ObJeetive To report a simple and efficient method to label two different types of retinal ganglion cells (RGCs) in mouse retina.Methods Eyeballs were harvested from normal adult C57 BL/6 J mouse,the retinas were isolated,four radial cuts were done,the retinas were pasted on the nitrocellulose membrane with the ganglion cell layer upturned.The immunofluorescence double staining and laser confocal nmicroscope was used to reveal conventional retinal ganglion cells and intrinsically photosensitive retinal ganglion cells (ipRGCs) using Brn3a and Melanopsin.Results The double staining results of whole mount retina showed that conventional RGCs and melanopsin immunopositive ipRGCs had a complementary distribution in mouse retina,these two subtypes of RGCs were predominantly present in the ganglion cell layer.The numbers of ipRGCs was just about 1％-2％ of conventional RGCs,and the axons of ipRGCs toward the direction of the optic disc,several dendrites toward the inner plaximem layer.Conclusion The immunofluorescence double staining of whole mount retina is a simple,stable and efficient method to label two different types of mouse RGCs.

OBJECTIVE:To systematically review the efficacy and safety of insulin glargine versus insulin detemir in the treat-ment of type 2 diabetes,and provide evidence-based reference for clinical treatment. METHODS:Retrieved from PubMed,EM-Base,Cochrane Library,CBM,CJFD,VIP and Wanfang database,randomized controlled trials (RCT) about the clinical efficacy and safety of insulin glargine versus insulin detemir in the treatment of type 2 diabetes were collected. Meta-analysis was performed by using Rev Man 5.2 software after data extraction and quality evaluation by Cochrane 5.1.0. RESULTS:A total of 18 RCTs,in-volving 3 638 patients were included. Results of Meta-analysis showed there was no significant difference in reducing glycosylated hemoglobin[MD=0.08,95%CI (-0.01,0.17),P=0.09];fasting blood glucose level in insulin glargine group was significantly lower thaninsulin detemir,the difference was statistically significant [MD=0.15,95%CI(0.03,0.27),P=0.02]. And there was no significant difference in the incidence of hypoglycemia [OR=0.97,95%CI(0.91,1.03),P=0.25];the degree of body mass gain ininsulin detemir was significantly lower than insulin glargine group [MD=-0.95,95%CI(-1.06,-0.85),P=0.003],but the in-cidence of injection site reactions was significantly higher than insulin glargine group [OR=2.28,95%CI(1.16,4.50),P=0.02],the differences were statistically significant. CONCLUSIONS:The insulin glargine has better efficacy,than insulin detemir with lower incidence of injection site reactions but higher degree of body mass gain than insulin detemir in the treatment of type 2 diabetes.

Objective To investigate the effecof arthroscopiminimally invasion fothe local excision of benign bone tumo.MethodEighpatientwith benign bone tumofrom Augus2011 to Decembe2012 were treated by arthroscopimini-mally invasive operation ,including 3 caseof osteochondrom,2 caseof bone cys,1 case of non-ossifying fibromand 2 caseof osteoid osteom.The lesion location wadetermined by the preoperative imaging dat,the arthroscope and plane cuttewere placed into the lesion arethe tumowaperformed the local lesion resection undethearthroscopimonitoring ,then the plasmwaadopt-ed to treathe resected wound surface of tumo,the bone defectwere implanted by allogeneibone .ResultEighcasewere re-covered well afteoperation .No postoperative complicationwere found .The approach incision ＜3 and the wound length ＜1 .5 cm .Two casewere filled with allogeneibone .There were no recurrence ,fracture odyfunction during the average follow up peri-odof 8-12 month.The postoperative X-ray showed thathe lesion wacompletely resected and the bone defeclesion wacom-pletely filled with allogeneibone .Conclusion The resection of deep benign bone tumoundethe arthroscopimonitoring can maximize to retain the local anatomistructure .

Objective To study the estradiolNGF regulatory cascade in retinal endothelial cells. Methods Retinachoroids vascular endothelial cell line RF/6A from rhesus was cultured in M199 medium contain 20% FBS.mRNA of ER,NGF and its receptors,TrkA and P75 NTR were detected with RTPCR.The cells were incubated with NGF,VEGF,antibodies against NGF or VEGF,and K252a(100nmol/L),the specific inhibitor of TrkA,separately or in different combination.MTT based cell counting assay was used to study the viability of the cells.The apoptosis was evaluated by FACS,mass migration by wound healing assay,and tubogenesis by AngioMatrix assay. Results We amplified the specific fragments of cDNA of ER,NGF and NGF receptor TrkA using RTPCR.10?nmol/L1??mol/L estradiol augments the proliferation and increases the viability of RF/6A in a dosage dependent manner.In the wound healing based migration assay,we found the similar alteration.This effects of estradiol was partially blocked by NGF neutralized antibody and K252a.Apoptosis rates were at the similar level among the groups.For the tubogenesis of RF/6A,we found no augmentation by NGF,and no blocked augmentation by estradiol.Conclusion NGF,first identified as the survival factor of nerve system,now also seemed to be an activator of retinal endothelial cell,is under the regulation of estrogen.The proliferation and migration,but not the tubogenesis of retinal endothelial cells are regulated by this regulatory cascade.

Objective The purpose of this experiment is to study the in vivo differentiation and myelin formation of rat striatal neural precursor cells after transplantation into homogeneous retina,observe the order of myelination and its influence on the structure of retina,establish an animal model of CNS myelin formation in vivo. Methods Passage cultured striatal neural precursor cells from embryonic Sprague-Dawley rats were transplanted into the vitreous cavity of neonatal rats.In different stages after transplantion,myelin formation in retina was observed under light and electron microscope and analysed with different stained methods.Results Bundles of myelin appeared in parts of retina 4 weeks later.The distribution and morphology of myelined area expanded with prolonged survival time after cell transplantation.Oligodendrocyte wrapped the naked axons and formed normal myelin limited in the nerve fiber layer.Myelination influenced the distribution of local retinal ganglion cells.Conclusion Striatal neural precursor cells could differentiate into oligodendrocytes and formed myelin after transplanted into retina and the naked axons in retina promoted the myelin formation.This model provides a new method to study the myelin formation and myelin-axon interaction in vivo.

BACKGROUNDTobacco-specific 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is the most important carcinogen in cigarette. Models induced by NNK are widely used in investigations about the mechanisms of pulmonary neoplasia and chemoprevention studies. The aim of this study is to explore the pulmonary precancerous lesions induced by NNK and its possible mechanisms.

METHODSFifteen Wistar rats were divided into two trial groups, in which the high-dose group was instilled with iodized oil including 10 mg (50 mg/kg) NNK into the left lower lobar bronchus, and the low-dose group received 5mg ( 25mg/kg) NNK. Another 15 Wistar rats were instilled only with iodized oil as control group. All rats were examined immediately after instillation and followed up periodically by pulmogram. The pulmonary tissues of rats were pathologically examined, and the expression of AE1/AE3, PCNA and p53 was detected by immunohistochemical method.

RESULTSThe pulmograms showed that the iodized oil localized at the bottom of left lobe and disappeared 107 days later. In trial group, 10 of 15 rats (67%) had nodus at the bottom of left lobe. All of rats in trial group (15/15) displayed atypical hyperplasia in alveolar region, showing single or multiple layers of proliferative epithelial cells along intact alveolar septa with irregular and non-discrete margins of lesion, but continuous alveolar spaces were not obliterated by proliferative epithelial cells. Ten of 15 rats in trial group showed severe atypical hyperplasia of glandular epithelium with occasional infiltrating to muscular layer. All of those atypical hyperplasia cells showed positive AE1/AE3 expression. The positive rate of PCNA was 90% (9/10) and 100% (5/5) in low-dose group and high-dose group respectively, which was significantly higher than that in control group (13%, 2/15) (P=0.000, P=0.001). The positive rate of p53 expression was 50% (5/10) and 60% (3/5) in low-dose group and high-dose group respectively, which was significantly higher than that in control group (0) (P=0.005, P=0.009). However, there was no remarkable difference in PCNA and p53 expression between low-dose group and high-dose group (P > 0.05).

CONCLUSIONSTransbronchial instillation of iodized oil including tobacco-specific NNK can induce pulmonary lesions as atypical hyperplasia of alveolar cell and glandular epithelium in Wistar rats. This model can be used in experimental studies about tobacco-related lung cancer.

Objective To explore the clinical application effect of multiple end-to-side anastomosis in free anterolateral thigh perforator flap transplantation.Methods From January,2013 to October,2017,29 cases were applied the technology of multiple end-to-side anastomosis to the same recipient vessel in anterolateral thigh perforator flap transplantation for wound repair.Fifteen cases treated with multiple end-to-side anastomosis on flap arteries,and 2-3 arteries were anastomosed,with the average of 2.13.Nine cases were treated with venous multiple end-to-side anastomosis,and 2-4 veins were anastomosed,with the average of 3.11.Five cases treated with arterial and venous multiple end-to-side anastomosis,and 4-6 vessels were anastomosed,with the average of 5.20.Distribution of wounds:10 cases with hands and wrists wounded,15 cases with forearms and elbow joints wounded,and 4 cases with lower legs and feet wounded.The soft tissue defect size was 5 cm×13 cm-11 cm×27 cm,and the flap area was 6 cm× 15 cm-12 cm×29 cm.Postoperative followed-up was performed every 3-6 months to review flap survival.Results All flaps of 29 cases survived.Venous congestion occurred in 2 flaps within 48 h after the operation,among which,1 was overcomed after released the dressing and sutures,and the other underwent surgical exploration.The venous end-to-side anastomotic stomas were unobstructed,and hematoma was formed.After the hematoma compression was removed,circulation was recovered and the flap survived.With followed-up for 6 months to 2.5 years,both donor site and recipient site of the flaps healed well and the injured distal limbs had no hemodynamic disorder.Conclusion The application of multiple end-to-side anastomosis to the same recipient vessel for free transplantation of anterolateral thigh perforator flaps is safe and reliable.

Objective To investigate the effect of anti-inlammation and antiulcer in rats of Erkou powder and Erkou film.Methods The rats were divided into six groups according to random number table:control group,Guilin watermelon frost group,Erkou powder low dose group,Erkou powder high dose group,Erkou film low dose group and Erkou film high dose group,each with 12.Guilin watermelon frost group was given 0.7 g/kg,Ekou powder low dose group and high dose group were respectively given 14.56,29.12 mg/kg,Erkou film low dose group and high dose group were given 19.25,38.5 mg/kg by intragastric administration.Control group was given the same volume of normal sodium by intragastric administration.To observe the inhibitory actions of paw edema induced by carrageenan of big rats,the increase of abdominal cavity capillary permeability in rats due to acetic acid and effect of oral ulcer healing on aceti acid-cautery type oral ulcer rats.Results Compared with the control group,1 h and 3 h after injection of 1％ carrageenan,the paw edema of rats were significantly reduced in Guilin watermelon frost group,Erkou powder high dose group and Erkou film high dose group (P＜0.01 or P＜0.05).Compared with the control group,the absorbance (0.62 ± 0.08,0.62 ± 0.17,0.64 ± 0.15 vs.0.82 ± 0.24) of peritoneal lavage fluid in Guilin watermelon frost group,Erkou powder high dose group and Erkou film high dose group were significantly decreased (P＜0.05).Compared with the control group,the diameter of ulcer area (3.16 ± 0.68 mm,3.12 ± 0.45 mm,3.22 ± 0.59 mm,3.23 ± 0.70 mm vs.3.90 ± 0.72 mm) of rat in Guilin watermelon frost group,Erkou powder low dose group,Erkou powder high dose group and Erkou film high dose group were significantly decreased (P＜0.01 or P＜0.05) in the third day.Compared with the control group,the diameter of ulcer area (1.93 ± 0.49 mm,1.39 ± 0.42 mm,2.01 ± 0.45 mm,1.98 ± 0.61 mm vs.2.69 ± 0.74 mm) of rat in Guilin watermelon frost group,Erkou powder low dose group,Erkou powder high dose group and Erkou film high dose group were significantly decreased (P＜0.01 or P＜0.05) in the fifth day.Compared with the control group,the healing time of oral ulcer (7.20 ± 0.67 d,6.64 ± 0.67 d,7.27 ± 0.90 d,7.27 ± 0.90 d vs.8.37 ± 1.12 d) of rat in Guilin watermelon frost group,Erkou powder low dose group,Erkou powder high dose group and Erkou film high dose group were significantly shortened.Conclusions Both Erkou plastics and Erkou powder have the effects of resisting inflammation and promoting the healing of oral ulcers.

Objective:To study the mechanism of Yangyin Yiqi mixture on the clinical symptoms in convalescent period of different types of pneumonia based on network pharmacology, so as to provide more options for the prevention and treatment of pneumonia with Traditional Chinese Medicine (TCM).Methods:Based on the TCM theory of "treating different diseases with the same method", the Integrative Pharmacology-Based Research Platform of Traditional Chinese Medicine (TCMIP) V 2.0 was used to screen the active components, action targets and disease targets of the herbs in Yangyin Yiqi mixture. The disease-target-medicine network map was constructed by Cytoscape software and the Reactome enrichment analysis of key targets were carried out.Results:Seven different types of clinical sympotms of pneumonia in convalescent period were established, including fatigue, tachypnea, thirst, palpitations, hyperhidrosis, poor appetite and cough. After the screening, a total of 276 active components and 177 key targets were obtained, 37 common targets of this herb and pneumonia were obtained. The key targets were associated with cell metabolism, apoptosis, inflammatory response, immune response, and stress response, etc. There are 17 common targets between Polygonatum and 7 clinical symptoms in the recovery period of different types of pneumonia. Reactome enrichment analysis showed that the pathways involved in the core genes of Yangyin Yiqi mixture in the treatment of the main symptoms of different types of pneumonia in the recovery period were mainly enriched in: by regulating genes and protein translation to affect the virus or cell function, immune regulation and inflammatory response and regulate the function of nervous system, and prevent viral canceration.Conclusions:The effective components in Yangyin Yiqi mixture may regulate phosphatidylinositol 3-kinase (PI3K), interleukin-4, interleukin-13 (IL-4, IL-13) and inhibitory kappa protein (IκBα) by intervention of PIK3CA, MAPK3, BCL2 and NF-κB2 and other targets to promote the recovery of pneumonia. This study could provide a theoretical basis for the prevention and treatment of pneumonia in the recovery period.