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Objective To deepen the understanding of chronic eosinophilic leukemia (CEL).Methods The course of diagnosis and treatment in a case of FIP1L1/PDGFRα fusion gene negative CEL was reported. Flow cytometry was used to analyze the immunophenotype of the cells in peripheral blood and pleural fluid. Karyotype was analyzed with G-banding. The expression of FIP1L1/PDGFRα fusion gene was detected by RT-PCR technique. Routine pathological examination of the tissues from bone marrow, lung and spleen were performed. Result A sixteen-year-old girl had severe anemia, fever, splenomegaly,thrombocytopenia and dominant hypereosinophilia lasting for 22 months. Trephine biopsy showed a hypercellular marrow with eosinophilic proliferation and moderate reticular fibrosis. Eosinophilic infiltration was found in lung and spleen and embolism was also found in spleen. She had a clonal chromosomal abnormality t(5;12)(q31;p13). The expression of FIP1L1/PDGFRα was negative. An abnormal clone of T cells expressing CD3-,CD4-,CD8- was found in peripheral blood and pleural fluid, in which the cional T cell accounted for 5.43% and 1.66% of the total lymphocytes respectively. The patient was refractory to treatment with hydroxyurea, prednisone and interferon alpha. She had poor response to a combination of therapy with low dose cytosine arabinoside, mitoxantrone, vincristine, cyclophosphamide, methotrexate and prednisone. She did not respond to imatinib and died of multiple organ failure. Conclusion The present case fulfilled the WHO diagnostic criteria of FIP1L1/PDGFRα(-) CEL which did not respond to routine treatment and imatinib. Allogenic stem cell transplantation should be considered as early as possible in this case. It is noteworthy that clonal CD3-,CD4-,CD8- T-cell abnormality is related to the pathogenesis of CEL.
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Objective To express and purify recombinant human platelet glycoprotein Ⅵ extracellular domain and prepare the polyclonal antibody against it.Methods Human platelet glycoprotein Ⅵ extracellular domain fragment (123~268 residues) was amplified by polymerase chain reaction and cloned into the prokaryotic expression vector pGEX-3x.The recombinant plasmid was constructed and expressed in E.coli after induction with isopropyl-?-D-thiogalactopyranoside (IPTG).The fusion protein was identified by Western blot analysis after purification by affinity chromatography.Rabbits were immunized with the purified fusion protein, and the collected rabbit antiserum was evaluated by sandwich ELISA, Western blot and flow cytometry.Results The coding sequence of GPVI extracellular domain was successfully inserted into pGEX-3x.Sequencing result showed that the cloned gene was identical as reported.After induction, a Mr 42kd fusion protein was expressed and confirmed by Western blot, which was identical to that expected.The titers of the antisera were up to 1∶[KG-*2]128. Sandwich ELISA result demonstrated that the prepared antibody recognized GPVI in human platelet. Western blot and flow cytometry revealed that the prepared antibody reacted with GPVI of platelet lysate and the native GPVI on human platelet surface.Conclusions Using purified prokaryotic expressed the fusion protein as antigen, the specific antibody was elicited in the immunized animals. The prepared polyclonal antibodies react specially with GPVI on human platelet surface and can be used for further studies of GPVI.
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<p><b>OBJECTIVE</b>To detect the methylation pattern of the p15(INK4B) gene and to explore its significance in the pathogenesis of acute leukemia (AL) and leukemic transformation of myelodysplastic syndromes (MDS).</p><p><b>METHODS</b>A total of 49 AL cases and 22 MDS cases were analyzed by methylation specific polymerase chain reaction (MSP) for methylation patterns in CpG islands of the p15(INK4B) gene.</p><p><b>RESULTS</b>Hypermethylation of the p15(INK4B) gene was found in 90% (26/29) of newly diagnosed AL, including 46% with complete methylation and 54% with partial methylation. All 3 evolved AL from MDS and 9 relapsed AL showed a methylated p15(INK4B) gene and the proportion of complete methylation was 67% and 56% respectively. Only 5 of 11 (45%) AL in remission, including 2 in complete remission (CR) and 3 in partial remission (PR), were partially methylated. The frequency of p15(INK4B) gene methylation in newly diagnosed or relapsed AL was significantly higher than that in AL in the remission stage (P = 0.002) p15(INK4B) gene methylation was found in 5 of 13 (38%) low-risk MDS (RA/RAS) patients and 80% of them showed only partial methylation. However, p15(INK4B) gene methylation was found in all 9 cases in the high-risk group (RAEB/RAEB-T), including complete methylation in 56%, significantly different from the low-risk MDS group (P = 0.002).</p><p><b>CONCLUSIONS</b>Hypermethylation of the p15(INK4B) gene occurs frequently in leukemia and high-risk MDS. It is possible that hypermethylation of this gene is related to the pathogenesis and development of AL and MDS. It may be used as a gene marker to detect minimal residual disease, relapse of AL and leukemic transformation in MDS.</p>
Subject(s)
Humans , Cell Cycle Proteins , Genetics , Cyclin-Dependent Kinase Inhibitor p15 , Cyclin-Dependent Kinase Inhibitor p16 , Genetics , DNA Methylation , Leukemia, Myeloid, Acute , Genetics , Myelodysplastic Syndromes , Genetics , Precursor Cell Lymphoblastic Leukemia-Lymphoma , Genetics , Tumor Suppressor ProteinsABSTRACT
Objective To establish a method of competitive polymerase chain reaction combined with restrictive endonucleases to measure the methylation quantitatively in MDR1 promoter region.Methods One sense primer and two anti-sense primers were designed to amplify a fragment of MDR1 gene promoter region, which was located between -102 and +186 bp containing a methylation site. The internal reference DNA fragment, witch was less 30bp than target fragment was made by three times of polymerase chain reaction(PCR)using different anti-sense primer and then subcloned into the Pbluescriptsk+ plasmid. The genomic DNA digested by Hpa, a methylation-sensitive restrictive endonuclease and competitive internal reference DNA were competitively amplified for MDR1 promoter in the same tube by PCR. The PCR products were electrophoresed on agarose gel, stained by ethidium bromide and subjected to image analysis scanner. The amount of target fragment was calculated as following, the optical density ratio of target fragment to competitive internal reference fragment multiplied the amount of competitive internal reference DNA. The ratio of PCR products amplified from HpaⅡ digested DNA and undigested DNA was named the methylation rate.Results The genomic DNA serially diluted with optimal amount of competitive internal reference DNA were co-amplified for MDR1 promoter. The significant positive correlation between the ratio of two products and the amount of genomic DNA was demonstrated. The correlation coefficient was 0.992, P
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Objective To explore the efficacy of calmodulin antagonist berbamine(BBM) in down-regulating survivin mRNA. Methods Human breast cancer cell line MCF7 and its adriamycin-resistant counterpart MCF7/ADR were used in the study. The cells were cultured with different concentration of BBM for 72 hours. The mRNA expression level of survivin gene in both MCF and MCF7/ADR cells was detected by semi-quantitative RT-PCR. Results After treating MCF7 and MCF7/ADR cells by 20?mol/L BBM, the mRNA expression level of survivin gene decreased from 0.43?0.02 to 0.21?0.04 in MCF7 cells, and from 0.57?0.05 to 0.45?0.04 in MCF/ADR cells(P
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The livers from 5 week 14～32 human fetuses were studied. The results are summarized as follows:1. Two kinds of liver cells revealed by EM: the "light" and "dark" cells. The light ones are larger in size, more numerous in number, containing larger mitochondria and obvious RER and SER, but less free ribosomes than the dark ones. Both of them may contain hemosiderin granules and the configuration of mitochondria enclosed by circular or semicircular RER cisternae.2. Bile canaliculus: Beside canaliculi between two adjacent liver cells, canaliculi situated between 3 or 4 liver cells are not infrequently revealed. That is due to the fetal liver cells are arranged in groups rather than in cords. The stereoscopic configurations of bile canaliculus and the tight junction surrounding it are demonstrated more clearly in freeze-fracture micrograph than that of the TEM. In addition, one intracellular bile canaliculus is found in the freeze-fracture preperation.3. The blood sinusoid and hemopoietic focus: In the 4-week fetus, the endothelium of the sinusoid can hardly be recognized While its lumen is quite large. Many erythrocytes accompanied by their immature components can be seen among it. As to the 32-week fetal liver, the endothelium and Kupffer cell may be easily recognized but the lumen of the sinusoid dimenishes enormously. The hemopoietic foci are localized extra-sinusoidially and in close contact with liver cells. Their volume exceeds one half of the fetal liver lobule before the age of 28 weeks, then it dimenishes gradually. In the space of Disse a fat storage cell is revealed.4. The portal canal : The mensenchyma and immature erythrocytes in the portal canal dimenishes with the increase of the fetal age. The portal canal is surrounded by the pigmented limiting plate cells of the liver lobules. The interlobular bile duct is differentiated from the limiting plate cells. Its epithelium is composed of both light and dark cells. The former is more numerous and contains many large mitochondria in its apical portion, while the latter shows higher electron density. Microvilli are located on the surfaces of both kinds of cells and finger like processes in the enlarged intercellular spaces are revealed.
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A 14-week human fetal kidney was studied by the method of freeze-fracture, the results were as follows:1. All the nuclei of various cells at this stage of development possess distinct nuclear pores with the same diameter and they are distributed randomly on the nuclear membrane. The intramembraneous particles on the PF of both the inner and outer nuclear membranes are more numerous than that of the EF. The morphological features of the nuclear pores vary according to the plane of their fracture face; they appear as dimples on the EF and as valcano mouth on the PF. The interior of the nucleus usually contains homogeneously dispersed particles, but no such structures were seen in thin sections. In some nuclei a round vesicle was revealed.2. The cell membrane of various cells, at this stage, shows special structures to manifest their degree of differentiation. In the less differentiated epithelial cells of the renal tubules, the cell membrane is straight and the intramembraneous particles are randomly distributed, the intromembraneous particles on PF are more than on EF, comparetively well developed tight junctions are located at the latexal surface near the apical portion of the cell while in the differentiated proximal tubules, there are many microvilli on their luminal surface, but their lateral cell membrane is still straight without interdigitations. In some tubules small processes near the basal part may be seen, however, basal fold is still absent. The basal membrane of the renal tubules is very prominent. As to the epithelial cells of the viseral layer of the renal corpuscle, gap and tight junctions are revealed while they are columnar in shape, but as they differentiated into podocyte with primary and secondary processes, the structure of tight junctions become simple, less prominent and discontinuous.3. Cytoplasm: In the process of cell differentiation the amount of cytoplasm increases and the ratio of nuclear end cytoplasmic volume decreases. The orgauelles are less in the undifferentiated cells, so the structure of the cytoplasm of them is much simpler than that of the more differentiated ones. The compartmentation phenomenon of the cytoplasm of differentiated cells become distince. In the freeze-fracture micrographs the membraneous structures, such as mitochondria, Golgi complex and secretion granules etc. are more stereoscopic than those in thin sections, but the opportunity to reveal the fine structures of various cells is minimized due to only one fracture face for each specimen and what structure will be fractured is beyond the control of the operator.
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Objective To explore the efficacy of calmodulin antagonist berbamine(BBM)on multidrug resistance(MDR)reversal and its mechanism. Methods Human breast cancer cell line MCF7 and its adriamycin-resistant counterpart MCF7/ADR were used in the study.The cells were cultured with ADR and different concentration of BBM. MTT assay was used to analyze the effect of BBM on cell growth inhibition.According to the MTT assay,the 50% inhibitory concentration(IC 50 ),the multiples of drug resistance and increased sensitivity of ADR were calculated.The concentration of intracellular ADR and expression level of P-glycoprotein(P-gp)were detected by flowcytometry(FCM).The mRNA expression level of mdr1 gene was detected by semi-quantitative reverse transcriptase polymerase chain reaction(RT-PCR)with ?-actin as internal reference. Results The IC 50 of ADR in MCF7 and MCF7/ADR cells were(0.98?0.06)?mol/L and(101.20?5.72)?mol/L,respectively.The resistant multiple of MCF/ADR cells to ADR was 103 folds higher than that of MCF7 cells.BBM increased the chemo-sensitivity of ADR in MCF7/ADR cells with dose-dependent relationship,i.e.when 5*!?mol/L ,10*!?mol/L and 20*!?mol/L BBM was added into the culture the chemo-sensitivity of ADR was increased to 2.76,5.88,and 28.26 folds(P