Objective To evaluate the effects of rigorous surveillance and retroperitoneal lymph node dissection (RPLND) in the treatment of low-risk patients with clinical stage Ⅰ nonseminomatous germ cell testicular tumors (NSGCT) after radical orchiectomy.Methods The data of 71 patients with clinical stage Ⅰ NSGCT were analyzed retrospectively in Hunan Provincial Tumor Hospital,Xiangya Third Hospital of Central South University and Hunan Provincial People's Hospital between Feb,2001 and Apr,2012.Excluding lymphatic and vascular invasion,percentage of embryonal carcinoma＞50％ and increasing tumour markers (AFP/β-HCG) following orchiectomy,46 low-risk patients out of 71 patients with clinical stage Ⅰ NSGCT were selected and divided into rigorous surveillance group (30 cases) and RPLND group (16 cases) according to different therapeutic methods after radical orchiectomy.Univariate analysis was used to confirm variables associated with disease progression,and the disease free survival rates (DFSR) were compared by using Kaplan-Meier analysis.Results Five cases were lost,and 41 cases were followed up for an average of 61 months (range,15-147 months),with 58 months in rigorous surveillance group (range,19-147months) and 65 months in RPLND group (range,15-144 months).The survival rate was 100％ in 2 groups.The DFSR was 89％ (24/27) and 86％ (12/14),respectively,and there was no significant difference between the 2 groups (x2 =0.08,P=0.78).The DFSR was 83％ in patients with small amout of embryonal (percentage of embryonal carcinoma ＜ 50％),and 92％ in patients without embryonal carcinoma,and there was no significant difference between the 2 groups (x2=1.07,P=0.30).Also there was no significant difference between the patients less than 15 years and patients more than 15 years (x2=1.59,P =0.21).Conclusions There is no significant difference in recurrence rate and DFSR between rigorous surveillance group and RPLND group.Low-risk patients with clinical stage Ⅰ NSGCT may achieve satisfactory prognosis with surveillance after radical orchiectomy.

OBJECTIVETo investigate the mechanism of decreasing the K562/ADM cells chemosensitivity by up-regulating expression of PTEN gene.

METHODSK562/ADM cells were transient transfected with pGFP-PTEN or vector. The level of PTEN in K562/ADM cells was assayed by Western blot and RT-PCR. Cell viability on K562/ADM was determined by MTT assay. Cell apoptosis by flow cytometry. Activity of caspase-3 by Caspase Colorimetric Assay Kit. The proteins expression of LC3-I/II, Beclin1, p-Akt, p-p70S6K by Western blot. The autophagic vacuoles by MDC stain and Electron microscopy.

RESULTS(1) The mRNA and protein levels of PTEN in K562/ADM cells transfected with pGFP-PTEN were significantly increased compared with the control (untreated and transfected with empty vector). (2) Enhanced expression of PTEN by gene transfection resulted in a reversal of resistance to ADM. Compared with empty vector group, cell viability decreased from (94.07 ± 2.6)% to (53.83 ± 4.2)%, the cell apoptotic rate increased from (11.89 ± 1.7)% to (43.69 ± 2.3)%, meanwhile, pretreated with caspase-3 inhibitor (Z-DEVE-FMK) didn't completely inhibit the cytotoxicity of ADM to K562/ADM cells. (3) After treated with ADM for 12 and 24 h, the activities of caspase-3 in PTEN-transfected K562/ADM cells increased compared with those in pGFP-transfected K562/ADM cells \[(2.27 ± 0.13) vs (1.19 ± 0.14)\] at 12h, \[(3.15 ± 0.08) vs (1.48 ± 0.05)\] at 24 h (P < 0.05). (4) The protein levels of LC3-II and Beclin1 in K562/ADM cells transfected with pGFP-PTEN were increased by 83% and 18% respectively, and the protein levels of p-Akt and p-p70S6K were declined by 96% and 87% respectively, compared with those in K562/ADM cells transfected with pGFP plasmid. (5) The upregulation of PTEN in K562/ADM cells improved the number of autophagic vacuoles compared with the empty vector group.

CONCLUSIONThe upregulation of PTEN expression increases the chemosensitivity of K562/ADM to ADM, which may related with the inhibition of PI3K/AKT/mTOR pathway induced by PTEN gene transfection.

Apoptosis ; Drug Resistance, Multiple ; Drug Resistance, Neoplasm ; Gene Expression Regulation, Leukemic ; Genetic Vectors ; Humans ; K562 Cells ; Leukemia ; drug therapy ; PTEN Phosphohydrolase ; genetics ; metabolism ; Transfection ; Up-Regulation

Objective To investigate the effect of microRNA-223-3p (miR-223-3p) on megakaryocytic differentiation and maturation, and explore the potential mechanism .Methods The endogenous expression of miR-223-3p during megakaryocyte ( MK) differentiation was detected by real-time PCR.Flow cytometry further indicated that alteration of miR-223-3p in human cell lines exerted effects on MK differentiation and maturation .By performing integrative bioinformatic analysis, the potential miR-223-3p target gene, MYH10,was identified.Real-time PCR, luciferase reporter assay and flow cytometry revealed that MYH10 was a direct target of miR-223-3p.Results Endogenous expression of miR-223-3p was in-creased with the differentiation and maturation of MK .The expression of megakaryocytic surface markers CD41 and CD61 and the ploidy were significantly increased in K562 and Meg-01 cells after transfection with miR-223-3p mimics.The expression of MYH10 decreased with the increase in miR-223-3p.Using a luciferase reporter assay ,we demonstrated that MYH10 was a direct target of MiR-223-3p.Furthermore, direct downregulation of MYH10 promoted MK polyploidization . Conclusion MiR-223-3p might regulate the polyploidization of MK by targeting MYH10.

Objective To investigate the impact of the initial fluid resuscitation with different ratio of crystalloid and colloid on the prognosis of patients with moderate severe acute pancreatitis (MSAP).Methods A retrospective analysis was made by reviewing the clinical data of 72 patients with the diagnosis of MSAP from January 2015 to July 2017 in Shanghai Changhai Hospital.According to crystalloid-colloid ratio,which was the total volume of crystalloid fluid versus colloid fluid in the first 7d at admission,patients were randomly divided into low crystalloid-colloid ratio group (＜ 4.5),middle crystalloid-colloid ratio group (4.5-7.5),and high crystalloid-colloid ratio group (＞ 7.5).The parameters of the fluid resuscitation,the cases progressing into severe acute pancreatitis (SAP),the incidence of multiple organs dysfunction syndrome (MODS) and mechanical ventilation,pancreatic necrosis and infection rate,30-day mortality,the duration of systemic inflammatory response syndrome(SIRS) and the time reaching full amount of enteral nutrition were analyzed.Results There was no statistically significant difference in gender,age,etiology and APACHE Ⅱ score within 24 h at admission in each group,which were comparable.Within the first 7 d,there were no statistic difference in the total volume of fluid infusion and the speed of resuscitation in the three groups.While the total fluid volume in the first 24 h and 72 h [(3 095 ± 1 253) ml vs (2 524 ± 751) ml,(8 005 ± 7 269) ml vs (6 667 ± 1 498)ml],the total volume of crystalloid fluid in the first 7 d [(14 485 ± 3 917) ml vs (11 544 ±2 639) ml],crystalloid-colloid ratio (12.7 ± 4.9 vs 6.0 ± 1.0),the cases of SAP (12 vs 4),MODS (41.7 ％vs 16.0％) in high ratio group were significantly higher than those in middle ratio group,but the total volume of colloid fluid was significantly lower [(996 ± 528) ml vs (1 968 ± 574) ml].In addition,the duration of SIRS [(16.5 ± 15.2) d vs (8.2 ± 6.4) d],and the time reaching full amount of enteral nutrition [(7.2 ±3.6) d vs (4.8 ± 2.4) d] in high crystalloid-colloid ratio group were higher than those in middle crystalloid-colloid ratio group (all P ＜ 0.05).Comparing with middle crystalloid-colloid ratio group,there were no significant difference in the mechanical ventilation rate,pancreatic necrosis and infection rate and 30-day mortality in high ratio group.The total volume of colloid fluid was significantly higher [(3 680 ± 1 310) vs (1 968 ±574)] and the crystalloid-colloid ratio was significantly lower [(3.2 ±0.9) vs (6.0 ± 1.0)] in low ratio group than that in middle ratio group,and there were no statistical differences on other parameters.Conclusions For the patients with MSAP,early fluid resuscitation with the crystatloid-colloid ratio of (4.5-7.5) can decrease the incidence of SAP and MODS,shorten the duration of SIRS,and promote the recovery of intestinal mucosal barrier function.

Objective To investigate the correlation between the changes of serum S100B protein level in acute phase and the scores of mini-mental state examination (MMSE) in patients with brain concussion, and evaluate the role of serum S100B protein level in the prognosis of cognition disorders after brain concussion. Methods The serum S100B protein level was determined by an enzyme linked immunosorbent assay (ELISA) in 126 cases of brain concussion 6 and 12 h, and 3 d after admission, and these data were compared with those in 96 cases of moderate head trauma without transitory loss of consciousness (admitted to our hospital at the same period, control group). MMSE was performed 1 and 14 d and 3 months after injury, and the correlation between the changes of serum S100B level in acute phase and MMSE scores was observed. Results As compared with that in control group,the serum S100B protein level in patients with brain concussion was significant higher at 6 and 12 h after admission(P＜0.05). The serum S100B protein level at 6 h, but not at 12 h and 3 d after admission, was closely associated with the MMSE scores 1 and 14 d and 3 months after injury. Conclusion Early elevation of S100B within 6 h of admission in patients with brain concussion, obviously correlating with cognitive impairment, may serve as an important prognostic marker in predicting clinical outcome of cognition disorders after brain injury.

Objective To investigate the effective treatments of combined injury with craniocerebral firearm wound in dogs immersed by seawater under maritime environment. Methods Models of combined injury with craniocerebral firearm wound, including craniocerebral gunshot wound,open chest injury, open abdominal injury, open trauma of extremities and burn injury, were established in 60 healthy adult mongrel dogs. Animal models after being wounded were immersed by the seawater for 30 min, and then, they were equally randomized into conventional treatment group and comprehensive treatment group; 30 dogs in the conventional treatment group were given routine treatment and the other 30 dogs in the comprehensive treatment group were given lukewarm glucose liquid, β-aescin, naloxone hydrochloride, levofloxacin and re-warming treatments besides the conventional treatment. Transcranial Doppler ultrasound, blood gas analysis, measurement of plasma osmotic pressure and intracranial pressure (ICP) monitoring were performed on the dogs of the 2 groups; and the treatment efficacy of the 2groups were compared. Results Low incidence rate of brain vasospasm was noted and TCD indicated that blood flow speed approached normal in the comprehensive treatment group 3 h after the treatment.The plasma osmotic pressure and the indicators of metabolic acidosis reached normal levels in the comprehensive treatment group 12 h after the treatment. The ICP significantly decreased in the comprehensive treatment group 24 h after the treatment. Survival rate in the comprehensive treatment group (70%) was significantly higher as compared with that in the conventional treatment group (53%)7 d after the treatment (P＜0.05). All the indexes in the comprehensive treatment group were better than those in the conventional treatment group (P＜0.05) Conclusion Early infusion of lukewarm hypotonic solution can significantly reduce the osmotic pressure, correct the electrolyte balance, help the re-warming and prolong the survival rate. Naloxone possesses protective effect on brain. The β-aescine sodium can diminish viscosity, slow down brain edema progress, obviously reduce ICP and improve brain tissue oxygen metabolism. In a word, comprehensive treatment in effective in treating combined injury with craniocerebral firearm wound.

OBJECTIVETo evaluate the application of health assessment instruments in Chinese medicine.

METHODSAccording to a pre-defined search strategy, a comprehensive literature search for all articles published in China National Knowledge Infrastructure databases was conducted. The resulting articles that met the defined inclusion and exclusion criteria were used for analysis.

RESULTSA total of 97 instruments for health outcome assessment in Chinese medicine have been used in fundamental and theoretical research, and 14 of these were also used in 29 clinical trials that were randomized controlled trials, or descriptive or cross-sectional studies. In 2 152 Chinese medicine-based studies that used instruments in their methodology, more than 150 questionnaires were identified. Among the identified questionnaires, 51 were used in more than 10 articles (0.5%). Most of these instruments were developed in Western countries and few studies (4%) used the instrument as the primary evidence for their conclusions.

CONCLUSIONUsage of instruments for health outcome assessment in Chinese medicine is increasing rapidly; however, current limitations include selection rationale, result interpretation and standardization, which must be addressed accordingly.

Databases, Factual ; Humans ; Medicine, Chinese Traditional ; Outcome Assessment (Health Care) ; methods ; Randomized Controlled Trials as Topic ; Research Design

Objective To compare the clinical efficacy and safety of levetiracetam tablet and carbamazepine tablet in the treatment of children's intractable epilepsy.Methods A total of 96 children with intractable epilepsy were randomly divided into control group and treatment group with 48 cases per group.Control group was given carbamazepine 4-8 mg · kg-1 · d-1,tid,oral.Treatment group was given levetiracetam 4 mg · kg-1,bid,the maximum dose was 16 mg · kg-1 at the speed as 4 mg · kg-1 with every 2 weeks.Two groups were treated for 8 months.The clinical efficacy,neurocognitive function test [verbal intelligence quotient(VIQ),performance intelligence quotient (PIQ),total intelligence quotient(TIQ) and short-term visual memory],and adverse drug reactions were compared between two groups.Results After treatment,the total effective rates in treatment and control groups were 87.50％ (42 cases/48 cases) and 79.17％ (38 cases/48 cases) with significant difference (P ＜ 0.05).After treatment,the main indexes in treatment and control groups were compared:VIQ were (106.97 ± 5.65) and (95.25 ± 3.28) points,PIQ were (116.45 ± 5.16) and (103.61 ± 2.74) points,TIQ were(119.92 ± 4.69) and(95.20 ± 3.24) points,short-term visual memory were (18.45 ± 2.17) and (13.84 ± 1.81) s,the differences were statistically significant (all P ＜ 0.05).The adverse drug reactions of two groups were based on emotional,drowsiness,palpitations and dizziness,also,the incidences of adverse drug reactions in treatment and control groups were 12.50％ and 16.67％ without significant difference (P ＞ 0.05).Conclusion Levetiracetam tablet has a definitive clinical efficacy in the treatment of children's intractable epilepsy,which is better than carbamazepine tablet.Levetiracetam tablet can improve the cognitive ability for children's intractable epilepsy,without increasing the incidence of adverse drug reactions.

Objective:To investigate molecular mechanisms underlying the inflammatory response induced by Cutibacterium acnes ( C. acnes) biofilms in human primary keratinocytes. Methods:A C. acnes biofilm model was established in vitro, and confocal fluorescence microscopy was performed to examine its three-dimensional structure. The cultured human primary keratinocytes were divided into 3 groups: a dimethyl sulfoxide (DMSO) control group (treated with 0.01% DMSO alone), a C. acnes suspension group (co-incubated with C. acnes suspensions), and a C. acnes biofilm group (co-incubated with C. acnes biofilms). Real-time fluorescence-based quantitative PCR (RT-qPCR) was performed to determine the relative mRNA expression of interleukin (IL) -6, IL-8, and tumor necrosis factor (TNF) -α in the groups after 6-hour culture, enzyme-linked immunosorbent assay to detect the free protein levels of IL-6, IL-8, and TNF-α in the groups after 24-hour culture, and Western blot analysis to determine the protein expression of Toll-like receptor 2 (TLR2) in keratinocytes. In addition, some human primary keratinocytes were pretreated with key molecular blockers targeting the TLR2/mitogen-activated protein kinase (MAPK) /nuclear factor (NF) -κB signaling pathway (C29, ST2825, BAY11-7082, SB203580, U0126-EtOH), and then co-incubated with C. acnes biofilms; the DMSO control group and the C. acnes biofilm group receiving no pretreatment were simultaneously set as negative and positive controls, respectively. The mRNA and free protein expression levels of IL-6, IL-8, and TNF-α were then detected in the above groups. One-way analysis of variance was used for comparisons among multiple groups, and the Bonferroni method was used for multiple comparisons. Results:Confocal fluorescence microscopy demonstrated a three-dimensional C. acnes biofilm structure resembling a lawn, and the biofilm grew well. RT-qPCR and ELISA showed significant differences in the mRNA and free protein expression levels of IL-6, IL-8, and TNF-α among the C. acnes biofilm group, C. acnes suspension group and DMSO control group (mRNA: F = 89.70, 312.17, 46.09, respectively, all P < 0.001; free protein: F = 886.12, 634.25, 307.01, respectively, all P < 0.001) ; in detail, the mRNA and free protein expression levels of IL-6, IL-8, and TNF-α were significantly higher in the C. acnes biofilm group than in the C. acnes suspension group and DMSO control group (all P < 0.001) ; the C. acnes suspension group showed significantly increased expression levels of IL-6 mRNA and TNF-α free protein compared with the DMSO control group ( P < 0.001, = 0.003, respectively), while there were no significant differences in the expression of IL-6 free protein, TNF-α mRNA, or IL-8 mRNA and free protein between the 2 groups (all P > 0.05). Western blot analysis showed that the TLR2 protein expression was significantly higher in the C. acnes suspension group and C. acnes biofilm group than in the DMSO control group. After the pretreatment with molecular blockers targeting the MAPK/NF-κB signaling pathway and co-incubation with C. acnes biofilms, the mRNA and free protein expression levels of IL-6, IL-8 and TNF-α were all significantly lower in the C29 group, ST2825 group, BAY11-7082 group, SB203580 group, U0126-EtOH group, as well as in the DMSO control group compared with the C. acnes biofilm group (all P < 0.05) . Conclusion:The C. acnes biofilms exhibited a strong ability to induce inflammatory responses in human keratinocytes, possibly through the activation of the TLR2/MAPK/NF-κB signaling pathway.

OBJECTIVETo discover the techniques for ex vivo generation and cryopreservation of erythroid progenitor cells (EPCs)derived from umbilical cord blood (UCB)mononuclear cells (MNCs).

METHODSUCB was chosen as the source of EPCs. Erythrocytes were precipitated by hydroxyethyl starch (HES). MNCs were separated by Ficoll density gradient centrifugation. Erythroid progenitor cell were generated from MNC ex vivo in suspension culture supplemented with stem cell growth factor, insulin growth factor, erythropoietin, Fms- liketyrosinekinase ligand, transferrin and dexamethasone. Cell maturation was evaluated by morphologic analysis and CD71/CD235a expression profiling. In vitro induced cells were cryopreserved using different cryopreservation media. The cell survival rate, phenotype and proliferation curves were detected after cell thawing.

RESULTSWith the extension of culture time, the total number of cells increased significantly accompanied with the elevation of CD71 and CD235 positive populations. After 14- day inducing, the cells reached to approximately 110 times of the starting number with the cell viability as (88.92±0.95)%. The percentages of cell surface markers were (86.77±9.11)% for CD71 and (64.47±16.67)% for CD71/CD235, respectively. With the extension of inducing time, wright- Giemsa staining showed that the middle erythroblasts appeared mostly at day 10, and the late erythroblasts were seen at day 14. The red pellets were present at day 14, which indicated the more production of hemoglobin. Colony forming assay showed that erythroid colonies at induction day 7 were higher than that for non-induced cells (326.00±97.96vs 61.60±20.03 per 2 000 cells). With the extension of culture time, the number of erythroid colonies decreased. Induced EPCs were preserved with different cryopreservation solutions, in which 10% DMSO were better than 5% DMSO. Additionally, 10% DMSO + 2% HSA showed no different with 10% DMSO + 5% HSA. Combined 50% plasma with 2% HSA was more effective.

CONCLUSIONSThis non- serum culture media could effectively induced and expanded EPCs, and 10% DMSO + 2% HSA + 50% plasma appeared to be a desirable cryopreservation solution for EPCs from UCB.

Cell Culture Techniques ; Cell Differentiation ; Cell Survival ; Cells, Cultured ; Cryopreservation ; methods ; Erythroblasts ; cytology ; Erythroid Precursor Cells ; cytology ; Fetal Blood ; cytology ; Humans ; Leukocytes, Mononuclear ; cytology ; Umbilical Cord