ,superoxide(SOD) ,lipid peroxide(LPO) ,TNF-α and IL-6 were measured in real-time according to the condition of the patient. Results The monitoring biomarkers obviously changed with injury severity which endangered the situation of patients after trauma (P < 0.05) , especially in the patients with ischemia , hypoxia, shock, iniection, SIRS, and MODS (P < 0.01). The persistence of extremely elevated levels of biomarkers meant the organ failure and fatality of patients after trauma, and there was a obvious differece between those cases and cases without elevation of biomarkers ( P < 0.01). Conclusions The outcome of patients after trauma is closely correlated with injury severity,infection and MODS,and the levels of biomarkers including ABL, SOD, IPO, TNF-a and IL-6 are useful indicators of outcome measure.

Objective To evaluate the physiological variables,which precisely and reliably reflected the effect of emergency fluid therapy for severely traumatized patients, in order to set up the ultimate criteria of optimal goal in fluid resuscitation. Method A total of 149 patients with severe trauma were given fluid resuscitation and were stratified into 3 groups with different severities of trauma as per ISS (injury severity score) and APACHE Ⅱ . Of all patients, heart rate (HR), systolic blood pressure (SBP), oxygen saturation of arterial blood (SaO2), blood gas analysis, arterial blood lactate (ABL), oxygen saturation of central venous blood (SCVO2) or oxygen saturation of mixed venous blood (SVC2), urine output, base excess (BE) and oxygenation index (OI = PaO2/FiO2) were measured and calculated. These variables were compared between groups to find out the significant differences and the relationship to response time to fluid therapy as well as complications and outcomes. Results Within 24 hours of fluid resuscitation, 127 patients reached the therapeutic goal in respect of systemic hemodynamics improved including the variables of SBP, HR and urine output, and the optimal goal of fluid therapy in 112 patients was estimated with cellular oxygen available found in the levels of ABL, BE and OI measured. These two sets of criteria (clinical signs vs laboratory findings) for determining the therapeutic goal showed significant difference in length of time taken for reaching the goal of treatment ( P < 0.05). There were significant differences in APACHE Ⅱ scores between those reaching the therapeutic goal within 24 hours and those taking longer time over 24 hours reaching the therapeutic goal or the death (P < 0.01). The duration of persistence in abnormal systemic hemody-namics and laboratory findings was longer in patients with complications or injured to death than that in survivors (P <0.05 -0.01). Conclusions In addition to the stability of vital signs, tissue perfusion and cellular oxy-genation should be taken as ultimate criteria of successful fluid resuscitation for severely traumatized patients judged by means of measuring the ABL, BE and OI variables.

Objective To prospectively study the relation between transforming growth factor beta-1 (TGF-?_1), V_(20) and lung function (PFTs) and radiation pneumonia (RP), as well as to set up a prediction model of RP. Methods From Jan 2004 to Dec 2005, 121 valid patients with esophageal carcinoma or lung cancer were treated with conventional thorax radiotherapy(RT) by 15 MV X-ray beams to a total D_T 60-68 Gy over 30-34 fractions in 42-46 days. All patients received chest CT scanning before RT. Dose volume his- togram(DVH) and V_(20) were obtained through 3-dimensional TPS. Serum TGF-?_1 and PFTs of the patients were measured both before and after RT as well as on the 20th day after the beginning of RT. RP was diag- nosed basing on contrasted CT and clinical symptoms. Results RP was diagnosed in 32 of all 121 pa- tients. The results of Logistic Regression Statistic showed that V_(20) and TGF-?_1 ratio (after RT/before RT) significantly influenced the incidence of RP. Patients with V_(20)≥30% had more RP than patients with V_(20)

Objective:To study the protein leakage of mouse lens treated with hydrogen peroxide in vitro, and to identify and carry out bioinformatics analysis of the leaked proteins. Methods:Intact binocular lenses of 42 healthy male C57BL/6J mice were enucleated following sacrifice, and 28 lenses were arbitrarily combined as one sample, with 3 samples in total.Mouse lenses were cultured with medium containing 2 mmol/L hydrogen peroxide for 24 hours to establish a cataract model in vitro.Blank medium M-199 was taken as control sample.Supernatant was subjected to polyacrylamide gel electrophoresis (PAGE). The structure of the supernatant proteins was identified by liquid chromatography coupled with tandem mass spectrometry, and the high-abundant proteins were quantitatively analyzed by label-free quantitative mass spectrometry.The biological information of the identified proteins was analyzed by METASCAPE database.Content of cytokines in the supernatant was determined using the multiplex bead immunoassay system.This study protocol was approved by an Ethics Committee of The First Affiliated Hospital of Chongqing Medical University (No.2019-173), and the use and care of experimental animals complied with the ARVO statement. Results:After 24-hour culturing, the lenses became turbid, and the capsules were intact.The protein concentration in the supernatant was (3.73±0.59)μg/μl.PAGE analysis of the supernatant protein profile revealed that protein bands were below 35 000.A total of 675 proteins were identified with mass spectrometry.Sixteen crystallin proteins accounted for (86.1±0.8)% of the total protein abundance; the biological processes those proteins participated in mainly included cell-cell adhesion, cell transformation, oxidation-reduction, inhibition of apoptosis, and protein translocation; the molecular functions of those proteins mainly involved the activity of hydrolase, oxidoreductase, catalysis, translation initiation factor, and GTPase; the subcellular localization of proteins were mainly in cytoplasm, extracellular exosome, the nucleus, the plasma membrane, and the intercellular space.The results of an antibody-based multiplex bead immunoassay presented 9 cytokines in the supernatant, which included monocyte chemotactic protein-1 and transforming growth factor-β 2 highly expressed in intraocular fluid. Conclusions:There are protein leakages in lens treated with hydrogen peroxide in vitro, and it may have influences on metabolism and/or functions of other intraocular tissues.

This paper summarized the experience of Professor FANG Dingya in staged treatment of Sjögren's syndrome from the perspective of dryness toxin. It is believed that the cause of Sjögren's syndrome is externally-contracted dryness， consumption of essence and fluid， congenital and acquired essence deficiency， depleted essence and insufficient blood， and the core mechanism is internal accumulation of dryness toxin. The treatment can be divided into three stages， that is dryness toxin transforming into fire-heat， damp-heat and phlegm-stasis， from the perspective of dryness metal qi transformation. It is emphasized to dispel pathogen mainly， to clear and moisten with yin-nourishing medicinals in supplementation， and to treat by stages based on syndrome differentiation. For dryness toxin with fire-heat， it is suggested to moisten dryness， resolve toxins and subdue fire， with self-made Runzao Jiedu Decoction （润燥解毒汤） in modification. For dryness toxin with damp-heat， the method of nourishing yin， clearing heat and draining dampness should be used， and Chunze Decoction （春泽汤） in modification is suggested. For dryness toxin with phlegm-stasis， it is recommended to unblock collaterals， disperse phlegm and dissipate stasis， with self-made Sanyu Xiaotan Decoction （散瘀消痰汤） in modification.

Objective To analyze the clinical features of the multiple trauma patients combined with spine and spinal cord injuries.Methods A retrospective study was performed in 143 multiple trauma patients combined with spine and spinal cord injuries admitted to our department between March 2004 and March 2009.The parameters including injury cause,segment of injuries,associated injuries,complications,treatment methods and outcomes were analyzed.Results Falling and traffic accidents were the main causes for the injuries of spine and spinal cord,accounting for 53.8%(77 cases)and 38.5%(55 cases),respectively.The injured segments involved 101 lumbar vertebrae(50.8%),61 thoracic vertebrae(30.7%),29 cervical spines(14.6%)and 8 sacrococcygeal vertebrae(4.0%).The associated injuries were located at chest(163 regions,38.6%),abdomen(84 regions,19.9%),head and neck(77 regions,18.3%),extremity(65 regions,15.4%),face(17 regions,4.0%)and body surface(16 regions,3.8%).The early complications included electrolyte disturbances in 33 patients (16.8%),respiratory infection in 30(15.3%)and abdominal distention in 19(9.7%).The late complications were malnutrition in 26 patients(13.3%),amyotrophy in 23(11.7%)and deep vein thrombus in 11(5.6%).Treatment methods were operations and expectant treatments in 106 patients (74.1%)and 37(25.9%)respectively.According to American Spinal Injury Association(ASIA)scale,there were 20 patients(14.0%)at grade E before treatment and 53(37.1%)at grade E after treatment.Of all,12 patients were died of mainly multiple organ failure(MOF),cerebral hernia and malnutrition,with mortality rate of 8.39%.There showed an increase of complication and mortality rate with increase of ASIA grade(P ＜ 0.05).Conclusions The spine and spinal cord injuries in patients with multiple trauma are mainly caused by high energy injuries and characterized by high injury severity,complex associated injuries,multiple complications,difficult management and high mortality rate.