Calorie restriction refers to a dietary regimen low in calories without malnutrition.During the recent 70 years,the benefit of calorie restriction regimen has been explored extensively.Work in widely diverse species,from model organism to rodents even human beings,has demonstrated that calorie restriction is an effective nutritional intervention for retarding aging and preventing chronic diseases.

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 investigate the relationship between the relevant platelet parameters and the thromboelastographic in-dexes with the the occurrence and development of diabetic vascular complications so as to early find the high-risk group of vascular complications or early patients .Methods According to the WHO diagnostic criteria for diabetes ,268 patients with diabetes and 80 retired people with the physical examination (normal control group) were selected as the research subjects .268 cases were divided into the group A(HbA1c <6 .2% without vascular complications) the group B(HbA1c >6 .5% without vascular complications) and the group C(HbA1c >8 .0% with vascular complications ) .The Siemens ADVIA2120 fully automatic blood analyzer was adopt-ed to detect the platelet parameters of mean platelet volume (MPV) ,platelet distribution width(PDW) ,large-platelet(L-PLT) , MPM and mean platelet concentration(MPC);The TEG@ 5000 coagulation analyzer was adopted to perform the thromboelastogra-phy for detecting the values of R ,K ,Angle A and MA .All obtained data were statistically analyzed .Results L-PLT in the group B and the platelet parameters in the group C had statistical differences compared with the normal control group (P<0 .05) .The val-ues of R ,K ,Angle A in the group B and the values of R ,K ,Angle A and Ma in the group C had statistical differences compared with the normal control group (P<0 .05) .Conclusion Relevant platelet parameters and the thrombelastogram detection have the important significance to help to the clinical diagnosis of diabetic angiopathy ,guide the treatment and the condition monitoring ;the thrombelastogram is more sensitive than the detection of the relevant platelet parameters in early finding the patients with diabetic angiopathy .

Objective To explore the mediating effects of resilience between family function and geriatric depression. Methods By convenient sampling, 212 elderly people who were from the communities in Yuexiu, Guangxhou city were analyzed by using Geriatric Depression Scale (GDS-30) and Family Concern Index Questionnaire (APGAR) and Connor-Davidson Resilience Scale (CD-RISC) and the self-designed General Condition Questionnaire, and a mediating model was proposed and the impacts of resilience on family function and geriatric depression were tested. Results The incidence rate of depression among the community elderly was 23.6% (50/212), and the average score of APGAR was 7.87 ± 2.83, and the average score of CD-RISC was 83.66 ± 12.88. The scores of GDS-30 showed significantly negative correlation with the scores of APGAR (r=-0.582,P<0.01)and the scores of CD-RISC (r=-0.425, P<0.01), and there was positive correlation between the scores of APGAR and the scores of CD-RISC (r=0.335, P < 0.01). The mediating model had high degree of fitting, and level of path had statistical significance (P < 0.01). Resilience was the mediator between family function and geriatric depression. Family function had direct effects (0.50) on geriatric depression and indirect effects (0.11) through resilience, and the combined effects of family function and resilience determined 45%changing range of geriatric depression. Conclusions Family function was a direct predictor of geriatric depression, and resilience had mediating effects between family function and geriatric depression. Community mental health work should pay enough attention to the effects of family function on the life of the aged, and increased the care to the elderly with family dysfunction, while promote and develop family-centered mental health education, propaganda and psychological guidance, so that improves resilience of the aged and help the aged positively coping and maintain good mental health status.

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.

Objective To investigate the endoscopic prevalence of erosive esophagitis (EE) among 13 hospitals in Guangdong province of China. Methods Retrospectively reviewed all the cases (63459 cases) that received oesophagogastrodeuodenoscopy in 13 main hospitals in Guangdong province of China in 2003. Los Angeles criteria for classification of erosive esophagitis were employed as the basis of analysis. Results One thousand two hundreds and sixty-three patients (age range 3-90yr, mean 50. 2 ?17. 1 ) were found to have EE. The overall prevalence of EE was 1. 99% (1263/63459). The prevalence of EE in A, B, C, and D grade were 0. 94% , 0. 69% , 0. 21% and 0. 14% respectively. Age correlated positively on endoscopic grading of EE (F=22. 932, P

Objective To investigate the effect of KRAS gene mutation and clinical factors on postopera-tive prognosis of rectal cancer patients and to explore their value in prognosis. Methods A total of 130 cases of rectal cancer patients from January to December 2010 were collected in the study. The tumor tissues sample was used to detect the KRAS gene mutation and 5-year follow-up was conducted. The correlation between KRAS gene mutation and clinical pathological features was analyzed.The clinic pathological factors that may affect the progno-sis were analyzed by survival analysis. Results Forty-five patients had mutations in No.2 expressed region of KRAS,with a mutation rate of 34.6%.KRAS gene mutation and stronger positive expression of EGFR(P<0.05), and multiple metastasis of tumor(P<0.05)were strongly coupled.The average survival of patients with wild-type KRAS gene was 57.5 months and that of patients with KRAS gene mutation 58.9 months but no significant differ-ence was observed(P>0.05).The TNM by high staging,multiple metastasis,lung metastasis and liver metasta-sis of cancer cells was closely related with poor postoperative prognosis of patients(P<0.05).The average surviv-al of postoperative patients in stage Ⅳ was 49months. Conclusions KRAS gene mutation in patients with rectal cancer after surgery is related with stronger positive expression of EGFR and multiple metastasis of cancer.TNM by high staging and metastatic sites affects the prognosis. The survival of rectal cancer after surgery in patients with stage Ⅳ are prolonged but the relation between KRAS genovariation and patients′ postoperative prognosis can not be determined.

ObjectiveTo investigate the effect of HBsAg on the expression of interferon-α (IFN-α) in peripheral blood plasmacytoid dendritic cells (pDCs) induced by the stimulator of interferon genes (STING) signaling pathway activated by cyclic GMP-AMP (cGAMP). MethodPeripheral venous blood was collected from healthy adults and the patients with chronic hepatitis B virus (HBV) infection who attended the outpatient service or were hospitalized in Department of Infectious Diseases, The Affiliated Hospital of Xuzhou Medical University, from February to December 2016, and peripheral blood mononuclear cells (PBMCs) were isolated and extracted. After the STING agonist cGAMP was added to PBMCs, ELISA was used to measure the levels of IFN-α, interferon-β, and tumor necrosis factor-α in supernatant. PBMCs from healthy adults were pre-incubated with HBsAg and then stimulated by cGAMP, and supernatant was collected to measure IFN-α. The magnetic-activated cell sorting method was used to remove pDCs from PBMCs, and after culture with cGAMP, ELISA was used to measure the level of IFN-α in supernatant. PBMCs from healthy adults were stimulated by HBsAg and/or cGAMP, and then flow cytometry was used to measure the frequency of pDCs. The independent samples t-test was used for comparison of continuous data between two groups. ResultsPBMCs from the patients with chronic HBV infection stimulated by cGAMP in vitro had a significantly lower level of IFN-α than healthy controls (469.72±18.95 vs 599.90±84.06, t=4.868, P=0.001). PBMCs from healthy adults co-cultured with HBsAg and stimulated by cGAMP had a significantly lower level of IFN-α than those in the non-HBsAg group (448.5±52.0 vs 571.0±30.8, t=4.500, P=0.011). Compared with PBMCs containing pDCs, PBMCs without pDCs stimulated by cGAMP had a significant reduction in the level of IFN-α (164.50±40.73 vs 339.50±35.33, t=6.482, P=0.001). Compared with PBMCs from healthy adults stimulated by cGAMP, PBMCs pre-incubated with HBsAg and then stimulated by cGAMP had a significant reduction in the frequency of pDCs (0.12%±0.04% vs 0.24%±0.04%, t=5.176, P=0.014). ConclusionHBsAg can inhibit the expression of IFN-α induced by the STING pathway in pDCs activated by cGAMP.