Objective In this study ,we constructed a series of recombinant plasmids carriers expressing shRNA targeting hTERT and Bi‐1 gene .These recombinant plasmids carriers were transfected into CNE‐2Z cell lines using Lip and continuously in‐duced the expression of shRNAs .Furthermore ,the shRNAs caused the degradation of mRNAs homologous in sequence with the target genes ,which lead to a sequence‐specific gene silencing .Methods The CNE‐2Z cells was divided into untreated group ,pEG‐FP‐N1 group and pEGFP‐N1/Lip group .Flow cytometry(FCM ) was applied to determine the transfection efficiency .The changes of hTERT and Bi‐1 gene expression were detected by Real‐time RT‐PCR and Western blotting .Results The best transfection effi‐ciency between plasmid and Lip was 2 .5 μg plasmid and 6 .25μL Lip .Conclusion We constructed several shRNA recombinant eu‐karyotic expression plasmids successfully .The recombinant plasmid can inhibit the expression of hTERT and Bi‐1 gene specifically and effectively .

Polymeric micelles have exhibited attractive properties as drug carriers, such as high stability in vivo and good biocompatibility, and been successfully used to dissolve various drugs of poor aqueous solubilities. In this study, we developed a new type of polymeric micelles with mannose-mediated targeting and pH-responsive drug release properties for anticancer drug delivery. The polymeric micelles were prepared from an amphiphilic polymer, poly (glycidyl methacrylate)-g-mannose (PGMA-Mannose). An anticancer drug, doxorubicin (DOX), was encapsulated into the micelles during the micellization, and could be released rapidly under acidic condition. The specificity of cellular uptake of the micelles by two different cell lines was studied using confocal laser scanning microscopy and the MTT assay. DOX-loaded micelles were efficiently trapped by mannose-receptor-overexpressing cancer cells MDA-MB-231, whereas mannose- receptor-poor cells HEK293 showed much lower endocytosis towards the micelles under the same conditions. Thus, DOX-loaded micelles displayed higher cytotoxicity to MDA-MB-231 cancer cells as compared with free DOX. The present study demonstrates that PGMA-Mannose micelles are a promising targeted drug delivery system for cancer therapy.
Cell Line, Tumor ; Doxorubicin ; pharmacology ; Drug Delivery Systems ; HEK293 Cells ; Humans ; Lectins, C-Type ; metabolism ; Mannose ; chemistry ; Mannose-Binding Lectins ; metabolism ; Micelles ; Receptors, Cell Surface ; metabolism

Objective To explore the safety and effectiveness of diagnostic ultrasound associated with microbubbles to open the blood brain barrier(BBB).Methods Microbubbles were injected through caudal vein,the rat head was radiated by GE Vivid 7 diagnostic ultrasound immediately.The radiated depth was located in the basal ganglia assisted by magnetic resonance imaging(MRI)scanning.The degree of BBB opening was evaluated by enhanced MRI and Evans blue dyeing.The safety was inspected by observation of cell morphology under hematoxylin eosin(HE)staining.Results After the rat head radiated by diagnostic ultrasound with microbubbles,signal enhancement of the radiated area was observed on post contrast T1-weighted images.Red fluorescence of Evans blue was detected by fluorescence microscope in the same area.Normal cellular morphology and structural integrity were showed by HE staining.Conclusions The BBB of rat could be opened targetedly and noninvasively by diagnostic ultrasound associated with microbubbles.This may provide a new strategy for the drugs and stem cells treatment in the central nervous system diseases.

Objective To investigate the effects ofYangyin Yiqi Mixture on pulmonary fibrosis caused by bleomycin in rats;To discuss its possible mechanism.Methods SD rats were randomly divided into normal control group, model group, dexamethasone acetate group, low-dose, middle-dose and high-doseYangyin Yiqi Mixture groups. Bleomycin was used to establish pulmonary fibrosis rat models through endotracheal instillation. One day after molding, each medication group received gavage with relevant medicine. Lung tissues were taken on the 7th, 14th and 28th day. Masson staining was used to observe pathological change;alkalinehydrolysis was used to detect HYP content;Western blot was used to detect the protein expressions of MMP-2 and TIMP-1.Results Compared with the normal control group, on 7th day, collagen in lung tissue of rats in model group markedly increased;the content of HYP in lung tissue significantly increased (P<0.01). On 28th day, collagen was deposited diffusely and the alveolar was destroyed;content of HYP in lung tissue increased significantly (P<0.01). Compared with model group, deposited collagen in lung tissue was alleviated, and the content of HYP were remarkable reduced in middle and high doseYangyin Yiqi Mixture groups and displayed dose-dependent. Western blot results showed that the protein expressions of MMP-2 and TIMP-1 increased quickly compared with the low expression of normal lung tissue in the 7th day of molding (P<0.05), and reached high expression in the 28th day. Compared with the model group, the level of MMP-9 in high-dose Yangyin Yiqi Mixture group decreased significantly in the 14th day;the expression of TIMP-1 decreased significantly in the 7th, 14th and 28th day (P<0.05).ConclusionYangyin Yiqi Mixture can effectively resist pulmonary fibrosis, which may be realized by inhibiting the expressions of TIMP-1 and MMP-9, regulating and promoting collagenic hydrolysis and restraining deposition of collagen.

Objective To compare the clinical efficacy of different sized incisions for phacoemulsification combined with vitrectomy in the treatment of age-related cataract associated with maculopathy. Methods From January 2016 to October 2016, a total of 60 eyes of 60 senile cataract patients with maculopathy were prospectively divided into the micro incision group and the standard incision group according the random number table,with 30 cases in each group.The micro incision group received a 1.8 mm micro incision for phacoemulsification combined with 23G vitrectomy and the standard incision group underwent a 2.8 mm incision for phacoemulsification combined with 23G vitrectomy.The intraoperative conditions and the postoperative complications of the two groups at 1 d, 1 and 6 months after surgery were compared. The intraocular pressure (IOP), best corrected visual acuity (BCVA), surgically induced astigmatism (SIA), dry eye symptoms (Sx), break-up time (tBUT), schirmer test (ST)-Ⅱwere respectively measured at 1 and 6 months after surgery.Results The operation procedures were similar in two groups,and there were no significant differences in the operative time and the incidence of intraoperative complications(P>0.05).There were no significant differences in IOP and the incidence of postoperative complications between two groups at 1 d, 1 and 6 months after surgery (P>0.05). At the time point of 1 month after operation,there was no significant difference in BCVA between two groups,but the level of SIA in micro incision group was lower:0.4 ± 0.2 vs.0.6 ± 0.3,t=3.038,P=0.004;the level of Sx micro incision group was lower: (1.2 ± 0.8)score vs. (1.8 ± 1.1) scores, t = 2.416, P = 0.019; the level of tBUT was higher:(5.2 ± 1.6)s vs.(3.8 ± 1.3)s, t = 3.454,P = 0.001;the level of ST-Ⅱwas higher: (4.7 ± 2.1) mm vs. (3.5 ± 1.8), t = 2.376, P = 0.021; there were significant differences. At 6 months after operation,there were no significant differences in the indexes of BCVA,SIA,Sx,tBUT and ST-Ⅱbetween two groups(P>0.05).Conclusions The different sized incisions for phacoemulsification combined with vitrectomy in the treatment of senile cataract associated with maculopathy are similarly safe and effective. However, the micro incision surgery can improve the surgically-induced astigmatism and dry eye in the early stage,with a shorter postoperative recovery time.

Objective To explore the influence of delivery mode and feeding pattern on cytomegalovirus (CMV) infection on infants born ≥ 32 gestational weeks,and to observe the outcomes after CMV infection.Methods In this retrospective study,378 pregnant women with positive CMV IgG and negative CMV IgM,and their offsprings (384 cases,including six pairs of twins),who got visited at five hospitals of our collaboration group during March 2013 and February 2016,were enrolled.Serum samples were retrieved from a previous study of these participants for CMV IgM and IgG detection with enzyme-linked immunosorbent assay.All participants were divided into exclusive artificial feeding (EAF) and breastfeeding groups (BF),and the latter included exclusive breastfeeding (EBF) and mixed feeding (MF).T or Chi-square or Fisher's exact tests were performed for statistical analysis.Results (1) Among the 378 pregnant women,there were 186 mothers and 190 infants (4 pairs of twins) in BF group,and the other 192 mothers and 194 infants (2 pairs of twins) in EAF group.The percentage of male infants were 54.7％(104/186) and 56.2％(109/194) in the BF and EAF group,respectively.The mean birth age was (38.9± 1.4) and (38.7± 1.7) weeks,and the age at followingup was (9.8± 2.2) and (10.5± 2.9) months,respectively.(2) The CMV IgG positive rate of infants in BF group was higher than in the EAF group [62.6％(119/190) vs 29.9％ (58/194),x2=41.403,P＜0.001].CMV IgG levels in infants were higher than the mothers [(537.1 ±249.5) vs (416.2±241.2) U/ml,t=4.609,P＜0.001].In infants with positive CMV IgG,the positive rates of CMV IgM were similar in the two groups [21.0％(25/119) vs 19.0％ (11/58),x2=0.101,P=0.751].(3) The positive rate of CMV IgG in vaginally born infants was higher than those born by caesarean section [55.2 (95/172) vs 38.7％ (82/212),x2=10.472,P=0.001].Further analysis in the EAF group showed that those infants born vaginally had a higher positive rate ofCMV IgG than those born by caesarean section [42.9％ (33/77) vs 21.4％ (25/117),~=10.231,P=0.001],while this figure did not show statistical difference in the BF group.(4) Infants with positive or negative CMV IgG were in similar age and gender proportion,as well as their height and weight.Among 36 infants with both positive CMV IgG and IgM,three failed in alanine aminotransferase (ALT) test due to hemolysis.However,among the other 33 cases,15.1％ (five cases) presented with lightly elevated ALT (42-107.2 U/L),which was similar to those infants with positive CMV IgG and negative CMV IgM (14/98,14.3％) and those with both negative CMV IgG and IgM (20/144,13.9％),(x2=0.036,P=0.982).Conclusions Although breastfeeding and vaginal birth may increase CMV infection rate in neonates and infants,but no obviously adverse prognosis was reported in those born over 32 gestational weeks.So we should encourage vaginal birth and breastfeeding in these population.

Breast milk is the best food for infants. However, worrying about the transmission of pathogens to their offspring, mothers who have ongoing infections are usually hesitated on breastfeeding, or even unwillingly give up breastfeeding. This article summarized some basic knowledge and evidence on breastfeeding when maternal infections occur and emphasized that there is no risk of hepatitis virus transmission during breastfeeding. After taking appropriate measures, mothers with transmittable diseases through breastfeeding can still breastfeed their babies. This review provides reasonable medical advice on whether to breastfeed when maternal infection occurs in order to increase breastfeeding rate.

Objective To investigate the effect of Genistein,a tyrosinase inhibitor,on retinal neovascularization in mice.Methods Thirty-six 7-day-old C57BL/6J mice were randomly assigned into hyperoxiainduced group,Genistein group,DMSO group and normal control group.The mice in the hyperoxia-induced group,Genistein group and DMSO group were fed in a static chamber with the oxygen volume fraction (75±2)％ for 5 days and then sent back to natural environment for 5 days to establish retinal neovascular models,and 1 μl Genistein diluted by 5％ dimethyl sulfoxide (DMSO) (400 mg/L) and 1 μl DMSO were intravitreally injected in the 12-dayold mice of Genistein group and DMSO group,respectively.The mice in the normal control group were bred in natural environment.The fluorescence angiography was carried out in 17-day-old mice (2 mice in each group) to prepare the whole retinal flatmounts,and the morphology of retinal vessels was observed under the fluorescence microscope.The other mice in various groups were sacrificed and the retinas were collected.The expressions of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) in mRNA and protein levels were detected by realtime fluorescence quantitative PCR and Western blot,respectively.The use and care of the mice complied with regulations for the management of laboratory animals.Results Retinal vessels were normal in the mice of normal control group.In the mice of the hyperoxia-induced group and DMSO group,retinal vessels were tortuous,and neovacularization and non-perfusion areas were visible.In the Genistein group,retinal vessels were clearly visible,but non-perfusion areas were exhibited.The relative expression levels of VEGF mRNA in retinas were 0.64±0.25,0.37±0.23,0.03±0.02 and 0.04±0.02,and the relative expression levels of bFGF mRNA in retinas were 21.40±3.07,17.22±2.63,0.52±0.25 and 0.67±0.23,in the hyperoxia-induced group,DMSO group,Genistein group and normal control group,and the expressions of VEGF and bFGF in mRNA level in the hyperoxia-induced group and DMSO group were significantly higher than those in the Genistein group and normal control group (all at P＜0.05).The protein expression levels of VEGF and bFGF were 1.01 ±0.05 and 0.97±0.06 in the hyperoxia-induced group,1.06±0.07 and 1.03±0.08 in the DMSO group,0.73±0.05 and 0.76±0.07 in the Genistein group,0.52±0.05 and 0.56± 0.05 in the normal control group.The expressions of VEGF and bFGF in protein level in the hyperoxia-induced group and DMSO group were significantly higher than those in the Genistein group and normal control group (all at P＜0.05).Conclusions Genistein can inhibit hyperoxia-induced retinal neovascularization may be by downregulating the expressions of VEGF and bFGF in retinas.

Objective To compare the diagnostic accuracies of deep convolutional neural network (CNN) and dermatologists for pigmented nevus and seborrheic keratosis.Methods CNN network ResNet-50 was trained with 5 094 dermoscopic images of pigmented nevus and seborrheic keratosis using transfer learning,so as to establish a CNN two-classification model.Then,this model was applied to the automatic classification of 30 dermoscopic images of pigmented nevus and 30 dermoscopic images of seborrheic keratosis.Meanwhile,in combination with clinical photos of skin lesions,95 experienced dermatologists who had received dermoscopy training gave their diagnosis for the above 60 dermoscopic images.The diagnostic accuracies were compared between the two methods,and misclassified images were further analyzed.Results The CNN automatic classification model had the diagnostic accuracies of 100％ (30/30)and 76.67％ (23/30) for pigmented nevus and seborrheic keratosis respectively,and the total accuracy was 88.33％ (53/60).The average diagnostic accuracies of 95 dermatologists were 82.98％ (25.8/30) and 85.96％ (24.9/30) for pigmented nevus and seborrheic keratosis respectively,and the total accuracy was 84.47％ (50.7/60).There were no significant differences in the diagnostic accuracies for pigmented nevus or seborrheic keratosis between the CNN automatic classification model and 95 dermatologists (x2 =0.38,P ＞ 0.05).The dermoscopic images misclassified by CNN were divided into 3 categories:special-type lesions with high pigment content and marked keratosis,typical skin lesions with interference factors,and typical skin lesions without definite reasons for misclassification.Conclusions The performance of CNN automatic classification model is similar to that of experienced dermatologists in the two classification of pigmented nevus and seborrheic keratosis.The reasons for misclassification by CNN still need to be explored by dermatologists and professionals in artificial intelligence.

Objective:To optimize the challenge scheme for establishing a stable mouse model of Artemisia annua pollen-induced allergic rhinitis. Methods:BALB/c mice were subcutaneously injected with 0.1 ml allergen extract containing 20 μg/ml Art a1 from Artemisia pollen on 1 d, 4 d and 7 d. One week after the sensitization, these mice were divided into three groups and intranasally challenged with Artemisia annua pollen allergen extract containing 500 μg/ml Art a1 for 7 (7 d group), 10 (10 d group) and 14 (14 d group) consecutive days, respectively. The first challenge was followed by another 7 days of challenge every four weeks. Blank control group was set up through sensitizing and challenging BALB/c mice with normal saline. Behavioral changes and nasal pathological changes were observed. The changes in humoral and cellular responses were also detected. After the first challenge cycle was decided, the challenge frequency was further optimized. Results:After the first challenge, the allergic symptoms of mice in 10 d group were significantly severe than those in 7 d and 14 d groups, and the levels of serum specific IgE antibody in 10 d and 14 d groups were significantly higher than that in 7 d group. After the second challenge, the mice in the three model groups still had obvious allergic symptoms as compared with the blank control group. There were obvious pathological changes in the nose, including epithelial cell proliferation, turbinate enlargement and inflammatory cell increase. Moreover, the level of serum specific IgE antibody increased significantly and the proliferation of antigen-specific IL-4 and IL-6 lymphocytes was significantly up-regulated, especially in 10 d and 14 d groups. The frequency of challenge had a great impact on the stability of the allergic model. The allergic symptoms of sensitized mice challenged every two weeks were significantly severe than those of mice challenged every four weeks and the level of serum antigen-specific antibody was also higher.Conclusions:This study optimized the first challenge cycle and challenge frequency for establishing a mouse model of Artemisia annua pollen-induced allergic rhinitis, which provided reference for the establishment of drug efficacy evaluation system for desensitization therapy.