Objective To amplify natural killer (NK) cells in vitro and explore its killing effect on ovarian cancer cells.Methods (1) The separation of NK cells and identification.A total of 20 ml peripheral blood of one healthy volunteer was collected in Nov.2015,Peking University People's Hospital.The peripheral blood mononuclear cells of normal volunteers were isolated,cultured in vitro and amplificated cultivation for 14 days with K562 cells transfected and expressing interleukin 21 (IL-21-K562) as nourish cells.The number and dynamic state of the growth cells were monitored during the cultured process.Cells were harvested and counted after 14 days cultured.The NK cells phenotypes were detected by flow cytometry.(2) The killing effect of NK cells on ovarian cancer cells:the ratio of effector cells (NK cells) and target cells (ovarian cancer cells and its control) was 50∶ 1,20∶ 1,10∶ 1,5∶1 or 1 ∶ 1,NK cells killing effect on ovarian cancer cells was detected by the lactate dehydrogenase (LDH) release experiments.Results (1) The results of NK cells establishment and phenotypic characterization:the cells were induced in vitro for 14 days by amplification culture.With the extension of incubation time,the number of NK cells increased constantly,from 2.0× 107 on day 0 to 5.1 × 109 on day 14.Obvious amplification of the total number of cells were detected for 255 times.Living cells unstained by trypan blue eventually reached 95％ above.Before and after the induction and amplification in vitro,the percentage of NK cells (CD3-CD56+cells) in CD3-cells were 2.33％ and 85.32％,respectively (P＜0.01),which covered the whole lymphocytes 1.06％ and 69.42％,respectively (P＜0.01),which showed that NK was the main cell type in the amplificated lymphocytes.(2) The killing rate of NK cells on ovarian cancer cells in vitro:the results detected by LDH release experiments showed that NK cells could performed strong nonspecific killing effect on ovarian cancer cell lines SKOV3,HOC1A,3AO and CAOV3,as well the normal ovarian cell line T29 and NK sensitive cell line K562,and the killing effect increased significantly along with the increase of effector cells and target cells ratio (P＜0.01).When the ratio was 1 ∶ 1,the killing rate was 37％ for K562,while the rate of killing of other cells was around 10％ (P＜0.05).When the effect-target ratio was 20∶1 and 50∶ 1,in addition to CAOV3 cells (more than 70％),NK cells had a kill rate of more than 80％ for other ovarian cancer cells lines and their control cell K562 and T29 cells (P＞0.05).Conclusion NK cells could be established in vitro and have a good non-specific killing effect on ovarian cancer cells.

Objective To explore the dynamic variation rule of drainage fluid parathyroid hormone ( dPTH) , serum parathyroid hormone ( PTH) and serum calcium after thyroidectomy .According to the variation rule, the survival , function and prognosis of the parathyroid which retained at the original place can be predicted . Methods From Apr.2012 to Aug.2012, 90 patients who underwent thyroidectomy in Thyroid Surgery Center of the First Affiliated Hospital of Kunming Medical University were chosen as the research object .All of the objects'operations were performed by the same surgeon team and they were divided into four groups according to different surgical methods ( group A:bilateral thyroidectomy group , group B:bilateral thyroidectomy and central neck dis-section group , group C: bilateral thyroidectomy and functional neck dissection group , and group D: unilateral thyroidectomy group ) .Parathyroid retention situation during operation and the occurrence of hypocalcaemia after operation were recorded .Blood samples were taken between 7am and 8am in the 4 consecutive days after opera-tion to detect serum calcium and serum parathyroid hormone .All of the 90 patients had the drainage tube and their drainage fluid were taken for testing dPTH .The levels of serum calcium , PTH and dPTH were analyzed by statistical analysis of repetitive measure analysis of variance ( ANOVA) .Results The mean postoperative maxi-mum serum calcium was in group D and the minimum was in group C .There was no obvious difference in terms of serum calcium between group A and group B .However , the serum calcium showed an upward trend in each group.The mean postoperative maximum PTH was in group D and the difference has statistical significance com -pared to the other 3 surgical methods.The minimum was in group B and group C .However, it showed an upward trend with time in each group .The difference of dPTH in each group had no statistical significance and it showed a downward trend in all the 4 groups.Low serum calcium and hypocalcaemia occurred to 22 cases and 13 cases respectively after operation .The low serum calcium cases in each group were 12, 3, 4 and 2 respectively and hy-pocalcaemia cases in each group were 4, 3, 1 and 1 respectively.Conclusions By monitoring dPTH, PTH and serum calcium after thyroidectomy , the survival and function of parathyroid retained at the original place can be e-valuated comprehensively .Furthermore, it also helps to estimate prognosis .dPTH at a high level after operation is a direct evidence that parathyroid retained at the original place survives .Low PTH and high dPTH after thyroid-ectomy illustrates the operation just affects the way that PTH secreted into blood and the parathyroid retained at the original place can still secrete large amount of PTH .PTH will return to normal gradually with reconstruction and healing of microcirculation around parathyroid .Persistent low serum PTH after operation , low dPTH after 24 hours and the early advent of hypocalcaemia suggest the parathyroid retained at the original place was injured seri -ously and its blood supply was damaged obviously and more than one parathyroid were affected .Secretion function of parathyroid will remarkably decrease .

0.05).But there was less operation time and less volume of bleeding in the group one ( P

OBJECTIVE:To prepare Puerarin polymeric micelles and establish a method to determine its entrapment efficiency. METHODS:Puerarin polymeric micelles were prepared by film dispersion method. The polymeric micelles and free drug were sepa-rated by centrifugal-millipore filter filtration method. The entrapment efficiency of puerarin polymeric micelles was determined by HPLC. Diamonsil C18(2)column was used with 1% citric acid solution-methanol(65∶35)at the flow rate of 1 ml/min. The detec-tion wavelength was set at 250 nm,and column temperature was room temperature. RESULTS:The prepared polymeric micelles were spherical and spherical-like in shape with a mean particle size of 54.12 nm,polydispersity index of 0.122,Zeta potential of -13.60 mV;the linear range of puerarin was 2-10μg/ml(R2=0.999 4)with average recovery rate of 99.2%(RSD=0.9%,n=3). The re-covery rate of free drug was 95.3%(RSD=1.7%,n=3). The mean entrapment efficiency and drug-loading amount of puerarin were(35.5±2.12)% and(0.3±0.07)%,respectively(n=3). CONCLUSIONS:Film dispersion method is suitable for the prepara-tion of Puerarin polymeric micelles. Established method is convenient,accurate and reliable for the content and entrapment efficien-cy determination of Puerarin polymeric micelles.

Objective:To analyze phylogenetic structure and molecular characteristics of H5N6 avian influenza virus (AIVs) isolated from live poultry market (LPM).Methods:Oropharyngeal and cloacal swabs from poultry, and environmental samples were collected from LPM in Urumqi in December 2018, AIVs were isolated and identified by inoculation of chicken embryo, hemagglutination test and RT-PCR, the viral whole genome was amplified with the universal primers of influenza A virus, and then sequenced, pairwise sequence alignments, phylogenetic and molecular characteristics analysis were performed by BLAST, Clustal W, MEGA-X and DNAStar software.Results:Six strains of H5N6 AIVs were isolated from poultry samples, the identity between the viral genes was high (99.4%-100.0%), so the isolates were the same source. BLAST analysis revealed that the viral NP sequence had the highest identity (99.7%) with H5N6 AIVs isolated from poultry in Suzhou, while the sequence of the remaining 7 viral genes had the highest identity (99.0%-100.0%) with H5N6 AIVs isolated from environment in Guangdong during 2017 to 2018. Phylogenetic analysis showed that the viral HA belonged to Clade 2.3.4.4C, and the viral HA, NA, PB1, PA, NP, and MP were all clustered together with H5N6 AIVs isolated from mink in Eastern China in 2018, while the PB2 and NS were clustered together with H5N6 AIVs isolated from environment in Guangdong from 2017 to 2018. The HA cleavage site contained multiple basic amino acid residues, which was highly pathogenic AIVs (HPAIVs). S137A and T160A mutations of HA could increase binding to human-type receptor SAα2, 6-Gal. Additionally, the viral multiple mutations, including 59-69 deletion in NA, the L89V, G309D, R477G, I495V, I504V, D391E, and A661E in PB2, as well as the P42S, D92E, and 80-84 deletion in NS1, could enhance the viral virulence and pathogenicity to mammals. Conclusions:The 6 strains of H5N6 HPAIVs isolated from LPM have relatively close genetic relationship with H5N6 AIVs isolated from mink in Eastern China and environment in Guangdong during 2017 to 2018, the viral multiple mutations could increase its pathogenicity to mammals, which could pose a potential risk to public health.

Objective To study the influence of different microembolism on left ventricular systolic synchronism in pigs by detecting the real-time dypyridamole stress contrast echocardiography (RT-MCE).Methods Eighteen miniature pigs were randomly divided into three groups and underwent microembolization injection procedure through the middle of anterior descending coronary artery with different numbers of microsphere injection,as:group A(dosage 50 thousands,3 pigs),group B (dosage 120 thousands,8 pigs) and group C (dosage 150 thousands,7 pigs).The peak values and the time-to-peak circumferential strain(Circ.Strain),radial velocity (Radial Vel.) and radial strain (Radial Strain) were obtained both at mitral valve level and papillary muscle level at left ventricle short axis views using two-dimensional speckle tracking imaging(STI)analyzed by Philips Q-Lab 8.1 workshop,respectively.Results No significant difference in the presence of contraction synchrony was observed using RT-MCE.The time-to-peak Circ.Strain of microembolism related segments were prolonged at 1 week after microembolism detecting with dypyridamole stress RT-MCE (P＜0.05,both intro-group and inter-group).While time-to-peak radial strain were extended since 6 hours after the intervention to 1 week after the procedure.Conclusions Dypyridamole stress RT-MCE can be used to measure the myocardial perfusion accurately.The elongation of time-to-peak circ.strain and radial strain were developed with time in microembolism related segments.

Objective To assess the effect of ultrasound contrast agent SonoVue on the dimensions and systolic function of left and right ventricle in pigs. Methods Sixteen pigs were randomly assigned to two groups. Intravenous injection of 1 ml of SonoVue were given in study group, and repeated 20 min later. The control group was given the same doses of saline. Before and after the administration of contrast agent, the end-diastolic dimension (LVEDD, RVEDD). end-systolic dimension ( LVESD, RVESD) and fractional shortening(LVFS,RVFS) of left and right ventricle were measured. The time to reach the extreme value of these parameters and the time to return to the baseline were recorded. Results There was no significant difference regarding the parameters at baseline between the two groups. After injection of SonoVue,RVEDD significantly increased from (25. 88 ± 1. 38) mm at baseline to its maximum of (33. 26 ± 0. 99)mm( P < 0. 05). Accordingly,RVFS significantly increased from (26. 90 ± 1. 92) % to (33. 92 ± 2. 53) % ( P <0. 05). Meanwhile,LVEDD remarkably decreased from (38.10 ± 1. 39)mm at baseline to its minimum of (26.25 ± 0. 65)mm( P <0. 05) and LVFS remarkably decreased from (36. 24 ± 1. 93) % to (29.13 ± 3.00) % ( P < 0. 05). There was no change in the control group after administration of the saline. When SonoVue was given repeatedly, the maximum RVEDD and RVFS was (29. 98 ± 1. 23) mm and (31. 09 + 1.90) % , respectively, which had less increase compared to the first time. Minimum LVEDD and LVFS was (31. 91 ± 1, 64)mm and (32. 17 ± 2. 31)%,respectively,with less decrease compared with which at first injection. It took (10. 15±0. 59) min for the right and left ventricle to reach the extreme value and (9.00± 0. 56) min to return to the baseline at the first injection. The time used for the right and left ventricle to reach its peak change and back to baseline after second injection of SonoVue were shorter [(8.73± 0.55) min and (6.89± 0.43) min, respectively,both P <0.05]. Conclusions Administration of SonoVue was associated with acute, transient dilation of right ventricle and compression of left ventricle. The influence of SonoVue on the right and left ventricle became less at it second injection.

Objective: To evaluate the outcome and postoperative reaction of dural substitute (Neuropatch) applying in the treatment of Chiari I malformation(CMI) associated with syringomyelia(SM). Methods:Forty patients of CMI associated with SM were operated in our department from Jul. 2002 to Jul. 2004. All patients underwent posterior cranial fossa decompression and duraplasty. They were divided into two groups, 20 patients being repaired with Neuropatch (Neuropatch group), and the others with autologous fascia lata (fascia group). There were 6 males and 14 females in Neuropatch group and 10 males and 10 females in fascia group. The operations were performed under general anesthesia via suboccipital approach and the extent of posterior cranial fossa decompression ranged from 20 cm2 (5 cm×4 cm) to 35 cm2 (5 cm×7 cm). The removal of posterior arch of atlas depended on the extent of tonsillar herniation, and the dura was opened in Y shape. The Neuropatch was cut into triangular shape, and the same sized autologous fascia lata was used in fascia group. The patches were sutured tightly to the dura matter in each group. The incision was closed layer by layer and drainage was used, if necessary. Antibiotics and hormone were routinely used. The duration of operation, postoperative fever were evaluated, the outcome of the operation was evaluated by Tator scale, and the data were analyzed with statistic software SPSS 10.0. Results: There were12 patients (60%) who suffered from postoperative fever in the Neuropatch group, and 9 patients (45%) in the fascia group(χ2=0.902,P=0.342). Seventeen patients in each group were improved postoperatively. The duration of operation, postoperative fever and antibiotics used were compared between the two groups. No significant difference was found, but the duration of postoperative fever and the time of hormone used were different. There were no postoperative infections that occurred after the follow up for 1 to 2 years, except for one patient in fascia group who developed infective granuloma and recovered later by treatment. Conclusion: Neuropatch is a useful dural substitute for the repair of dural defects in the treatment of CMI associated with syringomyelia.

Objective To explore the changes of neutrophil elastase (NE) and surfactant protein A (SP-A) in acute lung injury(ALI) rats,and the effect of antioxidant. Methods Sixty healthy mature Wister rats were divided into 2 groups, the control group and treatment group. The rats in two groups all received peritoneal injection of E. coli to establish the ALI animal model. 30 minutes after injection of E. coli,the rats in treatment group were injected reduced glutathione from vena caudalis. The levels of NE in blood and expressions of SP-A in lung tissue were detected at 3,6 and 12 hours after injection of E. coli. Results ALI symptom appeared 3 hours after injection of E. coli in the control group, obvious after 6 hours, the rats vomi-ted pink secretion after 12 hours. Lung edema and bleeding were found by pathologic examination. No obvious symptom was found in treatment group after 3 hours, slight tachypnea after 6 hours, slight edema in pulmonary tissue after 12 hours. After administration of reduced glutathione,levels of NE at 3,6 and 12 hours in the treatment group were lower than those in the control group,and indicated statistical significance in 6 and 12 hours(P <0. 05) ;Levels of SP-A in 3,6 and 12 hours in the treatment group were higher than those in the control group, and indicated statistical significance in 3,6 and 12 hours (P < 0. 05). Conclusion Dysfunction of pulmonary surfactant is secondary in ALI, degradation of SP-A is the one of reasons, the application of reduced glutathione as antioxidant, could effectively suppress NE to decompose basosexine elastin of cells and destroy surface active protein, has protective effect on ALI.

Objective:To compare the clinical and prognostic characteristics of ovarian endometrioid carcinoma (OEC) patients with synchronous endometrial lesions and patients with pure OEC.Methods:A retrospective review of the medical records of patients received initial treatment and a postoperative pathological diagnosis of OEC at Peking University People′s Hospital between August 1998 and December 2017 were performed. According to the inclusion criteria, a total of 56 patients with OEC were included in the study, including 13 patients concurrent with simultaneous endometrial lesions (Group A) and 43 patients with pure OEC (Group B).Results:Patients with synchronous endometrial lesions accounted for 23% (13/56). Mean age of Group A at diagnosis was (44.9±8.3) years old, 2/13 of patients were postmenopausal, and no one had a history of hypertension, the first symptom of 5/13 people was irregular vaginal bleeding. Mean age of Group B patients at diagnosis was (52.7±10.2) years old, 53% (23/43) of patients were postmenopausal, and 28% (12/43) patients had the history of hypertension, the first symptom of 4 (9%, 4/43) people was irregular vaginal bleeding. The differences of age, menopause status, history of hypertension and initial symptoms between the two groups were statistically significant (all P<0.05). There were no significant differences in fertility history, dysmenorrhea history, age of menarche, history of endometriosis, preoperative and postoperative CA 125 level, International Federation of Gynecology and Obstetrics (FIGO) stage, tumor grade, metastatic site and platinum-based chemotherapy drug resistance between the two groups (all P>0.05). The overall 5-year survival rate of OEC patients was 91.6%, and the overall 5-year progression-free survival rate was 76.6%. Among them, the 5-year survival rate of the OEC concurrent with simultaneous endometrial lesions group was 80.2%, and the pure OEC group was 93.4%; the 5-year progression-free survival rate of the OEC concurrent with simultaneous endometrial lesions group was 74.1%, and the 5-year progression-free survival rate of the pure OEC group was 77.3%. There were no significant differences between the two groups (all P>0.05). Multivariate analysis showed that the independent factors for the prognosis of OEC patients were FIGO stage ( P=0.006) and residual lesion size ( P=0.020). Conclusions:OEC patients have a high proportion of simultaneous endometrial lesions. OEC with simultaneous endometrial lesions are younger than patients with pure OEC. Synchronous endometrial lesions do not affect the prognosis of patients with OEC.