Objective To search for a good method for generation of hematopoietic stem cells (HSC) by embryonic stem cells (ESC),and to investigate the potential of HSC derived from ESC to reconstruct hematopoiesis in vivo.Methods Using a three-step method to induce a mice ESC line, E14.1-into HSC.And identifying HSC by flow cytometry analysis cell markers with CD34 +/Sca-1+, then HSC (1×109L-1)from ESC differentiation were injected into severe combined immunodefieency (SCID) mice for observing teratoma formation.To validate function of HSC by colonngenic cells assay and to reconstitute the hematopoiesis in lethally irradiated mice.Results Combining to use more hematopoietic stimulating factor to availably promote the El4.1 cell into embryoid body (EBs) with abundant hematopoietic progenitors.EBs were induced after 14 day to fast differentiate for HSC with peak percentages of CD34+/Sca-1+ cells reached to (13.72±2.07)%.To harvested ceils from EBs by day 14 for second -step HSC differentiation, percent of CD34+/Sca-1+ cells rise to(24.62±2.50) % after day 16 induction.Cloning forming units (CFU) analysis showed that more Erythro -myeloid cloning generation was observed at this period.Cells obtained in the second step are subsequently plated on monolayer of mice bone marrow stromal cells, in the presence of TPO, FLt3 ligand and superoatant of mice fetal liver stromal cells, cultured for additional 15 days, followed fast expansion of CD34+/Sca-1+ to maximally (58.64±4.20 ) % with more CFU-E, CFU-GM and CFU-GEMM population.Wright-Giemsa stain showed that its had the character of hematopoietic progenitors.Cells from the third-step were injected into SCID mice, but no teratomas were recovered in 2 mices after 6 weeks.Positive selection of CD34+/Sca-1+ cells by magnetic sorting from the third - step differentiation were transplanted into 7 lethally irradiated female mice while predominant hematopoietic reconstitution were observed in 10 days after transplantation, with 71.4% successful engraftment rate.And 3 recipients showed that the cell population of the peripheral blood leukocytes,red cells, hemoglobin approached normal index at 40 days after transplant, hut followed relative'slow renew in platelet count.Survival rate of transplant group is 43.0%, compared to 100% mortality in control mice.Karyotyping assays confirmed female mice with XY.Conclusions The results showed that the three-step differentiation and the culture conditions described here good support differentiation of ESC into HSC.HSC derived from ESC were safe without teratomas formation in body, and its can reconstruct hematopoiesis.

Objective To investigate the expressions of microRNA-21 in cholangiocarcinoma tissues and the relationship between epithelial-mesenchymal transition (EMT) and the prognosis of patients.Methods Forty-one samples of cholangiocarcinoma and 10 samples of adjacent tissues from 10 patients who received radical resection of cholangiocarcinoma at the Provincial Hospital of Anhui Medical University from January 2005 to January 2010 were collected.The expressions of microRNA-21,E-cadherin and N-cadherin were detected by in situ hybridization and immunohistochemistry,and effect of their expressions on the prognosis was analyzed.Enumeration data were analyzed using chi-square test.The correlation between microRNA-21 and EMT markers was analyzed using the Spearman correlation coefficient.The survival curve was drawn by Kaplan-Meier method,and the survival rate was analyzed using the Log-rank test.Results The expression rate of microRNA-21 in the cholangiocarcinoma tissues was 63％,which was significantly higher than 30％ of that in the adjacent tissues (x2 =0.324,P ＜ 0.05).The expression of microRNA-21 was closely related with the tumor differentiation degree,lymph node metastasis,perineural invasion (x2 =6.365,0.552,11.896,P ＜ 0.05),but not with gender,age,tunor location and tumor type (x2 =0.322,0.588,0.510,0.256,P ＞ 0.05).The expressions of E-cadherin and N-cadherin were related with lymph node metastasis and perineural invasion (x2 =4.630,5.512;6.600,7.152,P ＜0.05),but not with gender,age,tumor location,tumor differentiation degree and tumor type (x2 =0.266,0.013,0.067,0.666,0.003; 1.036,0.997,1.808,2.997,0.812,P ＞0.05).A positive correlation between the expression of microRNA-21 and EMT related markers E-cadherin and N-cadherin was detected (r =0.373,0.614,P ＜0.05).The results of survival analysis showed that the overall survival rate and tumor-free survival rate of patients with low expression of microRNA-21 were significantly higher than those of high expression of microRNA-21 (x2 =3.999,4.376,P ＜ 0.05).Conclusion Over expression of microRNA-21 in cholangiocarcinoma and metastatic lymph nodes may accelerate the invasion and metastasis of cholangiocarcinoma through inducing EMT,microRNA-21 might predict the prognosis of patients.

0.05).Conclusion There may be no significant difference in sensory block,motor block and prevalence of adverse effects between injection rates of 0.27 mL/s and 0.04 mL/s in spinal anesthesia with 20 mg isobaric ropivacaine.

Objective To establish a risk assessment model for laparoscopic partial nephrectomy (LPN) with segmental renal artery clamping (SRAC).Methods In the first stage,107 patients who underwent LPN with SRAC from December 2009 to September 2011 were retrospectively reviewed.There were 63 men and 44 women,aged from 11-80 years,mean (58.1 ±10.1) years.The blocking program of target arteries was dependent on the experience of the surgeon and CTA.After multiple Logistic regression analysis,variables used to build a nomogram were selected using a backward elimination scheme.Then,a model for a clamping program customized to the patient was designed.In the second stage,the surgical outcomes of 141 patients who subsequently underwent LPN-SRAC with the applied model from October 2011 to June 2014 were compared with those of the first stage patients.There were 84 men and 57 women,aged from 51-75years,mean (59.9 ± 8.6) years.Outcomes of two stages were compared.Results Five potential predictors were initially assessed including segmental renal artery angle,target artery diameter,and distance to the abdominal aorta,renal hilum,and kidney midline.The regression equation was then set up with Logistic regression analysis.Compared with the patients in the first stage,success rate of clamping in the second stage,with the help of the new SRAC model,was improved from 74.8％ (175/234) to 94.2％(227/241) (P ＜ 0.001),the total operative time was decreased from (88.6 ± 10.9) min to (80.2 ±16.6) min (P＜O.001),and operative blood loss was reduced from (198.7 ±111.6) ml to (168.5 ±117.8) ml (P =0.042).No obvious differences were observed in warm ischemia time,postoperative hospitalization,R.E.N.A.L nephrometry score,or number of final clamped branches.Conclusion The model for assuring clamping success was helpful in designing an SRAC program.

Objective To explore the effects and safety of endoscopic parallel placement of double metal stents on unresectable hilar malignant obstruction.Methods The clinical data of 11 patients with malignant hilar obstructive jaundice due to advanced carcinoma who were treated with parallel placement of double biliary stents from January 2011 to September 2012 were retrospectively analyzed.Results Out of 11 patients,10(90.9％) were successfully embedded with double biliary stents and 4 were dead during the follow-up.There was no sign of stent occlusion during the follow-up period.The survival time ranges from 128 to 185 days.One case was lost during the follow-up and 5 others are still alive.Conclusion The endoscopic parallel placement of double biliary stents is effective and safe for patients with unresectable malignant hilar obstruction.

Objective To study the in vivo expression and biodistribution of Ad5-Fluc (Adenovirus carrying firefly luciferase genes) in mice.Methods The recombinant Ad5-Fluc virus was constructed and infected to BALB/c or nude mice through three different routes.The protein expression level,tissue distribution and the characteristics of infection were analyzed by in vivo bioluminescence imaging technology.Results Compared to other two routes,the BALB/c mice infected through muscular route had the longest expression cycle (over 60 days) and the highest expression level,while the virus was transferred into the liver and spleen after infection.The nude mice had a significantly extended expression cycle than BALB/c mice.Moreover,the characteristic of liver tropism was eliminated after Ad5 F35 infection in mice,while maintained similar expression efficiency.Conclusion Due to the highest expression efficiency,the muscular route would be the optimal route for Ad5 vector based vaccination.In addition,Ad5F35 virus could become an ideal alternative vaccine vector for eliminating the liver tropism.

Objective To know the prevalence of chronic obstructive pulmonary disease(COPD) in patients with obstructive sleep apnea-hypopnea syndrome(OSAHS) in Kunming Yunnan Province, and the clinical symptom of OS.Methods Retrospective study of 4 636 cases of patients with snoring, excluding COPD in addition to chronic respiratory disease and assess the patient`s condition.The test included AHI, BMI, Epworth sleepiness scale, lung function.The index of OS was confirmed by AHI>5 times/h and FEV1/FVC<70%.Results During the period of 2006 to 2012, he prevalence of COPD in patients with OSAHS was 10.1% [95% confidence interval (CI) 9.1%-11.1%] in Kunming Yunnan Province.And male`s OS prevalence rate is higher than the female(male 10.2%;female 9.7%).The OS patients` average age was 56.9±14.1, the mean AHI was (47.46±26.79) times/h, and the average FEV1/FVC was 60.09%±23.57%.Polysomnographyresults show that patients suffered obvious hypoxiaat night in both OSAHS group and OS group, but it was more significant in OS group.Pulmonary function test showed that OS patients have more serious chronic obstructive pulmonary disease.Conclusions The prevalence of COPD in OSAHS patients was high in Kunming, Yunnan Province, and the prevalence rate in old group reached more than 24%.In addition, the sympotms of patients with OS were more severe than those with only OSAHS or COPD in lung function and hyoxemia.

Objective To determine the effect of mild hypothermia on neonatal piglet cardiac hemodynamic function after hypoxia-ischemia (HI).Method Twenty five 7-day-old piglets were used for hypoxic ischemic brain damage (HIBD) model by the method of temporary occlusion of the bilateral carotid arteries and followed by mechanical ventilation with low concentration of oxygen (FiO_2=6%) for 30 minutes.The piglets were randomly divided into three groups:group A (normothermia with body temperature to 39℃,n=9),group B (body temperature to 36℃for 72 hours,n=8),and group C (body temperature to 34℃for 72 hours,n=8).Mild hypothermia was initiated at 4 hours after HI,the systolic and diastole function were evaluated by Doppler echocardiography at pre-HI,post-Hi 4 hours and post-HI 72 hours.Results There were no significant differences in left ventrieular ejection time/left ventrieular ejection time (LPEP/LVEF),right ventricular ejection acceleration time/right ventricular ejection time (RACT/RVET) and CO at post-HI with hypothermia 72 hours in three groups,but the heart rate decreased in B and group C group.Compared with nonnothermia,mild hypothermia treatment showed no significant differences in MAP,LPEP/LVET,RACT/RVET,CO,SV at post-HI with hypothermia 72 hours.Conclusions Body temperature decreased by 3～5℃for 72 hours will not aggravate hemodynamic abnormity.

OBJECTIVEA large body of experimental and clinical observations indicates that disturbances in cerebral blood flow (CBF) and impaired cerebrovascular autoregulation are important in the pathogenesis of germinal matrix-intraventricular hemorrhage (GMH-IVH) and periventricular leukomalacia (PVL), the 2 most important forms of brain injury in pretmature infants. Near-infrared spectroscopy (NIRS) has been used recently to estimate CBF in human newborns. The objectives of this study were to evaluate the correlation of NIRS estimations and cerebral blood flow in newborn piglets, which in turn may help provide the ideal NIRS estimation reflecting the changes of cerebral blood flow and cerebrovascular autoregulation.

METHODSTen newborn piglets, aged 1 - 3 days, were randomly assigned to one of the following groups: normal control group (n = 6) and hypotension group (n = 4). Hypotension was induced by withdrawing blood from an arterial catheter. We NIRS was used to determine quantitative changes in cerebral concentrations of oxygenated hemoglobin (DeltaHbO(2)) and deoxygenated hemoglobin (DeltaHHb), then calculated NIRS estimations DeltatHb (DeltaHbO(2)+DeltaHHb) and DeltaHbD (DeltaHbO(2)-DeltaHHb). Cerebral blood flow (CBF) was determined by colored microspheres, and mean artery blood pressure (MABP) measured by arterial catheter pressure transducer was recorded simultaneously. Linear regression methods were used to analyze the relationships between NIRS estimations, CBF measured by micropheres, and MABP.

RESULTSThe correlation of NIRS estimations and CBF was quantitated by calculating coherence scores. A coherence of 1.0 indicates perfect correlation, a coherence of 0 indicates a complete lack of correlation. In the norm group, the experimental study showed strong correlations beween DeltaHbD, DeltatHb and changes in global CBF (GCBF), cerebral cortex CBF (CBFc), coherence scores r(1a) = 0.409, r(1b) = 0.440, r(2a) = 0.394 and r(2b) = 0.400, respectively, P < 0.05. In the hypotension group, the decrease of CBF was significant when the MABP dropped to 35 mm Hg (P < 0.05). With the decreasing MABP, there was a notable increase of DeltaHHb (P < 0.01), a modest increase (P < 0.05) at the beginning and then a marked fall (P < 0.01) of DeltaHbO(2) and DeltatHb was noted when the MABP dropped to 35 mm Hg. DeltaHbD decreased in parallel with the decline in CBF determined by colored microspheres, DeltaHbD varied with CBF during hypotensive episodes. Notably, there was a very strong correlations between DeltaHbD and changes in CBF (coherence scores GCBF r(3a) = 0.890, CBFc r(3b) = 0.887, P < 0.01); Importantly, decreases in DeltatHb did not correlate significantly with decreases in CBF during hypotension (coherence scores GCBF r(4a) = 0.395, CBFc r(4b) = 0.375, P > 0.05). Concordant changes (correlation coefficient > 0.5) in DeltaHbD, CBF and MABP, consistent with impaired cerebrovascular autoregulation, were observed in newborn piglets when MABP was less than 35 mm Hg. When MABP was more than 35 mm Hg, newborn piglets with intact cerebrovascular autoregulation in which CBF are maintained constant despite alternations in MABP have shown inconsistent changes in DeltaHbD, CBF and MABP (correlation coefficient < 0.5).

CONCLUSIONDeltaHbD signal is more sensitive to changes in CBF than DeltatHb signal, in terms of cerebral hemodynamic changes both in normal and hypotensive conditions, while DeltatHb in normal condition. The lower limit of CBF autoregulation in newborn piglets aged 1 - 3 days was 35 mm Hg, and correlation between NIRS estimation (DeltaHbD) and MABP could be used to identify cerebrovascular autoregulation in newborn piglets.

Animals ; Animals, Newborn ; Blood Flow Velocity ; drug effects ; Cerebrovascular Circulation ; physiology ; Homeostasis ; Hypotension ; physiopathology ; Oxygen ; metabolism ; Spectroscopy, Near-Infrared ; methods ; Swine

OBJECTIVETo study the change in regulation of cerebral blood flow (CBF) and cerebral metabolism rate (CMR) during selective head cooling (SHC) in newborn piglets.

METHODSSixteen newborn piglets, aged 5 approximately 7 days, were randomly assigned to one of the following three groups: SHC in normal piglets (n = 4), SHC after hypoxia-ischemia HI (n = 6) and normal temperature after HI (n = 6). Nasopharyngeal temperature was respectively reduced to 35 degrees C and then 32 degrees C in SHC group. HI was induced by using temporary occlusion of the bilateral carotid arteries and mechanical ventilation with low concentration of oxygen (6%) for 30 minutes. CBF was measured with color microspheres method. Mixed venous blood and arterial sample were collected at the same time from superior sagittal sinus and forelimb artery respectively for blood gas analysis, blood glucose and lactate measurement. Cerebral oxygenation metabolism rate (CMRO(2)), cerebral glucose metabolism rate (CMR(Glu)) and cerebral lactate production (CLP) were calculated.

RESULTSIn normal piglets, CBF, CMRO(2) and CMR(Glu) were significantly decreased at 35 degrees C and 32 degrees C respectively, while CLP did not change. When nasopharyngeal temperature decreased to 35 degrees C, The ratios of CBF/CMRO(2), CBF/CMR(Glu) and CBF/CLP did not significantly change, coupling of CBF and CMR was maintained. While nasopharyngeal temperature decreased to 32 degrees C, the ratios of CBF/CMR(Glu) and CBF/CLP significantly decreased. Maintained newborn piglets normal temperature after HI, the CBF and CMRO(2) were significantly reduced, while CMR(Glu) and CLP were markedly increased. The ratio of CBF/CMRO(2) increased, CBF/CMR(Glu) and CBF/CLP decreased. Uncoupling of CBF and CMR was observed. During SHC after HI, the same decrease was observed in CBF and CMR(Glu) and CLP was markedly reduced. The ratio of CBF/CMRO(2), CBF/CMR(Glu) and CBF/CLP were restored at 35 degrees C and 32 degrees C as compared to the same time point in normal temperature, respectively.

CONCLUSIONCBF and CMR decreased in normal piglets during SHC and coupling of CBF and CMR was maintained. After HI, CBF was reduced and the disturbance of cerebral oxygenation metabolism occurred. CBF and CMR was uncoupled. SHC after HI can improve CMR and correct uncoupling of CBF and CMR.

Animals ; Animals, Newborn ; metabolism ; Blood Glucose ; Brain ; blood supply ; metabolism ; Cerebrovascular Circulation ; Cold Temperature ; Hypothermia, Induced ; Hypoxia-Ischemia, Brain ; metabolism ; Lactic Acid ; blood ; Oxygen Consumption ; Swine ; metabolism