Objective To investigate the correlation of polymorphism of 8473 (T/C,rs5275) in the 3′-untranslated region of cyclooxygenase-2 (COX-2) gene with bladder cancer. Methods A case-control study on the relation between COX-2 polymorphism and bladder cancer was performed in this study. The Taqman SNP genotyping assay was used to study the COX-2 rs5275 polymorphism. Results The differences in allele or genotype distributions of COX-2 rs5275 between cases and controls (all P < 0.05) were significant. A significantly reduced risk was revealed in bladder cancer patients carrying the TC genotype (adjusted OR =0.178;95%CI:0.119 ~0.264),CC genotype (adjusted OR = 0.087; 95%CI:0.058 ~ 0.129) or (TC/CC) genotype (adjusted OR =0.122;95%CI:0.082~0.181) compared to the control group. Significant difference in genotype distribution of the COX-2 rs5275 site was found associated with sex and smoking (adjusted OR:2.125,0.476;95%CI:1.500 ~3.010,0.325 ~ 0.696);No corelation was found between genetype TT or TC/CC and the pathological features of bladder cancer (P>0.05). Conlusion The separate effect of rs5275 polymorphism of COX-2 is associated with the susceptibility of bladder cancer, the TC/CC genotypes may be a protective factor.

Objective To explore the effect and mechanism of TGF beta1/smad3 signaling pathways on apoptosis in mouse pulmonary fibrosis.Methods Fifty-four healthy male C57BL/6 mice were randomly divided into three groups:normal control (n=18),pulmonary fibrosis model (n =18) and TGF-β1/smad3 inhibitor group (n=18).Six mice in each group were randomly killed on days 7,14 and 28.Hematoxyli~eosin and Masson staining were adopted to evaluate the severity of pulmonary inflammation and fibrosis.The content of hydroxyproline (Hyp) in the lung tissues was detected by alkaline hydrolysis technique.The apoptosis was observed by tunnel apoptosis assay kit.P-smad3 and caspase3 protein expressions were assessed via Western blot.Results Lung in model mice versus normal control showed alveolar inflammatory change in 7 days and significant pulmonary fibrosis in 28 days(P＜0.05).Meanwhile,apoptosis index,hydroxyproline content,caspase3,and phosphorylated Smad3 were obviously higher in model mice than in control group (P ＜ 0.05).Compared with model group,TGF-β1/smad3 inhibitor group showed that alveolitis and pulmonary fibrosis degree,hydroxyproline content,cell apoptosis index,the expressions of p-smad3 and caspase3 were decreased at same time point (P ＜ 0.05).Conclusions TGF beta1/smad3 signaling pathways may participate the abnormal apoptosis during the development of pulmonary fibrosis,and TGF-β1/smad3 inhibitor SB431542 could inhibit this process.

Objective To observe the effects of Yishen Tongluo Decoction (YTD) on the renal mRNA expression of transforming growth factor-β1 ( TGF-β1) and collagen Ⅳ ( ColⅣ) in membranous nephropathy ( MN) rats. Methods SD rats were randomly divided into normal group, model group, benazepril group (in the dosage of 10 mg·kg-1·d-1) , YTD group ( in the dosage of 20 g·kg-1·d-1) . The rats in various groups were given intragastric administration of corresponding agents. At the end of the fourth week, 24-hour urinary protein quantity, albumin ( ALB) , total protein ( TP) , triglyceride ( TG) , total cholesterol ( TC) , blood urea nitrogen ( BUN) , and creatinine (Cr) levels were observed. The mRNA expression levels of TGF-β1 and ColⅣ in renal tissue of rats were detected by immunofluorescence method, electron microscopy and real-time polymerase chain reaction (PCR) method. Results In the model group, urinary protein quantity in rats was increased, serum levels of TP and ALB were significantly lowered, serum levels of TC and TG were significantly increased, renal pathological changes were present, and mRNA expression levels of TGF-β1 and ColIV in renal tissue were up-regulated (P<0.05 compared with normal group). Compared with the model group, 24-hour urinary protein quantity, TC and TG levels were significantly lowered, TP and ALB levels were significantly increased, rat renal injury was relieved, mRNA expression levels of TGF-β1 and ColIV in renal tissue were down-regulated in the treatment groups ( P<0.05) . However, the differences between benazepril group and YTD group were insignificant ( P>0.05) . Conclusion The therapeutic mechanism of YTD for MN is probably related with the inhibition of mRNA expression of TGF-β1 and ColⅣin renal tissue.

Objective To explore the impact of folate on MeCP2 expression and cervical cancer cells growth.Methods Cervical cancer cell lines Caski (HPV16-positive) and C33A (HPV-negative) were treated with different concentrations of folate.MTT,flow cytometry,Western blott and real-time PCR were used to detect the cells’ viability,apoptosis,the expression of MeCP2 protein and mRNA expressions respectively.Results The inhibitions of both cell growth were upgraded with the folate concentration increasing.The differences were significant between the experimental groups and the control group.With increasing of folate concentration,apoptosis ratio of C33A and Caski increased gradually (C33A:r =0.965,P ＜ 0.001; Caski:r =0.973,P ＜ 0.001) and the expression of MeCP2 protein downgraded gradually,presenting significantly negative correlations between them (C33A:r =-0.952,P ＜ 0.001; Caski:r =-0.947,P ＜ 0.001).There was significantly difference for mRNA expression in different concentration groups of Caski and C33A (C33A:F =77.041,P ＜ 0.001; Caski:F =59.885,P ＜ 0.001).In the same concentration group,the expression of MeCP2 protein and mRNA were higher in Caski than that of C33A,and the difference was significant in the concentration of 500 μg/ml group.There was a negative correlation between the expression of MeCP2 protein and cells’ apoptosis ratio (C33A:r =-0.970,P ＜ 0.001; Caski:r =-0.93,P ＜ 0.001).Conclusion Folic acid can inhibit the growth of cerical cancer cells,promote apoptosis and reduce the expression of MeCP2.The aberrant high-expression of MeCP2 can inhibit apoptosis of Caski and C33A.

· AIM:To investigate the prevalence and related factors of dry eye in primary school pupils in Lanzhou,Gansu Province.· METHODS:From October to November 2016,1347 pupils in two primary schools in Lanzhou,Gansu Province,were randomly selected as subjects.Every pupil was carried on the questionnaire of dry eye and eye inspection to confirm the diagnosis of dry eye.Besides,the prevalence and influencing factors of dry eye in pupils were analyzed by chi-square test,Mann-Whitney U test and logistic regression model.· RESULTS:A total of 1 268 pupils took part in this study and the inclusion ratio was 94.14％;271 individuals were diagnosed as dry eye,and the prevalence rate was 21.37％.Statistical analysis showed that the risk factors of dry eye were male,senior pupils,often using eye drops,poor reading habits,wearing contact lenses,video terminals last for a long time,learning pressure.· CONCLUSION:Dry eye has become one of the main diseases that plagued pupil's life and learning.It should cause wide attention.Considering the above factors,rational use of eye and improve lifestyle will help to reduce the damage to eye of pupils.

OBJECTIVEAcute monocytic leukemia (AML)-M5 is the common type of acute myeloid leukemias in children. Studies have shown that there are abundant lipopolysaccharide (LPS) receptor (designated as CD14) molecules on the cell membrane of M5 cells and they play an important role in the diagnosis of M5, since they can be recognized and bound by mouse-anti-human CD14 monoclonal antibody (McAb). However, mouse-originated antibodies are largely not suitable for clinical application due to the severe side effects, thus "humanized antibody" is desired. As the first step for developing humanized antibody, the construction and expression of single chain antibody (scFv) with functional protein are necessary. The present study aimed to express ZCH-7-2F9 ScFv (scFv(2F9)) in eukaryotic cells and obtain the scFv(2F9) protein with a high biological activity.

METHODSFour primers were synthesized to construct the eukaryotic expressional vector, which included SfiI and EcoRI enzyme cleaving site, 6 x His and stop code TGA sequences. scFv(2F9) gene was amplified through splicing by overlap extension (SOE) using the high fidelity Taq polymerase. Positive recombinants (pSectag2A/scFv(2F9)) were identified through enzyme cleaving and sequenced before expression and were transformed into Chinese hamster ovary (CHO) cells for expression. Western-Blot and flow cytometry (FCM) were carried out to determine the relative molecular mass (Mr) and binding activity of scFv(2F9).

RESULTSThe cloned scFv(2F9) gene was identified to be functional by sequencing and expressing. The interested protein could be detected in the culture supernatant of transformed CHO cells with an Mr of 31 000. The blocking test showed that the positive cell percentages, the mean fluorescence intensity (MFI) and the peak of channel (peak Ch) were reduced by 90.02%, 63.30% and 63.38%, respectively after blocking with scFv(2F9), while those were 4.55%, 10.09% and 5.02% after blockage using the supernatant from the CHO cells transfected with blanked vector pSectag2A.

CONCLUSIONSThe scFv(2F9) against human CD14 antigen was successfully expressed in eukaryotic cells and showed a high biological activity, which may be useful for the further studies on its humanized antibodies.

Amino Acid Sequence ; Animals ; Antibodies, Monoclonal ; genetics ; isolation & purification ; Base Sequence ; CHO Cells ; Child ; Cloning, Molecular ; Cricetinae ; Cricetulus ; Humans ; Lipopolysaccharide Receptors ; immunology ; Molecular Sequence Data ; Single-Chain Antibodies ; genetics ; isolation & purification

OBJECTIVETo construct a prokaryotic vector of ZCH-7-2F9 single chain antibody (ScFv2F9) and to obtain the ScFv2F9 protein with biological activity for further studies.

METHODSPrimers were synthesized according to the gene sequence of ScFv2F9, four tandem glycin and one serine (G4S) 3linker and multiple cloning site(MCS) of pIVEX2.3-MCS vector, which included NdeI and SmaI enzyme cleaving sites. ScFv2F9 gene was amplified through splicing by overlap extension (SOE) using the high fidelity Taq polymerase. Then the gene was cloned to pGEM-T easy and pIVEX2.3-MCS vectors. Positive recombinants (pIVEX2.3-MCS/ScFv2F9) were identified through enzyme cleaving and sequenced before expression. The recombinant plasmids were transfected into E.coli BL21star(DE3)plysS for expression. After purification with Ni+resin and renaturation in vitro, the relative molecular mass (Mr) and the binding activity of the interesting protein were determined by SDS-PAGE and flow cytometry (FCM), respectively.

RESULTThe cloned ScFv2F9 gene was identified to be functional by sequencing and expressing. The interesting protein was detected in inclusion body with a Mr of 31 000. The blocking test showed that the positive cell percentage, the mean fluorescence intensity (MFI) and the peak of channel (peak Ch) were reduced by 11.73%, 11.96% and 31.57%, respectively after once blockage by scFv2F9 protein, and 26.44 %, 21.75 % and 42.11 % after blockage twice.

CONCLUSIONThe ScFv2F9 against human CD14 antigen has been successfully expressed in prokaryotic cells with partial biological activity, which lays the foundation for further studies on its immunotoxin and other kinds of engineered antibodies.

Antibodies, Monoclonal ; Biotechnology ; methods ; Cells, Cultured ; Cloning, Molecular ; Gene Expression ; Genetic Vectors ; Humans ; Lipopolysaccharide Receptors ; immunology ; Prokaryotic Cells ; metabolism ; Recombinant Fusion Proteins ; genetics

In order to provide the evidences for CD19 as a better antibody targeting molecule for B lineage acute leukemias than CD20 through the multi-parameter flow-cytometry analysis of leukemia cells, the samples from 321 patients with acute leukemia (AL) were immunophenotyped by multi-color flow cytometry and CD45/SSC gating strategy followed by the analysis of CD19 and CD20 expression. The results showed that the positive rate of CD19 (115/116, 99.1%) in 116 cases with B lineage acute lymphoblastic leukemia (B lineage ALL) was significantly higher than that of CD20 (33/116, 28.4%) (P < 0.01); in 17 patients with B lineage/Myeloid (B/My) acute mixed lineage leukemia (AMLL), the former positive rate (17/17, 100%) was also higher than the latter (5/17, 29.4%) (P < 0.01). Both of the two antigens were negative in 29 patients with acute T lymphoblastic leukemia and 7 patients with T/My AMLL. The positive rates of CD19 and CD20 in 152 patients with acute myeloid leukemia (AML) were 7.2% and 2.0%, respectively. The difference of the fluorescence intensity between the two antigens on the cells from each patient with B lineage ALL or B/My AMLL was statistically significant (t = 20.68, P < 0.001). The specificity of CD19 and CD20 in B lymphocytic lineage was 92.3% (132/143) and 92.7% (38/41), respectively, while the sensitivity was 99.2% (132/133) and 28.6% (38/133), respectively, the former sensitivity was significantly higher than the latter (chi(2) = 144.018, P = 0.001). It is concluded that CD19 continuously and steadily express on almost all subtypes of B lineage leukemic cells with homogeneous pattern while only a small number of leukemias express CD20. Both the specificity and sensitivity of CD19 were very high with a much broader reaction pattern than that of CD20 on this group of diseases. These indicate that CD19 may be a better antibody targeting molecule than CD20 for patients with B-lineage acute leukemia.
Acute Disease ; Adolescent ; Adult ; Antigens, CD19 ; biosynthesis ; Antigens, CD20 ; biosynthesis ; Bone Marrow Cells ; immunology ; metabolism ; pathology ; Cell Lineage ; Child ; Child, Preschool ; Female ; Flow Cytometry ; Humans ; Immunophenotyping ; Infant ; Leukemia ; blood ; immunology ; pathology ; Leukocytes, Mononuclear ; immunology ; metabolism ; pathology ; Male ; Middle Aged ; Tumor Cells, Cultured

The aim of this study was to detect the presence of human AML leukemia stem cells (LSC) in childhood patients with acute leukemia (AL) and analyze the correlation between LSC concentrations and minimal residual disease (MRD) levels in AML cases after remission. The multi-parameter flow cytometry (FCM) and a panel of monoclonal antibody combination were used to detect the AML LSC or AML LSC immunophenotype-identical cell (AML LSC-IPIC) concentrations in childhood AML or ALL leukemia both at new diagnosis and at remission and correlated AML LSC to the MRD levels at different time points after remission. The results indicated that the AML LSC or AML LSC-IPIC concentrations [in average 166 (range 14 - 1459)/100 000 mononuclear cells (MNCs)] in AML at initial diagnosis were significantly higher than those in ALL [7 (range 0 - 560)/100 000 MNCs, p < 0.017] and control [0 (range 0 - 6)/100 000 MNCs, p < 0.017], respectively. The AML LSC concentrations in AML at non-CR were in average 36 (range 5 - 224)/100 000 MNCs. No statistical difference (p > 0.05) was found between the AML LSC or AML LSC-IPIC concentrations in AML (in average 6 (range 0 - 41)/100, 000 MNCs) and ALL [10 (range 0 - 105)/100, 000 MNCs] after CR. The significantly negative correlation was noticed between AML LSC concentrations and MRD levels. It is concluded that the AML LSCs exist in newly diagnosed AML, which are significantly reduced when complete remission has achieved, but the low levels of these populations still remain. The phenotypically similar (CD34(+)CD38⁻CD123(+)) AML LSC populations have also been found in the bone marrow from ALL patients, but their concentrations are not significantly different when CR has achieved. The significantly negative correlation between AML LSC concentrations and MRD levels is observed in AML patients after remission.
Adolescent ; Child ; Child, Preschool ; Female ; Flow Cytometry ; Humans ; Immunophenotyping ; Leukemia, Myeloid, Acute ; immunology ; metabolism ; Male ; Neoplasm, Residual ; immunology ; metabolism ; Neoplastic Stem Cells ; cytology ; Tumor Stem Cell Assay

This study was aimed to evaluate the therapeutic efficacy of bortezomib combined with autologous peripheral blood hematopoietic stem cell transplantation (autoPBSCT) for patients with multiple myeloma (MM). 5 patients underwent autologous hematopoietic stem cell transplantation. Bortezomib treatment was supplied for patients before autoPBSCT and in the conditioning of transplantation, it was also used in maintaining treatment. Patients with transplantation adopted bortezomib plus melphalan conditioning regimen. The number of infused MNC and number of CD34(+) cells were 4.06×10(8) (4.09×10(8) - 4.37×10(8))/kg and 3.98×10(6) (2.49×10(6) - 8.2×10(6))/kg respectively. The results showed that hematopoiesis was reconstituted in 5 patients, with a neutrophil cell count more than 0.5×10(9)/L at day 14 (13 - 25 days) after transplantation and platelet count more than 50×10(9)/L at day 28 (21 - 41 days) after transplantation. Transplantation-associated death was not observed. 5 patients were disease-free survival. In conclusion, treatment of bortezomib combined with autologous peripheral hematopoietic stem cell transplantation is an effective method for patients with multiple myeloma. Use of bortezomib after transplantation might still be favourable to MM patients, for survival prolongation and life quality improvement.
Adult ; Boronic Acids ; therapeutic use ; Bortezomib ; Combined Modality Therapy ; Humans ; Male ; Middle Aged ; Multiple Myeloma ; therapy ; Peripheral Blood Stem Cell Transplantation ; Pyrazines ; therapeutic use ; Transplantation Conditioning ; methods