Objective To investigate the effect of hydrogen-rich saline on apoptosis in hippocampal neurons induced by cerebral ischemia-reperfusion in rats and PI3K/Akt/FoxO1 signaling pathway.Methods The rat model of focal cerebral ischemia-reperfusion was established by thread-occlusion of the middle cerebral artery in rats.SD rats were randomly divided into sham operation group (Sham group), ischemia-reperfusion group (I/R group) and hydrogen-rich saline treatment group (HRS group), 10 rats in each group.At 24 h after reperfusion, the serum levels of IL-6, TNF-a and IL-1β were detected by ELISA.Histological changes of the hippocampus were observed by pathology using HE staining.Apoptosis in brain tissues was observed by TUNEL staining.The expression changes of p-PI3K, Akt, caspase-3 and FoxO1 proteins were detected by Western blot assay.Results Compared with the sham group, pyramidal cells were arranged loosely in the I/R group and a large number of pyramidal cells were necrotized, and the amount of apoptotic hippocampal cells was increased.The levels of IL-1β, IL-6 and TNF-α were significantly increased (P< 0.05), as well as the expression of p-PI3K, Akt and caspase-3 in the brain tissue.However, the expression of FoxO1 protein was decreased.There were significant differences between the two groups (P< 0.05).Compared with the I/R group, the inflammatory factors were significantly decreased in the HRS group.The expressions of p-PI3K, Akt and caspase-3 were also significantly decreased, while the expression of FoxO1 protein was increased (P< 0.05).Conclusions Hydrogen-rich saline can reduce brain injury caused by ischemia-reperfusion, and its mechanism may be related to PI3K/Akt/FoxO1 signaling pathway.

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 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 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.