Objective To investigate the expression of Toll-like receptor 4 (TLR4) and myeloid derived suppressor cells(MDSC) in bone marrow cells in children with acute myeloid leukemia (AML),and to detect its relationship with the clinical features,the effect of chemotherapy and prognosis.Methods Twenty-nine cases of children with AML were collected from June 2013 to March 2014 in People's Hospital of Wuhan University,in which 11 cases of low-risk group,10 cases of middle-risk group,8 cases of high-risk group;and 17 cases of non blood disease was as the control group.The expressions of TLR4 and MDSC were detected by using reverse transcription-polymerase chain reaction (RT-PCR),Western blot methods,immunohistochemical staining,and flow cytometry,respectively,in the bone marrow cells of 29 children with AML.Results The mRNA and protein expression of TLR4 in the initial treatment group was higher than those in the complete remission group(t =3.092,3.393,all P ＜ 0.05).The mRNA and protein expression of TLR4 in the relapse group was higher than those in the complete remission group(t =4.013,4.279,all P ＜ 0.05).The positive expression rates of MDSC in the above 3 groups were (29.77 ± 1.39) ％,(5.19 ± 0.65) ％,(38.62 ± 3.54) ％,respectively,compared with the control group [(1.32 ± 0.27) ％] and there was significant difference(all P ＜0.05).The positive expression rates of TLR4 and MDSC in the initial treatment group,relapse group and complete remission group were significantly higher than those in the control group,with significant differences (initial treatment group TLR4:t =3.559,P ＜ 0.05;MDSC:t =3.727,P ＜ 0.05;relapse group TLR4:t =4.043,P ＜ 0.05;MDSC:t =4.125,P ＜ 0.05;complete remission group TLR4:t =2.798,P ＜ 0.05;MDSC:t =3.469,P ＜ 0.05).Pearson rank correlation analysis showed that there was a positive correlation between the expression of TLR4 and MDSC (r =0.673,P ＜0.01).Conclusions The expressions of both TLR4 and MDSC play an important role in onset,progression,curative effect and prognosis in children with AML,and the two may play an importment role in synergistic effect.

Objective To investigate the molecular mechanism underlying lymphocyte functionassociated antigen-1 (LFA-1) / intercellular adhesion molecule-1 (ICAM-1) mediated anti-neoplastic effects of cytokine induced killer (CIK) cells. Methods Lymphocytes isolated from peripheral blood of children leukemia were induced with interferon-gamma (IFN-y), anti-CD3 monoclonal antibody (CD3McAb) and interleukin-2 (IL-2) and co-cultured with dendrite cells (DC) to generate DC-CIK cells. When treated with LFA-1 monoclonal antibody, cytotoxicity of DC-CIK cells against leukemia cell lines was measured by the MTT assay, while RT-PCR and Western blotting were used to determine mRNA and protein expressions of GATA-3 and T-bet in DC-CIK cells, respectively. IL-12, IFN-γ and tumor necrosis factor-α (TNF-α) levels released by DC-CIK cells were quantified by ELISA. Results Induced DC-CIK cells were regular, round and transparent with variable cell volume and cellular aggregation. When treated with mouse anti-human LFA-1 monoclonal antibody, the cytotoxicity decreased mostly towards B95 cells under administration of 20 μg/ml LFA-1 monoclonal antibody in comparison with the control group(t =10.138, P ＜0.05). It led to a highest elevation of GATA-3 mRNA and protein levels (t =16.386, P ＜ 0.05; t =22.652, P ＜ 0.05) and a most decrease of T-bet mRNA and protein levels (t =17.728, P ＜0.05; t =17.452, P ＜0.05) under 20 μg/ml LFA-1 monoclonal antibody in B95 cells group in comparison with the control group. The expression levels of IL-12,IFN-γ, and TNF-o in supernatant were the lowest under 20 μg/ml LFA-1 monoclonal antibody in B95 cells group in comparison with the control group (t =21.621, P ＜0.05; t =13.739, P ＜0.05; t =15.278, P ＜0.05).Conclusion GATA-3 and T-bet were implicated in the LFA-1/ICAM-1 mediated anti-neoplastic effects of DC-CIK cells via activation of the Th1 pathway, with high secretion of Th1 cytokines, such as IL-12, IFN-γ and TNF-α.

ObjectiveTo discuss the value of interrupted circular suture in hemostasis of placenta previa during cesarean section. Methods We summarized 54 caesarean section patients with placenta previa. Results The hemostasis was succeeded in all of the 9 patients and uterus was retained without postpartum complications. The duration of operation was obviously shorter than that of hysterectomy( P＜0.05). Bleeding and blood transfusion were less than that of hysterectomy, but without statistical difference (P＞0.05). ConclusionInterrupted circular suture is one of the efficient methods in controlling postpartum bleeding during caesarean section with placenta previa.

Objective To explore the feasibility and advantages of transvaginal myomectomy(TVM).Methods A total of 65 patients with uterine myoma were treated with TVM from August 2002 to August 2004.The myomectomy was performed through a vaginal incision,which was transversely made through anterior or posterior fornix of the vagina.The uterus was exposed outside the incision with a pulling suture.The muscular layer covering the myoma was incised and the myoma was removed.Then the incisions of the uterus and vagina were closed respectively by using absorbable sutures.Results The TVM was successfully completed in all the 65 patients.The surgical time was 25~140 min(mean,56 ?19 min),the postoperative bleeding amount was 60~650 ml(mean,170?45 ml),and the length of hospital stay after operation,2~5 d.A follow-up was carried out for 2~12 months(mean,3.6?2 months) in 58 patients.The flow of menstrual cycle recovered to normal levels in 40 patients and was less than normal levels in 2 patients.Pressure symptoms of adjacent organs disappeared,and no residual tumors were detected on B-ultrasonography.Conclusions Transvaginal myomectomy is a safe and reliable procedure with little invasion and quick recovery.

BACKGROUND: Clinical observation demonstrates that accelerated fracture healing or lower limb heterotopic ossifications always occur in patients with paraplegia. It indicates that peripheral nervous system may play an important role in fracture healing process.OBJECTIVE: To observe bone histomorphometery parameter, callus formation and biochemical change during the process of fracture healing of unilateral lower limb denervated tibia.DESIGN: Self-control animal experiment.SETTING: Tianjin Hospital.MATERIALS: Totally 36 six-month-old healthy male Wistar rats, with mean body mass of 210 g, were used in this experiment.METHODS: This experiment was carried out at Animal Experimental Center of Tianjin Hospital from March 2001 to March 2004. Denervated tibia fracture model and innervated tibia fracture model were made in the same rat. Animals were executed under anaesthetic status at week 2 and week 4 after fracture. Bilateral tibias were chosen to take radiografts.Biomachamical strength was measured and non-decalcification sections were prepared to perform bone histomorphometery observation.MAIN OUTCOME MEASURES: ① Comparison of wet weight of bilateral tibias and callus of rats between two groups after fracture. ②X-ray plain film scoring. ③ Biomechanical testing of tibial samples. ④ Histomorphological observation of fracture healing RESULTS: ① Wet weight of bilateral tibia and callus of rats in denervated group was much higher than that in innervated group at weeks 2 and 4 after fracture [(0.94±0.15) vs (0.76±0.14) g, (1.06±0.26)vs (0.81±0.10) g,P ＜ 0.05]. ②In X-ray plain film scoring, callus formation was significantly increased in denervated group (P ＜ 0.01). ③In biomechanical testing of three-point bending of tibial sample, callus intensity was significantly lower at weeks 2 and 4 after fracture in denervated group than in innervated group[ (9.88±8.49)vs ( 16.62±13.38 ) N, ( 12.77±7.55 )vs (20.19±10.60) N,P ＜ 0.05]. ④Bone histomorphometery showed that compared with innervated group, mineralized bone trabecula width of denervated group was significantly reduced (P ＜ 0.05), osteoid width was increased , osteoclast index and bone absorption area were significantly increased (P ＜ 0.05), and there were no significant difference of fibroblast index and bone formation area between two groups; Compared with innervated group, mineralized deposition rate in the denervated group was significantly reduced (P ＜ 0.05), the mature time of osteoid was elongated (P ＜ 0.05).CONCLUSION: Peripheral nervous system may play an important role during early and middle period of fracture healing. Intact innervation is essential for normal fracture healing.

OBJECTIVE Topreparealipidderivativeofgemcitabine(Gem)anditspolymericmi-celles to overcome the disadvantages of Gem.METHODS N-benzyl-3′-acetyl-gemcitabine(BAG)was synthesized.A BAG-loaded poloxamer polymeric micelle (BAG∶poloxamer 188 =10∶1 ,mol/mol)was prepared using an injection method.The micelles were characterized with a laser particle size and elec-tric charge instru ment and negatively-stained trans mission electron microscopy.Hu man breast cancer cells MCF-7 were cultured with Gem or BAG polymeric micelles of 5,10,20,30,50,70,90 μmol·L-1 for 24,48 and 72 h,respectively.The inhibitory rate of cells was measured with an MTT method.The MCF-7 cytotoxicity of BAG polymeric micelles was investigated.A pharmacodynamic study was per-formed on the mice bearing mouse hepatocellular cancer cells H22.Intravenous (iv)and oral (ig)ad-ministration was used at the dose of Ge m 40 mg·kg -1 or BAG polymeric micelles 62 mg·kg -1 .The mice were administered on the 1 st,4th and 7th day and sacrificed on the 8th day.Tumor inhibitory rates were measured.RESULTS TheBAGstructurewasidentifiedbythinlayerchromatograph,1Hand13C NMR,infrared ray chromatograph and mass spectrum.The appearance of BAG micelles was a slightly blue suspension.The micelles were spheres according to the electron microscopic observation.Their size was 62.82 nm and the zeta potential was -18.8 mV.The half inhibition concentration (IC50)of Gem and BAG polymeric micelles was 40.6 and 90.0 μmol·L-1 ,5.0 and 14.9 μmol·L-1 ,5.0 and 1 3.6 μmol·L-1 at 24,48 and 72 h,respectively according to the MTT results.According to the in vivo results,compared with the tumor model group,Gem (ig),Gem (iv)and BAG polymeric micelles (iv and ig)had significant effect on the tumor weight of H22 cell xenograft mice (P<0.01 ).As for anti-tumor efficiency,BAG polymeric micelles (ig)were better than Gem (ig)(P<0.05);BAG polymeric micelles (iv)were better than BAG polymeric micelles (ig)(P<0.05),and BAG polymeric micelles (iv)were almostequaltoGem(iv).CONCLUSION ThelipidderivativeofGemcanbeloadedinthepoloxamer 1 88 polymeric micelles.BAG polymeric micelles show in vitro MCF-7 cell inhibition and in vivo inhibition of mouse H22 xerografts;iv or ig.BAG polymeric micelles (ig)show better anti-tumor effect than Gem (ig),indicating that BAG polymeric micelles are a promising novel anti-tumor oral preparation.

The effects of tetrandrine (Tet) on blood pressure, plasma renin activity (PRA) and the contractility of the papillary mascle and portal vein were studied in rats. After 4 d administration of Tet 30 mg/kg, 2/d, ig, blood pressure was decreased markedly in anesthetized male SD rats, but there were no effects on heart rate and PRA. A single dose of Tet 15 mg/kg iv reduced blood pressure and heart rate significantly, while did not change PRA. This single dose produced similar hypotensive effect in rats with and without pretreatment of Tet 30 mg/kg, 2/d, 4d, indicating the absence of tolerance. Tet inhibited the paced papillary muscle contractility and the spontaneous portal vein contractility, and the EC50 were 5.33?10-6mol/L and 4.25?10-5 mol/L, respectively. So the vascular selectivity of Tet is 0.12.

Aim To develop a model for screening DPPIV inhibitor from Chinese Herbal medicine.Methods With Gly-Pro-PNA as a substrate,DPPIV activity was assayed by the chromogenic substrate.The concentration of DPPIV and Gly-Pro-PNA,temperature,pH and reaction time were optimized.Results The optimal enzymatic reaction system was as follows:DPPIV 0.5 U?L-1,Gly-Pro-PNA 0.262 ?mol?L-1,37℃,pH 8.2,reaction for 60 min.Further more,many kinds of herbal abstracts were screened and some had the DPPIV inhibitory function,such as Leech,Poria cum Radix Pini and Buddleja officinalis Maxim.Conclusion The model can be used to screen for DPPIV inhibitors in vitro.

Objective To investigate the cytotoxicity and mechanism of killing tumor of cytokine-induced killer (CIK) cells in vitro.Methods Mononuclear cells were acquired freshly from bone marrow of children with leukemia,and the cells obstained were induced into dendritic cells by adding granulocyte-macrophage colony-stimulating,IL-4,TNF-? and other cytokines.Lymphocytes cells were isolated freshly from peripheral blood of children with leukemia by Ficoll-Hypaque density centrifugation,and the cells obstained were induced by IFN-?,IL-2 and CD3McAb.The DC cells and CIK cells were co-cultured for 10-25 days,then DC-CIK cells were obtained.Phenotypes of DC-CIK were analyzed by flow cytomery.The cytotoxicity of DC-CIK against a variety of leukemic cell lines was investigated by MTT technique.When treated with mouse-anti-human LFA-1 monoclonal antibody,the expression of GATA-3 and T -bet in the levels of mRNA and protein were mea-sured by using RT-PCR and Western Blot technique,respectively.Results In the first 0-6 days,DC-CIK induced slowly,the proliferation of DC-CIK got 100-fold at the 13th day,cells were rapidly proliferating in the first 13-21 days.The maximum proliferation of DC-CIK reached at the 22nd day.The phenotypes of CD3,CD11a,CD54,HLA-DR were expressed highly; CD3/CD56,CD25,CD28,CD69,FasL were expressed moderately on DC-CIK.The expression of CD16 was not increased.DC-CIK possessed the cytotoxicity against tumor cells of B95,Jhhan and M07e.The effect was stronger to B95,there was no significant difference when the efficiency target ratio was 12.5:1.0 or 25:1,the cytotoxicity reached about 50% and 60%,respectively,against tumor cells of B95.However,it was not obvious to Jhhan and M07e.When the efficiency target ratio was 12.5:1.0 or 25:1,the cytotoxicity reached to 27.21%,25.13%,33.05%,29.72%,respectively,against tumor cells of Jhhan and M07e.When treated with mouse-anti-human LFA-1 monoclonal antibody,the expression of GATA-3 in the level of mRNA was up-regulated(t=3.425,4.523 Pa

Objective To investigate the biological activity of cytokine-induced killer(CIK)cells in vitro.Methods Lymphocytes isolated from peripheral blood in leukemic children were induced with interferon-?(IFN-?),anti-CD3 monoclonal antibody(CD3McAb)and interleukin-2(IL-2)and co-cultured with dendritic cells(DC)to generate DC-CIK cells.The morphology and immunophenotype of these cells were determined by electron microscopy and flow cytometry,respectively.Cytotoxicity of DC-CIK cells against leukemia cell lines was measured by the methyl thiazolyl tetrazolium (MTT) assay.Interleukin-12(IL-12),tumor necrosis factor-?(TNF-?)levels released by DC-CIK cells were quantified by enzyme-linked immunosorbent assays.Results Induced DC-CIK cells were regular,round and transparent with variable cell volume and cellular aggregation.At the 0th-4th day,its amplification was very slow,and it increased quickly at the 5th-8th day,it reached its peak amplification at the 9th-10th day,at approximately 100-fold.The main effector cells in this population were CD3+CD8+ cells and CD3+CD56+ cells.DC-CIK cells were cytotoxic to B95 cells,K562 cells and HL-60 cells,with the highest cytotoxicity towards B95 cells.The expression levels of IL-12 and TNF-? in supernatant were very high.Conclusions DC-CIK cells induced with cytokines displayed powerful amplification and strongly killing activities in vitro.It suggested that DC-CIK cells induced with cytokines may play killing activities through Th1 pathway in vitro,as a result of high secretion of Th1 cytokines,such as IL-12 and TNF-?.