Objective:To construct a retroviral vector carrying both enhanced green fluorescent protein(EGFP) and human insulin gene for transfecting the bone marrow mesenchymal stem cells (BMSCs),and to observe the treatment effect of transfected BMSCs after transplanted into diabetes mice.Methods:Two gene segments of insulin-IRES-EGFP and IRES-EGFP were obtained by overlap extension PCR technology,and then the 2 segments were cloned into retroviral vector (pMSCV).The vectors were used to transfect BMSCs after packaged with PT67 cells.The expression of EGFP was observed by inverted fluorescence microscope and the expression of insulin gene was examined by RT-PCR in the infected BMSCs.The transfected BMSCs with 2 viruses were transplanted into diabetic mice separately.The blood glucose levels and body weights of mice were examined in 2 groups,and the results were compared with those of normal control group.Results:The recombinant retroviral vectors pMSCV-insulin-IRES-EGFP and pMSCV-IRES-EGFP were successfully constructed.The BMSCs infected by both vector stably gave out green fluorescence under microscope; the cells infected with pMSCV-insulin-IRES-EGFP also stably expressed human insulin gene.The blood glucose level of the mice transplanted with BMSCs transfected with pMSCV-insulin-IRES-EGFP was significantly lower than that transplanted with BMSCs transfected with pMSCV-IRES-EFGP (P

Objective To observe the effects of differentiation of mature islet cells of mice on marrowmesenchymal stem cells (mMSCs). To provide transplant source for islet cell transplantation in the treatment of diabetes. Methods The culture, isolation and passage of mMSCs was performed by using patch wall, cell shape was observed by confocal microscope, and flow cytometry analysis was used to determine their biological characteristics. The type Ⅳ collagenase was injected into common bile duct to digest the pancreas, and then gradient centrifugation was used to isolate islet cells. The transwell co-culture system was used for third generation of mMSCs and isolated islet cells, then inversion microscope was used to observe the cell growth and morphological changes, immunochemistry methods was applied to detect the expression of insulin in mMSCs, and insulin release test was performed to determine the secretion of insulin. The control group consisted of cultured mMSCs alone. Results The cells from mouse bone marrow were found to be in long spindle shape with large volume after 48 hours in culture. One week later the cells grew in the form of colony with serial subcultivation. The cell surface molecules including Sca-l, CD29, CD44, CD105 were positive with high level of expression;while the cell surface molecules including CD34, CD45 were negative, all of these results confirmed that the ceils were mMSCs. After 7 days of coculture with mice islet cells, part of mMSCs cells were positively stained by insulin immunohistochemisty, the insulin secretion was (16.83±0. 15)μIU/ml.Conclusions After cocultured with islet cells, mMSCs isolated from mouse bone marrow could differentiate into islet like cells. These cells may be used in the islet cells transplantation in the treatment of diabetes, which provided a solution to the problems of donor-shortage and immunologic rejection.

(0.05)). (Conclusions) PFC and serum TNF-? ,NO could be early observation indicators of acute rejection in minipig pancreas transplatation,and should have important significance in the diagnosis of acute rejection.

Background and purpose:PARP-1 is closely related to malignant tumors. This study aimed to ex-plore the clinical significance of serum level of PARP-1 protein in onset and progression of gastric cancer.Methods:The serum samples from 145 patients with gastric cancer and 112 healthy check-up cases were collected. The serumHP spec-ificity IgA and PARP-1 protein levels were detected using enzyme-linked immunosorbent assay method. The correlation of serum PARP-1 protein levels with clinical characteristics of gastric cancer was analyzed.Results:Compared with healthy people, serum PARP-1 protein levels were significantly higher in gastric cancer patients [(407±139) pg/mLvs(258±120) pg/mL,P=0.014). Serum PARP-1 protein levels were significantly higher inHp(+) gastric cancer patients than those in patients withHp (-) (P<0.001). Serum PARP-1 protein levels were positively correlated with family gastric cancer history (P=0.033) and alcohol intake history (P=0.015) in gastric cancer patients. Compared with serum protein PARP-1 negative patients, PARP-1 protein positive patients had a significantly shorter cancer-free survival (P=0.011). However serum PARP-1 protein level was not found to be an independent risk factor for the overall survival of gastric cancer patients using multivariate COX regression.Conclusion:High expression of serum PARP-1 protein may be involved in the pathogenesis and progression of gastric cancer. Inhibition of PARP-1 may be potential new target for the treatment of gastric cancer.

Objective To analyze the etiology of pseudoachalasia.Methods Patients who were diagnosed as having pseudoachalasia in Endoscopy Center of The Affiliated Zhongshan Hospital of Fudan University from September 2010 to June 2015 were retrospectively analysed.Results A total of 12 patients were enrolled in this study,6 males and 6 females.The median age,median disease duration and median Eckcardt score was 54.5(24.0～71.0) years old,2.5(0.2～ 10.0) years and 4(3～9),respectively.Seven cases were caused by malignant tumors,2 had benign tumors,and 3 peptic stricture.Conclusion The most common cause of pseudoachalasia is malignant tumors.Accurate diagnosis relies on the comprehensive medical history and complete examinations.

Objective To study the expression of cyclooxygenase-2(COX-2)in hepatic inflammatory reaction and the effects of COX-2 on immunologic function of rats with sepsis.Methods Fifty-four Wistar rats were divided into sham group(n=6),sepsis group(n=24),and NS-398 intervention group(n=24).All rats were subjected to cecal ligation and puncture or sham operation.The expression of COX-2 mRNA in rat hepatic tissue was determined by RT-PCR,serum levels of IL-6,IL-10 and TNF-α were detected by ELISA,and percentage changes of CD4+,CD8+ cells by flow eytometry.The pathological changes of liver were observed at the same time.Results (1)Severe pathologic injuries of liver were observed in sepsis group,while not in NS-398 intervention group.(2)The expression of COX-2 mRNA was up-regulated in sepsis group and NS-398 intervention group,and the expression value was higher in sepsis group than that in NS-398 intervention group.The expression of COX-2 mRNA was the lowest in sham group.(3)The level of IL-6 was higher in sepsis group than that in sham group and NS-398 intervention group(F=125.582,134.712,54.760,121.441,P＜0.05).(4)The level of IL-10 was higher in NS-398 intervention group than that in sham group and sepsis group(F=39.064,34.382,51.115,8.174,P＜0.05).(5)The levels of TNF-α in sepsis group and NS-398 intervention group were increased,and the difference between the 2 groups had no statistical significance(x2=5.600,6.162,7.136,7.200,P＞0.05).(6)The ratio of CD4+to CD8+ was higher in NS-398 group than that in sepsis group(F=17.448,15.055,30.068,64.210,P＜0.05).Conclusions COX-2 plays an important role in the development of sepsis by changing the dynamic equilibrium between pro-inflammatory and anti-inflammatory cytokine and that between CD4+and CD8+.

Objective To study the effectiveness of family-integrated-care (FICare) for preterm infants with moderate to severe bronchopuhnonary dysplasia (BPD) in neonatal intensive care unit (NICU).Method Preterm infants with moderate to severe BPD in Hunan Children's Hospital from January 2015 to March 2016 were selected as the objects.These infants were assigned into two groups (FICare group and control group) in the base of whether the parents agreed to implement FICare.If the infant was enrolled into FICare group,the parents need to spend 3 hours in ward every day,and participate in nursing services under the guidance of nurses.The nursing services included bathing,cuddling,skin care,breast-feeding,and so on.If the infant was enrolled into control group,these nursing services were implemented by nurses.Oxygen exposure time,breast-feeding rate,time of begin oral feeding,time of total oral feeding,weight growth rate during hospitalization,weight of discharge,length of stay,readmission rate within 30 days,the rate of parents that mastered basic care knowledge and skills when discharge from hospital,the rate of satisfaction,and the rate of follow-up within 30 days of the two groups were compared.Result There were 106 cases in our study,54 in the FICare group and 52 in the control group.Compare the FICare group and the control group,there were statistical differences between two groups in the follow aspects (all P ＜ 0.05):oxygen exposure time [(57.1 ±20.9) d vs.(71.4 ±32.6) d],breast-feeding rate (77.8％ vs.44.2％),time of total oral feeding [(46.1 4 19.6) d vs.(59.4 ± 30.2) d,length of stay [(65.8 ± 18.4) d vs.(84.3 ±35.0) d],the rate of parents that mastered basic care knowledge and skills when discharge from hospital (96.3％ vs.82.7％),the rate of satisfaction (94.4％ vs.84.6％),the rate of follow-up within 30 days (92.6％ vs.73.1％).Conclusion FICare could significantly reduce oxygen exposure time of preterm infants with moderate to severe BPD,could improve the level of parents mastered basic care knowledge and skills of preterm infants,especially in critically ill preterm children,and could shorten length of stay of them.FICare is beneficial to the healthy growth of premature infants with BPD.

Objective To investigate the efficacy of combined application of laparoscope and multiple endoscopes for the treatment of extra-hepatic bile duct stones.Methods The clinical data of 3780 patients with extra-hepatic bile duct stones who were admitted to the First Hospital of Lanzhou University from March 1998 to June 2010 were retrospectively analyzed.According to the condition of bile duct stones,laparoscope,choledochoscope and duodenoscope were applied separately or combinately.All patients were divided into A,B and C groups.Patients in group A were treated by laparoseopy,choledochoscopy or duodenoscopy;patients in group B were treated by choledochoscopy+duodenoscopy.duodenoscopy+laparoscopy or laparoscopy+choledochoscopy;patients in group C were treated by laparoscopy+duodenoscopy+choledochoscopy.The efficacies of different treatment approaches were analyzed by comparing the results of imaging examination and follow-up.Results The curative rate and complication rate of the group A were 89.54%(1276/1425)and 6.73%(86/1276),respectively.Of the 149 patients in the group A who were failed in the treatment,83 patients were transferred to the group B,and 66 patients were transferred to the group C.The curative rate and complication rate of the group B(including 83 patients transferred from the group A)were 95.93%(1719/1792)and 4.07%(70/1719),respectively,and 73 patients who were failed in the treatment were transferred to the group C.The curative rate and complication rate of the group C(including 139 patients transferred from the group C)were 99.75%(783/785)and 0.26% (2/783),respectively,and 2 patients who were failed in the treatment received open surgery.Conclusion Combined application of laparoscope and endoscopes could raise the success rate of stone clearance and decrease the postoperative complications.

Objective To investigate the influence of streptozocin (STZ)'s dose on the inductive effect of diabetes in C57BL/6J mice, and investigate the dose-effect relationship and the optimal dose range. Methods 145 C57BL/6J mice were randomly divided into 9 diabetic groups (group A to group 1, n = 15 in each group) and I control group (n = I0) to receive intraperitoneal injection of STZ with the dosages of 30, 60, 80, 100, 120, 150, 180, 210, 240 mg/kg and same amount of buffer solution,respectively. Changes of blood glucose, body weight, survival rate at 45 day and serum insulin level were monitored, and the relationship with STZ doses was analyzed. Pancreas and kidneys of the mice were removed for morphological examination, and immunohistochemistry was used for determination of insulin in pancreas and CD<,68> in kidneys. Results Compared with control group, blood glucose in group C ～G increased significantly; body weigh, insulin level decreased significantly (P < 0.05), and the STZ dose was positively correlated with mean blood glucose (r = -0.984, P < 0.05) and was negatively correlated with mean serum insulin levels (r = 0.994, P <0.05). The diabetes modeling rates in group C ～ G (86.7% ～ 100%) were higher than those of group A and B (0 and 40%, P<0.05). At the 45th day, the survival rates of group C ～G (46.7% ～ 73.3%) were higher than those of group H and 1 (13.3% and 0, P <0.05). There was no obvious injury of pancreas and kidneys in group B, whereas, in group C and G, pancreatic island atrophy and decreased insulin secretion were observed; deposits of extracellular matrix and macrophage increased in the mesangium were also present. Conclusions 80 ～ 180 mg/kg of STZ dose was ideal for establishing diabetes model in C57BL/6J mice. Within this range, the modeling rate and survival rate was higher, and target organs injury was typical. The STZ dose was positively correlated with blood glucose and negatively correlated with serum insulin levels.