MicroRNA-193a regulating Wilms'tumor gene 1 to promote mouse podocyte apoptosis in diabetic nephropathy / 解剖学报

Objective To explore the effect of microRNA( miR) -193a on the apoptosis of mouse podocytes in diabetic nephropathy (DN) and its mechanism. Methods The DN model was replicated by culturing podocytes with high glucose in vitro and intraperitoneal injection of streptozocin (STZ) in mice in vivo. The cells or 60 mice were randomly divided into normal control (NC) group, model control group, and miR-NC inhibitor group, miR-193a inhibitor group, miR-NC mimic group and miR-193a mimic group. Flow cytometry, immunohistochemistry, TUNEL, Real-time PCR, Western blotting were used to examine the apoptosis of DN mice and mouse podocytes. Results The expression of Nephrin and Podocin in podocytes was weakened in DN mice and renal podocytes induced by high glucose, the apoptotic rate increased significantly, miR-193a was highly expressed, the levels of cleaved-Caspase-3 and Bax protein increased significantly, the level of Bcl-2 protein decreased significantly, and miR-193a inhibitor could improve this process. Wilms' tumor gene 1 ( WT1 ) mRNA and protein expression levels were significantly reduced in DN mice and podocytes cultured with high glucose. WT1 protein expression increased significantly after miR-193a inhibitor intervention, and WT1 protein expression was significantly reduced after miR-193a mimic transfection. Up-regulating WT1 could reduce the effect of miR- 193a on the apoptosis of mouse podocytes induced by high glucose. The dual luciferase reporter experiment confirmed the targeting relationship between miR-193a and WT1. Conclusion MiR-193a down-regulates the expression of WT1 and promotes apoptosis of DN podocytes.

Progress in Newcastle disease virus against tumor / 生物工程学报

Newcastle disease virus is paramyxoviridae, Avian mumps virus genus type I, and infects more than 250 species of birds, causing huge losses on poultry farming worldwide. Numerous experiments have demonstrated that Newcastle disease virus has oncolytic activity on tumor cells and can selectively replicate in cancer cells. Thus, Newcastle disease virus is a potential therapeutic agent for cancer treatment. Some human clinical trials achieved good results. In this review, we summarized research progress of the relationship between the structural protein of Newcastle disease virus and virulence, anti-tumor and autophagy of Newcastle disease.

Prediction of outcome in acute myeloid leukemia by measurement of WT1 expression as a basic marker of minimal residual disease / 中华血液学杂志

Objective@#To probe the potential utility of Wilms tumor 1 (WT1) as a marker of minimal residual disease (MRD) in acute myeloid leukemia (AML) to estimate the relapse-predicting cut-off value.@*Methods@#Quantitative assessment of bone marrow WT1 mRNA level was preformed using real-time quantitative reverse transcription polymerase chain reaction (RQ-RT-PCR) assay. The expression levels of WT1 dynamically measured with RQ-RT-PCR were retrospectively analyzed in 121 AML cases (not including acute promyelocytic leukemia) achieving complete remission (CR) after induction therapy followed by consolidation therapy. By comparing WT1 levels of patients with different post-therapy outcomes, the investigators used the receiver operating characteristic (ROC) curve to determine WT1 threshold so as to predict their clinical relapses. Then prognoses and the significance of intervention were analyzed between WT1 positive and negative patients according to the cut-off value of WT1.@*Results@#According to ROC curve, WT1 level higher than 2.98% predicted the possibility of relapse. For simplicity and clinical application, 3.00% was used as the cut-off value of WT1 level for relapse. WT1 levels in 41 patients at diagnosis were detected, meanwhile 3 patients whose WT1 levels at diagnosis below 3.00% were excluded, then the median WT1 level of the rest 38 patients at diagnosis was 44.09% (range 7.19%-188.06%) . The median WT1 level in remission was 0.48% (352 samples, range 0-8.41%) . The median WT1 level at diagnosis was higher than that in remission. Excluding the 3 patients with WT1 level at diagnosis under 3.00%, the relapse rate of WT1 positive group (>3.00% during consolidation phase and follow-up) and WT1 negative group (≤3.00%) was 70.0% (14/20) and 12.2% (12/98) respectively (P<0.001) . The median time from WT1 positivity to clinical relapse was 58 days.@*Conclusions@#WT1 expression level above 3.00% was associated with markedly high risk of relapse, which could be as a useful marker for monitoring MRD following consolidation therapy.

Experimental study of WT1 specific CD8+T cells in the treatment of breast cancer / 天津医药

Objective To investigate the feasibility of Wilms’tumor gene 1 (WT1)-specific CD8+T cells from periph?eral blood for the treatment of breast cancer by detecting the killing activity of WT1 specific CD8+T cells on breast cancer cells. Methods Flow cytometry was used to detect WT1-specific CD8+T cells in the peripheral blood of 20 samples from HLA-A2 seropositive healthy donors, which were isolated by WT1/MHC streptamer magnetic beads and cultured. The func?tion of WT1-specific CD8+ T cells were analysis by cytotoxicity assay. Results Twelve of 20 healthy donors had naive WT1-specific CD8+T-cell frequencies of>0.5%, and 4 of 20 even>1.0%of all CD8+T cells. After positive selection by magnetic cell separation, a purity of up to 80%can be achieved. WT1 specific CD8+T cells can specifically kill breast can?cer cell line with WT1 polypeptide. Conclusion WT1 specific CD8+T cells can be detected in peripheral blood of healthy volunteers. WT1 specific CD8+T cells have killing effect on breast cancer cells, suggesting the feasibility of adoptive immu?notherapy for breast cancer.

Prognostic Implications of Expression of the Wilms Tumor 1 (WT1) Gene in Acute Leukemia (Experience from South Egypt).

Background: Wilms’ tumor (WT1) gene expression has been reported in the majority of acute leukemia patients at diagnosis and has been evaluated as a prognostic and minimal residual disease (MRD) marker but its role is still controversial. Methods: Real-time quantitative polymerase chain reaction was used on bone marrow samples from 100 newly diagnosed adults and pediatrics acute leukemia patients (50 AML and 50 ALL patients). WT1 expression were examined at diagnosis and at the end of induction. Results: WT1 was expressed in (14%) ALL and in (36%) AML patients. We found no statistically significant impact of WT1 expression at diagnosis on response p= 0.054, 0.057, DFS (P = 0.591, 0.858), or OS (p= 0.339, p= 0.331) in ALL and AML patients respectively. Persistence of WT1 expressions at the end of induction didn't show any effect on relapse rate in AML however, it showed significant results in ALL p=0.045. Conclusion: Our results suggest that WT1 expression in patients with acute leukemia doesn't have any implication on response or survival however, significant association was found in predicting ALL relapse but the small sample size should be considered.

Denys-Drash syndrome, Septated Vagina And Low Level Of Anti-mullerian Hormone In male neonate

There is a wide variety of genital abnormalities observed in patients with Denys-Drash syndrome (DDS). WT1 is thought to influence the genes related to genital development and mutations in this gene have been associated with DDS. DDS should be considered in the differential diagnosis of newborns with genital anomalies. In contrast to other conditions with 46,XY disorders of sex development, individuals with DDS often have duplicated genital organs (a double vagina, cervix or uterus). A double uterus has not yet been reported with 1390G>A (Arg464 Asn) mutation. However, duplicated genitals have been reported with other genetic mutations in patients with DDS. The duplicated genitals in DDS may be associated with low anti-Mullerian hormone (AMH) secretion. Measurement of the AMH levels may add to our understanding of variations in genital development and their abnormalities in disorders such as DDS. In conclusion, this is first case of low level of AMH and double uterus in 1390G>A (Arg464 Asn) mutations of DDS male.

The expressions of ER, PR, C-erbB-2, E-Cad, p53 and Ki-67 in breast cancer and their clinical significance / 中国基层医药

Objective To explore the expressions of ER,PR,C-erbB-2,E-Cad,p53 and Ki-67 in breast cancer and their relationship with clinicopathology.Methods The expressions of ER,PR,C-erbB-2,E-Cad,p53 and Ki-67 proteins were detected in 80 cases of patients with breast cancer using immunohistochemical Streptavidin-Perosidase(SP) method,and were analyzed with clinical and medical records.Results The positive expression rates of ER,PR,C-erbB-2,E-Cad,p53 and Ki-67 in breast cancer were 60.0％,57.5％,45.0％,80.0％,48.8％ and 82.5％,respectively.In addition to C-erbB-2 which had correlation with lymph node metastasis,other five indicators showed no significant correlations with patient age,lymph node metastasis,tumor size and pathological grade.Conclusion There is a certain correlation in the expressions of ER,PR,C-erbB-2,E-Cad,p53 and Ki-67 in breast cancer.Combined detection is valuable in the treatment and prognostic evaluation of breast cancer.

Prediction of HLA-A2.1 restricted CTL epitope of SSX gene family and affinity assay of these epitopes with MHC-Ⅰ molecule / 第三军医大学学报

Objective To explore the immunotherapeutic strategy based on SSX tumor gene family by the CTL epitope prediction of the members of SSX tumor gene family and affinity assay of the epitope combining with MHCⅠ molecule.Methods The CTL epitope in the members of SSX gene family were predicted with the predictive software of HLA-A2.1 restricted CTL epitope.The 4 predicted epitope peptides were synthesized: P1(AMTKLGFKA),P4 (AMTKLGFNV),P5(AMTKLGFKV) and P6(VMTKLGFKV).After the affinity between these peptides and MHC-Ⅰ molecule were examined respectively,the combining stability between these peptides and the MHC-Ⅰ molecule were investigated respectively.Results As compared with positive control peptide HBcAg18-27,all of the synthesized peptides except P1 showed high affinity with MHC-Ⅰ molecule.The combining stability assay indicated that dissociation complex 50(DC_(50)) for P1,P4 and the positive control HBcAg 18-27 was all above 8.0 h,but DC_(50) for P5 and P6 was between 2 h and 4 h.Conclusion Two ideal peptides were screened out from the predicted CTL epitope peptides by in vitro MHC-Ⅰ combining affinity and stability assay,which provided the experimental data for the further identification of these epitope peptides in vivo.

Gene mutation in pituitary adenomas of human / 医学研究生学报

The single colonel composition of pituitary adenomas of human attests to the molecular basis of pituitary tumorigenesis. This review offers a specific overview of the involvement of alternation of tumor oncogenes and tumor suppressor genes (TSGs) in the pathogenesis of human pituitary adenomas. Mutations of gsp and H ras oncogenes genes and amplification of PKC have been identified in pituitary adenomas. Especially the mutation of gsp has been detected in 40% human GH secreting pituitary adenomas. The role of TSGs(tumor suppressor genes) including MEN 1, p53, Rb and p16 in pituitary adenoma formation and progression has yet be observed. Providing evidence proves that activation of oncogenes and inactivation of tumor suppressor genes seem to be at least one mechanism by which human pituitary adenomas origin and progress.

Detection of Mutations in the Wilms' Tumor Gene (WT1) in Leukemias / 中国医师杂志

Objective To analyze mutations in the Wilms tumor gene (WT1) in Ieukemogenesis.Methods WT1 gene in peripheral blood was determined by PRC-SSCP technique,in 32 cases of acute leukemia,included 9 cases of acute granulocytic leukemia,8 cases of chronic granulocytic leukemia(mean age-33 years) and 16 specimens of normal subjects were also detected.Results Mutations in the WT1 gene in 3 of 32 leukemias were found .WT1 mutations were found in 11% of cases of acute lymphoblastic leukemia and in 13% of cases of acute myeloid leukemia,in which they were associtated with a poor response to chemotherapy.Conclusions The mutations in Wilms tumor gene WT1 are associated with leukemogenesis and its therapy,which WT1 transcripts may prove a significant tumor marker, as a MRD monitor in evaluating remission status and early relapse,and may be useful in prognosis of acute leukemia.

The Expression of GFP Gene in Transformed and Tumor Cells / 上海第二医科大学学报

ObjectiveTo search the appropriate experimental conditions for using green fluores-cent protein (GFP) as a reporter gene in tumor gene therapy. Methods The plasmids carrying mu-tated GFP gene were transfected into the eukaryotic cells to observe transient gene expression. Thesestudies were conducted to 1. directly determine GFP expression and express stability in the COS- 7cells, 2. compare the transfection efficiency of two plasmids pcDNA3 - EGFP, pSVKa - S65T with dif-ferent promoters in different tumor cell lines, 3. determine two genes expression in a single cell usingLacZ cotransfected with GFP by FACS. ResultsFluorescence could be detected in intact viable cellsunder different sets of conditions. The expression of GFP might last two weeks or more and the ex-pressed fluorescence was stable. The transfection rate of pcDNA3 - EGFP expressed was different inthree tumor cell lines examined. But pSVK3 - GFP expressed similarly in four tumor cell lines exam-ined. FACS showed the probability of two genes entering a single cell is above >85% at the ratio 1:4.ConclusionThe above data indicate that the GFP can be visualized continuously and directly for geneexpression in living cells. GFP may also be used for quicklly selecting cells carrying a target gene.

Monitoring of WT-1 gene expression in peripheral blood of patients with acute leukemia by semiquantitative RT-PCR; possible marker for detection of minimal residual leukemia

The expression of the WT-1 gene which is found exclusively in human leukemic blasts frequently disappears from bone marrow of leukemia patients in complete remission (CR). Using semiquantitative RT-PCR, we investigated the expression of the WT-1 gene in peripheral bloods (PBs) of 33 patients with acute leukemia (AML 26; ALL 7) and monitored its expression after achievement of CR. None of the 6 normal controls expressed detectable levels of WT-1 transcripts (< 10(-4), background level), whereas 31 (93.9%) of 33 patients expressed variable levels of WT-1 transcripts (range, 10(-4) to 10(1)) at diagnosis. The level of WT-1 expression was not different between AML and ALL. By monitoring WT-1 gene expression in PB of 31 patients during CR, 5 patients relapsed (two from the 18 patients with undetectable levels of WT-1 gene expression and three from the 13 with WT-1 gene expression in low levels). Three of the 5 relapsed patients showed preceding reappearance or rise of WT-1 gene expression. From these results, we reconfirmed that the WT-1 gene is a pan-acute leukemic marker, which can be used to monitor minimal residual leukemia (MRL) after chemotherapy or in patients with CR.

Detection Wilm′s Tumor Gene Expression in Peripheral Blood and Its Clinical Significance of Acute Lymphocytic Leukemia Children / 实用儿科临床杂志

Objective To establish a real time reverse transcription polymerase chain reaction method for detecting WT1 and to understand the expression levels of WT1 in acute lymphocytic leukemia(ALL) of children through examining peripheral blood of leukemia children.Methods Thirty ALL patients, 13 non-leukemia Children and 18 normal children were included in this study. The method of real time RT-PCR detecting the expression of WT1 was established. The expression levels of WT1 gene were tested by this method.Results The expression levels of WT1 in 13 ALL with newly diagnosed patients were (105-106)copies/?g RNA, 12 with partial remission were (102-104)copies/?g RNA and 12 with complete remission were (0-102)copies/?g RNA.Conclusions Significant expression levels of WT1 in ALL are higher than those in non-leukemias and normal children.WT1 could be a marker for detecting minimal residual disease and evaluating therapy efficacy in ALL.