0.05),but significant difference existed between two groups at 6 and 12 months after treatment(P

Objective To study the role and mechanism of microRNA-16(miR-16)in the proliferation,invasion and apoptosis of ovarian epithelial carcinoma cells in vitro.Methods The SKOV-3 cells were transfected with miR-16 mimics or negative control RNA(NC)by lipofectamine 2000.The expression of miR-16 was detected by real-time reverse transcription(RT)-PCR in SKOV-3 cells,and western blot was used to detect the expression of vascular endothelial growth factor(VEGF),matrix metalloproteinase-2(MMP-2)and bcl-2 protein.Methyl thiazolyl tetrazolium(MTT),5-ethynyl-2'-deoxyuridine(EdU)and transwell assay were used to determine the proliferation and invasion abilities.And the rate of apoptotic cell was detected by flow cytometry method.Results(l)The expression level of miR16 in the transfection cells group was significantly higher than that in NC group(125.93 ± 15.30 versus 0.78 ± 0.16,P ＜ 0.01).(2)The rclative expression level of VEGF protein in transfection cells,NC and blank control group was 0.58 ± 0.05,1.22 ± 0.03,1.20 ± 0.03,MMP-2 protein was 0.63 ± 0.03,1.16 ±0.03,1.21 ± 0.03,and bel-2 protein 0.52 ± 0.03,1.19 ± 0.05,1.28 ± 0.06,respectively.The level of VEGF,MMP-2 and bcl-2 protein in the transfection group were lower than those in other control groups,and there were significantly differences among them(all P ＜0.01).(3)After transfected 4 days,the inhibition rate of cell proliferation in the transfection group was dramatically higher than that in NC group[(37.2 ±6.2)％ versus(3.6 ± 3.2)％,P =0.001].(4)The percentage rate of proliferative cells in the transfection,NC and blank control group was(12.3 ± 0.8)％,(23.4 ± 1.8)％,(31.1 ± 4.9)％.And it was lower in the transfection group(P ＜ 0.05).(5)Decreased cells via the transwell member in the transfection group(6 ± 3)were detected as compared with NC group(40 ± 9)and blank control group (48 ± 8,P ＜ 0.01).(6)Twenty-four hours after cultured in serum starvation and hypoxia,the rate of the viable and late apoptotic cells in the transfection group were significantly higher than those in NC group and blank control group[the rate of viable apoptotic cell was(16.9 ± 2.1)％,(10.3 ± 1.7)％ and(9.0 ±0.8)％ respectivcly,P＜0.01;the rate of late apoptotic cell was(13.4±3.3)％,(3.2 ±1.8)％ and (0.7 ±0.6)％ respectively,P ＜ 0.01].After cultured 48 hours,total apoptotic cells in the transfection group was significantly more than those in other groups(P ＜ 0.01).Conclusion miR-16 might inhibit the proliferation,invasion of ovarian epithelial carcinoma cells and enhance their sensitivity to apoptotic stimuli via downregulation of the expression of VEGF,MMP-2 and bcl-2 protein.

ObjectiveTo investigate the role and mechanism of microRNA-21 (miR-21) in the proliferation and apoptosis of ovarian epithelial carcinoma cells. MethodsA short-hairpin RNA specifically targeting miR-21 plasmid was constructed, and the recombinant was identified by restriction endonuclease analysis and DNA sequencing. Three experimental groups were included, transfection group (transfected with pSIREN-miR-21 ), negative control group ( transfected with pSIREN-miR-21-neg) and blank control group (without transfection plasmid ). The expression of miR-21 was detected by stem-loop real-time reverse transcription (RT)-PCR in OVCAR3 cells ,and western blot was used to detect the expression of programmed cell death 4 ( PDCD4 ) protein. Tethyl thiazolyl tetrazolium (MTT) and flow cytometry method were used respectively. ResultsRecombinant plasmid (pSIREN-miR-21) was constructed successfully and identified by restriction endonuclease analysis and DNA sequencing. The relative expression level of miR-21 in cells transfection, negative control and blank control group was 0.26 ± 0.08, 1.26 ± 0.21and 1.00 respectively. The level of miR-21 in the cells in transfection group was significantly lower than those in the negtive control and blank control group(P ＜0. 01 ). The gray scale of PDCD4. protein was 1443 ±33,858 ± 19 and 846 ± 16 in the transfection group, negative control and blank control group respectively. The value of PDCD4 in transfection group was higher than other control groups, and there were significantly difference among them( P ＜0. 01 ). Moreover, the optical density of the cells in transfection group was 0. 661 ±0. 015,significantly lower than those in two control groups (0. 848 ± 0. 150 for negative control, 0. 935 ± 0. 133 for blank control,P ＜ 0. 01 ). Forty-eight hours after tranfection, the rate of viable apoptotic cell was significantly higher than negative control and blank control group [(25.821 ± 0. 763 )％ vs.(0. 010 ± 0. 003 ) ％ vs. (0. 238 ± 0. 023) ％ ; P ＜ 0. 01];72 hours after tranfection, the rates of viable apoptotic cell and necrotic cell were all higher than the two control groups [the rate of viable apoptotic cell was ( 30. 480 ±0. 821 ) ％, ( 7. 792 ± 0. 312 ) ％ and ( 7. 033 ± 0. 257 ) ％ respectively ( P ＜ 0. 01 ) ; the rate of necrotic cell was (3.558 ±0.211)％, (1.557 ±0.067)％ and (1.049 ±0.028)％, respectively (P＜0. 01)].ConclusionmiR-21 might play an important role in the proliferation and apoptosis of ovarian epithelial carcinoma cells through negatively control the expression of PDCD4.

Objective:To investigate the effects of ovarian cancer ascites-derived exosomes on the stem cell properties and invasion ability of ovarian cancer stem-like cell (OCS-LC).Methods:(1) A2780 cells were induced into OCS-LC in serum-free medium, and authenticating their stem-like properties by sphere-forming test, differentiation test and CD 133 marker detection. (2) Exosomes from ascites and A2780 cell were extracted by ultracentrifugation, then authenticating them. The morphology of exosomes was observed by the transmission electron microscope, exosome particle size was measured by nanoparticle tracking analysis (NTA). The expressions of heat shock protein 70 (HSP-70), CD 63 and CD 9 were detected by western blot. (3) The exosomes from ovarian cancer ascites and tumor cell supernate were co-cultured with OCS-LC. The groups were divided into control group, ascites-derived exosomes (ADE) group (ADE+OCS-LC group), and cells-derived exosomes (CDE) group (CDE+OCS-LC group). The sphere-forming ability was evaluated by sphere-forming cycle, maximum sphere diameter and sphere-forming rate of each group; the expression of CD 133 was detected by immunofluorescence staining under microscope; quantitative real-time (qRT)-PCR was used to detected the expression levels of octamer-4 (Oct-4), Nanog mRNA of the signature genes in the stem cells of each group; the metastasis ability of each group was measured by transwell assay. Results:(1) Identification of OCS-LC: sphere-forming experiment showed that the suspension of OCSC single cells was grown in serum-free medium in secondary sphere-forming. Differentiation function experiment showed that OCS-LC were differentiated into adherent A2780 cells by changing the growth mode in serum containing medium. Flow cytometry showed that the proportion of CD 133 positive ( CD133+) cells in OCS-LC group was (18.9±0.9)%, significantly higher than that of control group (0.6±0.5)% ( t=38.570, P<0.01). (2) Under transmission electron microscope, clear lipid bilayer structure was observed in ADE and CDE, and one side presented a concave hemispheric or cup like structure. NTA showed that the diameter of exosomes mainly ranged from 30 to 100 nm, with an average diameter of 67.2 nm. Western blot analysis showed that the specific protein molecules HSP-70, CD 63 and CD 9 were positive. (3) Three groups′ OCS-LC could continue to grow into spheres, and the group of ADE+OCS-LC showed two growth modes, most of the cells continued to grow into spheres, while a small part of cells grew in adherent differentiation. The sphere-forming rate of OCS-LC in the control group, ADE+OCS-LC group, and CDE+OCS-LC group were (1.05±0.20)%, (4.15±0.10)%, and (10.45±0.25)%, the sphere-forming cycle of OCS-LC in the three groups were (15.3±1.5), (10.3±0.6), and (6.7±0.6) days, and the maximum diameters of OCS-LC were (100.3±3.2), (145.2±5.1) and (170.0±2.1) μm, respectively. And the differences were statistically significant (all P<0.05). After co-culture of exosomes with OCS-LC, the sphere-forming ability of cells in the group of CDE+OCS-LC was prior to ADE+OCS-LC group (all P<0.05). Immunofluorescence staining showed that the number of CD 133 green fluorescent chromophore cells in OCS-LC groups [(46.2±2.1)%, (58.4±2.2)%] was significantly higher than that in the control group [(26.6±1.5)%] after the addition of exosomes in co-culture, the positive rate of CD 133 was higher than that in the control group( F=187.588, P<0.05). The qRT-PCR results showed that the expression levels of Oct-4 mRNA in ADE+OCS-LC and CDE+OCS-LC groups were 3.46±0.24, 4.03±0.31, compared with that in control group (1.04±0.12), the differences were statistically significant ( F=134.932, P<0.05). The mRNA expression levels of Nanog were 1.57±0.32, 2.66±0.15, which were significantly higher than that in the control group (1.00±0.07), and the differences were statistically significant ( F=49.329, P<0.05). And the expression of both in CDE+OCS-LC group increased more significantly than ADE+OCS-LC group (all P<0.05). The number of invasive cells in the three groups of OCS-LC were: control group 30±5, ADE+OCS-LC group 102±4, CDE+OCS-LC group 210±7, and there were statistically significant differences among three groups ( F=820.800, P<0.05). Compared with the control group, the number of invaded cells in the co-culture group were significantly increased ( P<0.05), and the CDE+OCS-LC group had the higher cell invasion ability then the ADE+OCS-LC group ( t=23.202, P<0.05). Conclusions:Exosomes derived from ovarian cancer ascites could enhance and maintain the stemness of OSC-LC, and promote the invasion of tumor cells. Moreover, CDE is superior to ADE.

Objective To investigate the expression of long intergenic non-protein coding RNA-regulator of reprogramming (Linc-ROR) in high-grade ovarian serous cancer, and explore the relationship between Linc-ROR expression and biological function of high-grade ovarian serous cancer. Methods A total of 34 high-grade ovarian serous cancer tissue samples and 19 normal fallopian tube tissue samples were collected between June 2014 and February 2016. Real-time reverse transcription (RT)-PCR was used to detect the Linc-ROR mRNA expression in different samples. The relationship between Linc-ROR expression level and ovarian cancer International Federation of Gynecology and Obstetrics (FIGO) stage, lymph node metastasis was analyzed. Constructed Linc-ROR small interference RNA (siRNA) and pIRES2-EGFP-Linc-ROR plasmid, then Linc-ROR siRNA and pIRES2-EGFP-Linc-ROR plasmid were respectively transfected into SKOV3 cells. Cell proliferation, migration and invasion ability were assessed by cell counting kit-8 (CCK-8), wound healing assay and transwell invasion assay. Results (1) The expression level of Linc-ROR mRNA was significantly higher in high-grade ovarian serous cancer than normal fallopian tube tissues (4.31± 0.38 vs 1.03 ± 0.21; t=25.842, P<0.01). With the progression of FIGO stages, the expression of Linc-ROR was increased (F=95.702, P<0.01), and it was associated with lymph node metastasis (t=7.397, P<0.01). (2) The results of RT-PCR showed that the expression level of linc-ROR in Linc-ROR-i group was significantly lower than that in Linc-ROR-NC-i group (0.30 ± 0.11 vs 1.02 ± 0.10; t=15.269, P<0.01). The expression level in Linc-ROR-p group was significantly higher than that in Linc-ROR-NC-p group (8.90 ± 0.45 vs 1.03 ± 0.17;t=21.934, P<0.01). The CCK-8 assay showed that when the cells were cultured for 3, 4, 5 and 6 days, the A value in Linc-ROR-i group was significantly lower than that in Linc-ROR-NC-i group (P<0.05). And the A value in Linc-ROR-p group was significantly higher than that in Linc-ROR-NC-p group (P<0.05). Wound healing assay showed that, after 48 hours incubation, migration rate of cells in Linc-ROR-i group was significantly less than that in the Linc-ROR-NC-i group [(52±4)%vs(67±5)%;t=5.720,P<0.01]. The migration of cells in Linc-ROR-p group was significantly greater than that in the Linc-ROR-NC-p group [(84±4)%vs(66±4)%;t=7.330,P<0.01]. Cell transwell invasion assay showed that, after 48 hours of incubation, the number of invasive cells in Linc-ROR-i group was lower than that in Linc-ROR-NC-i group (74 ± 3 vs 104 ± 3; t=15.810,P<0.01). And the number of invasive cells in Linc-ROR-p group was higher than that in Linc-ROR-NC-p group (217 ± 4 vs 108 ± 5; t=38.060, P<0.01). Conclusion Highly expressed Linc-ROR could enhance the proliferation, migration and invasion ability of high-grade ovarian serous cancer cells, which may be one of the important molecules in the occurrence and development, invasion and metastasis of high-grade ovarian serous cancer.

Objective To valuate the applicability of multiplex ligation‐dependent probe amplication (MLPA ) for detection of chromosomal unbalance .Methods Aneuploid and subtelomeric MLPA technology were used to analyze 6 samples with chromosom‐al unbalances ,confirmed by karyotype analysis .Results All samples were identified to have abnormal signal of corresponding probes .The extent of unbalances contained subtelomeric region .Conclusion MLPA technology could have important role in diag‐nosis of chromosomal unbalance ,which could complement conventional karyotype analysis .

Objective To explore the risk factors influencing the prognosis of patients with acute poisoning by analysis of clinical data of 356 patients in order to provide the scientific evidence for planning therapeutic strategies in ICU.Methods The clinical data of 356 patients with acute poisoning were collected during the period from January 1,2005 through December 30,2009,and the clinical findings from close observation were filled into the tables of specially designed “ Clinical observation of acute poisoning patients”.Some risk factors of 356 cases with complete clinical data were studied by single-factor analysis and Logistic multiple regression,such as gender,age,mode and cause of poisoning,kind of poison agents,time elapsed from poisoning to admission into the hospital,time elapsed from poisoning to admission into ICU,length of hospital stay,cardiopulmonary resuscitation,mechanical ventilation,APACHE Ⅱ score.Results Three hundred fifty-six patients with complete data were divided into survival group (n =260) and death group (n =96).Univariate analysis showed the length of hospital stay (5.72 ± 4.37) d,APACHE Ⅱ score (10.27 ±7.77),time elapsed from poisoning to admission into ICU (17.16 ± 31.22)h in the survival group,and the length of hospital stay (3.53 ± 5.79) d,APACHE Ⅱ score (18.78 ±8.66),time elapsed from poisoning to admission into the ICU (37.21 ±67.35) h in the death group (P ＜0.05 or P ＜ 0.01).The differences in rates of CPR,mechanical ventilation and kind of poison agents between the two groups were statistically significant (P ＜ 0.05).Multivariate Logistic regression analysis revealed that the length of hospital stay,APACHE Ⅱ score,rates of cardiopulmonary resuscitation,mechanical ventilation and kind of poison agents were positively correlated with prognosis of patients with acute poisoning (P ＜ 0.05).Model to predict mortality was established:Y =-0.817-0.137X1 +0.140X3 + 2.133X4 + 1.039X5-0.291X6.Conclusions Hospital stay,APACHE Ⅱ score,cardiopulmonary resuscitation,mechanical ventilation and kind of poison agents were independent risk factors for predicting prognosis.APACHE Ⅱ score system and Logistic regression analysis can be used to evaluate the severity and prognosis of patients with acute poisoning.

Objective To measure the diameter of cochlear nerve(CN) in normal hearing children with different ages,and evaluate the diameter variations with age.Methods A total of 156 normal-hearing children were assessed with 3D-FIESTA sequence scanning of the inner ear.All the subjects were divided into 13 groups according to age,witha group of ≤6 months,a group of ≤1 year,and 11 groups from one year to 12 years.Each group had 12 cases,including 6 boys and 6 girls.The diameter of CN was measured at the entrance of the cochlea,the middle of internal auditory canal(IAC) and the fundus of IAC respectively on the axial and oblique sagittal images of 3.0 T MRI by two independent observers.The average values measured by them were the final results.The intraclass correlation coefficient was used to determine the consistency between the two independent observers.The t test was used to access differencesin CN diameter by sex and sides.One-way analysis of variance(ANOVA) and two-way ANOVA were used for comparisons among different groups.Spearman correlation coefficient was applied to find a correlation between the CN diameter and age.Results The diameters of normal-hearing children's CN at the middle of the IAC,IAC fundus and the entrance of the cochlea were (1.12 ± 0.08),(1.05 ± 0.06),(0.87 ± 0.14) mm respectively,and there was significant difference among the three measuring points (F=527.57,P＜0.05).The diameters of the CN had no significant differenc (P＞0.05) in age-groups,gender and sides(P＞0.05),and there was no correlation between the diameters of normal children's CN and age.Conclusions The diameters of normal-hearing children's CN change with different points of the IAC,of which the maximum value is at the middle of the IAC,followed by the IAC fundus,and the entrance of the cochlea is at the minimum,more over the normal size doesn't change with age.

Objective To determine the effects of non-thermal biological effect of low frequency pulsed electromagnetic field (LFPEF) with 15 Hz and 30 mT on ulcer wound during the plateau stress ulcer cure.Methods Thirty male Sprague-Dawley rats were assigned to three equal groups:control,normal cure group (NC) and exposure treatment groups (ET).The plateau rats stress ulcer models were constructed with the combination method of a hypoxic and low-pressure acclimation and the stimulation of absolute ethyl alcohol irrigating stomach.The LFPEF equipment generated the LFPEF of 15 Hz,40％ duty cycle and 30 mT to expose and treat the rats' stomach of ET group with the frequency of 3 hours each day.After 6 days of exposure treatment,the stress ulcer cure situation of each group was evaluated by the methods of macroscopic observation,the gastric juice pH values detection and ulcer index (UI) measurement.Results After 6 days of model,the macroscopic observation showed that the ulcer of NC group was worse than the that of ET group (P＜0.05).The pH values and UI of ET group were significantly different with those of NC group,and closed to those of the control group.Conclusion The nonthermal biological effect of LFPEF could promote the plateau stress ulcer cure.

Objective To investigate the safety and effectiveness of different intravenous anesthesia methods for pediatric ERCP . Methods Data of 45 children undergoing ERCP at the Affiliated Hangzhou Hospital of Nanjing Medical University from August 2013 to July 2016, including intravenous anesthesia,the procedure of ERCP, adverse reactions and the waking time were retrospectively studied. Results A total of 45 patients in two groups under intravenous anesthesia successfully underwent ERCP . Seventeen patients ( 37. 8%) whose body weights were over 20 kg and the duration of surgery was predicted less than 30 minutes received deep sedation without airway intubation. Twenty?eight patients ( 62. 2%) with an initial weight of less than 20 kg and the duration of surgery was predicted more than 30 minutes received general anesthesia with airway intubation. In patients with deep sedation, the mean time of waking was 7. 2±6. 3 minutes, body movement reaction occurred in 1 case ( 5. 9%) and with transient decreasing of pulse blood oxygen ( beyond 95%) occurred in 2 cases ( 11. 8%) . In patients receiving endotracheal anesthesia with intubation, the mean waking time was 10. 5±8. 7 minutes without adverse reactions associated with anesthesia. Conclusion Both deep sedation and general anesthesia with airway intubation are safe for pediatric ERCP. However, general anesthesia with airway intubation is an ideal method ensuring the airway safety and oxygen supply for children less than 20 kg undergoing first?time ERCP or the duration of surgery lasting over 30 minutes.