Objective To express the mouse peroxisome proliferator activated receptor ? 2 (mPPAR? 2) in NIH3T3 cells induced by the recombinant retrovirus vector. Methods mPPAR? 2 gene digested from the recombinant plasmid pcDNA3/mPPAR? 2 containing the target gene segment was subcloned into retrovirus vector pGCEN to generate the recombinant retrovirus pGCEN/mPPAR? 2. The recombinant retrovirus pGCEN/mPPAR? 2 and pGCEN were used to transfect PA317 cells with LipofectAMINE, and anti G418 clones of PA317 cells were selected and viral supernatants were harvested and used to infect NIH3T3 cells. The expressing products were identified with immune flurescence assay (IFA) and Western Blot. Results The recombinant retrovirus pGCEN/mPPAR? 2 was constructed, and 5?10 4 CFU/ml and 6?10 5 CFU/ml of pGCEN/mPPAR? 2 containing and pGCEN containing viral supernatants were obtained respectively. mPPAR? 2 was expressed in NIH3T3 cells mediated by the recombinant retrovirus vector. Conclusion This work is the basis for the foundation of adipocyte differentiation model in vitro and further researching on the molecular mechanism of adipocyte differentiation induced by PPAR? 2.

Pem gene is one of the homeobox gene. Unlike members of Hox gene family, Pem gene is unique located at the proximal end of the X chromosome and its expression has been detected in several immortalized and transformed cells, placenta, embryos and reproductive tissues. Numeral studies showed that its expression is controlled by hormone such as androgen. This review discussed the possible role of Pem in regulating genes involved in the differentiation and development of extraembryonic tissue, spermatogenesis and sperm maturation.

Objective To conclude the practical nursing experiences of the infantile actue necrotizing fasciitis treated with vacuum sealing drainage(VSD) and continuous irrigation.Methods 21 cases of the infantile actue necrotizing fasciitis treated with with VSD and continuous irrigation were reviewed from January 2009 to December 2014.The nursing experiences of observation for the state of the illness,the safety management of VSD,the management of medication,mental nursing and health education were summarized.Results 21 infants all recovered and were discharged.Postoperative follow up 6 months later were conducted.No obvious disabilities of the extremities were found.Among them,8 cases were treated with VSD and continuous irrigation for 2 weeks and then had routine wound dressing changing for 10 days,the wound almost healed when discharged.13 cases were treated with VSD and continuous irrigation for one week and then had routine dressing changing for 12 to 13 days.The wound healed when discharged.No skin necrotizing,no scar of the incision and no other complications were detected.Conclusions Using VSD and continuous irrigation to treat the actue necrotizing fasciitis in infants are of great significance for the nurses to reduce the postoperative complication and promote rehabilitation doing as follows:turning over with the postoperative nursing of VSD in a standard manner,observation and management of the incision area,maintenance of the irrigation and VSD;observation and assessment of the infant's condition,management of the medication,mental nursing and health education.

Objective To investigate the effect of Buyanghuanwu decoction(BYHWD) on the expression of growth-associated protein 43(GAP-43) and synaptophysin(SYP) after focal cerebral ischemia in mice.Methods Focal cerebral ischemia was induced by 30 min of middle cerebral artery occlusion followed by reperfusion.BYHWD (20 g/kg) was administered orally 24 h after ischemia and once a day.The neurological score and the corner test were used to evaluate sensorimotor function on 1,3,7,and 14 days after ischemia.The expression of GAP-43 and SYP in the ischemic boundary zone was determined by immunohistochemistry 14 days after ischemia.Results Compared with the model group,BYHWD significantly ameliorated neurological dysfunction at 3,7 and 14 days and reduced the number of right turn at 7 and 14 days after ischemia(P ＜ 0.05).The average optical density of GAP-43 and SYP was 0.217 ±0.012 and 0.278 ±0.019 at the boundary zone in model group 14 days after ischemia,respectively.BYHWD also significantly increased the immunoreactivity of GAP-43 (0.250 ± 0.013)and SYP(0.323 ± 0.017) (P＜ 0.01).Conclusion BYHWD promotes axonal regeneration and synaptic plasticity in the ischemic boundary zone,which may contribute to functional recovery after focal cerebral ischemia.

AIM: To study the effect of Liuwei Dihuang Wan (LWDHW) on the expression of immune-associated genes in the aged rats using cDNA microarray. METHODS: Forty 20-months-old rats were divided into two groups equally. One group was treated with LWDHW for 5 weeks,another was untreated as control. Some biochemical assays were used to confirm the physiological differences between two groups. The mRNA from the spleens of twenty 4-months-old young rats were extracted and divided equally into two parts. Each part was conjoined with mRNA from drug treated aged rats and that from untreated control aged rats,respectively,and two experimental combinations for reverse transcription and cDNA microarray were formed. By comparing two groups of data from two combinations,the effects of LWDHW on the expression of the associated genes were evaluated. RESULTS: 13 down-regulated genes and one up-regulated gene were identified in the untreated control old rats,whose expression did not change obviously in treated old rats compared with young rats. The expressions of another two sequence were up-regulated distinctly in treated old rats compared with young rats. CONCLUSIONS: 16 immune-associated genes expressions were affected markedly by LWDHW. It will be helpful for understanding the molecular mechanism of LWDHW in ageing.

BACKGROUND: Bone marrow mesenchymal stem cells (BMSCs) can grow in host myocardium, differentiate under myocardial condition, improve cardiac function. However, biological characteristics of BMSC differentiation are still unclear presently.OBJECTIVE: To study the expression and electrophysiological characteristics of BMSC/n vitro connexin-43 following 5-azacitidine (5-aza) treatment.DESIGN, TIME AND SETTING: The cytological in vitro controlled study was perormed at the Heart Center, Hebei Provincial People's Hospital from July 2007 to February 2009.MATERIALS: A total of 24 male pigs aged 2 months were purchased from Exparimental Animal Center, Hebei Medical University.METHODS: Bilateral femoral bone marrow was obtained from pigs under sterile condition. BMSCs were harvested by Percoll density gradient in vitro. At passage 2, BMSCs were treated with 10 μmol/L 5-aza, and incubated in DMeM without inductor 24 hours later. Indices were measured I, 2, 3 weeks following induction. A control group was set up, which was not treated with 5-azacitidine. Following bone marrow extraction, experimental pigs were anesthetized to obtain ventricular muscle. Normal ventricular muscle cells were isolated and cultured by tissue block enzyme digestion method.MAIN OUTCOME MEASURES: Expression of connexin-43 was measured by immunohistochemical staining (ABC method). Ito current density and action potential were determined by patch clamp technique.RESULTS: At 1 and 2 weeks following 5-aza induction, some BMSCs were positive for connexin-43, with the presence of brown particles surrounding nuclei. At 3 weeks, positive rate of connexin-43 was 95%. The area with large cell density was presented with similar structure to normal myocardium. At +80 mV, compared with normal myocardial cells, Ito current density was significantly reduced in BMSCs following 1 and 2 weeks and in the control group (P < 0.05). Ito current density was significantly increased to a normal levels in BMSCs 3 weeks following induction (P > 0.05). No action potential was detected in BMSCs following 1 and 2 weeks of 5-aza, and action potential could be determined 3 weeks following induction, which was identical to normal myocardial cells.CONCLUSION: Through induced by 5-aza for three weeks, BMSCs have the similar expression of connexin-43 and electrophysiological characteristics as normal myocardium.

BACKGROUND: Previous studies have confirmed that embryonic stem cells call be induced to differentiate into insulin-producing cells, but the induction process takes a long time. Most of the processes take about one month.OBJECTIVE: Activin A, all-trans retinoic acid (ATRA), basic fibroblast growth factor (bFGF) and nicotinamide were applied in vitro in combination to observe whether mouse embryonic stem cells could be induct to differentiate into insulin-producing cell in a relatively short time.DESIGN: Cell observation experiment.SETTING: Shanghai Institute of Endocrine and Metabolic Diseases, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China.MATERIALS: This study was performed at Shanghai Institute of Endocrine and Metabolic Diseases from October 2004 to February 2006. Two mice of clean grade and of 12.5-14.5 days of gestational age were provided by Shanghai SLAC Laboratory Animal Co., Ltd (Permission No. 2004A034). The protocol was performed in accordance with ethical guidelines for the use and care of animals. Mouse embryonic stem cell lines were supplied by Dr Changxian Zhang (CNRS UMR5641, France). Activin A was the product of the R & D Corporation. ATRA and nicotinamide were supplied by the Sigma Corporation, USA. BFGF was supplied by Gibco Corporaion. METHODS: Head and viscera were removed from embryos of the pregnant mouse. The remaining tissues were cut into pieces and digested with trypsin. Cell suspension was centrifuged and inoculated at 3×108L-1. The cells could be used as mouse feeder layer after 2-3 times of passage. The mouse embryonic stem cells (ESCs) were inoculated onto the feeder layer in knockout Dulbecco's modified Eagle medium (DMEM) supplemented with leukemia inhibitory factors (LIF). ESCs were passaged at 1:3-1:6 after 2-3 days of culture. Culture medium with serum was added into the culture dishes to terminate the digestion. Cell fluid was centrifuged and supernatant was discarded. The sediments were prepared into suspension and inoculated at 2.5×104 with LIF-free culture medium. After 24-48 hours, embryonic bodies (EBs) were collected and replated in 1% Matrigel-coated dishes. When began to adhere to the dishes, EBs were cultured in 10% FBS/DMEM supplemented with 100μg/L activin A for 24 hours. Then EBs were switched to 10% FBS/DMEM for 6-8 hours as an interval. After this interval. EBs differentiated were cultured in 10% FBS/DMEM with 10<-6mol/L RA for another 24 hours followed by culture in 10% FBS/DMEM supplemented with 10μg/L bFGFs for 3-5 days. Finally, EBs differentiated were cultured in DMEM/F12 supplemented with N2 supplement, B27 supplement, 1μg/L laminin, 10μg/L bFGFs, and 10mmol/L nicotinamide for 3-5 days. Dithizone (DTZ) staining, inununofluorescent staining and reverse transcription-polymerase chain reaction (RT-PCR) were applied to detect insulin expression in the differentiated cells.MAIN OUTCOME MEASURES: Induction of ESCs, DTZ staining and immunofluorescent staining as wel as RT-PCR detection.RESULTS: Mouse ESCs growing on a feeder layer formed many colonies with clear boundary and dense structure. However, there was no obvious outer limit between these ESCs. EBs began to adhere to the dishes, which were coated with matrigel, on the 2nd day. After activin A and ATRA interval induction, EBs spread, and most of the living cells were epithelial cell-like when cultured in 10% FBS/DMEM supplemented with 10μg/L bFGFs. After culturing in DMEM/F12 supplemented with N2, B27, nicotinamide, bFGFs and laminin, the cells formed small clusters. The insulin-producing cells were stained dark red with DTZ, and the cells stained with primary antibody to insulin were insulin-positive. After 2 weeks of induction of activin A, ATRA, bFGFs and nicotinamide, the insulin-producing cells expressed insulin 2, Pdxl, Nkx6.1, Nkx2.2, PP, IAPP, Glut2, Somastatin, Hnf3β and Neuro D mRNA but did not express insulin 1 mRNA.CONCLUSION: Mouse ESCs call be induced to differentiate into insulin-producing cells by activin A, ATRA, bFGFs and nicotinamide in vitro. Induction time call be shortened to 2 weeks.

AIM: To study the interrelation between the structure and the function of artificial ribozyme M_1GS. METHODS: Ribozyme M_1GS-T_7, which targeted the mRNA segment of HCMV UL54 gene, was constructed. The secondary structure of M_1GS was simulated under different temperatures (20 ℃, 37 ℃ or 55 ℃, at which the secondary structure of M_1GS was relatively stable) and the interrelation between the secondary structure and the cleavage activity of M_1GS was analyzed under different temperature in vitro. To investigate the interrelation between the structure and the function of ribozyme M_1GS further, mM_1GS-T_7 was designed, in which some mutation sites was added, according to the result of temperature change experiment and the simulated secondary structure, showing that were the same structures at 37 ℃ as that of M_1GS-T_7 at 55 ℃. RESULTS: In temperature change experiment, the cleavage activity of M_1GS-T_7 was highest at 55 ℃. The result of mutant experiment showed that the mutant type was more active than M_1GS-T_7 at 37 ℃. CONCLUSION: The cleavage activity of M_1GS, which has some certain secondary structure, is higher than others. There is some interrelation between the structure and the function of M_1GS.

AIM:pUL23,the product of human cytomegalovirus (HCMV) gene UL23 was identified as one of tegument proteins. The aim of this research is to investigate the function of pUL23 during HCMV life cycle.METHODS:GAL4 yeast two-hybrid assay was performed to screen the human fetal kidney cDNA library to obtain host cell protein molecules which interact with pUL23 of HCMV. Then the GST-pulldown experiment was applied to confirm the protein interactions identified by yeast two-hybrid.RESULTS:ATPase inhibitory factor 1 (ATIF1) was selected from host cells using yeast two-hybrid assay. GST-pulldown experiments in vitro further confirmed the interaction between ATIF1 and pUL23.CONCLUSION:pUL23 of HCMV can interact with ATIF1 in host cell,which may provide the evidence for understanding the function of pUL23 in the life cycle of HCMV.

Objective To investigate the effect of bibliotherapy in soothing the postoperative pain in children and relieving the perioperative anxiety of children and their care givers. Methods Hospitalized children and their care givers from August 2007 to March 2009 were studied. 153 cases from August 2007 to May 2008 were assigned to the control group and the other 153 cases to the intervention group. Routine surgical nursing were applicated in the control group by introduction of perioperative nursing procedures.Bibliotherapy were applicated in the intervention group on the basis of the control group-using "bibliotherapy materials for hospitalized children" which was designed by ourselves and correspond with the theory of bibliotherapy to interfere in the 153 cases in the intervention group. The variance of preoperative anxiety of care givers and perceptions of postoperative pain of children between the two groups were compared with scales of mYPAS, STAI and FLACC and Wong- Baker Facial Scale. Results The scores of mYPAS in children of the intervention group and the control group was (35.875+4.441)and(46.796+8.606 )respectively and the variance was significant. The scores of STAI in care givers of the two groups was(38.125+4.371 )and (49.901 +7.420) respectively and revealed significant variance. The scores of Wong-Baker and FLACC in children of both groups 1 hour after operation were compared and revealed no statistical significance. The scores of Wong-Baker and FLACC in children at 6 hours and 24 hours postoperative were compared subjectively and objectively and revealed statistic significance. Conclusions Bibliotherapy can ameliorate the anxiety level of both children and their care givers, relieve the perception of postoperative pain in children and improve their comforts. Bibliotherapy thus conduces to the recovery of postoperative children.