Objective:To develop specific anti-AML T cells in vitro,and to study their biological characteristics and functions.Methods:Peripheral blood or bone marrow mononuclear cells (MNCs) were isolated from 12 patients with AML,and co-cultured with cytokine combinations in 96 well plates to be induced into dendritic cells (DCs).Cell morphology was observed under an inverted microscope during the first week and immunophenotype was detected by flow cytometry at day 7.Cytokine combination was replaced with high dose IL-2 at day 7 to promote specific anti-AML T cells.T cell phenotype was detected after 4 to 5 weeks.Lactate dehydrogenase (LDH) release assay was used to determine T cell killing activity.Results:After being cultured with cytokine combinations,the typical dendritic appearance with delicate membrane projections was observed.CD80,CD86 and HLA-DR markers were significantly upregulated(P

OBJECTIVE To investigate the effect of root saponins of Panax notoginseng(RPNS) on different voltage-dependent calcium and potassium ion channels. METHODS By using the two-elec?trode voltage clamp (TEVC), the effect of RPNS 0.01, 0.06, 0.1, 0.6, 1 and 4 g · L- 1 was investigated on Cav1.2,and the effect of RPNS 1 g · L-1 was evaluated on Cav2.1,Cav2.2,Cav3.1, KCNH2,KCNQ1,KCNQ1/KCNE1 and BK channel. All the ion channels examined were expressed in Xenopus laevis oocytes. RESULTS TEVC suggested that the effect of RPNS on Cav1.2 exhibited the concentration-response relationship and its EC50 was 0.048 g · L-1. Compared with cell control,TEVC also showed that RPNS 1 g·L-1 had obviously inhibitory effect on Cav1.2,Cav2.2 and Cav3.1,and the inhibitory rate of RPNS 1 g · L-1 on the peak current of Cav1.2,Cav2.2 and Cav3.1 was(57.1 ± 8.6)%, (17.2 ± 0.7)% and(50.2 ± 7.7)%(P<0.01),respectively. RPNS 1 g · L-1 had obviously activated effect on BK channel,and the activated rate of RPNS 1 g·L-1 on the peak current of BK channel was(37.9± 2.7)%(P<0.01). RPNS 1 g·L-1 showed no significant effect on Cav2.1,KCNH2,KCNQ1 and KCNQ1/KCNE1. CONCLUSION RPNS may effectively inhibit Cav1.2 and Cav3.1,activate BK channel,but have little effect on Cav2.1,Cav2.2,KCNH2,KCNQ1 and KCNQ1/KCNE1.

AIM: To investigate the feasibility and efficacy of tissue plasminogen activator (tPA) injection into the optic nerve as a treatment for retinal vein occlusion in rabbits. METHODS: Rose Bengal-mediated laser-induced retinal vein occlusions were produced in rabbit eyes. Fluorescein angiography (FA) was performed on each eye 3 days before laser irradiation and 30 minutes after laser irradiation.The treatment group (n=20 veins) received intra-optic nerve injection of tPA (12.5μg in 0.05mL BSS) and the controls (n=24 veins) received 0.05mL BSS. FA was repeated to determine the recanalization of the vessel at 3 and 7 days after treatment, followed by histological examination.RESULTS: Rose Bengal-mediated laser-induced retinal vein occlusions were successfully developed and confirmed by FA. The incidence of the recanalization of the vessels in treatment animals was 70.0%, while 16.7% in the control animals (P=0.001). CONCLUSION: Intra-optic nerve tPA injection increased the incidence of recanalization of the occluded vessels. Although further studies are needed, our data suggested that injection of tPA into the optic nerve may have a potential benefit in the treatment of central retinal vein occlusion.

AIM: To investigate the safety of tissue plasminogen activator (tPA) intra optic nerve injection in rabbits. METHODS: Group 1 and 2 (6 eyes in each group) received injection of tPA 25μg and 12.5μg in 0.1mL balanced saline solution (BSS). Group 3 (6 eyes) received injection of 0 1mL BSS. Six eyes in group four as a normal control received no injection. The eyes were examined with slit lamp biomicroscope, indirect ophthalmoscope, visual evoked potentials (VEP) and electroretinography (ERG) at 1, 3, 7, 14 and 28 days after injection. RESULTS: No evidence of optic nerve or retinal toxicity or physical damage were revealed by ophthalmoscopy, VEP, and ERGs after the injection of tPA into the optic nerve. The means of the latency of the first peak of the VEP were 24.6±1.5, 24.1±1.9, 24.0±2.0 and 24.6±1.3mS respectively for the above specified groups (P=0.4112). The means of the amplitude of the first peak of the VEPs were 124±42, 145±41, 132±48 and 117±29μV respectively (P=0.0649). The means of the latency of a-waves were 6.0±0.4, 5.9±0.4, 5.9±0.5 and 5.8±0.3 mS respectively (P=0.6279). The means of the amplitude of a-waves were 110±14, 112±15, 110±16 and 108±11μV respectively (P=0.7248). The means of the amplitude of b-waves were 151±12, 148±14, 144±16 and 141±20μV respectively (P =0.0957).CONCLUSION: Injection of tPA upto 25μg in 0.1mL into optic nerve is well tolerated.

Objective To evaluate the effects of early tracheotomy and delay tracheotomy on the prognosis of patients with severe craniocerebral injury.Methods Data of randomized controlled test (RCT) of early tracheotomy on the prognosis of the patients with severe craniocerebral injury were retrieved by using Conchrane Library,PubMed,Embase,OVID,CBM,CNKI,Wanfang and VIPdatahase.The retrievaled time was limited from their building to March 2016,and all the index of the obtained references was accessed.Two researchers independently screened the literature in accordance with the established criteria for inclusion or exclusion.The incidence of VAP,mechanical ventilation time and ICU length of hospital stay,mortality were considered as evaluation indexes.The methodology quality of the included references was evaluated,and the Meta-analysis was performed by using Review Manager 5.3 software.Results Finally,4 randomized controlled studies (RCT) were enrolled,including 220 patients.Meta-analysis results showed that:early tracheotomy did not have any effects on VAP incidence rate (P=0.15) and fatality rate (P=0.20) of the patients with severe craniocerebral injury,there was not any statistically difference;but there was a statistically difference between the mechanical ventilation (P =0.01) and ICU hospitalization time (P =0.05).Conclusion Early tracheostomy could not significantly reduce the incidence of VAP and fatality rate of the patients with severe craniocerebral injury,but it can reduce the mechanical ventilation time and ICU hospitalization time.

OBJECTIVETo screen the lentiviral vector carrying siRNA and capable of significantly suppressing the ROCK2 gene expression in the corpus cavernosum smooth muscle cells of spontaneously hypertensive rats (SHR).

METHODSWe designed and synthesized 4 siRNA fragments targeting the ROCK2 gene and packaged them into lentiviral vectors. We collected corpus cavernosum smooth muscle cell samples from 5 male SHRs and randomly divided them into groups A (non-transfection control), B (GFP lentiviral transfection), C, D, E, and F (lentiviral transfection with siRNA fragments 1 -4 targeting the ROCK2 gene). Each group consisted of 5 samples and each sample 3 x 10(4) cells. At 48 hours after transfecting MOI = 80 into the SHR corpus cavernosum smooth muscle cells, we detected the expression of GFP under the fluorescent microscope and the mRNA expression of the ROCK2 gene by RT-PCR.

RESULTSThe transfection efficiency of the SHR corpus cavernosum smooth muscle cells was > 50%. Compared with group A, the expression of ROCK2 mRNA in the corpus cavernosum smooth muscle cells showed no remarkable change in group B (P > 0.05) but was inhibited very significantly in C ([43.91 +/- 8.19]%), D ([47.15 +/- 6.64]%), and F ([25.7 +/- 6.03]%) (P < 0.01), and significantly in E ([16.81 +/- 5.94]%, P < 0.05).

CONCLUSIONWe successfully constructed 4 lentiviral vectors carrying siRNA targeting the ROCK2 gene, all of which can significantly suppress the ROCK2 expression in the SHR corpus cavernosum smooth muscle cells, and one has a highly strong inhibitory effect.

Animals ; Gene Silencing ; Genetic Vectors ; Lentivirus ; genetics ; Male ; Myocytes, Smooth Muscle ; Penis ; cytology ; RNA, Messenger ; metabolism ; RNA, Small Interfering ; Random Allocation ; Rats ; Rats, Inbred SHR ; Transfection ; methods ; rho-Associated Kinases ; genetics

Objective To investigate the therapeutic effects of early enteral nutrition by gastroscopeguided naso-jejunal feeding tube placement on the intestinal endotoxemia of patient with severe acute pancreatitis (SAP).Methods Fourty-three patients were randomized into two groups:patients receiving early enteral nutrition (EN) by gastroscope-guided naso-jejunal feeding tube placement (24 cases) and those receiving total parenteral nutrition (TPN) ( 19 cases).The serum endotoxin(ET),albumin (ALB) and amylase (AMY) levels were measured.Abdominal distension and other complications were observed in the two groups.Results The abdominal pain and distension relief time,intestinal bleeding,infectious complications of EN group were significantly improved compared with that of TPN group ( P ＜ 0.01 或 P ＜ 0.05 或 P ＜ 0.001 ).The serum ET levels of EN group was much lower ( [ 0.19 ± 0.11 ] EU/ml) than that of TPN group ( [ 0.85 ± 0.28 ] EU/ml)on day 14 post-treatment (t =10.456,P ＜ 0.001 ).The serum AMY levels were decreased significantly in two groups after treatment,and the difference between two groups was not significant (t =3.l17,t =1.889,P ＞0.05 ).The serum ALB recovery of two groups was not significantly different ( P ＞ 0.05 ).Conclusion Gastroscope-guided Naso-jejunal feeding tube placement for early enteral nutrition can protect intestinal mucosa,reduce complications,alleviate symptoms of patients with SAP,which are benefitial factors for the treatment of intestinal endotoxemia in patients with SAP.

AIM:To investigate the characteristics of Ets-1 and VEGF expression and distribution in the experimental diabetic rat retina.METHODS:Diabetes was induced by intraperitoneal injection of streptozotocin (STZ).At 4 weeks after STZ-injection,animals were sacrificed.Total proteins were isolated from retinas of experimental and control eyes and were assessed by Western blot analysis.Frozen cross sections of eyeballs with 14um thickness were used to perform double immunoffuorescence staining with anti-Ets-1 and anti-VEGF antibodies.RESULTS:Both Ets-1 and VEGF expression were up-regulaled in the diabetic retina,the distribution of Ets-1 and VEGF was identical to each other,and the two proteins were almostlocalized in all retinal layers.CONCLUSION:Ets-1 might contribute to the pathologic progress of the diabetic retina induced by VEGF.

AIM: To determine the involvement of Ets-1 in the pathological progress of the experimental diabetic retina. METHODS: Diabetes was induced by intraperitoneal injection of STZ. Total RNA and Total proteins were isolated from retinas of experimental and control eyes at 4 weeks after STZ-injection and were assessed by Northern blot analysis and Western blot analysis, respectively.RESULTS: Expression of both Ets-1 mRNA and Ets-1 protein was significantly increased in the experimental diabetic rat's retina after STZ-injection compared with the control group (P＜0.001).CONCLUSION: Our results indicated that Ets-1 was involved in the pathological progress of experimental diabetic retina.Further studies should be conducted to focus on the relationship between Ets-1 and VEGF in the diabetic retina.

AIM:To investigate the role of lipopolysaccharide binding protein (LBP) for diagnosis and prog-nosis prediction in the septic patients.METHODS:A total number of 80 ICU patients were enrolled.The patients were divided into systemic inflammatory response syndrome ( SIRS) group and sepsis group, the patients in sepsis group were di-vided into non-survivor sub-group and survivor sub-group.We collected the serum samples and analyzed acute physiology and chronic health evaluation ( APACHE) II score on the first day of the patients hospitalized in ICU.In addition, we also selected 10 healthy volunteers and collected their serum samples.The serum concentrations of LBP, C-reactive protein ( CRP) and procalcitonin ( PCT) were measured by ELISA.ROC analysis of LBP, CRP, PCT and APACHE II score was conducted to discriminate among critically ill patients with sepsis and predict the prognosis of the patients with sepsis.RE-SULTS:The levels of the 4 indicators in the septic patients were higher than those in the patients of SIRS (P<0.05).In addition, serum LBP and APACHE II score in the non-survivor sub-group were higher than those in the survivor sub-group (P<0.05), whereas no difference of the PCT and CRP levels between survivors and non-survivors with sepsis was ob-served.LBP levels greater than 26.84 mg/L had 97.1% sensitivity and 95.9% specificity to discriminate between SIRS and sepsis.LBP levels greater than 54.16 mg/L had 85.2%sensitivity and 80.0%specificity for prognosis of unfavorable outcome.CONCLUSION:LBP level was more accurately correlated with diagnosis or prognosis prediction than CRP or PCT in patients with sepsis.