Objective To evaluate the gene amplification and protein expression of HER-2 in gastric carcinoma with or without neuroendocrine differentiation (NED), and to explore the difference in HER-2 gene profile between these two neo?plasms. Methods Cases of gastric carcinoma with NED (n=70) and cases of gastric carcinoma without NED (n=150) were retrospectively reviewed. Gene amplification and protein expression of HER-2 genes in gastric carcinoma with or without NED were detected by combination of immunohistochemical method (IHC) and double color silver dye for in situ hybridiza?tion (DISH). Prognosis of gastric cancer patients with NED were predicted using Kaplan-Meiers survival analysis. Results Expression rates of HER-2 in gastric cancer with and without NED are 20.00%and 21.33%respectively. Amplification of HER-2 in gastric cancer with and without NED are 8.57%and 14.67%respectively. Gene amplification and protein expres?sion of HER-2 between gastric cancer with NED and without NED showed no statistical difference. Chromosome 17 multi-body positively correlated with gene HER-2 amplification in Gastric carcinoma with NED. Postoperative survival period in patients of gastric carcinoma with NED and HER-2 amplification was shorter than that in patients of gastric carcinoma with?out NED but with HER-2 amplification. Gastric carcinoma with or without NED, HER-2 gene amplification, lymph node me?tastasis and operation method obviously correlate with prognosis of gastric carcinoma patients (P<0.05). Conclusion The gastric cancer with NED is a special type of gastric cancer, there was no difference of gene amplification and protein expres?sion of HER-2 gene between gastric carcinoma with NED and without NED. Poor prognosis would be expected in gastric can?cer patients with NED and HER-2 amplification.

Objective:To investigate the effect of miR-218 on the proliferation and invasion of breast cancer cells.Methods: The expression of miR-218 in breast cancer tissues and breast cancer cell lines was detected by qPCR.The relationship between the expression of miR-218 and the clinicopathological parameters of breast cancer were analyzed.Double luciferase assay was used to detect the relationship between miR-218 and SNX4.MTT assay and invasion assay were used to detect the proliferation and invasion of breast cancer cells after overexpression of miR-218.MTX assay and invasion assay were used to detect the recovery level of SNX4 on the proliferation and invasion of breast cancer cells.The effect of miR-218 on the tumorigenesis of breast cancer cell lines was examined by tumorigenesis in nude mice.Results: The expression level of miR-218 in breast cancer tissue and MCF-7 cell line was higher.The expression of miR-218 was associated with pathological stage of breast cancer and lymph node metastasis.SNX4 may be the target of miR-218.Overexpression of miR-21 could inhibit the proliferation and invasion of breast cancer cells.Overexpression of SNX4 could reverse the inhibitory effect of miR-218 on breast cancer cells.Overexpression of miR-218 could inhibit the breast cancer cell line in nude mice tumorigenic ability.Conclusion: miR-218 can up-regulate the expression of miR-218 in breast cancer,and miR-218 can regulate the expression of SNX4 in breast cancer cell proliferation and invasion.

5-Flucytosine (5-FC) could be changed to 5-fluorouracil (5-FU) by cytosine deaminase (CD), the latter is able to kill cancer cells. However, there is no efficient method to deliver the CD gene into the tumor cells, which hampers the application of the suicide gene system. In this experiment, for the first time, the NDV has been utilized as a vector to deliver the CD gene into the cancer cells, the virus can infect the cancer cells specifically, replicate and assemble, while the cytosine deaminase is expressed. Then the CD converts the prodrug 5-FC into 5-FU to achieve the purpose of inhibiting tumor. Firstly, the whole genome of E. coli JM109 was extracted, and the CD gene was obtained by cloning method. Then the CD and IRES-EGFP were ligated into the pEE12.4 expression vector to become a recombinant pEE12.4IE-CD eukaryotic expression plasmid. The human liver cancer cells were transfected with the plasmid. The cells were treated with different concentrations of 5-FC, MTT method was used to determine the killing effect of CD/5-FC system on the human liver cancer cells. The cell deaths were 18.07%, 42.98% and 62.20% respectively when the concentrations of prodrug were at 10, 20 and 30 mmol x L(-1). In 5-FC acute toxicity experiment, Kunming mice were injected with different concentrations of 5-FC at intervals of 1:0.5. The LD50 of 5-FC through iv injection was determined by improved Karber's method, the LD50 was 507 mg x kg(-1) and the 95% confidence limit was 374-695 mg x kg(-1). According to the maximum LD0 dose of the LD50, the maximum safe dose was 200 mg x kg(-1). Body weight and clinic symptoms of the experimental animals were observed. These results laid the foundation to verify the antitumor effect and safety of CD/5-FC system in animal models. The CD gene was ligated into the NDV (rClone30) carrier, then the tumor-bearing animal was established to perform the tumor inhibiting experiment. The result showed that the recombinant rClone30-CD/5-FC system has a high antitumor activity in vivo. To summarize, CD gene has been cloned and its bioactivity has been confirmed in the mammalian cells. It is the first time in this study to utilize the recombinant NDV to deliver the CD gene into the tumor cells; our result proves the rClone30-CD/5-FC system is a potential method for cancer therapy.

Objective To evaluate the protective effect of astragaloside Ⅳ against ultraviolet B (UVB)-induced photodamage to human HaCaT keratinocytes,and to investigate its mechanisms.Methods Culturedimmortalized human HaCaT keratinocytes were divided into four groups:blank control group receiving untreated,UVB group irradiated with 50 mJ/cm2 UVB,astragaloside Ⅳ group treated with astragaloside Ⅳ,UVB + astragalosideⅣ group treated with astragaloside Ⅳ for 24 hours before and after 50 mJ/cm2 of UVB radiation.The concentration ofastragaloside Ⅳ ranged from 10 to 200 mg/L in cell proliferation assay,and according to the results of proliferationassay,20 mg/L was determined as the optimal concentration in the other assays.At 24 hours after UVB radiation,cellcounting kit-8 (CCK8) assay was performed to evaluate cellular proliferative activity,flow cytometry to determineintracellular reactive oxygen species (ROS) levels,and Western blot to measure the expression levels of p53,p38,matrix metalloproteinase-9 (MMP-9) and high mobility group Al (HMGA-1) protein in HaCaT cells.ResultsCompared with the control group,astragaloside Ⅳ at 10 and 20 mg/L had no inhibitory effect (F =1.32,P ＞ 0.05),while astragaloside Ⅳ at 50,100 and 200 mg/L showed significantly inhibitory effect (F =20.20,P ＜ 0.05),on theproliferation of HaCaT cells.In addition,cellular proliferative activity in the UVB group was significantly lower thanthat in the control group (F =99.00,P ＜ 0.01).Compared with the UVB group,cellular proliferative activityincreased to different degrees in HaCaT cells treated with both UVB and astragaloside Ⅳ of 10-200 mg/L (F =19.08,P ＜ 0.01),with the strongest increase observed in those treated with UVB and astragaloside Ⅳ of 20 mg/L.Further experiments revealed reduced intracellular ROS levels in the UVB + astragaloside Ⅳ (20 mg/L) groupcompared with the UVB group (t =21.12,P ＜ 0.01).Western blot assay showed that the expression levels of p53,p38,MMP-9 and HMGA-1 protein were significantly higher in the UVB group than in the control group (all P ＜0.01),but significantly lower in the UVB + astragaloside Ⅳ (20 mg/L) group than in the UVB group (all P ＜ 0.01).Conclusion Astragaloside Ⅳ can effectively protect keratinocytes from UVB-induced photodamage.

OBJECTIVE:To analyze intervention effect of Drug Rational Usage Guidelines System(DRUGS)on antibacterial agents use in department of obstetrics and gynecology in our hospital. METHODS:The application of antibacterial agents in depart-ment of obstetrics and gynecology in our hospital during Jan.-May(before intervention)and Jun.-Nov. 2012(after intervention)were extracted in respects of drug name,preoperative medication duration,perioperative additional condition,postoperative drug with-drawal time,drug combination,usage and dosage,average hospitalization stay,hospitalization cost. The intervention effects were analyzed. RESULTS:After intervention,the type of antibacterial agents were more in line with national regulations;the proportion of type Ⅰ incision surgery without antibacterial agents increased from 57.8% to 75.2%;the prophylactic application of antibacteri-al agents in type Ⅱ incision surgery within 0.5-2 h increased from 80.2% to 97.0%. The rate of reasonable antibacterial selection, drug combination,usage and dosage increased from 76.9%,64.9%,71.3% to 89.3%,84.6%,90.2%,respectively. The average hospitalization stay and antibacterial cost per capita decreased significantly. There was statistical significance among above indica-tors before and after intervention(P<0.05 or P<0.01). CONCLUSIONS:DRUGS effectively change irrational use of antibacterial agents in department of obstetrics and gynecology,which provide a new method for the management of antibacterial agents.

Objective To study the clinical,pathological and genetic features of myofibrillar myopathy caused by BAG3 gene mutation.Methods The clinical features and pathological findings of a patient with myofibrillar myopathy were analyzed.Genomic DNA of the patient was extracted from peripheral blood and the next generation sequencing was performed to explore the mutation of genes about myopathies.Results The patient presented with nine-year-old onset myopathy characterized by progressive difficulty for squatting,rigid spine and muscle atrophy in the limbs symmetrically.Peripheral neurogenic damages were found on electromyography.On muscle biopsy,myogenic and neurogenic damages with rimmed vacuoles appeared,and the deposited materials were positive for sarcoglycan,dystrophin-R and dystrophin-C.There was a reported heterozygous mutation in the exons of the BAG3 gene (c.626C ＞ T).Conclusion There is no specificity of clinical manifestation in myofibrillar myopathy,and the diagnosis of this disease mainly depends on muscle biopsy and genetic screening.

Objective To establish a simple and reliable method for isolation and cultivation of epidermal stem cells from neo-natal rat skin basal layer .Methods The single cells were dissociated with twice trypsinization form neonatal rat skin .Thereafter we purified the basal layer stem cells with differential velocity adherent technique with collagen Ⅳ ,and the slow adherent cells were cultured as negative control cells .Both basal layer stem cells and control cells were cultivated with keratinocyte serum-free medium (K-sfm) .Stem cells were identified with β1-integrin and Keratin 19 by co-immunofluorescence assay ,and colony forming assay was executed to evaluate the proliferation potential of stem cells .Results The polygonal cells grew like flagstones ,with doubling time of approximately 24 hours .Both the morphology and growth properties of cells were in accordance with the character of basal layer stem cells .Co-immunofluorescence identification showed the cells were positive for the expression of β1-integrin and Keratin 19 . Basal layer stem cells had stronger clone forming ability in vitro compare with control group .Conclusion The results indicate that two-procedure trypsinization plus differential velocity adhesion is an ideal method for basal layer stem cells separation followed with vigorous vitality and reliable phenotype .

Aim To investigate the effect of baicalin on cell proliferation and cell migration in human skin SCC A431 cell line. Methods The A431 cells were incu-
bated with 50 mg·L-1 baicalin. The protein level of cofilin-1 was assayed by Western blot. Cofilin-1 specific siRNA fragment was designed , synthesized and trans-
fected into A431 cells. The proliferative activity and migration ability of cells were assessed by CCK8 assay and scratch wound healing assay separately. ResultsWestern blot results showed that baicalin treatment in-hibited the cofilin-1 protein expression to 49.3% com-pared with the control group. Single baicalin treatment and cofilin-1 silencing could drease the A431 cell growth and migration. And cofilin-1 silencing signifi-
cantly enhanced the efficacy of baicalin. Conclusions Baicalin could significantly inhibit the tumor cell's growth and migration in the A431 cell line. And cofi-lin-1 might become the potential target gene to enhance the effect of anticancer drugs.

Background The pathogenesis of diabetic retinopathy (DR) might be related with oxidative stress and its mechanism has not been fully elucidated.Grape seed proanthocyanidin extracts (GSPE),a powerful antioxidant,plays roles in some systemic diseases.However,the effect and mechanism of GSPE in DR has not been illuminated clearly.Objective This study was to investigate whether GSPE has a protective effect on the retinas of diabetic subjects and explore its mechanism.Methods Thirty SPF adult Wistar rats were divided into the control group,diabetic group and diabetes + GSPE group according to the randomized number table.Diabetic models were induced by intraperitoneal injection of 100 mg/kg strebtozotocin (STZ) prepared with citrate buffer,and only the equal amount of citrate buffer was used in the same way in the control group.GSPE solution was intragastrally used 250 mg/kg daily in the rats of the diabetes+GSPE group from injective day through 8 weeks,and distilled water was used in the same way in the rats of the control and diabetic groups.The rats were sacrificed in the eighth week after injection and the retinas were isolated.The morphology of the retina were examined by hematoxylin and eosin stain.Retinal homogenatewas prepared for the assay of superoxide dismutase (SOD),glutathione peroxidase (GSH-Px) activity and malonaldehyde (MDA) content.The expressions and location of nuclear erythroid related factor 2 (Nrf2) was determined by immunochemistry,and the expressions of Nrf2 and heme oxygenase-1 (HO-1) were quantitatively analyzed by Western blot.The apoptosis of retinal cells was detected by TUNEL.Results In the eighth week after modeling,the blood glucose levels were significantly higher and the body weight was lower in the rats of the diabetic group and diabetes+GSPE group compared with the control group (all at P＜0.01).Retinal structure was normal in the rats of the control group.However,loose tissue,irregular arrangement of cells and thickness decrease of retinal fiber layer,retinal ganglion cell layer and inner plexiform layer were exhibited in the rats of the diabetic group,while the morphology abnormality was slight in the rats of the diabetes+GSPE group.The SOD and GSH-Px activities and MDA content were significantly different among the 3 groups (F =11.010,P =0.001 ; F =12.072,P =0.000 ; F =25.224,P=0.000),and activities of SOD and GSH-Px in the retinas were significantly lower,and the MDA level was higher in the rats of diabetic group than those of the diabetes+GSPE group and the control group (all at P＜0.01).The relative expressing levels of Nrf2 were (165.5±29.4) ％ and (134.8 ±7.8) ％ in the diabetes+ GSPE group and diabetic group,with a significant difference between them (t=2.450,P=0.044).Compared with the diabetic group,the expressing level of the HO-1 was sigficantly increased ([170.2±22.0)％ versus [125.3±9.2] ％,t =2.360,P =0.002).TUNEL showed that the retinal apoptotic cells of diabetic rats were mainly located in the retinal fiber layer,RGCs layer,inner and outer nuclear layer,and the number of apoptotic cells was less in the diabetes+GSPE group compared with the diabetic group under the fluorescence microscope.Conclusions GSPE can play a protective role on diabetic rats by activating Nrf2 pathway and therefore improving retinal oxidative stress and decreasing apoptosis.

Objective: To identify choroideremia and retinitis pigmentosa(RP)using next-generation sequencing(NGS)technology. Methods: A cross-sectional study was adopted.The participants were two pedigrees that was suspected of RP in previous hospital treatments in He Eye Specialist Hospital between January 2017 and December 2018.The relevant medical and family history were collected, and the family tree was plotted.The visual acuity, intraocular pressure, fundus photography, electroretinogram(ERG), B-mode ultrasond, visual field, color vision, optical coherence tomography(OCT), and slit lamp assessment were performed on all subjects.Target region sequencing and Sanger sequencing were performed, deletion insertion of large fragments was verified by real-time quantitative PCR.This study followed the Helsinki Declaration and was approved by the Ethics Committee of Shenyang He Eye Hospital(NO. IRB[2017]k002.01) Results: Two known pathogenic mutations were identified in CHM gene: EX9 DEL and c. 715C>T.Furthermore, the clinical diagnosis of choroideremia was confirmed.The Family 1 proband Ⅱ1 with the EX9 DEL hemizygous mutation had a special clinical phenotype, "Star" small piece intact choroid was visible in the macular fovea of both eyes.The patients in pedigree 2 carried the pathogenic mutation c. 715C>T, the proband Ⅲ1 carried a hemizygous mutation and the reddish island area in the fovea macula was seen in both eyes.The mother Ⅱ2 and grandmother I2 of proband carried heterozygous mutations, the degree of fundus atrophy was lighter than that of the proband Ⅲ1, the fundus had a mottled appearance, the optic disc boundary was clear, the blood vessel thickness was moderate, and the exposed choroid was visible in the fundus atrophy area. Conclusions This study is the first to identify two known pathogenic mutations in CHM gene, EX9 DEL and c. 715C>T in Asian Manchu and Han populations.The clinical phenotypes of female carriers with the c. 715C>t pathogenic mutation are different from those of male patients.The NGS technology may become a powerful tool to distinguish choroideremia from RP.