BACKGROUND: Compared with the bone marrow mesenchymal stem cells, umbilical cord blood mesenchymal stem cells (UCB-MSCs) is an ideal source of tissue engineered seed cells, but the culture success rate is low.OBJECTIVE: To explore establish a stable reliable method to isolate and culture UCB-MSCs by optimizing medium choice,centrifugation speed and time, incubation density, choice of growth factor, first time of medium change.DESIGN, TIME AND SETTING: The cytological in vitro study was performed at the Department of Blood Transfusion, Bethune International Peace Hospital of Chinese PLA from January to October 2008.MATERIALS: A total of 20 samples of neonatal UCB by full-term uterine-incision delivery were supplied by Stem Cell Center,Bethune International Peace Hospital of Chinese PLA. Parturient and their family member signed the informed consent.METHODS: Under sterile conditions, UCB-MSCs were isolated by combination of density gradient centrifugation (1 500 r/min, totally 15 minutes) and different adherent time method. UCB-MSCs were incubated in DMEM/F12 medium, supplemented with 10% human UCB serum, 5 μg/L granulocyte-macrophage colony-stimulating factor (GM-CSF), 15 pg/L interleukin-3. MSCs at 1 ×1010/L were incubated in plastic flask coated with human UCB serum at 37℃ and 5% CO2 saturated humidity. The medium was changed following 3 days of culture. Non-adhered cells were removed. Subsequently, the medium was changed once every other 24 hours. When 80% confluence, UCB-MSCs were digested by the mixture of pancreatin-athylenediamine tetraacatic acid.MAIN OUTCOME MEASURES: Morphological changes of UCB-MSCs were observed by inverted phase contrast microscopy. Cell immunophenotypes were determined by flow cytometry.RESULTS: A small quantity of adherent round cells were determined after 24 hours, and adherent cells became more at 48 hours,with a few monopole spindle cells. Cell colonies were detected at day 7. Fibroblast-like cells arranged parallelly, presented whirlpool-shape and unclear boundary, with 80% 80% confluence 2-3 weeks following culture. At the second passage, these calls adhered at hour 12, and reached 80% 90% confluence at day 10. Flow cytometry showed that these calls were positive for CD29 and CD44, but negative for hematopoietic lineage marker CD34.CONCLUSION: MSCs can be successfully isolated from human UCB by using this modified method in vitro, with short culture cycle and high cell purification. Adherent cells have the same immunophenotype as bone marrow mesenchymal stem cells.

BACKGROUND:The homing ability of mesenchymal stem cells is closely associated with the effects of celltransplantation. Clarifying the mechanism of chemotaxis and migration wil contribute to enhance the clinical application of mesenchymal stem cells. OBJECTIVE:To investigate the effect of Cdc42 in the homing of human umbilical cord mesenchymal stem cells. METHODS:First, mesenchymal stem cells were isolated from human umbilical cord, and co-cultured with tumor necrosis factorα, interleukin-1β, and transforming growth factorβ. Western blot assay was used to test the level of Cdc42. Besides, Cdc42 siRNA was synthesized by chemical method to transfect the cells, and cellmigration and adhesion were measured by Transwel and Matrigel separately. Meanwhile, the activity of signal molecule, extracellular regulated protein kinase 1/2, was evaluated by western blot. RESULTS AND CONCLUSION:The results indicated that the inflammation factors induced the highly expression of Cdc42 in human umbilical cord mesenchymal stem cells, almost double level to controls. siRNA notably inhibited the migration and adhesion of human umbilical cord mesenchymal stem cells through Cdc42 down-regulation, and the extracellular regulated protein kinase 1/2 and phosphorylation form were also decreased simultaneously. In a word, we speculate Cdc42 plays a role in the chemotaxis of human umbilical cord mesenchymal stem cells in vitro.

Objective To explore the causes and management of postoperative complications of laparoscopic Roux-en-Y gastric bypass (LRYGB).Methods The retrospective cross-sectional study was conducted.The clinical data of 450 patients with metabolic diseases who underwent LRYGB between June 2004 and November 2016 were collected,including 283 (58 in hospital consultation) in the First Affiliated Hospital of Jinan University,140 in the Jihua Hospital Affiliated to Jinan University and 27 in the Zhengzhou Hospital of Jinan University.Observation indicators:situations of surgical completion,follow-up situations,occurrence,treatment and prognosis of complications.Follow-up using outpatient examination and telephone interview was performed to detect postoperative complications once at month 1,3,6 and 12 within 1 year postoperatively and once every year after 1 year postoperatively up to March 2017.Measurement data with skewed distribution were described as M (range).Count data were evaluated by the ratio,and comparison between groups was analyzed using the chi-square test.Results All the 450 patients with metabolic diseases underwent successful LRYGB,including 50 receiving LRYGB during surgical internship period and 400 receiving LRYGB after surgical internship period,without conversion to open surgery.All the 450 patients were followed up for 70 months (range,1-153 months).Twenty-seven patients had postoperative complications,with an incidence of 6.00％ (27/450).The incidence of postoperative complications was 20.00％(10/50) in 50 patients receiving LRYGB during surgical internship period and 4.25％ (17/400) in 400 patients receiving LRYGB after surgical internship period,with a statistically significant difference (x2 =16.86,P＜ 0.05).Of 27 patients with postoperative complications,1 was complicated with fulminant acute pancreatitis and died from multiple organ failure at day 15 postoperatively,5 with intra-abdominal bleeding,2 with anastomotic leakage,3 with gastrojejunal anastomosis stenosis,2 with gastrojejunal anastomosis ulcer,1 with improper anastomosis,1 with respiratory failure,1 with umbilicus infection,3 with internal hernia,2 with dumping syndrome,6 with weight-loss failure (1 refused to undergo revision surgery),and patients with postoperative complications were improved or cured by surgery or conservative treatment except one death.Conclusions The incidence of complications in patients receiving LRYGB after surgical internship period is significantly reduced,and complications needs to make the individualized treatment plan.

Objective　To investigate the relationship between multidrug resistance(MDR) and chemotherapy and the possibility of P-gp as a clinical prognostic indicator. Methods　P-gp, MDR1 gene product, was detected in 66 cases of relapsed lung cancer patients after chemotherapy and 131 lung cancer patients with no prior chemotherapy by immunohistochemistry. Results　The expressive level of P-gp in relapsed group after chemotherapy was much higher than that of non-chemotherapy group (P＜0.05). A significant difference of P-gp expression existed between before and after chemotherapy for squamous cell carcinoma and SCLC (P＜0.05), but not for adenocarcinoma. In non-chemotherapy group, lung cancer tissues with different histological types had different P-gp positive rates, which were adenocarcinoma, squamous cell carcinoma and SCLC in sequence from high to low (P＜0.05). In relapsed group after chemotherapy, there was no significant difference among the three types of lung cancer (P＞0.05). P-gp positive group had a significantly lower 3-year survival rate than negative group (P＜0.05). Conclusion　Chemotherapy may increase the incident rate of MDR in lung cancer. Adenocarcinoma is mainly primary MDR while SCLC and squamous cell carcinoma are mainly secondary to chemotherapy. P-gp expressive level can be used as a common clinical indicator for prognosis and chemotherapy in the patients with lung cancer.

Objective To investigate the neuroprotective effect and mechanism of liraglutide on diabetic rats. Methods 24 healthy male SPF Goto-Kakizaki (GK) rats with random blood glucose greater than 11.1 mmol/L were selected as the experimental group, and randomly divided into diabetes mellitus group ( n＝12) and liraglutide group (n＝12). Ten healthy male SPF Wistar rats with the same age and weight as GK rats were selected as normal control group. After adaptively feeded for 2 weeks, the liraglutide group was given liraglutide (400 μg·kg－1·d－1, subcutaneous injection), while the control group and diabetes mellitus group were given the same volume of saline, and continued to be administered for 8 weeks. After 10 weeks, data and biochemical indicators were recorded. Effects of liraglutide on learning and memory in diabetes mellitus rats were detected by Morris water maze test. HE staining observed the hippocampal neurons morphology. Western blotting method detected the expression of p- IκB kinase (IKK) β, p-NF-κB, NF-κB, Klotho, and PRX2 in hippocampus. Results Morris water maze test showed that liraglutide can improve the spatial learning and memory ability of diabetes mellitus rats. HE staining showed that liraglutide significantly reduced the pathological damage of hippocampal neurons of diabetes mellitus rats. Western blotting showed that liraglutide inhibited NF-κB signaling pathway in hippocampus of diabetes mellitus rats. The expression of Klotho protein in hippocampus of diabetes mellitus group was significantly lower than that of control group, while the expression of PRX2 protein was higher than control group (t＝8.298,－7.398,all P<0.01). The expression of Klotho and PRX2 protein in hippocampus of liraglutide group were higher than diabetes mellitus group (t＝－13.059, 14.113, all P<0.01). The expression of Klotho protein of liraglutide group was similar to that of control group ( t ＝ －1. 137, P>0. 05 ). The expression of PRX2 protein was significantly higher than control group (t＝－28.055, P<0.01). Conclusions Liraglutide may enhance the expression of antioxidant stress protein including Klotho and PRX2, by inhibiting NF-κB signaling pathway in hippocampus of diabetes mellitus rats, reduced oxidative stress and improved the injury of hippocampal neuronal in diabetes mellitus rats, which seems to play a neuroprotective effect, to prevent and delay the occurrence of diabetic encephalopathy.

Objective:To investigate the effect of Salvia miltiorrhiza injection combined with human adipose derived stem cells (hADSC) on the survival rate of autologous fat granules implanted in nude mice.Methods:A total of 24 healthy 8 weeks old female nude mice weighing (23±3) g were randomly divided into four groups ( n=6): Group A was given fat granules 0.5 ml; Group B: fat granules 0.5 ml+ hADSC; Group C: Salvia miltiorrhiza 0.5 g/(kg·d) + fat granules 0.5 ml, and Group D: Salvia miltiorrhiza injection 0.5 g/(kg·d) + fat granules + enrichment. 3 nude mice were randomly selected from each group for 15 days after transplantation and stained with conventional HE. Immunohistochemical staining was performed to count the number of microvessels. On the 30th day after surgery, the remaining 3 nude mice in each group were sacrificed. The specimens were stained with HE and the volume of each specimen was measured. Results:Graft appearance was observed by naked eye: 15 days after the operation, all the specimens were formed completely. The new capillaries were shaped on the surface of the capsule. The boundaries of the capsule and the surrounding tissue were obvious. The activity was good, the hardness of the texture was medium, and the loose connective tissue was connected to the surrounding tissue. On the 30 day after operation, the volume of the graft was smaller than that at the beginning of transplantation, and fat liquefaction and necrosis were seen in some tissues. Blood vessel density values of immunohistochemical sections of fat transplantation in each group 15 days after surgery were compared pairwisely. The differences between the groups were statistically significant ( P<0.05). Lsd-t method was used for pairwise comparison of fat graft volume values of each group 30 days after surgery, and the difference between each group was statistically significant ( P<0.05). Conclusions:The combined use of Salvia miltiorrhiza injection and hADSCs can effectively promote the reconstruction of the early vascular system of the fat granule transplantation and improve the survival rate of fat particles more effectively than the individual use.