Objective To investigate the expression of NKG2D in peripheral blood of patients with cervical cancer,CIN,hysteromyoma and health person,and the expression of the human MHC class I chain-related gene A(MICA)on the correspondent tumor tissues.To discuss the anti-cervical cancer mechanism of NKG2D-MICA and immune escaping of cancer.Methods Flow cytometry analysis was used to detect the expression of NKG2D in the peripheral blood of patients with cervical cancer,CIN,hysteromyoma and health person.The expressions of MICA in part of the correspondent tissues were examined by means of reverse transcription-polymerase chain relation(RT-PCR).Results The expression of NKG2D in the patients with cervical cancer,CIN,hysteromyoma and health person were(76.87?9.39)%、(81.84?7.94)%、(86.77?8.68)%、(93.968?4.9)%,respectively.Compared with the normal group,the NKG2D expression in the particular disease group was of statistical significance,however,it is not statistically significant in the comparison in the particular disease group.The rate of MICA mRNA expression in cervical cancer was significantly higher than that in hysteromyoma and health tissues,and its difference is of statistical significance.But it is not statistically significant for the normal group to compare with the other group.Conclusion The activity of NK cell and the anti-cancer cellular immunity level reduce in patients with cervical cancer.The decrease of the receptor NKG2D is a reason for the descend of the activity of NK cells.MICA mRNA expression increases in the cervical cancer,and it has the tendency of up-regulation with the progress of pathological changes.It is relative to malignant transformation from cervical squamous intraepithelial lesion to cervical cancer;the immune-escape of cervical cancer probably is relative to the down-regulation of NKG2D and the up-regulation of its ligand MICA.

OBJECTIVE:To assess the feasibility of production and effects of Tobramycin eye drops prepared in this hospital for treating ocular infections .METHODS: 77 patients with external ocular infections were randomly divided into two groups with imported Tobrex as control drug to compare the effects.Assessment was made according to Gwon project results.RESULTS: Overall improvement rates were 92.7% in Tobramycin eye drops group and 94.4% in control group without significant difference between two groups(P＞ 0.05) .CONCLUSION: The technology of Tobramycin eye drops production is simple, therapeutic effect is satisfactory, and the production in hospital is feasible.

Objective To analyze the influence of radial neck fracture on the upper limb function in older children,and to evaluate the curative effect of plate fixation in the treatment of Salter-Harris Ⅱ type of radial neck fracture.Methods The clinical data of 16 older children with radial neck fractures who treated by T plate internal fixation were retrospectively analyzed.16 cases were closed fractures,merging radial nerve injury in 1 case,3 cases of distal humerus fractures,1 case with rib fractures,fractures were Salter-Harris Ⅱ type,adopt steel plate internal fixa tion.Results 1 6 patients were followed up for 9-1 8 months.X-ray healing time was 6-9 weekson average 7.6 weeks.No malunion and no healing,no case of epiphyseal injuries.1 patient appeared steel block forearm supination.Postoperative patients with forearm pronation were greater than 90° supination were greater than 60 °.No radial nerve injury.Evaluation results were good.Conclusion Clinical treatment effect of open reduction and plate internal fixation in the treatment of older children with Salter-Harris Ⅱ type of radial neck fracture is satisfactory.Surgical treatment not only can enhance the fracture stability,but also can do the elbow joint function exercise to prevent dysfunction much earlier.

Objective: Proto-oncogene Zbtb7A has been characterized as a molecular switch in the process of cancer initiation and development.Our goal is to obtain the POZ domain of the Zbtb7A protein and prepare its polyclonal antibodies.Methods: We optimized the coding sequence of the POZ domain according to the codon bias of E.coli and synthesized the sequence with two-step PCR,which was then introduced into the pET-26b(+) vector to express the protein.The recombinant protein was analyzed by 15% SDS-PAGE and the corresponding band was cut out from gel.The minced gel slice that contained the POZ domain protein was injected to immunize rabbits.The collected rabbit antiserum was purified using the saturated ammonium sulfate method in combination with protein G antibody purification,and the purified polyclonal antibodies was evaluated by Western blot.Results: The optimized sequence of the POZ domain was correctly obtained and successfully constructed into the pET-26b(+) vector.After induction,an expected protein band about 14 KD was detected on 15% SDS-PAGE,and highly purified polyclonal antibodies were obtained,which were specifically bound to human Zbtb7A.Conclusion: The obtained recombinant protein of the POZ domain and its polyclonal antibodies can be used for further studies of Zbtb7A.

Objective To isolate and identify side population (SP) cells like cancer stem cell from human pancreatic cancer cell line SW1990, for the purpose of further evaluation of their biological characteristics. Methods Cell suspension was stained with Hoechst 33342 and PI. Then SP cells were analyzed in the fluorescence activated cell sorter. Cell growth viability was measured by MTT. Stem cell marker CD133 was determined by flow cytometry. Cloning forming efficiency was determined by cloning plating. Expression of ABCG2 protein was detected by Western blot analysis. Results The proportion of SP cells was 2.7%, however it could be completely blocked by verapamil. 9 days later, the value of A492 of SP cells was 2.1, the cloning forming efficiency was (38.7 ± 6.8) % , the positive rate of CD133 was 69.63%, which were significantly higher than cells 0. 5, ( 15.5 ± 2.8)%, 16.71% of corresponding non-SP( P <0.05). The expression of ABCG2 in SP cells was significantly higher than that in non-SP cells. Conclusions SP cells existed in human pancreatic cancer cells SW1990.

Objective To investigate the effects on self-renewal of pancreatic cancer stem cells by inhibiting hedgehog signaling pathway through cyclopamine. Methods PANC1 stem cells, PANC1 adherent cells and immortalized pancreatic ductal epithelial H6C7 cells were treated with 0.5, 1, 2, 5, 10 mol/L of cyclopamine for 24, 48, 72 h. The expression of Smo mRNA and Gli1 mRNA were detected by real-time PCR.Cell growth viability was measured by CCK 8. Cell cycle and apoptosis were determined by flow cytometry.Results Seventy-two hours after cyclopamine treatment, the Smo mRNA expressions of PANC1 stem cells,PANC1 adherent cells and H6C7 cells were 1,0.83 and 2.61; the expressions of Gli mRNA were 57.27,26.35,1; the inhibitory rates were ( 37.85 ± 13.69 ) %, ( 8.53 ± 4.43 ) %, (43.55 ± 28.98 ) %. Compared with PANC1, the expressions of Smo mRNA, Gli1 mRNA and the inhibitory rate of PANC1 stem cells significantly increased ( P ＜ 0.05 ). The proportion of G1 stage of PANC1 stem cells significantly decreased from (67.41 ±6.35)% to (36.53 ±6.03)% (P ＜0.05), and the apoptosis decreased from (10.95 ±5.68) % to ( 5.73 ± 1.42 ) % ( P ＞ 0.05 ). The proportion of G1 stage of PANC1 cells significantly decreased from ( 67.64 ± 6.88 ) % to ( 53.13 ± 1.10 ) % ( P ＜ 0.05 ); the apoptosis decreased from ( 12.08 ±4.12)% to (5.66 ± 1.33)% (P ＞0.05). While both the proportion of G1 stage and apoptosis of H6C7 cells was not significantly different. Conclusions Cyclopamine can inhibit the proliferation of PANC1 stem cells via blocking hedgehog signal pathway, and the mechanism may not be associated with cell apoptosis.

Objective To establish the method of suspension culture for stem cells from human pancreatic cancer cell line PANC1.Methods PANC1 cells were cultured in serum-free medium under floating-culture system.Tumor cell spheres were observed by optical microscope.Expression of CD133 and cell cycle were detected by flow cytometry.Cancer stem cells were induced to differentiate with 10%FBS,and expression of CK18,was evaluated with immunofluorescence microscope.Spheres cells were injected into the subcutaneous space of NOD/SCID rat and tumor formation was monitored weekly.Results PANC1 cells could form the stem cells spheres,and the rate of sphere formation was stable between 4%0 and 5%0 after 20 passages in vitro.The expression of CD133(5.91±0.7%)and proportion of G0/G1 phase cell(80.99±2.60%)was significantly increased in spheres cells compared with parental PANC1 cells(1.44±0.52%and 69.01±5.03%),and the difference was statistically significant(P＜0.05).When these spheres cells were cultured in media with serum,these cells gradually returned to the status of parental cells and expressed CK18,2×103 sphere cells injection could initiate tumor fornmtion in NOD/SCID rat.Conclusions Tumor spheres stem cellscould be generated under serum-free floating-culture system.The sphere cells possessed the capacities of self renew,difierentiation,and tumorigenic potential.

Objective To explore the prognostic value of N-terminal pro-brain natriuretic peptide ( NT-pro-BNP) levels in critically ill infants. Methods Eighty-one critically ill infants were enrolled from January 2013 to January 2014 in pediatric intensive care unit. The minimum of pediatric critical illness score ( PCIS) and the number of dysfunction organs were calculated within 24 hour after admission. According to PCIS,the critically ill infants were divided into extremely critical group(PCIS≤70,n=25),critical group (PCIS 71-80,n=30)and non-critical group(PCIS>80,n=26). According to the prognosis,the critically ill infants were divided into survival group (n=68)and death group(n=13). The serum NT-pro-BNP levels were determined on the first day,third day and convalescent phase. The relationships of serum NT-pro-BNP levels with PCIS and the number of dysfunction organs and prognosis were observed. Results The study showed statistical significances of serum NT-pro-BNP levels among the extremely critical group, critical group and non-critical group,whether on the first day,or on the third day and convalescent phase(P<0. 01). There were statistical significances of serum NT-pro-BNP levels among different stages of the disease in each group(P<0. 01). Compared with survival group,PCIS was significantly lower and the serum NT-pro-BNP levels and the number of dysfunction organs were significantly higher in death group. The serum NT-pro-BNP level on the third day was higher than that on the first day in death group ( P<0. 01 ) , while no significant difference was found in survival group. The serum NT-pro-BNP levels on the first day and the third day and PCIS were negatively correlated(r= -0. 59,P<0. 01;r= -0. 66,P<0. 01). The serum NT-pro-BNP levels on the first day and the third day and the number of dysfunction organs were positively correlated(r=0. 40,P<0. 05;r=0. 57,P<0. 01). Conclusion The serum NT-pro-BNP levels of the critically ill infants are correlated with disease severity,and can be useful for assessing the severity of critical illness.

Objective To explore the effects of pigmentary epithelium derived factor (PDEF) on the phenotypic and immunologic function of murine-derived dentritic cells(BMDCs) .Methods Mononuclear cells(MNCs) isolated from murine bone marrow were cultured in RPMI1640 medium containing rmGM-CSF and rmIL-4 for 5 d ,and were divided into five groups .MNCs were stimulated for 3 d with either 50 ,100 ,200ng/mL PEDF ,1 μg/mL LPS(positive control) or RPMI1640(negative control) .The expression of CD11c ,CD80 and CD86 on DCs surface were analyzed by the fluorescence activated cell sorting (FCM ) .The ability of PEDF-induced BMDCs to stimulated T cell maturation were determined by the CCK-8 method and the level of IL-12 in the culture supernatant was detected by ELISA .Results The PEDF-treated BMDCs expressed high levels of CD11c ,CD80 and CD86 ,enhanced the immunolog-ical activities of T lymphocyte and its secretion of IL-12 when compared with untreated DCs .Conclusion PEDF can significantly up-regulate the expression of DCs immunological labelled molecule in in vitro cultured murine and increase its immunological com-petence .