Development of an LB cloning system and its application in expression of fusion genes in Sphingomonas sp. WG / 生物工程学报

In order to overcome the challenges of insufficient restriction enzyme sites, and construct a fusion-expression vector with flexible fusion direction, we designed an LB cloning system based on the type IIS and type IIT restriction enzymes LguⅠ and BbvCⅠ. The LB cloning system is constructed by inserting the LB fragment (GCTCTTCCTCAGC) into the multiple cloning site region of the broad-host plasmid pBBR1MCS-3 using PCR. The LB fragment contains partially overlapped recognition sites of LguⅠ and BbvCⅠ. Therefore, the same non-palindromic sequence will be generated by these two restriction endonucleases digestion. This feature can be used to quickly and flexibly insert multiple genes into the expression vector in a stepwise and directed way. In order to verify the efficacy of the cloning system, two glycosyltransferase genes welB and welK of Sphingomonas sp. WG were consecutively fused to the LB cloning vector, and the recombinant plasmid was transferred into Sphingomonas sp. WG by triparental mating. The results showed that gene fusion expression has little effect on sphingan titer, but enhanced the viscosity of sphingan. The viscosity of the sphingan produced by recombinant strain Sphingomonas sp. WG/pBBR1MCS-3-LB-welKB was 24.7% higher than that of the wild strain after fermentation for 84 h, which would be beneficial for its application. In conclusion, the application of LB cloning system were verified using Sphingomonas sp. WG. The LB cloning system may provide an efficient tool for fusion expression of target genes.

Effects of inner-heating acupuncture on apoptosis of chondrocytes and expression of Caspase-3 and Caspase-9 in rats with knee osteoarthritis / 中国针灸

OBJECTIVE@#To investigate the effect of inner-heating acupuncture on apoptosis of chondrocytes and expression of Caspase-3 and Caspase-9 in rats with knee osteoarthritis (KOA).@*METHODS@#A total of 32 rats were divided into a normal group, a model group, a control treatment group and a treatment group by random number grouping method, 8 rats in each one. The rats in the normal group received no intervention. The rats in the remaining three groups adopted modified Videman method to develop KOA model, the ankle joint of left posterior leg was fully extended and fixed with a resin bandage for 6 weeks. After successful modeling, the rats in the model group received no intervention. The rats in the control treatment group were treated with medium-frequency pulse electrotherapy. The rats in the treatment group were treated with inner- heating acupuncture, 30 min each treatment, once a day, five days per week, and totally 3-week treatment was given. After 3 weeks, the damaged cartilage tissue was collected, and HE staining was used to observe the pathological changes of the cartilage tissue of the knee joint. ELISA was used to detect the content of cytochrome-C in the tissue homogenate supernatant. The chondrocytes in damaged cartilage tissue were isolated, flow cytometer was used to detect the changes of apoptosis and mitochondrial membrane potential. The mRNA and protein expression of Caspase-3 and Caspase-9 in chondrocytes were detected by real-time quantitative PCR (qRT-PCR) and Western blot (WB), respectively.@*RESULTS@#Compared with the normal group, the damage of cartilage tissue in the model group was significant, and the expression level of Cyt-C in the homogenate supernatant of damaged cartilage tissue was increased (<0.01); the chondrocyte apoptosis was increased significantly (<0.01); the chondrocyte mitochondrial membrane potential was decreased significantly (<0.01); the mRNA and protein expression of Caspase-3 and Caspase-9 was increased significantly (all <0.01). Compared with the model group, the cartilage injury in the control treatment group and the treatment group was significantly relieved; the expression level of Cyt-C in the supernatant of damaged cartilage tissue homogenate was decreased (both <0.01); the chondrocyte apoptosis was significantly reduced (both <0.01); the chondrocyte mitochondrial membrane potential was increased significantly (both <0.01). Moreover, the mRNA and protein expression of Caspase-3 and Caspase-9 was significantly reduced (all <0.01). Compared with the control treatment group, the treatment group was more effective in the treatment of KOA.@*CONCLUSION@#The inner-heating acupuncture could significantly improve the pathological changes of KOA rats, inhibit the apoptosis of chondrocytes, which may be closely related to the suppression of Caspase-3 and Caspase-9 expression.

Ellagic acid attenuates hypoxic-ischemic brain injury by alleviating autophagy / 中国病理生理杂志

AIM:To investigate whether ellagic acid (EA) attenuates hypoxic-ischemic encephalopathy (HIE) by down-regulating autophagy.METHODS:In vivo, Sprague-Dawley rats (n=17) were randomly divided into 3groups:5 rats for sham group, 6 rats for HIE group and 6 rats for HIE+EA pretreatment group.The rats in HIE+EA pretreatment group were treated with EA (10 mg/kg, 10 m L/kg, suspended in corn oil, ig).After 24 h of operation, the rats from each group were sacrificed and their brains were collected.TTC staining and HE staining were used to define the infarct areas and brain structure.The autophagy-related proteins beclin-1, P62, LC3-II/-I and Atg5 in the cortex in each group were compared by Western blot.In vitro, PC12 cells were divided into 3 groups:control group, Coand CoEA pretreatment group.Co800μmol/L was added to the PC12 cells to induce an anoxic environment.The PC12 cells were pretreated with EA at 8μmol/L and the cell viability was measured by CCK-8 assay.The production of reactive oxidative species (ROS) in the cells was detected by flow cytometry with DCFH-DA staining.MDC staining and TM-RE staining were applied to reflect the extent of autophagy and the state of apoptosis, respectively.The autophagy-related proteins in PC12 cells were also investigated.RESULTS:In HIE group, 7-day-old rats were given the operations and the their large infarct areas in the hemisphere were observed by TTC staining.HE staining displayed the injured hemispheres which contained few neurons, and exhibited edema status and serious structural damage.EA pretreatment decreased the infarct area and alleviated the damage to hemisphere with more visible neurons, compared with HIE group.Compared with sham group, the levels of autophagy-related proteins Atg5, beclin-1 and LC3-II/-I in the cortex were increased (P<0.01) , and P62 protein expression was decreased (P<0.01) in HIE group.Compared with HIE group, the protein expression of Atg5, beclin-1 and LC3-II/-I was decreased (P<0.01) and P62 protein expression was increased in HIE+EA pretreatment group (P<0.01).In vitro, compared with CoPC12 cells in CoEA pretreatment group showed a lower ROS level.Moreover, the cells in CoEA pretreatment group exhibited higher mitochondrial membrane potential than that in CoMDC staining in Coshowed high value of fluorescence and increased number of autophagosomes.EA pretreatment reduced the number of autophagosomes and the extent of autophagy to protect PC12cells.Furthermore, the protein levels of Atg5, beclin-1 and LC3-II/-I in Cowere higher (P<0.01) , and the protein expression of P62 was lower (P<0.01) than those in control group.In CoEA pretreatment group, the protein levels of Atg5, beclin-1 and LC3-II/-I were decreased (P<0.01) and the protein expression of P62 was increased as compared with Co (P<0.01).CONCLUSION:EA pretreatment attenuates autophagy to protect the neurons against HIE injury.

In Vitro Identification and Characteristics of CD34Leukemia Stem Cell in t(8;21) Acute Myeloid Leukemia / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To preliminarily identify the existence of CD34leukemia stem cell (LSC) in t(8;21) acute myeloid leukemia (AML) by in vitro test.</p><p><b>METHODS</b>Bone marrow samples collected from newly diagnosed t(8;21) AML patients were tested. LinCD34CD38(abbreviation, CD34CD38), LinCD34CD38(abbreviation, CD34CD38) and LinCD34CD38CD45SSC(abbreviation, CD34"LSC") cell fractions were gated by flow cytometry after staining with fluorescent antibodies. Cells in Gphase were identified through Hoechst 33342 and pyronin Y staining. Aldefluor reagent was used to test aldehyde dehydrogenase (ALDH) activity. The above-mentioned 3 cell fractions were sorted, and mRNA levels of AML1-ETO and WT1 were measured by real-time quantitative PCR.</p><p><b>RESULTS</b>The 3 tested samples displayed the same tendency in ratio of the cells in Gphase: CD34"LSC">CD34CD38>CD34CD38. The paired t-test of 53 patients showed that frequency of ALDHcells of both CD34CD38and CD34"LSC" cell fractions was significantly higher than that of CD34CD38(P<0.01), furthermore, the ALDHcell frequency was significantly higher in CD34"LSC" than that in CD34CD38(P<0.01). AML1-ETO mRNA levels of cells sorted from 3 patients were similar among the 3 cell fractions within each patient, whereas WT1 mRNA levels were significantly higher in CD34"LSC" than that in other 2 cell fractions.</p><p><b>CONCLUSION</b>CD34LSC may exist in t(8;21) AML, and may be more primitive than CD34LSC. These results promote the necessity to perform in vivo xenogeneic transplantation mice.</p>

Expression rhythm of autophagic gene in neurons of neonatal rats with hypoxia/ischemia and its regulatory mechanism / 中国当代儿科杂志

<p><b>OBJECTIVE</b>To investigate the expression of autophagic gene and circadian gene in the neurons of neonatal rats after hypoxic-ischemic brain damage and the mechanism of nerve injury induced by hypoxia/ischemia.</p><p><b>METHODS</b>Twelve Sprague-Dawley (SD) rats were randomly divided into hypoxic-ischemic (HI) group and sham-operation group, with 6 rats in each group. Ligation of the right common carotid artery and hypoxic treatment were performed to establish a model of hypoxic-ischemic brain damage. Western blot was used to measure the expression of the circadian protein Clock in the cortex and hippocampus. The neurons of the rats were cultured in vitro and randomly divided into oxygen glucose deprivation (OGD) group and control group. The neurons in the OGD group were treated with DMEM medium without glucose or serum to simulate ischemic state, and hypoxic treatment was performed to establish an in vitro model of hypoxic-ischemic brain damage. Western blot was used to measure the expression of autophagy-related proteins Beclin1 and LC3 and Clock protein at different time points. The changes in the expression of Beclin1 and LC3 were measured after the expression of Clock protein in neurons was inhibited by small interfering RNA technique.</p><p><b>RESULTS</b>The expression of autophagy-related proteins Beclin1 and LC3Ⅱ in neurons cultured in vitro displayed a rhythmic fluctuation; after OGD treatment, the expression of Beclin1 and LC3Ⅱ gradually increased over the time of treatment and no longer had a rhythmic fluctuation. Compared with the sham-operation group, the HI group had a significant reduction in the expression of Clock protein in the cortex and hippocampus (P<0.05). After OGD treatment, the neurons cultured in vitro had a significant reduction in the expression of Clock protein (P<0.05). Compared with the negative control group, the Clock gene inhibition group had significant reductions in the expression of Beclin1 and LC3Ⅱ (P<0.05).</p><p><b>CONCLUSIONS</b>Hypoxia/ischemia induces the disorder in the expression rhythm of autophagy-related proteins Beclin1 and LC3, and the mechanism may be associated with the fact that the circadian protein Clock participates in the regulation of the expression of Beclin1 and LC3.</p>

Design, synthesis and biological evaluation of the novel ligustrazine derivatives / 药学学报

Using ligustrazine as the leading compound, we designed and synthesized ten novel ligustrazine derivatives, whose structures were determined by 1H NMR, 13C NMR and MS. Inhibitory effects of the new compounds on the proliferation of A549, A549/DDP and HBE cells were detected by MTT assay. The inhibi-tory activity of the synthesized compounds on migration and invasion of A549 cells were evaluated through scratch assay and transwell assay. Mechanism of the inhibition on migration and invasion was investigated by Western blotting. In addition, the cell cycle was analyzed with flow cytometry. The results showed that, both compounds Z8 and Z10 have an anti-proliferative activity, and the potencies of inhibition of tumor-cell migration and invasion were attributed to the down-regulation of MMP-2 and MMP-9. The compounds Z8 and Z10 could also enhance G2/M arrest in A549 cell as revealed by cell cycle analysis.

Application and research progress of bone marrow mesenchymal stem cells in corneal injury repair / 国际眼科杂志(Guoji Yanke Zazhi)

Bone marrow mesenchymal stem cells (BMSCs) are a class of cells that can differentiate into different kind of corneal cells both in vitro and in vivo,which include corneal epithelial cells,limbal epithelial cells and corneal stromal cells.BMSCs could differentiate into corneal epithelial cells after transplantation,which can not only repair the damaged corneal,but also relieve inflammatory injury caused by the inflammatory cell infiltration.The other function of BMSC transplantation is to reduce the rejection after corneal transplantation by inhibiting cell damage and apoptosis.BMSC can also express a variety of factors on the carrier,these factors paly the important role in promoting the proliferation of limbal stem cells.These findings above provide a new direction for the fundamental study of ophthalmology,and put forward new clinical treatment ideas for corneal disease,both of them have broad protect for development,in this paper,the research status and progress of BMSC in the repair of corneal injury are reviewed.

Effects of PINK1 gene on cell apoptosis and cell autophagy in neonatal mice with hypoxic-ischemic brain damage / 中国当代儿科杂志

<p><b>OBJECTIVE</b>To study the effect of PINK1 (phosphatase and tensin homolog deleted on chromosome ten induced putative kinase 1) gene on cell apoptosis and cell autophagy in neonatal mice with hypoxic-ischemic brain damage (HIBD).</p><p><b>METHODS</b>Seventy-two wild-type C57BL/6 mice and 72 PINK1 gene knockout neonatal C57BL/6 mice were randomly divided into four groups: sham-operated wild-type (SWT), HIBD model wild-type (MWT), sham-operated knockout (SKO) and HIBD model knockout (MKO). HIBD model was prepared by low oxygen exposure for 2.5 hours after right carotid artery ligation. After 24 hours of hypoxia-ischemia treatment, TTC (2,3,5-triphenyl four azole nitrogen chloride) staining was used to measure brain infarct volume. The immunohistochemical staining was used to measure the expression of cell apoptosis protein cleaved-caspase-3 (CC3) in brain tissues. The TUNEL method was used to measure cell apoptosis. The immunofluorescence staining and Western blot were used to measure the expression of cell autophagy protein LC3.</p><p><b>RESULTS</b>Compared with the MWT group, the infarct volume of brain tissues was markedly reduced in the MKO group (P<0.05), the number of apoptotic cells and the cell apoptosis index were markedly decreased in the MKO group (P<0.05), the expression of apoptosis protein CC3 was significantly reduced in the MKO group (P<0.05), the expression of cell autophagy protein LC3 was significantly decreased in the MKO group, and the autophagy indicator LC3II/LC3I was also markedly reduced in the MKO group (P<0.05).</p><p><b>CONCLUSIONS</b>PINK1 gene knockout can protect neonatal mice from HIBD.</p>

Sensitivity of Plasmodium falciparum to Antimalarial Drugs in Hainan Island, China

Pyronaridine and artesunate have been shown to be effective in falciparum malaria treatment. However, pyronaridine is rarely used in Hainan Island clinically, and artesunate is not widely used as a therapeutic agent. Instead, conventional antimalarial drugs, chloroquine and piperaquine, are used, explaining the emergence of chloroquine-resistant Plasmodium falciparum. In this article, we investigated the sensitivity of P. falciparum to antimalarial drugs used in Hainan Island for rational drug therapy. We performed in vivo (28 days) and in vitro tests to determine the sensitivity of P. falciparum to antimalarial drugs. Total 46 patients with falciparum malaria were treated with dihydroartemisinin/piperaquine phosphate (DUO-COTECXIN) and followed up for 28 day. The cure rate was 97.8%. The mean fever clearance time (22.5+/-10.6 hr) and the mean parasite clearance time (27.3+/-12.2 hr) showed no statistical significance with different genders, ages, temperatures, or parasite density (P>0.05). The resistance rates of chloroquine, piperaquine, pyronarididine, and artesunate detected in vitro were 71.9%, 40.6%, 12.5%, and 0%, respectively (Ppiperaquine>pyronarididine>artesunate. The inhibitory dose 50 (IC50) was 3.77x10(-6) mol/L, 2.09x10(-6) mol/L, 0.09x10(-6) mol/L, and 0.05x10(-6) mol/L, and the mean concentrations for complete inhibition (CIMC) of schizont formation were 5.60x10(-6) mol/L, 9.26x10(-6) mol/L, 0.55x10(-6) mol/L, and 0.07x10(-6) mol/L, respectively. Dihydroartemisinin showed a strong therapeutic effect against falciparum malaria with a low toxicity.

Sensitivity of Plasmodium falciparum to Antimalarial Drugs in Hainan Island, China

Pyronaridine and artesunate have been shown to be effective in falciparum malaria treatment. However, pyronaridine is rarely used in Hainan Island clinically, and artesunate is not widely used as a therapeutic agent. Instead, conventional antimalarial drugs, chloroquine and piperaquine, are used, explaining the emergence of chloroquine-resistant Plasmodium falciparum. In this article, we investigated the sensitivity of P. falciparum to antimalarial drugs used in Hainan Island for rational drug therapy. We performed in vivo (28 days) and in vitro tests to determine the sensitivity of P. falciparum to antimalarial drugs. Total 46 patients with falciparum malaria were treated with dihydroartemisinin/piperaquine phosphate (DUO-COTECXIN) and followed up for 28 day. The cure rate was 97.8%. The mean fever clearance time (22.5+/-10.6 hr) and the mean parasite clearance time (27.3+/-12.2 hr) showed no statistical significance with different genders, ages, temperatures, or parasite density (P>0.05). The resistance rates of chloroquine, piperaquine, pyronarididine, and artesunate detected in vitro were 71.9%, 40.6%, 12.5%, and 0%, respectively (Ppiperaquine>pyronarididine>artesunate. The inhibitory dose 50 (IC50) was 3.77x10(-6) mol/L, 2.09x10(-6) mol/L, 0.09x10(-6) mol/L, and 0.05x10(-6) mol/L, and the mean concentrations for complete inhibition (CIMC) of schizont formation were 5.60x10(-6) mol/L, 9.26x10(-6) mol/L, 0.55x10(-6) mol/L, and 0.07x10(-6) mol/L, respectively. Dihydroartemisinin showed a strong therapeutic effect against falciparum malaria with a low toxicity.

Immunophenotypic and clinical characteristic analysis of NPM1 mutated acute myeloid leukemia with a normal karyotype / 中国实验血液学杂志

This study was purposed to compare the immunophenotypic and clinical characteristics of NPM1 mutated acute myeloid leukemia with a normal karyotype under the similar constituent ratio of FAB subtypes. Immunophenotyping and NPM1 gene mutation type-A,B and D and other leukemic related fusion genes were detected by multiparameter flow cytometry and real time RT-PCR or PCR, respectively. 77 AML patients with a normal karyotype (NK) and mutated NPM1 gene (NPM1m(+)AML) detected by immunophenotyping assay were included in this study. 55 cases without NPM1 mutation (NPM1m(-)AML) and with normal karyotype were served as negative control. The results showed that there was significant difference between NPM1m(+)AML and NPM1m(-)AML in terms of sex, white blood count, platelet counts, blast, WT1 expression level, FLT3-ITD mutation positive rate and response to treatment. The characteristic immunophenotype is lower expression of early differentiation-associated antigens (CD34, HLA-DR, CD117, CD38), lymphocytic antigens (CD7, CD4, CD19, CD2) and higher expression of CD33 and CD123 (P < 0.05). When above features was further analyzed between the M1/2 and M4/5 subgroups in NPM1m(+)AML patients, the M1/2 cases retained a higher frequency in women and a higher WT1 expression level (P < 0.05) . Monocytic differentiation-associated antigens including HLA-DR and lymphocytic antigens CD7 were higher expressed and CD117 was lower expressed in M4/5 subgroup (P < 0.05). It is concluded that under condition of similar constituent ratio of M1/2 and M4/5 subtype and normal karyotype, NPM1m(+)AML patients have higher WT1 expression level and better response to treatment. The major immunophenotype features of NPM1m(+)AML patients are lower expression of early differentiation antigens and lymphoid lineage antigens and higher expression of CD33 and CD123. Monocytic differentiation-associated antigens only higher are expressed in M4/5 cases when compared with M1/2 cases within NPM1m(+) AML patients.

Expression of PML-RARα is up-regulated during ATRA and arsenics combined induction without influence on long-term prognosis of acute promyelocytic leukemia / 中国实验血液学杂志

The early molecular kinetics during all-trans retinoic acid (ATRA) plus arsenic-based induction therapy and its prognostic value for acute promyelocytic leukemia (APL) remain unclear. This study was purposed to investigate the molecular and cytogenetic kinetics and its clinical significance in treatment of APL with ATRA plus arsenic-based induction. The molecular and cytogenetic kinetics was assessed by real-time quantitative RT-PCR and interphase fluorescence in situ hybridization (FISH) in 32 newly diagnosed APL patients. The results showed that the median PML-RARα transcript levels (PML-RARα/ABL) were very significantly up-regulated at 14 days of induction therapy compared with that of pre-treatment (40.10% vs 57.74%, P < 0.01), and then decreased at 28 days of induction therapy and at the end of consolidation therapy (6.97% and 0%), respectively. The total of 65.62% and 31.25% patients showed up-regulation of PML-RARα transcript at 14 and 28 days after induction, as compared with pretreatment. The PML-RARα copies per APL cell before treatment, and at 14 and 28 days after induction were calculated as 0.9, 2.2, 1.4 by the formula of PML-RARA/ABL(%)×2/APL cells (%). With the median follow-up time of 22 months, 32 patients were still in continuous clinical remission and no molecular relapse occurred. Up-regulation of PML-RARa expression during the induction had no effect on outcomes of APL patients. It is concluded that up-regulation of PML-RARa expression is a common event during induction therapy with ATRA plus arsenics. Up-regulation of PML-RARa expression during induction therapy hasn't influenced the long-term prognosis of APL.

Clinical analysis of 31 cases of neonatal purulent meningitis caused by Escherichia coli / 中国当代儿科杂志

<p><b>OBJECTIVE</b>Neonatal purulent meningitis is a severe infection responsible for high mortality and disabling sequelae. Escherichia coli is the main pathogen of neonatal purulent meningitis. This study explored the clinical characteristics and antibiotic resistance of Escherichia coli-induced neonatal meningitis.</p><p><b>METHODS</b>A retrospective chart review was performed. A total of 31 cases of neonatal purulent meningitis caused by Escherichia coli were identified in the neonatal intensive care unit between January 1, 2001 and December 31, 2011. The clinical characteristics and antibiotic sensitivity test results were analyzed.</p><p><b>RESULTS</b>Fever, poor feeding, lethargy and seizure were common clinical signs of neonatal purulent meningitis caused by Escherichia coli. Acute complications mainly included hyponatremia (17 cases), hydrocephalus (8 cases), subdural collection (2 cases), ventriculitis (2 cases) and cerebral infarction (1 case). Thirty neonates (97%) had increased CRP levels. Of the 31 patients, 14 cases were cured and 12 had adverse outcomes (5 patients died during hospitalization). Escherichia coli strains were resistant (>50%) to commonly used penicillins and cephalosporins between 2007 and 2011, presenting significantly higher resistance rates than between 2001 and 2006. The detection rate of extended spectrum β-lactamases (ESBLs)-producing strains between 2007 and 2011 increased significantly compared with between 2001 and 2006 (57% vs 0).</p><p><b>CONCLUSIONS</b>The clinical manifestations of neonatal purulent meningitis caused by Escherichia coli are non specific. The outcome is poor. Monitoring of CRP levels is valuable for the early diagnosis of neonatal purulent meningitis. The antimicrobial resistance rates of Escherichia coli are increasing, especially to cephalosporins. The percentage of ESBLs-producing strains is increasing over the years.</p>

The more, the less: age and chemotherapy load are predictive of poor stem cell mobilization in patients with hematologic malignancies / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Intensive treatment such as autologous peripheral blood stem cell (PBSC) transplantation is an important therapeutic strategy in many hematologic malignancies. A number of factors have been reported to impact PBSC mobilization, but the predictive factors varied from one study to another. This retrospective study assessed our current mobilization and collection protocols, and explored the factors predictive of PBSC mobilization in patients with hematologic malignancies.</p><p><b>METHODS</b>Data of 64 consecutive patients with hematologic malignancies (multiple myeloma, n = 22; acute leukemia, n = 27; lymphoma, n = 15) who underwent PBSC mobilization for over 1 year were analyzed. Four patients with response to treatment of near complete remission or better were administered granulocyte colony-stimulating factor (G-CSF) to mobilize PBSCs. Sixty patients received G-CSF followed by chemotherapy mobilizing regimens. Poor mobilization (PM) was defined as when ≤ 2.0'10(6) CD34(+) cells/kg body weight were collected within three leukapheresis procedures.</p><p><b>RESULTS</b>The incidence of PM at the first mobilization attempt was 19% (12/64). The PM group was older than the non-PM group (median age, 51 vs. 40 years; P = 0.013). In univariate analysis, there were no significant differences in gender, diagnosis, and body weight between the PM and non-PM groups. A combination of chemotherapy and G-CSF was more effective than G-CSF alone as a mobilizing regimen (P = 0.019). Grade III or IV hematopoietic toxicity of chemotherapy had no significant effect on the mobilization efficacy. Supportive care and the incidence of febrile neutropenia were not significantly different between the two groups. In multivariate analysis, age (odds ratio (OR), 9.536; P = 0.002) and number of previous chemotherapy courses (OR 3.132; P = 0.024) were two independent negative predictive factors for CD34(+) cell yield. PM patients could be managed well by remobilization.</p><p><b>CONCLUSION</b>Older age and a heavy load of previous chemotherapy are the negative risk factors for PBSC mobilization.</p>

Effect of different regimens on bone disease of multiple myeloma / 中华血液学杂志

<p><b>OBJECTIVE</b>To explore the difference of effects of two regimens (bortezomib and dexamethasone, BD; and thalidomide and dexamethasone, TD) on bone disease in multiple myeloma (MM).</p><p><b>METHODS</b>Forty patients with newly diagnosed and refractory or relapsed MM were treated with BD or TD regimens from Dec 2006 to Sep 2008. Bone pain score and X-ray examination were carried out before and after therapy. Serum levels of DKK-1, sRANKL, OPG and TRACP-5b were measured by ELISA before and 3 months after therapy.</p><p><b>RESULTS</b>Serum TRACP-5b concentration was significantly decreased in patients received TD regimen (5.94 U/L before therapy vs 4.84 U/L 3 months after therapy, P < 0.05), and so did for serum DKK-1 concentration in patients responded to BD regimen (35.11 µg/L before vs 32.03 µg/L 3 months after therapy, P < 0.05); for serum concentration of sRANKL in patients responded to BD regimen (1.05 pmol/L before vs 0.67 pmol/L 3 months after therapy, P < 0.05); and for serum concentration of TRACP-5b in responders to BD regimen (5.57 U/L before therapy vs 4.90 U/L 3 months after therapy, P < 0.05).</p><p><b>CONCLUSION</b>Bortezomib lowers levels of serum DKK-1 and RANKL in responders, thus leads to normalization of abnormal bone remodeling through the increase of bone formation and reduction of bone resorption. Thalidomide decreases bone resorption regardless of treatment response.</p>

Hyperthermia increases the sensitivity of RPMI 8226 cells to adriamycin / 中国实验血液学杂志

This study was purposed to explore the effect of hyperthermia on sensitivity of multiple myeloma cells RPMI 8226 to adriamycin (ADM) and its mechanism. The working concentration of ADM against RPMI 8226 cells was defined by MTT assay. RPMI 8226 cells were divided into 4 groups: control group, hyperthermia (42°C) group, chemotherapy (ADM) group and combination group (42°C + ADM), the survival rate of RPMI 8226 cells in 4 groups was detected by trypan blue exclusion, the inhibitory effect of hyperthermia on proliferation of RPMI 8226 cells was detected by MTT assay, the cell cycle distribution, apoptosis rate of cells, intracellular ADM concentration and P-gp expression level were measured by flow cytometry. The 1/4 IC(50) of ADM was defined as the working concentration in the experiment. The results indicated that the hyperthermia promoted the entering the cells from in G(0)/G(1) phase into S and G(2)/M phases, the expression of P-gp protein on cells in hyperthermia and combination groups was down-regulated, the intracellular ADM concentration in combination group obviously increased. It is concluded that the hyperthermia combined with ADM obviously enhance the inhibitory effect on proliferation of RPMI 8226 cells. The hyperthermia increases the sensitivity of RPMI 8226 cells to chemotherapy through down-regulating the expression of P-gp protein on cells and increasing the intracellular ADM concentration.

Genetic diagnosis and weight loss surgery of a case of Prader-Willi syndrome / 中华内分泌代谢杂志

To investigate the clinical features, genetic diagnosis, and treatment of a patient with Prader-Willi syndrome(PWS). For a case with clinically suspected PWS, methylation specific PCR(MSPCR)amplification was applied to CpG islands of SNRPN(exon α)gene locus in the 15q11-q13. Furthermore, the diagnosis was comfirmed by the method of bisulfite sequencing PCR(BSPCR). Metabolic status before and after the operation of sleeve gastrectomy were compared. Absence of amplification of paternal allele on chromosome 15q11-q13 was detected in the case by MSPCR, different from the normal control. Results of BSPCR further proved a full methylation of CpG islands in the SNRPN gene locus. Four months after sleeve gastrectomy, systemic metabolic status and ventricular function were improved. MSPCR and BSPCR were both consistent with genetic diagnosis of PWS. Weight loss surgery is expected to be a major therapy of this disease.

Application of fluorescence in situ hybridization to prenatal diagnosis of aneuploidy in 110 uncultured amniotic fluid samples / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To optimize the prenatal diagnosis platform by using domestically made fluorescence in situ hybridization(FISH) kit and to explore the clinical application of FISH to rapid prenatal diagnosis of a wide range of chromosomal abnormalities.</p><p><b>METHODS</b>Amniotic fluid samples from 110 pregnant women were studied with the rapid prenatal diagnosis method of FISH and the conventional cell culture method of karyotyping, the results from both methods were compared.</p><p><b>RESULTS</b>Four cases of trisomy 21, 1 case of trisomy 18, 58 cases of 46, XX, and 47 cases of 46, XY were detected by FISH in the 110 amniotic fluid samples. It is concordant with the results from conventional karyotype analysis. The concordance rate is 100%.</p><p><b>CONCLUSION</b>Domestically made FISH kit can be used to rapidly and accurately detect the most common chromosome aneuploidies by using less sample volume while the price is relatively low. FISH can be a reliable and rapid prenatal diagnostic tool as an adjunct to classical cytogenetic study. It can be used for rapid and accurate prenatal diagnosis of women with high risk of maternal serum screening.</p>

The clinical characteristics of newly diagnosed acute myeloid leukemia patients with NPM1 mutation / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate the clinical characteristics of newly diagnosed acute myeloid leukemia (AML) with NPM1 mutation.</p><p><b>METHODS</b>NPM1 mutation (including A, B, D mutation type) was detected in 206 patients with newly diagnosed AML by real-time quantitative RT-PCR.</p><p><b>RESULTS</b>The incidence of NPM1 mutation was 15.5% in total AML patients and 32.5% in normal karyotypes AML patients. The characteristics of 174 NPM1 wild type patients v.s. that of 32 NPM1 mutation patients was as follow, median age (46 vs 35 years old, P < 0.01), WBC counts (27 × 10(9)/L vs 8 × 10(9)/L, P < 0.01), BPC (82 × 10(9)/L vs 36 × 10(9)/L, P < 0.01), proportion of AML-M(5) (31.2% vs 5.8%, P = 0.01), incidence of normal karyotypes (92.6% vs 40.8%, P < 0.01), incidence of FLT3-ITD-positive (25.0% vs 7.5%, P < 0.01), CD34-positvie (23.3% vs 69.5%, P < 0.01), cases with fusion gene (0 vs 47.1%, P < 0.01). No statistic difference was found in sex, percentage of blasts in bone marrow, complete remission rate, overall survival between the two groups. Relapse-free survival in AML patients with NPM1-mutation and FLT3-ITD-negative tended to be higher than in those with NPM1-mutation and FLT3-ITD-positive.</p><p><b>CONCLUSION</b>It is necessary to detect NPM1 mutation and FLT3-ITD in newly diagnosed AML patients, especially in patients with high WBC and BPC, CD34-negative, normal karyotype, which might help to molecular classification and treatment.</p>

Relationship of immunophenotypic features with minimal residual disease detection and gene types in 221 cases of acute promyelocytic leukemia / 中国实验血液学杂志

This study was aimed to investigate the relationship of immunophenotypic features with minimal residual disease (MRD) detection and gene types in APL patients. Immunophenotypes were analyzed in 221 newly diagnosed APL patients by using four-color flow cytometry. Among of them, CD123 antibody was examined in 87 patients and the fused gene pml-raralpha were detected by PCR in 196 specimens simultaneously. The results of immunophenotyping demonstrated that the positive percentages of CD123, CD33 and CD9 in newly diagnosed APL patients were 100%, 99.1% and 96.0% respectively, and mean percentages of positive cells in positive patients were all around 90%. Although the positive rates of CD117, CD13, CD38 and CD64 were all above 96%, but the mean percentages of positive cells in different positive patients were diverse and average percentages of positive cells were about 70%. CD15, CD56 and CD11b were expressed in some patients, but CD34 and HLA-DR were rarely expressed in the majority of patients, and average positive percentages were all lower. Among 196 newly diagnosed APL patients, bcr1, bcr2 and bcr3 expressions were 63.3%, 4.6% and 32.1% respectively. The results showed a strong correlation of positive expression of CD34 with bcr3 isoform. When cut-off value was chosen as 20%, the proportions of CD34 positive patients in bcr3 and bcr1 cases were 15.4% (10/65) and 3.3% (4/121) separately, which had a significant difference (p < 0.05). When cut-off value was 10%, bcr3 cases had a significantly higher percentage of CD34 positive, compared with bcr1 cases (p < 0.001), which was 47.7% (31/65) and 5.8% (7/121) respectively. However, there was no statistically significant difference on the other antigens between the two groups. Bcr3 isoform was highly indicated when CD34 was positive and non- large side scatter (NL-SSC) was shown in APL cells. It is concluded that there is a unique characteristics of immunophenotyping, and antigens such as CD123, CD33 and CD9 are more applicable to the detection of MRD in APL patients. The positive expression of CD34 and NL-SSC are associated with bcr3 isoform, and the relationship between gene type and antigen expression can be suggested more accurately when the cut-off value is chosen as 10%.