Analysis and evaluation on Paeoniae Radix Alba from different cultivars by UPLC-Q-TOF-MS and HPLC / 中国中药杂志

In this study, an established ultra-high performance liquid chromatography coupled with quadrupole-time-of-flight mass spectrometry(UPLC-Q-TOF-MS) method was combined with multivariate statistical analysis to investigate the commonality and difference of main chemical components in the medicinal parts of Paeonia lactiflora from different cultivars; in addition, a high performance liquid chromatography(HPLC) method was established to simultaneously determine the content of eight active components in Paeoniae Radix Alba. Non-targeted analysis was carried out by UPLC-Q-TOF-MS on a Waters ACQUITY UPLC BEH C_(18)(2.1 mm×100 mm, 1.7 μm) column with a gradient elution of 0.1% aqueous formic acid(A)-acetonitrile(B) as the mobile phase at a flow rate of 0.2 mL·min~(-1). The column temperature was 30 ℃, and an electrospray ionization source was used to acquire mass spectrometry data in positive and negative ion modes. According to the accurate molecular weight and fragment ion information provided by multi-stage mass spectrometry and by comparison with reference substances and literature reports, thirty-six identical components were identified in Paeoniae Radix Alba from different cultivars with positive and negative ion modes. In the negative ion mode, two groups of samples were well separated; specifically, seventeen components with significant differences in content were screened and identified, and one component unique in "Bobaishao" was obtained. Quantitative analysis was conducted by high-performance liquid chromatography(HPLC) on an Agilent HC-C_(18)(4.6 mm×250 mm, 5 μm) column with a gradient elution of 0.1% aqueous phosphoric acid(A)-acetonitrile(B) as the mobile phase at a flow rate of 1.0 mL·min~(-1). The column temperature was 30 ℃ and the detection wavelength was at 230 nm. An HPLC method was developed for the simultaneous determination of eight active components(gallic acid, oxypaeoniflorin, catechin, albiflorin, paeoniflorin, galloylpaeoniflorin, 1,2,3,4,6-O-pentagalloylglucose, benzoyl-paeoniflorin) in Paeoniae Radix Albaa from different cultivars. Satisfactory linearity was achieved within the investigated linear ranges and with fine coefficients(r>0.999 0), and the methodological investigation showed that the method had good precision, repeatability and stability. The mean recoveries were 90.61% to 101.7% with RSD of 0.12% to 3.6%(n=6). UPLC-Q-OF-MS provided a rapid and efficient qualitative analytical method for the identification of the chemical components in Paeoniae Radix Alba, and the developed HPLC method was simple, rapid and accurate, which could provide a scientific basis for the evaluation of the germplasm resources and herbal quality of Paeoniae Radix Alba from different cultivars.

Reducing the consumption of personal protective equipment by setting up a multifunctional sampling station in the emergency department to screen for COVID-19 infection in Taiwan

In Taiwan, high-risk patients have been identified and tested for preventing community spread of COVID-19. Most sample collection was performed in emergency departments (EDs). Traditional sample collection requires substantial personal protective equipment (PPE), healthcare professionals, sanitation workers, and isolation space. To solve this problem, we established a multifunctional sample collection station (MSCS) for COVID-19 testing in front of our ED. The station is composed of a thick and clear acrylic board (2 cm), which completely separates the patient and medical personnel. Three pairs of gloves (length, 45 cm) are attached and fixed on the outside wall of the MSCS. The gloves are used to conduct sampling of throat/nasal swabs, sputum, and blood from patients. The gap between the board and the building is only 0.2 cm (sealed with silicone sealant). ED personnel communicate with patients using a small two-way broadcast system. Medical waste is put in specific trashcans installed in the table outside the MSCS. With full physical protection, the personnel conducting the sampling procedure need to wear only their N95 mask and gloves. After we activated the station, our PPE, sampling time, and sanitization resources were considerably conserved during the 4-week observation period. The MSCS obviously saved time and PPE. It elevated the efficiency and capacity of the ED for handling potential community infections of COVID-19.

Effect of microrna-132-3p overexpression onproliferation and osteogenic differentiation of mc3t3-e1 cells / 中国组织工程研究

BACKGROUND: Mechanical stress can influence the proliferation and differentiation of MC3T3-E1 cells and trigger differential expression of miR-132-3p. However, further research is warranted concerning whether tensile stress can influence the proliferation and differentiation of osteoblasts by regulating miR-132-3p. OBJECTIVE: To determine the expression of osteogenic differentiation markers and miR-132-3p in MC3T3-E1 cells under 12% cyclic stretch and to explore the effect of miR-132-3p on cell proliferation and differentiation. METHODS: MC3T3-E1 cells were loaded with 0% and 12% tensile stress, and alkaline phosphatase activity, osteocalcin mRNA and miR-132-3p expression levels were detected. MC3T3-E1 cells were transiently transfected with miR-132-3p mimics and a negative control transfection group was set up. The expression of alkaline phosphatase, osteocalcin and Runx2 mRNA in transfected cells were detected by qRT-PCR, and the effect of miR-132-3p on cell proliferation were detected by cell counting kit-8 assay. RESULTS AND CONCLUSION: The alkaline phosphatase activity and osteocalcin mRNA expression were down-regulated in MC3T3-E1 cells under 12% stretch stress (P < 0.01), and the expression of miR-132-3p was significantly increased (P < 0.05). QRT-PCR results showed the expression levels of osteogenic differentiation markers alkaline phosphatase activity, osteocalcin, and Runx2 mRNA in miR-132-3p mimics group were significantly decreased after intracellular transfection of miR-132-3p (P < 0.05). Compared with the negative control transfection group, the cell proliferation in the miR-132-3p mimic group was decreased at 24, 48, and 72 hours after transfection (P < 0.001), and the most obvious reduction was observed after 48-hour transfection. These findings indicate that 12% cyclic tensile stress can negatively regulate the proliferation and differentiation ability of MC3T3-E1 cells by overexpressing miR-132-3p.

Hematopoietic reconstitution after transplantation of uncontrolled-rate cryopreservation autologous peripheral blood hematopoietic stem cells using -80 °C mechanical freezer / 中国实验血液学杂志

<p><b>OBJECTIVE</b>This study was to identify the efficacy of -80°C cryopreservated peripheral blood hemato-poietic stem cell (PBHSC) transplantation for hematopoietic reanstitution in patients.</p><p><b>METHODS</b>The efficacy of 104 patients underwent autologous peripheral blood hematopoietic stem cell transplantation using uncontrolled-rate freezing and storage at -80°C was evaluated.</p><p><b>RESULTS</b>This cryopreservation method could effectively cryopreserve peripheral blood stem cells. Out of 104 patients only 2 patients died, other patients got hematologic reconstition satisfactorily, the median engrafement times of neutrophils and platelet were 12 and 14 days respectively, the activity of cells after rehabilitation was 94%, the mean recovery rates of CD34(+) cells and mononuclear cells (MNC) were 86% and 80.3% respectively. There were no significant influences on engrafement time in sex, chemotherapy circles and radiotherapy. The engrafement of leukocytes associated with amount of CD34(+) cells.</p><p><b>CONCLUSION</b>This simple uncontrolled-rate freezing PBHSC at -80°C is safe, effective and economic, and can meet clinical needs. As compared with the classical cryopreservation, there were no significant differences in hematopoietic reconstitution. Therefore, this method worth to popularize and apply in clinic.</p>

Association of XRCC1 genetic polymorphism with susceptibility to non-Hodgkin's lymphoma / 中国实验血液学杂志

The purpose of this study was to explore the association between X-ray repair cross-complementing group 1 (XRCC1)gene polymorphism and non-Hodgkin's lymphoma risk. A total of 282 non-Hodgkin's lymphoma (NHL) patients and 231 normal controls were used to investigate the effect of three XRCC1 gene polymorphisms (rs25487, rs25489, rs1799782) on susceptibility to non-Hodgkin's lymphoma. Genotyping was performed by using SNaPshot method. All statistical analyses were done with R software. Genotype and allele frequencies of XRCC1 were compared between the patients and controls by using the chi-square test. Crude and adjusted odd ratios and 95% confidence intervals were calculated by using logistic regression on the basis of genetic different models. For four kinds of NHL, subgroup analyses were also conducted. Combined genotype analyses of the three XRCC1 polymorphisms were also done by using logistic regression. The results showed that the variant genotype frequency was not significantly different between the controls and NHL or NHL subtype cases. Combined genotype analyses of XRCC1 399-280-194 results showed that the combined genotype was not associated with risk of NHL overall, but the VT-WT-WT combined genotype was associated with the decreased risk of T-NHL (OR: 0.21; 95%CI (0.06-0.8); P = 0.022), and the WT-VT-WT combined genotype was associated with the increased risk of FL(OR:15.23; 95%CI (1.69-137.39); P = 0.015). It is concluded that any studied polymorphism (rs25487, rs25489, rs1799782) alone was not shown to be rela-ted with the risk of NHL or each histologic subtype of NHL. The combined genotype with mutation of three SNP of XRCC1 was not related to the risk of NHL. However, further large-scale studies would be needed to confirm the association of decreased or increased risk for T-NHL and FL with the risk 3 combined SNP mutants of XRCC1 polymorphism.

Effect of low-molecular-weight heparin and urokinase on pulmonary arteries involved in pulmonary embolism / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Pulmonary embolism (PE) is a common and often fatal disease. Early after pulmonary thromboembolism, inflammation and associated intimal hyperplasia occur within the pulmonary arteries, similar to what is observed with chronic thromboembolic pulmonary hypertension. This study tested the hypothesis that thrombolytic and anticoagulant agents would have anti-inflammatory effects or inhibit intimal hyperplasia of involved pulmonary arteries.</p><p><b>METHODS</b>Seventy-two male New Zealand white rabbits were randomly divided into two groups (54 rabbits in the PE group and 18 in the sham group). Experimental PE was induced in 54 rabbits by femoral vein injection of autologous blood clots and confirmed with pulmonary angiography, and other 18 rabbits underwent sham operations. Fifty-four rabbits in the PE group were randomly divided into three groups: a control group (treated with normal saline), a low-molecular- weight heparin (LMWH) group (treated with LMWH), and a urokinase (UK) group (treated with UK). Arterial blood gas was analyzed at 2, 7, and 28 days (n = 6 per time point by random group division), then lung tissues were removed and were analyzed for pro-inflammatory cytokines and chemokines, and were stained for intimal hyperplasia.</p><p><b>RESULTS</b>The overall survival of rabbits undergoing PE was 100%. PE distribution detected on digital signal angiography (DSA) and histopathology was shown in 67% of rabbits (36/54) in the bilateral low lobar pulmonary arteries (PAs). The results showed that alveolar-arterial partial pressure of oxygen (PO2) difference (PA-aO2) significantly increased and PO2 decreased in the control group compared with the sham group. Compared with controls, the UK group had a decreased level of PA-aO2 on day 2 (P < 0.05), however, there was no significant difference in the LMWH group. Compared with controls, the LMWH group had a decreased level of monocyte chemoattractant protein-1 (MCP-1) in affected tissue and serum samples on days 7 and 28 (P < 0.05), and the UK group had decreased levels on days 2 and 7 (P < 0.05). Compared with sham group, all PE groups had an increased level of interleukin-13 (IL-13) and transforming growth factor-β (TGF-β) in unaffected lung tissue samples at days 2 and 7. IL-13 in affected lung tissue in the LMWH group was decreased at all time points compared with controls (P < 0.05). However, TGF-β in affected lung tissue of the LMWH and UK groups increased at day 28. There was less intimal hyperplasia in involved pulmonary arteries at days 7 and 28 in the LMWH group compared with controls; there was no statistical difference in the UK group compared with controls.</p><p><b>CONCLUSIONS</b>UK treatment can rapidly improve the V/Q mismatch in PE and appears a short-term anti-inflammatory benefit. However, LMWH maybe inhibit the later local inflammatory reaction and reduce intimal hyperplasia.</p>

HLA antigen compatibility between patients with hematologic diseases and their parents / 中国实验血液学杂志

This study was aimed to investigate and analyze the HLA antigen compatibility between patients with hematologic diseases and their parents so as to provide basis for selecting the suitable donors in haploidentical hematopoietic stem cell transplantation. The HLA low resolution for 174 families was typed and analyzed by using PCR-SSP. The results showed that 52.30% of patients with hematologic diseases possessed father and/or mother with HLA matching over haploidentity, 10.92% patients were over 8/10 matched with their father and/or mother. 11.49% were over semi-matched with both their father and mother. The rate of 6/10 matched pairs (28.16%), 7/10 matched pairs (16.1%) and 8/10 matched pairs (8.62%) were all beyond 5%; 9/10 (2.3%) and 10/10 matched pairs (1.15%) were all below 5%. It is concluded that with the matching degree increasing between two generations, HLA matching rate is decreasing. Over 50% and 10% patients were over HLA semi-matched and 8/10 matched with their father and/or mother, respectively. This high matching rate offered a big chance for success of haploidentical HSCT. Patients are more likely over semi-matched with their father and/or mother when they have high frequency and strong linkage HLA disequilibrium. High frequency and strong linkage disequilibrium in populations are main reason, and population concentrating and isolated living may be another reason for this phenomenon.

Idiopathic pulmonary vein thrombosis extending to left atrium: a case report with a literature review / 中华医学杂志(英文版)

Pulmonary vein thrombosis is a rare disease and is usually represented as a complication of atrial fibrillation, pulmonary tumors, and lobectomy. Although it is a potentially life threatening condition, the venous disease is easy to misdiagnose because of the non-specific symptoms. In this article, we present a 30-year-old patient who suffered from pulmonary vein thrombosis without any causes. He was diagnosed with other pulmonary disorders till the thrombus within the pulmonary vein extended into the left atrium. Left atrium mass resection and a left lower lobectomy were undertaken with relative urgency. The postoperative course was uneventful. The patient received a long course of oral anticoagulant therapy.

Clinical observation of icotinib hydrochloride for patients with advanced non-small cell lung cancer / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To explore the efficacy and side effects of icotinib hydrochloride in the treatment of patients with advanced non-small cell lung cancer (NSCLC).</p><p><b>METHODS</b>The efficacy and side effects of icotinib hydrochloride in treatment of 59 cases with stage IV NSCIC and followed-up from March 2009 to January 2012 were retrospectively analyzed.</p><p><b>RESULTS</b>Twenty seven patients (45.8%) showed partial response (PR), 17 patients (28.8%) achieved SD, and 15 (25.4%) had progressive disease. The objective response rate (ORR) was 45.8% (27/59), and disease control rate (DCR) was 74.6% (44/59). Among the 23 patients with EGFR mutation, ORR was 73.9% (17/23), and DCR was 95.7% (22/23). Thirty six patients (61.0%) achieved remission of symptoms to varying degrees. The main symptoms relieved were cough, asthmatic suffocating, pain and hoarseness. The major adverse events were mild skin rash (35.6%) and diarrhea (15.3%). Others were dry skin, nausea and stomach problems. The efficacy of icotinib hydrochloride were related to the ECOG performance status, smoking history, EGFR mutation and rash significantly (P < 0.05).</p><p><b>CONCLUSIONS</b>Monotherapy with icotinib hydrochloride is effective and tolerable for patients with advanced NSCLC, especially with EGFR mutation.</p>

Clinical analysis for 3 cases of HLA-matched between father and son and 1 case of post-hematopoietic stem cell transplant efficacy / 中国实验血液学杂志

Getting a HLA-matched donor is a key factor for successful hematopoietic stem cell transplantation. People are almost semi-matched with their parents, while a person HLA-matched with his/her father or mother was rarely seen, if so, usually whose father and mother are genetically related. HLA-low resolution for patients and their relatives were performed using PCR-SSP technique and three patients were found HLA-matched with their father in these results. One of them accepted hematopoietic stem cell transplantation using his HLA-matched father as his donor. The results showed that the chimerism was detected as stable complete donor chimerism, fusing gene of MLL-ENL was detected all negatively in the post-transplant period. This case got well hematopoietic reconstruction and GVHD didn't occur, so far he has survived for two years in health conditioning. It is concluded that people HLA-matched with his/her father or mother can be found when there is one identical haplotype of high frequency and strong linkage disequilibrium between father and mother. This case is valuable for hematopoietic stem cell transplantation development.

Anti-apoptosis competence and drug-resistance protein expression of tumor spheres from lung cancer cell line A549 / 肿瘤

Objective: To investigate the anti-apoptosis competence and the expression of glutathione- S-transferase-π (GST-π) of tumor spheres from lung cancer cell line A549. Methods: The tumor spheres were achieved from lung cancer cell line A549 cultured in serum-free medium. The apoptosis rates of both tumor spheres and primary A549 cells after treatment with cisplatin were detected by immunofluorescence assay with Annexin V-FITC+PI and JC-1 stainings, and the expressions of GST-π protein were also examined in the tumor spheres and the primary A549 cells as well as their grafted tumors in nude mice by Western blotting and fluorescence immunohistochemistry, respectively. Results: Immunofluorescence assay showed that the apoptosis rate was significantly higher in tumor spheres than that in primary A549 cells. Compared with the primary A549 cells and their corresponding grafted tumors, the expression levels of GST-π protein were significantly higher in the tumor spheres and their corresponding grafted tumors. Conclusion: Tumor spheres from lung cancer cell line A549 have a strong capability of drug-resistance, which is probably associated with the up-regulated expressions of drug-resistance proteins in these tumor spheres.

Study on the optimal waist circumference cut-off points of metabolic syndrome in Kazakh / 中华内分泌代谢杂志

Questionnaire-based survey, physical examination, and blood testing were conducted according to cluster random samplings in Kazakh residents in Xinjiang.2 760 samples were collected to analyze the association of different strata of waist circumference and clustering of metabolic syndrome (MS) components.Accoding to International Diabetes Federation standard, the prevalence of ≥1and ≥2 components of MS showed increasing trend with the increase of waist circunference, and odds ratio of clustering of MS components also increased significantly.The distance of receiver operating characteristic curve was the shortest and the prevalence of MS was 22.1% ;22.4% in men, and 21.9% in women;when the waist circumference was ≥91 cm for men, and ≥88 cm for women.

Needle sticking method / 中国针灸

Needle sticking method, which can be combined with multiple needling techniques, has been attached with great importance in recent years by doctors in clinic. Combining with the clinical experiences, the authors expounded the needle sticking method through its unified concept, differences between needle sticking method, which was an acupuncture technique, and stuck needle which was an accident during acupuncture, selection of needles, manipulations, mechanism of treatment, range of application, attentions and advantages of popularization. It is held that the technique can be widely applied for treatment of acute and chronic diseases of various departments with filiform needles. Easy to be manipulated, understood and mastered, the technique is without side effect and valuable to be popularized.

The relationship between phosphatidylinositol-3-kinases/protein-serine-threonine kinase signaling pathway and orthodontic tooth movement / 华西口腔医学杂志

<p><b>OBJECTIVE</b>To study the relationship between phosphatidylinositol-3-kinases(PI3K)/protein-serine-threonine kinase(AKt) signaling pathway and orthodontic tooth movement.</p><p><b>METHODS</b>Twenty-four rabbits were chosen to establish rabbit models for the study. The right maxillary teeth of each animal treated by orthodontics were as the test side, and the untreated left teeth were as the control side. The animals were sacrificed at 3, 5, 7, 14 d, respectively. The prepared tissue specimens were processed for the study. The changes of the expression of PI3K, AKt in periodontal tissues were detected by real-time quantitative-polymerase chain reaction (RQ-PCR) and Western blot techniques.</p><p><b>RESULTS</b>RQ-PCR showed that the expression of PI3K, AKt mRNA dramatically changed at 3 d. The expression of PI3K, AKt mRNA in the test side was higher than the control side, especially at 7 d, and then decreased. Compared with the control side, there was statistical significant difference in the test side(P < 0.05). The study obtained consistent conclusion from Western blot and RQ-PCR.</p><p><b>CONCLUSION</b>Expression of PI3K, AKt in rabbit periodontal tissues increase during orthodontic tooth movement, which prompts that PI3K/AKt signal pathways relate to orthodontic tooth movement and PI3K/AKt signal pathway involve in the periodontal tissue remodeling.</p>

Application of spectral karyotyping to cytogenetic analysis in acute myeloid leukemia / 中华血液学杂志

<p><b>OBJECTIVE</b>To evaluate the value of spectral karyotyping (SKY) in cytogenetic analysis of acute myeloid leukemias (AML).</p><p><b>METHODS</b>Nine AML patients were analyzed by R-banding and SKY. MLL, PML-RARalpha, AML1-ETO fusion genes were detected by dual fusion- fluorescence in situ hybridization (D-FISH).</p><p><b>RESULTS</b>All 9 samples were successfully hybridized. SKY identified structural aberrations including 9q -, t(15;17) and ins(10;17) (q22;p11p12) ; and some numeral abnormalities. The results of SKY confirmed those of R-band karyotyping and D-FISH; with more accurate localization.</p><p><b>CONCLUSION</b>SKY appears to be fairly stable, accurate and sensitive, for AML cytogenetic study.</p>

A recombination event occurring between HLA-A and -A loci from father's HLA haplotype chromosome / 中国实验血液学杂志

This study was aimed investigate the recombination event occurring between HLA-A and-A loci discovered from father's HLA haplotype chromosome in a family. Peripheral blood samples were collected from a family. HLA class I (-A, -B, and -Cw) and II (-DRB1 and -DQB1) alleles were amplified and typed by both low and high resolution PCR with sequence-specific primers (PCR-SSP) and sequence-based typing (SBT). The results showed that 2 haplotypes of the patient were A(*)3001-B(*)1302-DRB1(*)0701 and A(*)3001-B(*)5601-DRB1(*)1454 respectively, those of her father were A(*)3001-B(*)1302-DRB1*0701 and A(*)1101-B(*)5601-DRB1(*)1454. Family analysis demonstrated that the patient's A(*)3001-B(*)1302-DRB1(*)0701 came from her mother and A(*)1101-B(*)5601-DRB1(*)1454 came from her father, but the A of patient was A(*)3001 and B, DR were the same to her father. This showed that the chromosome exchange and recombination event of father's 2 haplotypes occurring between HLA-A and -A loci at meiosis. And recombinate haploid chromosome was completely inherited to his daughter 1. HLA typing and Paternity testing demonstrated that father was the natural father, and the recombination event occurring between HLA-A and -A loci of the daughter 1 with father's HLA haplotype chromosome. It is concluded that the HLA-A/A of father's HLA haplotype chromosome recombination event occurring between HLA-A an-A loci has been found in a family in China, which helps further study on the mechanisms of HLA recombination.

Allogeneic hematopoietic stem cell transplantation combined with imatinib for treatment of Philadelphia chromosome positive acute lymphoblastic leukemia / 中国实验血液学杂志

The study was purposed to explore the efficacy of allogeneic hematopoietic stem cell transplantation (allo-HSCT) combined with Imatinib for treatment of Philadelphia chromosome positive acute lymphoblastic leukemia (Ph(+)ALL) patients. From 2007 to 2008, 3 patients with Ph(+)ALL were treated with allogeneic hematopoietic stem cell transplantation and Imatinib, and the follow-up ended at Oct 21(st) 2009. 1 patient received HSCT from matched sibling donor and 2 patients from haploidentical related donors. All 3 patients achieved complete remission before transplantation and were treated with Imatinib for distinct time at different periods before and/or after transplantation. The level of bcr/abl mRNA was monitored using real-time PCR. The results showed that all 3 patients achieved stable engraftments without severe transplantation related complications. The level of bcr/abl mRNA declined and achieved zero level finally. In conclusion, the allo-HSCT combined with Imatinib is an effective therapy regimen for Ph(+)ALL patients.

Effect of demethylation treatment on the expression of inhibitory receptor KIR gene in NK-92MI cell line / 中国实验血液学杂志

The aim of this study was to analyze the promoter methylation patterns of inhibitory killer cell immunoglobulin-like receptor (KIR) which gene expression and the effect of demethylation treatment were studied, and to explore the possible regulation mechanism of inhibitory kir gene expression. The promoter methylation levels of kir2DL1 and kir2DL2/kir2DL3 in NK-92MI cell line were detected by bisulfite sequencing technique. Then NK-92MI cells were treated with 5-azacytidine to induce the demethylation of CpG islands. The levels of gene expression of kir were determined by RT-PCR. The results demonstrated that the methylation frequencies of CpG dinucleotides surrounding the promoter regions of kir2DL1 and kir2DL2/kir2DL3 genes were 25% to 88% and 5% to 80% respectively. DNA-demethylating treatment with 5-azacytidine resulted in re-expression of kir2DL1 gene and increased expressions of kir2DL1, kir2DL2 and kir2DL3 genes in NK-92MI cells. In conclusion, the promoter DNA methylation participates in the regulation of kir gene expression in NK-92MI cells.

Investigation on MS-PCR as a method for detecting minimal residual disease in acute leukemia / 中国实验血液学杂志

The aim of this study was to investigate the feasibility of monitoring minimal residual disease (MRD) of leukemia with methylation specified-polymerase chain reaction (MS-PCR). The HL-60 cells with Id4 gene complete methylation and Hek937 cells with Id4 gene complete unmethylation were mixed in accordance with different ratios of cells and were divided into 3 groups: group A (10% HL-60 + 90% Hek937), group B (1% HL-60 + 99% Hek937) and group C (0.1% HL-60 + 99.9% Hek937). The MS-PCR technique was used to detect the methylation status of Id4 gene in different ratios of leukemia cells. The results indicated that the methylation specific amplification of Id4 gene with 155 bp was observed in HL-60 cells showing complete methylation of Id4 gene; while the unmethylation specific amplication of Id4 gene with 156 bp was found in Hek937 cells, showing complete unmethylation. The methylation specific amplification of Id4 gene with 155 bp and unmethylation specific amplification of Id4 gene with 156 bp were simultaneously detected in A, B and C groups, which showed the expression of Id4 gene methylation. In conclusion, the MS-PCR technique can detect the Id4 gene methylation in leukemia cell sample containing 0.1% of HL-60 cells, moreover the Id4 gene methylation in various leukemia cells shows no significant difference, thereby use of MS-PCR to detect the Id4 methylation level may be a potential approach for monitoring of MRD. Id4 gene promoter methylation is a candidate of biomarker for MRD detection in acute leukemias.

Construction and identification of Kir2ds4 RNAi lentiviral vector / 中国实验血液学杂志

This study was aimed to construct a lentiviral vector of RNA interfered (RNAi)-kir2ds4 gene. In accordance with study-confirmed effective sequence of siRNA targeting kir2ds4 gene, the complementary DNA containing both sense and antisense oligonuctide of the targeting sequence was designed, synthesized and inserted into pSicoR-GFP vector containing U6 promoter and GFP sequence. The resulting lentiviral vector containing kir2ds4 shRNA was named as LV-sh kir2ds4, and confirmed by PCR and sequencing. 293T cells were co-transfected with lentiviral vector LV-sh kir2ds4 and packaging system. All virus stocks were produced by Lipofectamine 2000-mediated transfection. The titer of virus was tested according to the expression level of GFP. As a result, PCR and DNA sequencing demonstrated that the lentivirus RNAi vector of kir2ds4 was constructed successfully. The titer of virus tested by expression level of GFP was 6 x 10(8) TU/ml. It is concluded that the lentivirus RNAi vector of kir2ds4 has been successfully constructed.