Objective To identify a detailed biological characterization of mesenchymal stem cells (MSCs) isolated from hu‐man umbilical cord(UC) tissue regarding their morphology ,immunophenotype ,purity and proliferative capacity and establish a rea‐sonably cultured and amplified system .Methods After stripping off arteries and veins ,the remaining parts of umbilical cord were cut into 1 mm3 small sections and cultured with DMEM/F12 containing 10% fetal bovine serum .Adhere cells were obtained and the morphology of the cells was observed under inverted phase contrast microscope .The growth curves of them were drawn by CCK‐8 and the cell cycle and surface antigens (CD29 ,CD73 ,CD90 ,CD105 ,CD31 ,CD14 ,CD34 ,CD45 ,CD11b ,HLA‐DR) were detected by flow cytometry .Results Seven to ten days after primary culture ,adhere cells came out of fragments .The MSCs harvested were a high purity and mainly presented as a fibroblast‐like morphology .UC‐MSCs had a strong ability of proliferation through the cell growth curve .The special surface antigens CD29 ,CD73 ,CD90 ,CD105 were positive expression ,while CD31 ,CD14 ,CD34 ,CD45 , CD11b ,HLA‐DR were negative .More than 80% cells of MSCs were found at G0/G1 phase .Conclusion Human UC‐MSCs could be cultured and proliferated in vitro .

Objective To discuss the efficacy and cosmetic effect of laparoscopic thyroidectomy for patients with thyroid diseases. Methods The clinical data of 160 patients who underwent laparoscopic thyroidectomy through the anterior chest approach were analyzed. The operation was performed under a pressure of 8 mm Hg within the surgical space at the neck. After the thyroid was separated completely, the lesions were resected using an ultrasound knife. Results Laparoscopic thyroidectomy was completed in 157 of the patients including 6 cases of papillary adenocarcinoma. The other 3 patients were converted to open surgery because of hyperthyroidisms complicated with intraoperative hemorrhage (1) or thyroid carcinoma complicated with lymph node metastasis (2). No severe complications involving the trachea and parathyroid occurred in this series. One patient with thyroid carcinoma developed transient hoarseness after the operation; one patient with nodular goiter had postoperative subcutaneous hemorrhage and hydrops; both of them were cured spontaneously. Postoperative hospital stay ranged from 3 to 5 days (mean, 4 days). The patients were followed up for 3 to 24 months with a mean of 6.8 months, during which none of them had recurrence. All the patients were satisfied with cosmetic outcomes of the surgery. Conclusion Laparoscopic thyroidectomy via the anterior chest approach is safe and feasible for patients with thyroid diseases with good cosmetic results.

Objective To investigate the clinical significance of CD55,CD59 and Aeromonas hydrophila toxin variant (FLAER) in the diagnosis of anemia and paroxysmal nocturnal hemoglobinuria (PNH).Methods Collected 30 healthy controls,22 cases of PNH,33 cases of aplastic anemia (AA),37 cases of iron deficiency anemia (IDA),45 cases of megaloblastic anemia (MA),30 cases of hemolytic anemia (HA) and 31 cases of myelodysplastic syndrome (MDS) from January 2009 to March 2017,CD55,CD59 and FLAER negative cell ratio of peripheral blood neutrophil of them were detected by multipa rameter flow cytometry.Results The detection rates of FLAER in PNH,AA and MDS groups were higher than those of CD55 and CD59,but there was significant difference in AA (x2 =7.759,5.518,P=0.005,0.019＜0.05).The average CD55,CD59 and FLAER deletion rate in PNH and AA group were significantly higher than those in normal control group and other groups (t=2.163～17.890,P=0.000～0.038＜0.05).The number of FLAER in PNH group was higher than CD59 and CD59 was higher than CD55 with the statistically significant difference (t=2.503 ～ 6.308,P=0.000 ～0.016＜ 0.05).Conclusion CD55,CD59 and FLAER have important value in the diagnosis of PNH and differential diagnosis with other anemia diseases,and can also be used to detect the presence of MDS and AA in patients with PNH.FLAER outperforms CD59,CD59 outperforms CD55.

Objective To investigate the effect of immunocompetent cells and immune regulator on the apoptosis of human mesenchymal stem cells ( MSCs) and on mRNA expression of heme oxygenase-1. Methods MSCs were cultured by density gradient centrifugation and then identified by flow cytometry. RT-PCR was used to detect HO-1 mRNA expression and flow cytometry was used to analyze cell apoptosis after the stimulation of IFN-? and PHA-activated T cells. Results The mRNA expression of Heme oxygenase-1 was observed in MSCs and decreased after the stimulation of IFN-? and activated T cells. IFN-?,znpp-Ⅸ and combined these two caused obvious cell apoptosis in MSCs,with an apoptotic rate of ( 56. 50 ? 0. 16) % ,( 56. 85 ? 2. 27) % ,and ( 82. 53 ? 2. 65) % respectively. All of them had a significant difference compared with the normal MSCs [( 7. 56 ? 1. 43) % ,P

ObjectiveTo explore the feasibility, safety and clinical value of laparoscopic Rouxen-Y cystojejunostomy in the treatment of pancreatic pseudocyst. Method Four patients with pancreatic pseudocyst received totally laparoscopic pancreatic pseudocystojejunostomy. The data on intraoperative bleeding, operative time, postoperative time to get out of bed, time of first flatus/bowel motion, complication and duration of hospital stay were collected and analyzed retrospectively. ResultsAll operations were carried out successfully with laparoscopic surgery. The mean operative time was 90 min. The average intraoperative blood loss was 40 ml. The mean postoperative time to get out of bed was 1.5 d, and the mean time of first flatus/bowel motion was 2. 3 d. All patients recovered smoothly without any pancreatic fistula. The average hospital stay was 7 days. Fever, pancreatitis,adhesive intestinal obstruction and other complications did not occur. ConclusionsTotally laparoscopic Roux-en-Y pancreatic pseudocystojejunostomy was an efficacious, safe, and minimally invasive procedure.

Objective To investigate the expression of pSTAT5 in 7 pancreatic carcinoma cell lines,and the change of expression of pSTAT5 in pancreatic carcinoma cells SW1990 after growth hormone (GH) treatment, and explore its molecular mechanism. Methods Human pancreatic carcinoma cell lines (SW1990, Cap-1, Colo, Mia, AsPc, P3, PANC1) were cultured in vitro, and Western blotting was used to detect the expression of pSTAT5 in these cell lines. SW1990 in exponential growth phase was collected and nude Balb/c mice were inoculated with SW1990 cells. When tumors became palpable after inoculation, mice (normal saline group). 1 h, 2 h and 24 h after the last dose of GH treatment, the mice were sacrificed.Western blotting was used to detect the expression of pSTAT5 in SW1990 and inoculation tumor cells after GH injection. Results Positive expression of pSTAT5 was observed in all human pancreatic carcinoma cell lines (SW1990, Cap-1, Colo, Mia, Aspc, P3, PANC1). 5 minutes after GH (50 ng/ml) stimulation, the expression of pSTAT5 in SW1990 was 0.57 ±0.05, which was significantly increased; and it reached 0.64 ±0.04 at 10 minutes, then decreased to 0.39 ±0.03 at 15 minutes, however, it remained higher than that in the control group at 1 h (0.33 ± 0.02 vs 0.25 ± 0.06), and its expression at 2 h was 0.26 ± 0.03 and returned to the normal level. The expression of pSTAT5 in xenograft was not significantly changed. Conclusions GH could rapidly up-regulate the expression of pSTAT5 in SW1990 but the effect lasted for a relatively short period. GH had no significant effect on the expression of pSTAT5 in xenograft.

Objective To observe the effects of low dose irradiation (LDR) on proliferation,adipogenesis and osteogenic potential of human umbilical cord mesenchymal stem cells (hucMSCs).Methods hucMSCs were isolated from Wharton's jelly tissue of human umbilical cord by modified tissuepiece inoculation,and flow cytometry was used to detect the expression of specific marker in the hucMSCs.The hucMSCs were randomly divided into two groups:irradiation group undergoing irradiation with the doses 50,100,or 200 mGy respectively,and control group without irradiation.MTT method was applied to evaluate the proliferation of the hucMSCs at different time points with various doses irradiation.The third passage hucMSCs were randomly divided into two groups:irradiation group undergoing low dose irradiation of 200 mGy,and control group without irradiation,and then underwent induction by adipocytic and oesteocytic differentiation induction fluids respectively so as to differentiate into adipocytes and osteoblasts.Oil red O staining was used to detect the activity of alkaline phophatase (ALP),and RT-PCR was used to detect the mRNA expression of core binding factor alpha 1 in human osteoblast.Results After 9-12days,fibroblasts began to swim out of the tissue piece with a confluence rate of 80% 2 weeks later.Within 7 days the absorption values of the hucMSCs undergoing different irradiation doses 2,3,4,5,and 6 days later were all significantly higher than those of the control group(F = 159.17,448.81,265.15,183.93,and 181.83 ,all P <0.01),with the proliferation rates of the 100 mGy subgroup being the highest.After being induced liquid,vacuoles were observed in the irradiated group 2 days later.21 days later,the adipogenic rates of irradiated group was significantly higher than that of the control group (t = 28.25,P <0.01).The ALP activity increased in the irradiated group compared with control group (t=16.87,P <0.01) .The expression level of Cbf-α1 mRNA was up-regulated obviously (t = 14.16,P＜0.01).Conclusions LDR promotes the proliferation of hucMSCs,and accelerates the hucMSCs' differentiation into adipocytes and osteoblasts.

BACKGROUND:Cobalt chloride (CoCl2) may promote the proliferation of human umbilical cord-derived mesenchymal stem cels (hUC-MSCs) in a time- and concentration-dependent manner, and meanwhile, CoCl2 can regulate the expression of genes and proteins in hUC-MSCs. OBJECTIVE:To explore the effects of CoCl2 induced-hypoxia on the proliferation of hUC-MSCs and gene and protein expressions in hUC-MSCs, thereby establishing an effective method for MSCs culture and amplificationin vitro. METHODS: hUC-MSCs were extracted using tissue explant method. Under hypoxia conditions induced by CoCl2 (0, 100, 150, 200, 250 μmol/L) for different periods (0, 1, 2, 3, 4 days), flow cytometry was used to identify cel surface-associated antigens; cel counting kit-8 was used to detect cel proliferation; RT-PCR was used to determine levels of hypoxia inducible factor-1α, inducible nitric oxide synthase, stromal cel-derived factor-1, interleukin-6, transforming growth factor-β mRNA; western blot assay was used to detect protein expression of hypoxia inducible factor-1α. RESULTS AND CONCLUSION:The cels were positive for CD29, CD73, CD90, CD105, while negative for CD31, CD14, CD34, CD45, CD11b, HLA-DR. Moreover, the antigen expression was not affected by CoCl2 induced-hypoxia. CoCl2 induced-chemical hypoxia could promote the proliferation of hUC-MSCs in a time- and concentration-dependent manner. RT-PCR results showed thatunder hypoxia, hypoxia inducible factor 1α, inducible nitric oxide synthase and stromal cel-derived factor-1 mRNA expressions were significantly up-regulated, but interleukin-6 and transforming growth factor-β mRNA expressions were down-regulated significantly (P < 0.05). Additionaly, the protein expression of hypoxia inducible factor 1α was increased under hypoxia conditions. These findings indicate that CoCl2 induced-hypoxia environment may promote the proliferation of hUC-MSCs and the optimal concentration of CoCl2 is 200μmol/L. However, a higher concentration of CoCl2 (≥ 250μmol/L) inhibits the proliferation of hUC-MSCs, and the mechanism may be related to the increase of hypoxia inducible factor-1α at protein and mRNA levels.

Objective To investigate the biological characteristics of primary osteoporosis syndrome types from the perspective of mitochondrial DNA ( mtDNA) , thus to reveal the nature of osteoporosis and its traditional Chinese medical syndrome types. Methods A total of 210 osteoporosis women patients meeting the diagnostic criteria, inclusion criteria and exclusion criteria were collected from July of 2011 to October of 2013. The osteoporosis patients were differentiated into the syndrome types of yin deficiency of liver and kidney ( N=67) , yang deficiency of spleen and kidney ( N=70) and qi stagnation and blood stasis ( N=73) . And a total of 69 age-matched post-menopause non-osteoporosis patients were chosen as the control group, which were classified into the syndrome of harmony of Qi and blood. The peripheral blood was sampled for detecting mtDNA copy number with fluorescent quantitatitation PCR and for examining 8-hydroxy-2’-deoxyguanosine ( 8-OHdG) content by enzyme-linked immunosorbent assay (ELISA) . Statistical methods was used to analyze the correlation of bone mineral density (BMD) with mtDNA copy number and 8-OHdG content in different groups. Results The difference of mtDNA copy number was significant between the osteoporosis patients and non-osteoporosis patients (P<0.05), and was also significant among the three syndrome types of osteoporosis patients (P<0.05) . And 8-OHdG content showed the same features between the osteoporosis patients and non-osteoporosis patients (P<0.05) and among the three syndrome types of osteoporosis patients (P<0.05) . The correlation analysis results showed that mtDNA copy number was positively correlated with BMD, while 8-OHdG was negatively correlated with BMD in each group. Conclusion The mtDNA copy number and 8-OHdG content are correlated with the syndrome types of primary osteoporosis patients, and close correlation is shown between spleen-kidney yang deficiency and 8-OHdG, and between liver-kidney yin deficiency and mtDNA copy number.

Objective To investigate the protection effect of cyclosporine A on spatial memory following chronic cerebral hypoperfusion in rats and its possible mechanism.Me,ods Sixty SD rats were randomly divided into sham operation,vehicle,low-dose cyclosporine A,medium-dose cyclosporine A,and high-dose cyclosporine A groups.A chronic cerebral hypoperfusion model was prepared by permanent bilateral ligation of bilateral common carotid arteries.From 46 days after modeling,olive oil 1 ml/d was used for intragastric administration in the sham-operation group and the vehicle group.Cyclosporine A 3 mg/kg,6 mg/kg,and 12 mg/kg were administrated intragastrically in the low-dose,medium-dose and high-dose cyclosporine A groups,respectively,once a day for 14 days.The spatial memory was assessed using Morris water maze test.Reverse transcriptase polymerase chain reaction (RT-PCR) was used to detect the expression of NADPH oxidase 4 (NOX4) mRNA in the cerebral cortex.Immunohistochemical staining and Western blot were used to detect the expression of NOX4 protein in the cerebral cortex.Results The Morris water maze test showed that the escape latencies in all cyclosporine A groups were significantly shorter than the vehicle group (all P ＜0.05).Immunohistochemical staining showed that the percentages of the NOX4-positive cells in the sham-operation,vehicle,low-dose,medium-dose,and high-dose cyclosporine A groups were4.43％ ±0.37％,37.44％ ±4.76％,18.05％ ±2.91％,12.51％ ±3.4％,and 11.06％ ±1.74％,respectively (F =262.021,P ＜ 0.001),the vehicle group was significantly higher than the sham-operation group (P ＜ 0.01),and all cyclosporine A groups were significantly less than the vehicle group (all P ＜ 0.01).RT-PCR showed that the expression levels of NOX4 mRNA in cerebral cortex in the sham-operation,vehicle,low-dose,medium-dose,and high-dose cyclosporine A groups were 0.36 ± 0.03,1.04 ± 0.04,0.58 ± 0.02,0.49 ± 0.01,and 0.40 ± 0.02,respectively (F =1 324.941,P ＜ 0.001),all cyclosporine A groups were significantly lower than the vehicle group (all P ＜ 0.01).Western blot showed that the expression levels of NOX4 protein in cerebral cortex in the sham-operation,vehicle,low-dose,medium-dose,and high-dose cyclosporine A groups were 0.02 ± 0.01,0.27 ± 0.04,0.09 ± 0.02,0.06 ± 0.02,and 0.06 ± 0.01,respectively (F =222.692,P ＜ 0.001),all cyclosporine A groups was significantly lower than the vehicle group (all P＜0.01).Conclusion Cyclosporine A may improve spatial memory following chronic cerebral hypoperfusion in rats by down-regulation of NOX4.