Objective To investigate the better method to detect the occurrence of JAK2~(V617F) mutation in patients with myeloproliferative disorders(MPD).Methods Genomic DNA was extracted from peripheral blood cell samples of 16 case of chronic myelogenous leukemia(CML),22 cases of polycythaemia vera(PV),26 cases of essential thrombocythaemia(ET)and 12 cases of idiopathic myelofibrosis(IMF).Polymerase chain reaction-restriction fragment length polymorphism(PCR-RFLP) and Allele-specific PCR(AS-PCR)were conducted to identify JAK2~(V617F) mutation individually.The sensitivity of two methods was compared successfully and the results were confirmed by sequence analysis consequently.Results The occurrence of JAK2~(V617F) mutation in PV patients detected by AS-PCR(72.7%)was higher than that of PCR-RFLP(50%).The occurrence of JAK2~(V6l7F) mutation in ET patients detected by AS-PCR(42.3%) was higher than that of PCR-RFLP(34.6%).All of the positive samples detected by PCR-RFLP were included in the positive samples of AS-PCR.The results of two methods were all coincided with sequence analysis. Conclusion AS-PCR is more sensitive,simple and convenient than PCR-RFLR.Consequently,it may have clinical significance in large scale detection of JAK2~(V617F) mutation.

To evaluate the frequency of jak2v617f mutation and analyze its correlation with clinical features of primary thrombocythemia (PT) patients, the mutation was detected by allele specific polymerase chain reaction (AS-PCR), the clinical and laboratory data in 66 PT patients with and without jak2v617f mutation were compared and clinical features of these PT patients were retrospectively analyzed. The results showed that among 66 patients, 27 patients (41%) had the mutation. The patients carrying the mutation displayed higher bone marrow erythropoiesis (26.9%+/-9.4% vs 16.3%+/-8.7%, p<0.05), higher ratio of granulopoiesis/erythropoiesis (2.9+/-1.8 vs 5.2+/-2.9, p<0.05) and higher incidence of microvascular disturbances (29.6% vs 5.1%, p<0.05). However, the age, gender, leukocyte and platelet counts, hemoglobin level, bone granulopoiesis, splenomegaly, history of thrombosis and hemorrhage had no difference between patients with and without the mutation. It is concluded that the frequency of jak2v617f mutation in primary thrombocythemia patients is 41%. The presence of the jak2v617f mutation is associated with a higher bone marrow erythropoiesis in primary thrombocythemia patients at diagnosis.

Recent studies have shown that Janus tyrosine kinase 2 (JAK2) V617F mutation is found in nearly all patients with polycythemia vera (PV) and underlie the basis of PV molecular pathogenesis. Moreover, JAK2 V617F patients with essential thrombocythemia (ET) have been found to have some clinical features similar to PV. To determine whether the same is true in a different Chinese patient population, we employed Allele-specific polymerase chain reaction in combination with sequence analysis to investigate the point mutation in a series of Chinese patients with hematological malignancies. A total of 99 Chinese myeloproliferative disorder patients and 120 additional patients with acute myeloid leukemia, acute lymphoblastic leukemia and myelodysplastic syndromes were studied. The V617F mutation was detected in genomic DNA of peripheral blood samples of 16 of 23 PV patients (69.6%), 21 of 45 ET patients (46.7%) and 3 of 8 patients with idiopathic myelofibrosis (37.5%). There were striking differences in clinical features such as hemoglobin, hematocrit and neutrophils percentages between V617F positive and negative patients with ET. Hence, our data support the idea that JAK2 V617F mutation divides ET patients into two subtypes, with the V617F positive group showing phenotypic similar to that of PV.

To investigate JAK2V617F mutation and its clinical significance in patients with idiopathic myelofibrosis (IMF), genomic DNA was extracted from peripheral blood cell samples of 12 IMF cases. Allele-specific PCR (AS-PCR) was performed to identify JAK2V617F mutation, and the results were confirmed by sequence analysis. A retrospective study was performed to explore the correlation between JAK2V617F mutation and the clinical, hematologic features. The results showed that in follow-up for 2 to 15 months, the occurrence of the positive point mutation in 12 patients with IMF was 50%, and the half of these positive patients had thrombosis. Patients with JAK2V617F point mutation had a higher counts of platelets and megakaryocytes in bone marrow than those in patients without JAK2V617F point mutation. Out of other 6 IMF patients without JAK2V617F point mutation only 1 patient had thrombosis, and lower counts of platelets in peripheral blood and megakaryocytes in bone marrow. It is concluded that majority of IMF patients with positive JAK2V617F point mutation have typical clinical and hematologic features, higher incidence of thrombosis, and higher counts of platelets in peripheral blood and megakaryocytes in bone marrow.

Background O-6-methylguanine-DNA-methyltransferase (MGMT) is a specific DNA revising enzyme transferring alkylated groups from DNA to its cysteine residue to avoid the abnormal twisting of DNA. Therefore, it is one of the drug resistant genes targeted in the treatment of cancer. This study explored the protective effect of MGMT gene transferred into mammalian cells.Methods Mammalian expression vector containing the MGMT gene cloned from human hepatocytes by RT-PCR was constructed and transferred into K562 cells and human peripheral blood mononuclear cells (PBMCs) via liposome, then assayed for gene expression at RNA and protein levels. MTT assay was used to check the drug resistance of cells transfected with MGMT gene.Results MGMT gene was successfully cloned. Real-time PCR showed that the mRNA expression in gene transfected groups in K562 cell line and PBMC: were 13.4 and 4.0 times that of the empty vector transfected groups respectively. Results of Western blotting showed distinct higher expression of MGMT in gene transfected group than in other two groups. The IC50 values increased to 7 and 2 times that of the original values respectively in stable transfected K562 cells and transient transfected PBMC.Conclusion The alkylating resistance of eukaryotic cells is enhanced after being transfected with MGMT gene Which protein product performs the protective function, and may provide the reference for the protective model of peripheral blood cells in cancer chemotherapy.

OBJECTIVE To study the JAK2 V617F point mutation in myeloproliferative disorders (MPD) and explore the clinical significance. METHODS We used Allele-specific polymerase chain reaction (AS-PCR) in combination with sequence analysis to detect the mutation in genomic DNA of peripheral blood mononuclear cells from 20 chronic myelogenous leukemia (CML) patients, 23 polycythaemia vera (PV), 40 essential thrombocythaemia (ET), 8 idiopathic myelofibrosis (IMF), 3 hypereosinophilic syndrome (HES). RESULTS JAK2 V617F was found in 38 (51.4%) of 74 BCR/ABL-negative MPD including 16 PV, 18 ET, 3 IMF and 1 HES patients. All positive samples and 10 negative samples identified by AS-PCR were confirmed by sequence analysis. Mutation-positive patients with ET had significantly increased hemoglobin, hematocrit, and neutrophil proportion than those without the mutation. CONCLUSION JAK2 V617F mutation is the key molecular genetics feature of BCR/ABL-negative MPD. Detection of JAK2 V617F mutation will bring about a major impact to the diagnosis, classification and treatment of MPD.

目的 克隆烷化剂耐药基因MGMT,构建真核表达质粒并导入真核细胞进行表达,筛选稳定表达的细胞系.方法 从人肝组织中克隆MGMT基因后重组构建真核表达粒质pIRES2-MGMT-EGFP并鉴定.通过脂质体法将目的基因导入K562细胞,应用RT-PCR及Western blot检测目的基因的表达并筛选稳定转染的细胞克隆.结果 成功克隆MGMT并构建了真核表达质粒pIRES2-MGMT-EGFP.转染细胞后MGMT在mRNA及蛋白水平均有表达,而对照组则为阴性.结论 含烷化剂耐药基因MGMT真核表达质粒的构建及转染后表达的成功为烷化剂耐药基因的相关研究奠定了良好的基础.

Myeloproliferative Diseases (MPD) are a spectrum of pathogenetically related disorders of varying clinical manifestations, characterized by neoplastic expansion of relatively mature granulocyte, erythroid, megakaryocyte, or monocyte and eosinocyte lineage cells. Recently a novel point mutation affecting the Janus tyrosine kinase 2 (JAK2 V617F) was identified as pathogenetically mechanisms by multiple competing groups. To investigate its prevalence and clinical significance in Chinese patients with hematological malignancies, we introduced Allele-specific PCR (AS-PCR) combined with sequence analysis to screen JAK2 V617F mutation. A total of 98 Chinese MPD patients and 120 additional hematological malignancies including AML, ALL, MDS were analyzed for the JAK2 V617F mutation. 98 MPD patients were referred for PV (n=57), ET (n=18), IMF (n=12), HES (n=2) and CML (n=9). 2 ml peripheral blood samples of MPD and 5–10 ml bone marrow samples of AML, ALL, MDS at the time of initial diagnosis were obtained with informed consent and genomic DNA was isolated presently. In addition, peripheral blood samples from 20 healthy donors were also collected as control. All samples were first screened by AS-PCR. The positive samples were subsequently confirmed by sequence analysis. The results showed that JAK2 V617F mutation was detected in 43 of 57 PV patients (75.4%), 7 of 18 ET patients (38.9%) and 5 of 12 IMF patients (41.7%). None of the AML, ALL, MDS, CML was found JAK2 V617F. There is no statistical difference of JAK2 V617F positive ratio between PV, ET and IMF. Furthermore, the mutation was not detected in the HES patient and 20 healthy controls. There is no other mutations and polymorphisms throughout exon 12 of JAK2. To our knowledge, this is the first report of JAK2 V617F mutation in a number of Chinese patients with hematological malignancies especially BCR/ABL-negative MPD. The incidence of JAK2 V617F of our study is a little lower compared with other publications especially in PV patients. The main reason may be firstly attributed to ethnic difference. In addition, with more MPD patients introduced and more sensitive methods such as ARMS-PCR or Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) applied, more JAK2 V617F mutation will be identified.

生理条件下,人类白细胞抗原G(HLA G)主要在胎盘绒毛远端的滋养层细胞特异表达,参与抑制母体的免疫应答反应,使母体对胎儿半抗原耐受。近年来研究发现:HLA G表达并不局限于母婴之间的胎盘组织内,在乳腺癌、黑色素瘤、白血病和淋巴瘤等恶性肿瘤细胞表面亦普遍表达。本文就该领域最新研究进展作一综述。

目的克隆O6-甲基鸟嘌呤-DNA甲基转移酶(MGMT)基因,测序鉴定正确后构建含增强荧光蛋白(EGFP)的真核表达载体.方法利用RT-PCR从人正常肝细胞中克隆MGMT并与克隆载体pGEM-T载体相连接.经PCR、酶切及测序鉴定证明克隆成功后用限制性内切酶切下MG-MT片段,同时酶切载体pIRES2-EGFP.凝胶纯化回收后重组构建真核表达载体并鉴定.结果测序结果显示所克隆的编码序列与GeneBank公布MGMT cDNA序列一致.真核表达载体的PCR及酶切鉴定结果与预期结果一致.结论成功克隆了耐药基因MGMT并构建了含EGFP编码序列的真核表达载体pIRES2-MGMT-EGFP,为MGMT的进一步相关研究奠定了坚实基础.