BACKGROUND: Establishment of drug (neomycin or hygromycin) resistant feeder layer cells is necessary for screening the target gene positive clone of embryonic stem cell (ESC) transfected in vitro, and the establishment of drug-resistant NIH3T3 cell line is also important for the screening of other target ESC genes.OBJECTIVE: To obtain a feeder layer of positive cell clone of ESC transfected by pTet-on gene by means of the G418-resistant NIH3T3 cell line establishment.DESIGN: Cell culture and DNA examination.SETTING: Faculty of Laboratory Animal, China Medical University.MATERIALS: NIH3T3 cells were contributed by the Cell Biology Staff Room of China Medical University, and pWL/neo plasmid was a gift from Professor Jin Zhuang of Harvard University Medical College. G418,leukemia inhibitory factor (LIF, ESC GRO 106 U/mL) and DMEM were produced by GIBCO BRL Company, while mitomycin-C (MIT-C), DIG marking and kits were the products of Roche Company. Lipoetin was the product of Invitrogen Company, and bought from Shenyang Lianxing Biotechnology Co.,Ltd.METHODS: ①The pWL/neo plasmid containing neo gene was purified and transfected into NIH3T3 cells by lipoetin.②The transfected cells were further cultured in 25 mL medium containing G418 antibiotic of gradient concentration to observe the survival and apoptosis of cells and measure G418 minimal fatal dose (MFD) to NIH3T3 cells.③The transfected cells were subcultured, and the clone of single cell was selected to 24-pore culture board for screening amplification. At the same time, normal NIH3T3 cells were also selected and added with the selective culture medium at the same dose of G418, as screening negative control. ④The cells were planked with lower cell density, and further screened in DMEM medium containing G418 MFD; Meanwhile, normal NIH3T3 cells were taken as controls, and cultured by G418 contain MFD and by DMEM medium without G418 respectively. Light microscope was used to observe G418R NIH3T3 cells. ⑤G418R NIH3T3 cells and MEF were all applied as the feeder layer to culture the ES-D3 cells, which were observed by light microscope. Cell DNA was prepared, and evaluated by PCR and southern blot.MAIN OUTCOME MEASURES: ①G418 MFD to NIH3T3 cells. ②screening result of G418R NIH3T3 cells. ③growth of G418R NIH3T3 cells. ④growth of ES-D3 cells on G418R NIH3T3 cells.RESULTS: ①G418 MFD to NIH3T3 cells was defined as 500 mg/L.②Under the condition of G418 (500 mg/L), G418R NIH3T3 cell clones were successfully selected. ③The G418R NIH3T3 cells had no difference from normal NIH3T3 cells in morphology and propagation rate.④The ESC cultured in feeder layer present a colony growth, smooth limitation and remained an undifferentiation state. The resistant cell genomic DNA of G418R NIH3T3 cells was assayed with specific neo gene primer, and then neo gene DNA fragment was amplified; Southern blot analysis showed that neo gene fragment integrated into G418R NIH3T3 cells.CONCLUSION: The G418R NIH3T3 cells are established successfully,and the ESC cultured in the G418R NIH3T3 cell feeder layer can keep the characteristics of normal ESC.

Objective To explore the mediating effect and moderating effect of positive mental characters in the relationship between childhood abuse and depression among recruits.Methods 1925 recruits aged from 16 ～24 were investigated using the Childhood Trauma Questionnaire-28 item Short Form (CTQ-SF),Self-Rating Depression Scale (SDS) and Positive Mental Characters Scale for Recruits (PMCS-R) through random cluster sampling.Results The scores of emotional abuse,physical abuse,sex abuse,emotional neglect,physical neglect,depression,positive mental characters were 6.11±1.69,5.49±1.32,5.61±1.45,9.15±3.66,9.57±2.93,0.45±0.11,3.80±0.64,respectively.There were correlations among childhood abuse,depression and positive mental characters (P＜0.05).Emotional neglect,physical neglect and emotional abuse could explain 28.6％ of the total variance of depression (F=256.72,P＜0.05).The positive mental characters partially mediated the relationship between emotional neglect,physical neglect,emotional abuse and depression (mediating effect were 0.19,0.15 and 0.09,respectively),and it could only moderate the relationship between emotional neglect and depression (F=24.73,P＜0.05).Conclusion Childhood abuse not only directly but also indirectly affects depression through the positive mental characters;meanwhile the positive mental characters can change the relationship between childhood abuse and depression.

Objective To investigate the effects of interleukin-10 on mice intestinal fibrosis and epithelial-mesenchymal transition(EMT),and the relation between these effects and endoplasmic reticulum stress(ERS).Methods Forty-two IL-10 knockout(IL-10-/-)mice were divided into fibrosis model group(n=18),IL-10 treatment group(n=12)and solvent control group(n =12),another 18 wild-type mice were taken as negative control group.IL-10 and 0.9％ NaCl were intraperitonealy injected in IL-10 treatment group and solvent control group respectively since 12th week,and mice were sacrificed at week 14th and 16th,and no treatment to fibrosis model group and negative control group.The injury and fibrosis in mice colon tissue were detected with HE staining and Masson collagen staining.The expressions of collagen Ⅰ,glucose-regulated protein78(GRP78),C/EBP homologous protein(CHOP)and a-smooth muscle actin(α-SMA)and E-cadherin(E-cad)at mRNA level were determined by realtime PCR.The expression of α-SMA and E-cad in mice colon tissue was examined by immunohistochemical staining.Results At 16th week,the colonic tissue injury scores(7.00±0.90,7.17±1.17)and collagen area ratio(17.78％±4.15％,18.56％±3.81％)of fibrosis model group and solvent control group significantly increased compared with negative control group(1.50±1.38 and 9.11％±2.99％)and IL-10 treatment qroup(4.33±0.82 and 12.56％±1.39％)(F=36.150,F=11.280； P=0.000).At week 12th,14th and 16th,the expressions of GRP78,α-SMA,collagen Ⅰ in fibrosis model group and solvent control group significantly increased compared with negative control group(all P＜0.05),however the expression of E-cad significantly decreased(P＜0.05).The expression of CHOP mRNA in fibrosis model group(0.95％ ±0.12％)significantly increased compared with negative control group(0.21％ ± 0.12％)at week 12th(t=5.188,P=0.000),however there was no statistical significant difference in groups at week 14th and 16th(P＞0.05).At week 14th and 16th,the expressions of GRP78,α-SMA and collagen Ⅰ(at week 14th:0.73％±0.31％,1.18％±0.11％ and 1.10％±0.49％； at week 16th:0.57％±0.16％,0.81％ ±0.50 ％ and 0.76 ％ ± 0.25 ％)in IL-10 treatment group were significantly lower than that of fibrosis model group(P＜0.05).The expression of E-cad(at week 14th:0.73％ ±0.29％ ； at week 16th:0.97％ ±0.25％)significantly increased compared with fibrosis model group(at week 14th:0.37％±0.17％； at week 16th:0.35％±0.20％)(F=6.524,P=0.003； F=17.493,P=0.000).However at week 16th,the expression of α-SMA in IL-10 treatment group was lower than that of solvent control group(1.82±0.22)％(F=9.842,P=0.000),and the expression of E-cad significantly increased than in solvent control group(0.47 ± 0.25)％(F=17.493,P =0.000).Conclusion IL-10 may have a role in inhibiting EMT and reducing intestinal fibrosis in mice,which may be related to the regulation of ERS by IL-10.

Objective To investigate the effects of transient receptor potential cation channel 6 (TRPC6) on the expression of nephrin, desmin and caspase 9 and on the apoptosis of podocytes in a mouse model of podocyte injury induced by TGF-β1.Methods Conditionally immortalized mouse podocytes were cultured in vitro and divided into four groups: control, TGF-β1 treatment, TGF-β1+PGPU6/GFP/Neo-TRPC6-mus-581 (TRPC6 knockdown) and TGF-β1+PGPU6/GFP/Neo-NC (negative control).Real-time RT-PCR and Western blot analysis were performed to detect the expression of nephrin, desmin and caspase 9 at mRNA and protein levels, respectively.Flow cytometry was used to analyze the apoptotic rate of podocytes.DAPI fluorescent staining was used to observe the morphological changes of apoptotic podocytes.Results Green fluorescent protein (GFP)-expressing podocytes at 48 hours after transfection were significantly more than those at 24 hours after transfection.The level of TRPC6 in mouse podocytes transfected with PGPU6/GFP/Neo-TRPC6-mus-581 was significantly decreased as compared with that of the control group (P<0.05).No significant difference in the expression of TRPC6 was observed between the negative control group and the control group.Compared with the control group, the TGF-β1 treatment group showed increased expression of desmin and caspase 9 at both mRNA and protein levels (P<0.01), but decreased expression of nephrin at mRNA and protein levels at 48 hours after TGF-β1 intervention (P<0.05).The up-regulated desmin and caspase 9 and the down-regulated nephrin induced by TGF-β1 could be inhibited by the means of TRPC6 knockdown.The apoptosis rate of podocytes in TGF-β1 treatment group was (14.0±2.1)%, while that in TRPC6 knockdown was (10.90±0.56)% (P<0.05).The apoptosis rate of podocytes in negative control group was higher than that in TGF-β1 treatment group (P>0.05).More apoptotic cells with typical morphological features of apoptosis were observed after exposure to TGF-β1 for 48 hours.Conclusion TGF-β1 could induce the apoptosis of podocytes, inhibit the expression of nephrin and enhance the expression of caspase 9 and desmin, the possible mechanisms of which may be related to TRPC6 signal pathway.These changes in TGF-β1-treated podocytes could be alleviated by inhibiting the expression of TRPC6, which might have a protective effect on podocyte injury.

Objective To construct a 5-lipoxygenase (5-LO) transgenic mouse model of atherosclerosis.Methods Purified 5-LO fragment was injected into male pronucli and the firtilized eggs were transplanted into pseudopregnant mice.PCR and Southern blot were used to detect the genotype of DNA separated from the newborn mouse tail tissues.RT-PCR and Western blot analysis were used to detect the gene transcription and expression.Results PCR and Southern blot results showed that 7 of 25 mice were transgenic mice.Expression of 5-LO and FLAP was found in the bone marrow,spleen,kidney,and peritoneal cells.Results of RT-PCR and Western blot showed that No.9,20,24transgenic mice expressed a higher level of 5-LO and FLAP than those in the wild type C57BL/6 mice.The expression levels in bone marrow and peritoneal cells were significantly different(P<0.05).Conclusion A 5-LO transgenie mouse line has been established in this study and may be used for future study on the function of 5-LO gene.

OBJECTIVETo study the immune responses of lymphocytes after activated by dendritic cells (DCs) loaded with cytotoxicity T lymphocyte (CTL) epitope based peptide of human alpha-fetoprotein (hAFP, 218-226 LLNQHACAV).

METHODSGet high purity DCs by cultured plastic-adherent monocytes isolated from healthy donor of HLA-A2(+) peripheral blood with granulocyte-monocyte colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4) for 7 days. Stimulate self-lymphocytes with DCs that loaded with CTL epitope based peptide of hAFP under the culture medium contains interleukin-2 (IL-2) for 7 days. Analyse IL-12 and TNF in culture medium and also the specific lysis activity of lymphocytes against four strains of primary hepatocellular carcinoma cells.

RESULTSAfter stimulated by DC loaded with CTL epitope based peptide derived from hAFP, lymphocytes appeared a good characteristics and the culture medium of activated lymphocytes contained a high level Th1 type cytokines of IL-12 and TNF. Activated lymphocytes not only specifically lysed HLA-A2(+) HepG2 line but also had the cytotoxicity against other three primary hepatocellular carcinoma cell lines and T2 target cell loaded with peptide of hAFP.

CONCLUSIONSThe results of this research supply the basic materials for the DC based vaccine with HLA-A2 restricted peptide epitope derived from hAFP against AFP positive primary hepatocellular carcinoma.

Adult ; Animals ; Dendritic Cells ; immunology ; Epitopes ; HLA-A2 Antigen ; immunology ; Humans ; K562 Cells ; Mice ; Peptides ; immunology ; T-Lymphocytes, Cytotoxic ; immunology ; Tumor Cells, Cultured ; alpha-Fetoproteins ; immunology

Objective:To investigate the status and influencing factors of enteral nutrition support in late preterm infants (34-36 +6 gestational weeks) treated in different grades of hospitals in Beijing. Methods:This was a prospective study involving late preterm infants treated in 25 hospitals in Beijing from October 2015 to October 2017. Data about nutritional management and nutrition-related complications were recorded. Exclusive breastfeeding status of the infants by gestational age(GA) and hospital levels was analyzed. The achievement of full enteral feeding and the potential influencing factors were also analyzed. t-test, Mann-Whitney U test, analysis of variance (ANOVA), Kruskal-Wallis test and Chi-square test were used for statistical analysis. Logistic regression and Cox regression analysis were used in multivariate analysis. Results:(1) A total of 1 463 late preterm infants with GA of 35.6±0.8 (ranging from 34.9 to 36.1) weeks was enrolled in this study. Compared with the infants with GA of 35-35 +6 and 36-36 +6 weeks, those born at 34-34 +6 gestational weeks had longer hospital stay [10 (8-13) vs 8 (7-10) and 7 (6-9) d, both P<0.05], greater loss of minimum weight [4.3% (2.6%-6.3%) vs 3.8% (2.0%-5.6%) and 3.3% (1.9%-5.5%), both P<0.05], higher incidence of apnea [5.3% (20/369) vs 2.1% (12/566) and 1.3% (7/528), both P<0.05] and respiratory distress syndrome (RDS) [7.1% (28/369) vs 3.0% (17/566) and 3.2% (17/528), both P<0.05], and lower percentage of failure to regain birth weight at discharge [32.5% (120/369) vs 38.7% (219/566) and 47.9% (253/528), both P<0.05]. Only the incidence of premature rupture of membranes among all maternal complications during pregnancy had statistical difference between 34-34 +6, 35-35 +6 and 36-36 +6 GA groups [6.2% (23/369) vs 12.7% (72/566) and 11.9% (63/528), χ2=10.244, P=0.007]. (2) The rate of enteral feeding increment in hospital was 13.7 (10.5-17.3) ml/(kg·d) and 46.0% (673/1 463) of the infants were fed formula. The exclusive breastfeeding rate increased from 4.5% (66/1 463) during hospitalization to 14.4% (211/1 463) at discharge. The breastfeeding rate at discharge varied widely among the 25 hospitals ( χ2=327.893, P<0.001) ranging from 32% to 0. (3) Logistic regression analysis demonstrated that gestational diabetes mellitus ( OR=2.426, 95% CI: 1.075-5.437, P=0.033) and premature rupture of membranes ( OR=8.726, 95% CI: 1.193-63.802, P=0.033) were the prenatal risk factors influencing the exclusive breastfeeding in late preterm infants. Enteral nutrition achieving 150 ml/(kg·d) and 120 kcal/(kg·d) (1 kcal=4.184 kJ) were noted for 28.4% (416/1 463) and 19.2% (281/1 463) of the late preterm infants at discharge, respectively. Cox regression analysis showed that hospital grades ( HR=1.470, 95% CI: 1.030-2.098, P=0.034), the length of hospital stay ( HR=1.162, 95% CI: 1.097-1.231, P<0.001), birth weight ( HR=0.946, 95% CI: 0.898-0.995, P=0.003), exclusive breastfeeding ( HR=2.354, 95% CI:1.031-5.374, P=0.042), feeding intolerance ( HR=3.677, 95% CI: 1.201-11.253, P=0.023), parenteral nutrition ( HR=1.900, 95% CI: 1.379-2.616, P<0.001), and the rate of enteral feeding advancement ( HR=1.426, 95% CI: 1.369-1.484, P<0.001) were independent factors associated with full enteral feeding at discharge. Conclusions:Exclusive breastfeeding rate in late preterm infants is low and enteral nutrition support varies greatly in different hospitals. The rate of enteral feeding increment is slow for hospitalized late preterm infants and most fail to achieve full enteral feeding at discharge. Gestational diabetes mellitus and premature rupture of membranes are prenatal risk factors affecting breastfeeding of late preterm infants. Those with low birth weight, exclusive breastfeeding in hospital, feeding intolerance, parenteral nutrition support, longer hospital stay or rapid enteral feeding advancement are more likely to achieve full enteral feeding at discharge.

Objective:To investigate the value of N-terminal pro-brain natriuretic peptide (NT-proBNP) combined with bedside echocardiography in diagnosis and treatment of neonatal sepsis with cardiac dysfunction.Methods:A total of 56 children diagnosed with neonatal sepsis in the Neonatal Intensive Care Unit, Liaocheng People′s Hospital from July 2016 to July 2017 were enrolled and divided into 2 groups, namely, the cardiac dysfunction group (26 cases) and the non-cardiac dysfunction group (30 cases). Children with general infection (45 cases) hospita-lized at the same period were taken as the control group.The clinical characteristics, related laboratory indexes and prognosis were compared among 3 groups.The related factors of neonatal sepsis with cardiac dysfunction were analyzed by the multivariate Logistic regression approach, and the value of related indexes in the early prediction neonatal sepsis with cardiac dysfunction was analyzed by using the receiver operating characteristic curve (ROC). Results:The onset age of sepsis patients with cardiac dysfunction [63.0 h (30.5 h, 185.6 h)] was significantly earlier than that of the patients without cardiac dysfunction [65.0 h (34.5 h, 170.6 h)] and the control group [80.0 h (45.5 h, 202.3 h)] ( P<0.05). The main primary site of the disease was the lung, which was not statistically significant among the 3 groups ( P>0.05). The NT-proBNP level and the high sensitivity-C-reactive protein (hs-CRP)/albumin (ALB) ratio in the cardiac dysfunction group [20 230.6 ng/L (15 890.0 ng/L, 35 000.0 ng/L); 0.33(0.29, 0.81)] were significantly higher than those in the control group [7 324.5 ng/L (2 426.5ng/L, 13 890.0 ng/L); 0.06(0, 0.21)] (all P<0.05). The right ventricular diameter and the Tei index of the cardiac dysfunction group [(8.74±2.42) mm; 0.52±0.03] were significantly higher than those in the control group [(8.55±1.41)mm; 0.30±0.04], while the EF of the cardiac dysfunction group [(62.61±2.56)%] was significantly lower than that in the control group [(70.03±0.35)%] (all P<0.05). The ROC curve analysis showed that NT-proBNP and the Tei index could effectively predict sepsis with cardiac dysfunction.Specifically, NT-proBNP had a cutoff value of 12 291.5 ng/L, with sensitivity of 80%, specificity of 79%, and the area under ROC curve (AUC) of 0.81.The Tei index had a cutoff value of 0.45, with sensitivity of 74%, specificity of 77%, and the AUC of 0.78. Conclusions:NT-proBNP can be used as a marker of early cardiac dysfunction.Its combination with the Tei index of bedside echocardiography can quickly diagnose cardiac dysfunction of children with sepsis, better guide clinicians in drug use, improve cardiac function of patients and enhance the treatment effect.

Objective To measure and compare the cerebral blood flow(CBF)of children with autism spectrum disorder(ASD),global developmental delay(GDD),and ASD with GDD groups via arterial spin labeling(ASL)technique,and to evaluate the diag-nostic value of CBF values.Methods ASL images of ASD,GDD,and ASD with GDD groups of children were firstly acquired,and the CBF values of frontal lobe,temporal lobe,parietal lobe,occipital lobe,striatum and thalamus region of interest(ROI)were fur-ther measured,respectively.One-way analysis of variance or Kruskal-Wallis H test was used to compare the differences in CBF values among these three groups,and the receiver operating characteristic(ROC)curve was used to analyze the efficacy of CBF values in distinguishing ASD with GDD from without GDD.Results ASD with GDD had significantly lower CBF values in the left and right frontal lobes than those with ASD or GDD alone,and the differences were statistically significant(P<0.05).The CBF values in the left and right frontal lobes effectively distinguished ASD with GDD from without GDD[area under the curve(AUC)>0.7].Conclusion ASL technique can noninvasively assess CBF in children with or without GDD,helping to understand the pathophysiology of ASD with GDD and improving diagnostic accuracy.

ObjectiveTo explore the effect of Suanzaoren Tang combined with Ziwu Liuzhu acupuncture on the vertebral artery hemodynamics， inflammatory cytokines， and neurotrophic factors in the patients with cervical insomnia with syndrome of deficiency of both heart and spleen. MethodThe random number table method was employed to assign 164 patients with cervical insomnia with syndrome of deficiency of both heart and spleen treated in the First Clinical Medical School of Guangzhou University of Chinese Medicine from January 2018 to June 2021 into a control group and an observation group. The control group was orally administrated with 1-2 mg estazolam tablets before bed for 4 weeks， and the observation group with Suanzaoren Tang combined with Ziwu Liuzhu acupuncture for 4 weeks. The therapeutic efficacy and safety were observed. The Pittsburgh Sleep Quality Index （PSQI） score， polysomnography monitoring results， hemodynamics parameters of vertebral artery， and serum levels of inflammatory cytokines and neurotrophic factors were compared before and after treatment. ResultExcept 4 dropouts， the remaining 160 patients were included in this study， with 80 patients in each group. The observation group had higher total effective rate than the control group ［92.50% （74/80） vs. 80.00% （64/80）， χ²=5.270， P<0.05］. Compared with that before treatment， the therapies in both groups decreased the PSQI score， sleep latency time， awakening time， awakening times， serum levels of interleukin-1β （IL-1β）， C-reactive protein （CRP）， and tumor necrosis factor-α （TNF-α） （P<0.01）. Meanwhile， they increased the proportion of rapid-eye-movement （REM） sleep， the diastolic blood flow velocity （Vd）， systolic blood flow velocity （Vs）， and mean blood flow velocity （MFV） of vertebral artery， as well as the serum levels of brain-derived neurotrophic factor （BDNF） and glial cell-derived neurotrophic factor （GDNF） （P<0.05， P<0.01）. Moreover， the observation group had lower PSQI score， sleep latency time， awakening time， awakening times， and serum IL-1β， CRP， and TNF-α levels （P<0.01） and higher proportion of REM sleep， Vd， Vs， MFV of vertebral artery， and serum BDNF and GDNF levels （P<0.05， P<0.01） than the control group. ConclusionZiwu Liuzhu acupuncture combined with Suanzaoren Tang can improve blood circulation of vertebral artery， reduce the serum levels of inflammatory cytokine， and increase the serum levels of neurotrophic factors to improve the sleep quality of the patients with cervical insomnia with syndrome of deficiency of both heart and spleen.