Cancer stem cells are considered to be the origin of tumor relapse.Isolating and identifying them by special immune-phenotypes is the most commonly used method.The technique of isolating SP cells is very mature.More attentions are being paid to the isolation of cancer stem cells using differential expression activities of Wnt pathway on surface of tumor cells.Establishing stem cells niches to capture cancer tumor cells remains to be further evaluated.

The patient was a 38-year woman.Six years prior to presentation,she developed erythema and papules with occasional pruritus in both labium majora,which gradually confluenced into plaques with the formation of superficial erosion and ulcer;extension into the vulva and crissum occurred later.Half a year prior to presentation,a subcutaneous firm nodule measuring about 1.5cm in diameter with intact epidermis,developed on the right submaxilla,associated with mild swelling in the area;the nodule enlarged gradually.A subcutaneous induration measuring 0.5 cm in diameter was observed in the left chin 2 months later.She also reported a 20-year history of dry eye and dry mouth.The patient tested postive for antinuclear antibody cally,there was a focal infiltration of lymphocytes in tissue of minor salivary glands.The pathology of lesions on the right labium majus showed a dense dermal infiltration with S100-and CD1a-positive,irregulady-shaped histiocyte-like cells with abundant eosinophilic cytoplasm and few mitotic figures.Needle biopsy of the right submaxilla area showed tumor cells.A diagnosis of Langerhans cell histiocytosis and Sj(o)gren syndrome was made based on clinical manifestation,laboratory findings and histopathological features.Combination chemotherapy with cyclophosphamide,vincristine,corticosteroids and etoposide resulted in clinical improvement.

PurposeTo explore the relationship between expression of apoptosis-modulating proteins and chemotherapeutic efficacy in acute leukemia. MethodsImmunocytochemical method was used to detect the expression of Bcl-2、Bcl-XL、Bax and Bak in 36 cases of acute leukemia including previously untreated/ drug-sensitive group and refractory/relapse group. ResultsThe average positive cell rates of Bcl-2 and Bcl-XL in refractory/relapse group were (41.68 ± 14.39) % and (35.96 ± 9.95 ) %, while the rates in previously untreated/drug-sensitive group were (15.64 ± 8.51 )% and (12.91 ± 8.63 )%. Statistical analysis showed the average positive cell rates of Bcl-2 and Bcl-XL in refractory/relapse group were higher than those in previously untreated/drug-sensitive group (P ＜ 0.01 ). There was no significant difference in average positive cell rates of Bax and Bak between refractory/relapse group (25.28 ± 15.49) %, (15.53 ± 10.64) % and previously untreated/drug-sensitive group (21.55 ± 12.58)%, (13.23 ± 8.36)%. The Logistic regression of expression of Bd-2 、Bcl-XL、Bax and Bak to complete remission rate (CR) of 36 cases of acute leukemia showed that Bcl-XL was the most risk factor in reducing the CR.ConclusionsBcl-2 and Bcl-XL might play important roles in multi-drug resistance of acute leukemia and Bcl-XL was more important than Bcl-2.

Objective To investigate the characteristics of dysplasia in myeledysplastic syndrome (MDS). Methods 716 samples of adult patients with abnormal blood routine and unelear cause were collected between July 04, 2003 and March 14, 2007. Based on the gold diagnostic standard of WHO MDS classification, all eases were detected on cytomorphology, cytochemical stain, bone marrow pathological assay,cytogenetics, flow eytometry et al. The cytological study of bone marrow on some abnormal hematopaietie cells has a diagnostic value to determine clonal or non-clonal diseases and assess sensitivity and specificity. Results in the complicated various dysplasia of hematopeiefic ceils, the following characteristics can be the main basis of cytomorphological diagnosis: one of granular Auer bodies, micronuclens (MN), or nuclear budding, erythroid nuclear budding, megakaryocytes presented in peripheral blood, myeloblast or prorubricyte exhibited in peripheral blood, ringed sideroblasts>1%. The subordinate basis of cytomorphological diagnosis development of nuclei, ring-shaped nuclei, and aggregation of nuclear chromafin, erythroid multi-nuclei, odd nucleus, mother-daughter nucleus, nuclear fragmentation, vacuole, anisoeytosis and mieromegakaryocytes.Conclusion Cytomorphologic assay is the base for the diagnosis of MDS, however, it presents certain limit,especially when eytomnrphoiogical change does not possess specificity for early MDS. Hereby, it requires to combine other deteetion methods.

Objective To evaluate the DLC-l gene promoter methylation in acute lymphoblastic leukemia(ALL)in children by methylation specific-PCR(MSP),explore the its prognostic value.Methods Both pyrosequecing and MSP was used to detect DLC-1 gene methylation in bone marrow samples from 34 children with acute lymphoblastic leukemia,5 normal bone marrow samples and acute leukemia T cell line 6T-CET.DLC-1 mRNA expression in cells with、or without treatment with 5-aza-2'-deoxycytidine wag investigated by real time RT-PCR Results MSP analysis showed that DLC-1 promoter methylation Was identified in 21 of bone marrow samples from ALL patients,but was absent in 5 normal bone marrow specimens.These results were completely agreement with pyrosequencing analysis.In additional,the latter can give the quantitative analysis of methylation status in specific CpG sites.No association Was observed between DLC-1 methylation and patient’S clinical-biologic characteristics including age,gender,WBC count,FAB classification,immunology typing and clinical typing(P>0.05)In 18 ALL patients achieving complete remission(CR)witIl DLC-1 methylation,14 relapsed in 12 monthhs,while only 4 relapsed in 1 patients without DLC-1 gene metllylation.Treatment of the cell line 6T-CEM harboring methylation with 5-aza-2'-deoxycytidine increased DLC-1 expression in dose dependent manner. Conclusions This is the first report of high frequency of promoter methylation of DLC-1 in ALL It shows that DLC-1 methylation iS useful for predicting relapse of ALL.Pyrosequencing assay Call provide a sensitive,easy-to-use method for quantitative assessment of DLC-1 methylation.

Objective To establish a novel glucocorticoid (GC)-resistant human diffuse large B lymphoma(DLBCL) cell line Toledo/dexamethasone (DEX) by the exposure to DEX,and observe the biological characteristics of resistant and parental cell line,investigate the mechanisms of glucocorticoid-resistance.Methods Toledo/DEX was established by the exposure to DEX,the dose of which was increased gradually and intermittently for long periods of time.Toledo,in the logarithmic growth phage,was incubated in the culture medium containing DEX at the concentration of 1×10-8 mol/L at first.The medium without DEX was replaced after for 96 hours until the cell line re-entered the logarithmic growth phase.Repeat the above steps to acquire the ultimate concentration of DEX in the medium as 1.024 ×10-5 mol/L.The biological characteristics of resistant and parental cell lines were evaluated.Results Toledo/DEX was more invasive in the aspects of ultrastructure,tumorigenicity and drug sensitivity.Meanwhile,Toledo/DEX achieved some stable biological characteristics such as morphology,karyotypes and immunophenotype.Furthermore,GC receptor (GR) α and GR β protein expression analysis showed that GR was involved in the mechanism of the GCresistance.Conclusions Toledo/DEX is a drug-resistant cell line with a stable biology backgroud.These results may help shed light on the knowledge of GC-resistance and lay the groundwork for searching new therapeutics to reverse drug-resistance.

Objective Based on our previous established cohort of myelodysplastic syndrome (MDS), we investigated the potential effect of beta-tubulin (TUBB) gene in the transformation of MDS into acute leukemia Methods From our nested case-control study cohort of MDS patients, we chose 11 paired transformed and nontransformed MDS patients.TUBB gene expression was tested by quantitative real-time PCR.TUBB-siRNA transfection was used to down-regulate TUBB gene expression in SKM-1 cell line.The function of TUBB gene in SKM-1 cell line was evaluated by cell proliferation, soft agar clone formation and electron microscope.Results TUBB gene expression in MDS patients in transformed group were significantly higher than that in control group (2.91 ± 0.41 vs 0.90 ± 0.23, P ＜0.01).After TUBB-siRNA transfection, A450/630nm of SKM-1 cells at 24 h, 48 h and 72 h were 0.299 ± 0.045, 0.526 ± 0.034 and 0.652 ± 0.035, respectively, which were significantly decreased than those in negative-siRNA group (0.438 ±0.074, 0.858 ±0.064 and 0.974 ±0.044) (P ＜0.05).Soft agar clone formation in TUBB-siRNA group was (7.0 ±0.2)％, which was significantly reduced than that of negative-siRNA group (25.0 ± 0.2)％ (P ＜ 0.01).Electron microscope showed significant apoptotic signs in TUBB-siRNA group, including vacuoles in cytoplasm and karyorrhexis.Conclusion Our results indicate that TUBB gene may play a role in the transformation of MDS into acute leukemia by affecting the proliferation of malignant clones.

ObjectiveTo establish a simple and sensitive method to detect MPL515 mutations in peripheral blood of ET and PMF patients,and investigate the frequencies of the MPL515 and JAK2V617F mutations in Chinese patients.MethodsTotallv 261 patients of ET and 25 PMF cases were collected from Huashan Hospital of Fudan University and DNA samples were isolated from peripheral blood of these cases.SYBR GreenⅠreal-time PCR was used to detect JAK2V617F mutation.Taqman probe was designed to be specific for the three types of mutations ( MPl515wt,MPLW515L and MPIW515K).Real-time PCR was used to detect MPL515 mutations.Tbe results were confirmed by sequencing after T-A cloning.Results Among 261 ET patients,119 cases (45.6％ ) were identified as JAK2V617F mutation carriers and 7 cases (2.7％ ) were detected to be MPl515 mutation carriers,including 5 cases with MPLW515L,1 case with MPLW515K and 1 ease with MPLW515L + K.Additionally 10 cases with JAK2V617F(40.0％ ) and 3 cases with MPL515 ( 12.0％ ) were screened out in 25 PMF patients,including 1 case with MPLW515L and 2 cases with MPLW515L + K.One ET patient was found to harbor concurrent JAK2V617F and MPL515 mutations.ConclusionJAK2V617F mutation is the major molecular marker of ET and PMF,meanwhile MPL515 mutation is important and useful complement.

A high performance liquid chromatography-tandem mass spectrometric ( HPLC-MS / MS) method coupled with an immunoaffinity clean-up column was successfully developed for determination of aflatoxins (AFB1 , AFB2 , AFG1 , AFG2 , AFM1 and AFM2 ) and zeranols ( α-zeranol, β-zeranol, α-zearalenol,β-zearalenol, zearalanone and zearalenone ). The sample was extracted with methanol-acetonitrile (20∶ 80, V/ V) after enzymatic digestion by β-glucuronidase / sulfatase, and the extraction solution was passed through glassy fiber filter paper and then diluted with phosphate buffer solution (PBS). The reconstituted solution was cleaned up with IAC-AZ immunoaffinity column, and then analyzed by HPLC-MS / MS in multiple reaction monitoring (MRM) mode. The results indicated that the linear detection range was 0. 03-6. 0 μg / L for AFB2 and AFG2 , and 0. 05-20 μg / L for the rest compounds. The correlation coefficients were above 0. 999. The limits of detection (LOD) and limits of quantitation (LOQ) were 0. 01-0. 03 μg / kg and 0. 04-0. 09 μg / kg, respectively. The recoveries of the aflatoxins and zeranols were in the range of 73. 6% -98. 4% at the spiked levels of 0. 5, 1 and 5 μg / kg, and the relative standard deviations (RSDs) were in the range of 1. 9% -11. 2% . The method was proved to be simple and accurate, and suitable for the rapid determination of aflatoxins and zeranols in animal-originated foods.

Myeloproliferative neoplasm (MPN) is a kind of clone hematopoietic stem cell disease characterized by one or more myeloid cell lines hyperproliferation, including bcr-abl gene positive chronic myeloid leukemia (CML) and bcr-abl negative MPN, the later representing polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). A big stride has been made since the discovery of JAK2 and MPL gene mutations. However, the exact genetic basis of JAK2/MPL mutation double negative in MPN patients is still unclear. It has been reported recently that a new CALR mutation is discovered in the JAK2/MPL unmutated MPN patients who show unique clinical presentations, which provides a new diagnostic and prognosis-accessing criteria. The paper reviews CALR mutation and genetic mechanism mediating MPN.