Effect of Danhong Injection on platelet metabolites based on metabonomics technology / 中国中药杂志

The effect of Danhong Injection on the endogenous metabolites of rabbit platelets was analyzed by the liquid chromatography-mass spectrometry( LC-MS) based metabonomic approach. Anti-platelet aggregation was detected after Danhong Injection treatment and the changes of platelet metabolites were analyzed by metabonomics. Principal component analysis( PCA) and partial least squares discriminant analysis( PLS-DA) were performed to investigate the effect of Danhong Injection on endogenous metabolites of platelets,characterize the biomarkers,and explore the relevant pathways and the underlying mechanism. As demonstrated by the pharmacodynamic results,Danhong Injection of different doses and concentrations antagonized platelet aggregation in a dose-and concentration-dependent manner. In contrast to the control group,25 differential metabolites such as nicotinic acid,nicotinic acid riboside,and hypoxanthine were screened out after platelets were treated by Danhong Injection. These metabolites,serving as important biomarkers,were mainly enriched in the nicotinic acid-niacinamide metabolic pathway and purine metabolic pathway. This study explored the therapeutic mechanism of Danhong Injection from a microscopic perspective by metabonomics,which is expected to provide a new idea for the investigation of platelet-related mechanisms.

Analysis and comparison of metabolic processes of salvianolic acid A and salvianolic acid B in rats based on UHPLC-LTQ-Orbitrap MS technology / 中国中药杂志

The metabolites of salvianolic acid A and salvianolic acid B in rats were analyzed and compared by ultra-high-perfor-mance liquid chromatography with linear ion trap-orbitrap mass spectrometry(UHPLC-LTQ-Orbitrap MS). After the rats were administrated by gavage, plasma at different time points and urine within 24 hours were collected to be treated by solid phase extraction(SPE), then they were gradient eluted by Acquity UPLC BEH C_(18) column(2.1 mm×100 mm, 1.7 μm) and 0.1% formic acid solution(A)-acetonitrile(B) mobile phase system, and finally all biological samples of rats were analyzed under negative ion scanning mode. By obtaining the accurate relative molecular mass and multi-level mass spectrometry information of metabolites, combined with the characteristic cleavage law of the reference standard and literature reports, a total of 30 metabolites, including salvianolic acid A and B, were identified. Among them, there were 24 metabolites derived from salvianolic acid A, with the main metabolic pathways including ester bond cleavage, dehydroxylation, decarboxylation, hydrogenation, methylation, hydroxylation, sulfonation, glucuronidation, and their multiple reactions. There were 15 metabolites of salvianolic acid B, and the main biotransformation pathways were five-membered ring cracking, ester bond cleavage, decarboxylation, dehydroxylation, hydrogenation, methylation, sulfonation, glucuronidation, and their compound reactions. In this study, the cross-metabolic profile of salvianolic acid A and B was elucidated completely, which would provide reference for further studies on the basis of pharmacodynamic substances and the exploration of pharmacological mechanism.

Analysis of carnosic acid metabolites in rats by UHPLC-Q-Exactive MS / 中国中药杂志

A method of ultra-high performance liquid chromatography coupled with quadrupole/electrostatic field Obitrap high-resolution mass spectrometry(UHPLC-Q-Exactive MS) was established to comprehensively identify the metabolites of carnosic acid in rats. After oral gavage of carnosic acid CMC-Na suspension in rats, urine, plasma and feces samples were collected and pretreated by solid phase extraction(SPE). Acquity UPLC BEH C_(18 )column(2.1 mm×100 mm, 1.7 μm) was used with 0.1% formic acid solution(A)-acetonitrile(B) as the mobile phase for the gradient elution. Biological samples were analyzed by quadrupole/electrostatic field Obitrap high-resolution mass spectrometry in positive and negative ion mode. Based on the accurate molecular mass, fragment ion information, and related literature reports, a total of 28 compounds(including carnosic acid) were finally identified in rat samples. As a result, the main metabolic pathways of carnosic acid in rats are oxidation, hydroxylation, methylation, glucuronide conjugation, sulfate conjugation, S-cysteine conjugation, glutathione conjugation, demethylation, decarbonylation and their composite reactions. The study showed that the metabolism of carnosic acid in rats could be efficiently and comprehensively clarified by using UHPLC-Q-Exactive MS, providing a reference for clarifying the material basis and metabolic mechanism of carnosic acid.

Long term outcome of posterior lumbar pedicle screw fixation combined with isthmic bone graft fusion in the treatment of lumbar spondylolysis in young patients / 中国骨伤

OBJECTIVE@#To investigate the long-term effect of posterior lumbar pedicle screw fixation combined with isthmus bone grafting and fusion in young patients with spondylolysis.@*METHODS@#A retrospective study was carried out, consisting of 16 young patients with lumbar spondylolysis without spondylolisthesis treated by lumbar posterior pedicle screw fixation combined with isthmic bone grafting fusion from January 2006 to July 2014. There were 11 males and 5 females, aged from 18 to 21 years old, with an average age of 19.3 years old, and the course of disease ranged from 12 to 26 months, with an average of 22 months. All the patients suffered from lumbar pain and difficulty in getting out of bed. Preoperative CT confirmed 12 cases of L₅ isthmus fissure and 4 cases of L₄ isthmus fissure. Bone graft fusion was confirmed and internal fixation was removed after operation. Lumbar spondylolysis was evaluated by lumbago visual analogue scoring method at preoperative and postoperative time points. Lumbar isthmic fusion was evaluated by lumbar CT, and degeneration of fixed and adjacent segments of lumbar intervertebral disc was evaluated by lumbar MRI.@*RESULTS@#Of the 16 patients, 13 patients (26 sides) were followed up, with a mean duration of 96 months. The operation time ranged from 80 to 105 minutes, with an average of 95 minutes. The intraoperative bleeding volume ranged from 150 to 300 ml, with an average of 225 ml. All the patients were successfully operated without any complications related to the operation. VAS scores at each time point after operation were improved compared with those before operation(<0.01). Postoperative CT scans of lumbar spine showed osseous fusion at 6 to 14 months, with an average of 12 months. There were no changes of adjacent segment degeneration, fixed segment disc degeneration and protrusion on lumbar spine MRI, and no symptomatic recurrence or recurrent spondylolysis in the long term.@*CONCLUSIONS@#The posterior lumbar pedicle screw fixation combined with isthmic bone grafting and fusion is safe and effective in the treatment of young spondylolysis. The fusion rate is high and the interference of normal physiological range is reduced. The long-term effect is satisfactory.

Analysis of mRNA expression profiles of megakaryocytes from human cord blood CD34+ cells ex vivo expanded using Solexa sequencing / 中国医学科学院学报

<p><b>OBJECTIVE</b>To investigate the mRNA expression profiles of megakaryocytes (MKs) from human cord blood CD34+ cells ex vivo expanded using Solexa technique.</p><p><b>METHODS</b>CD34+ Cells were isolated using density gradient centrifugation and magnetic activated cell sorting. Cultures were stimulated with recombinant human thrombopoietin (100 ng/ml). After 12 days, the MKs fraction was separated from the non-MKs fraction using an anti-CD41 monoclonal antibody by immunomagnetic sorting. The mRNA expression of MKs and non-MKs was detected by Solexa sequencing.</p><p><b>RESULTS</b>We obtained 3 773 147 and 3 533 805 Tags from MKs and non-MKs, respectively. The amounts of unambiguous tags were 3 291 132 and 2 967 947 and those of distinct tags were 197 769 and 245 318. The expression of 1161 genes was up-regulated and that of 902 genes down-regulated. The expression of 2717 tags was up-regulated and that of 1519 tags down-regulated.</p><p><b>CONCLUSIONS</b>MKs and non-MKs have remarkably different mRNA expression profiles. The differential gene-encoded products may be involved in cellular development, adhesion, apoptosis metabolism, intra- and intercellular signal transduction, and immune response. Further studies on this topic may clarify the expression mechanism, signal transduction, and regulation mechanisms.</p>

Detection of fetal short tandem repeat loci in maternal plasma as gender-independent fetal DNA marker / 中国实验血液学杂志

The aim of this study was to investigate the feasibility of using fetal short tandem repeat (STR) loci in maternal plasma as gender-independent fetal DNA marker. DNA from maternal plasma sample was extracted using QIAamp DNA Kit. AmpF1 STR profiler box was used to amplify 9 different polymorphic short tandem repeat (STR) loci (D3S1358, VWA, FGA, D5S818, D13S317, D7S820, D8S1179, D21S11, D18S51), the multiplex fluorescent PCR was used to amplify the STR alleles of fetal DNA in 36 pregnant plasma samples of pregnant women at different pregnancy. Their husbands' DNA isolated from whole blood samples were amplified at the same time. The PCR products were electrophoresis by ABI Prism 377 sequencer, the results of electrophoresis were analysed by Genscan. The presence of fetal DNA in maternal plasma by Paternally inherited fetal alleles were detected. The results showed that paternally inherited fetal alleles were detected in 4 cases in early pregnancy (4/6), 19 cases in middle pregnancy (19/20) and 9 cases in late pregnancy (9/10) respectively, the paternally inherited fetal alleles in 4 of 36 cases could not be detected. It is concluded that fluorescent multiplex PCR can be used for amplification of male and female fetal STRs in maternal plasma to obtain genetic information, which may have implication for non-invasive prenatal diagnosis of certain hereditary diseases independent of the fetal sex.

Human cord blood adult stem cells differentiate into hepatocyte-like cells in vitro / 中国实验血液学杂志

The aim of this study was to investigate the feasibility of the human cord blood adult stem cells (ASCs) to differentiate into hepatocytes in vitro induced by combined stimulation with hepatocyte growth factor (HGF), stem cell factor (SCF) and leukemia inhibitory factor (LIF). The adult stem cells were obtained through density gradient centrifugation and magnetic activated cell sorting (MACS). The adult stem cells were cultured in DMEM with HGF (10 ng/ml)+SCF (10 ng/ml)+LIF (10 ng/ml) in induced group I. In induced group II the enriched cells were cultured in DMEM with SCF (10 ng/ml)+LIF (10 ng/ml) and the undifferentiated cells acted as the control group without the factors. The morphology of cells was observed by the inverted phase contrast microscopy; the expression of albumin (Alb), human hepatocyte cytokeratin (CK18) and alpha-fetoprotein (AFP) were detected by immunofluorescence, immunohistochemistry and RT-PCR assay in the 21-day culture. Alb secreted by hepatocytes in the medium was determined by radioimmunoassay (RIA) at day 7, 14, 21, 23 and 25. The results showed that the shapes of ASCs changed and their sizes and number increased in the course of culture in group I. After being induced for three weeks, the cells turned round and resembled hepatocyte-like cells. The mRNA for Alb could be detected by RT-PCR in the differentiated adult stem cells in group I, and the mRNA for AFP was poorly detected by RT-PCR at day 21. Alb and CK18 were positive through immunofluorescence and immunohistochemistry at day 21, compared with group II and the control group. In group I, Alb in the medium significantly increased, compared with control group, and reached the highest level at day 21, then decreased at day 23. It is concluded that under some definite inducing conditions, human cord blood adult stem cells can differentiate into hepatocyte-like cells and HGF plays a critical role during the course.

Effect of angiotensin II on differentiation of umbilical cord blood CD34+ cells into megakaryocytes / 中国实验血液学杂志

This study was aimed to investigate the effect of angiotensin II on differentiation of cord blood CD34+ cells into megakaryocytes in vitro. The CD34+ cells from eight fresh umbilical cord blood samples sorted by a high-gradient magnetic cell sorting system (MACS) were cultured in serum-free culture medium containing thrombopoietin (TPO) 50 ng/ml, IL-3 10 ng/ml, stem cell factor (SCF) 50 ng/ml and different concentrations of angiotensin II (0, 50, 100, 1000 microg/ml) for 14 days. Mononuclear cells (MNC) were counted by automatic cell analyzer. Cultured CD41+ cell and platelet counts in cultured system, and cell cycle were analyzed by flow cytometry. CD41 specific monoclonal antibody staining was observed by immunofluorescence microscopy. The results showed that as compared with the control group, the number of MNC not increased significantly (P > 0.05), but the number of CD41+ cells and platelets increased significantly in treatment group (P < 0.05). Cell cycle analysis revealed that the amounts of 4N cells increased and apoptosis cells obviously existed in treatment group (P < 0.05). After fluorescence staining, more CD41+ cells of different sizes were observed by means of fluorescence microscopy in both groups. It is concluded that angiotensin II can induce the cord blood CD34+ cells to differentiate towards megakaryocyte, and enhance the function of megakaryocyte to produce platelet.

Formation of platelets from cord blood CD34+ cells-derived megakaryocytes induced by S-nitrosoglutathione / 生理学报

To investigate the effect of S-nitrosoglutathione (GSNO), a nitric oxide donor, on platelet production from megakaryocytes differentiated from cord blood CD34(+) cells in vitro, the CD34 (+) cells from eight fresh umbilical cord blood samples by a high-gradient magnetic cell sorting (MACS) system were cultured in serum-free medium for 14 d with thrombopoietin (TPO) 50 ng/ml, IL-3 10 ng/ml, stem cell factor (SCF) 50 ng/ml and rHuGM-CSF 20 ng/ml. Then, CD61 (+) cells were purified by MACS system from these CD34 (+) cells, and were cultured in serum-free medium supplemented with TPO 50 ng/ml, IL-3 10 ng/ml and SCF 50 ng/ml in the presence (treatment group) and absence (control group) of GSNO for 30 min or 2 h. Platelet-sized particles were counted by flow cytometry; megakaryocyte structure was detected by scanning electron microscope. Aggregation of the thrombin-induced platelet particle was observed under inversion microscope. cGMP was assessed by commercial ELISA kit. The results showed that, compared with the control group, the number of platelet-sized particles significantly increased (P<0.05) in the treatment group, in which megakaryocytes presented significant pseudopod formation and extensive membrane blebbing. The platelet particle aggregation could be observed under microscope after thrombin induction. cGMP activity was significantly increased after treatment with GSNO (P<0.05). These results propose that GSNO can facilitate platelet production from megakaryocyte, and it may be partly through cGMP pathway.

Early experiences on living donor liver transplantation in China: multicenter report / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Because of the lack of brain death laws in China, the proportion of cadaveric organ donation is low. Many patients with end-stage liver disease die waiting for a suitable donor. Living donor liver transplantation (LDLT) would reduce the current discrepancy between the number of patients on the transplant waiting list and the number of available organ donors. We describe the early experience of LDLT in the mainland of China based on data from five liver transplant centers.</p><p><b>METHODS</b>Between January 2001 and October 2003, 45 patients with end-stage liver disease received LDLT at five centers in China. The indication and timing, surgical techniques and complications, nonsurgical issues including rejection, infection, and advantages of LDLT in the series were reviewed. Actuarial patient and graft survival rates were calculated by using the Kaplan-Meier product-limit estimate. Statistical analysis was completed by using SPSS 10.0.</p><p><b>RESULTS</b>All LDLT recipients were cirrhotic patients, except for one man with fulminant hepatic failure. Among the 45 cases of LDLT, 35 (77.8%) were performed in one center (the First Affiliated Hospital of Nanjing Medical University). The overall 1 and 3 year survival rate of the recipients was 93.1% and 92.0%, respectively. Of the 45 LDLT donors, there were 3 cases of biliary leakage, 2 subphrenic collections, 1 fat liquefaction around the incision and 1 biliary peritonitis after T tube removal. All donors recovered completely.</p><p><b>CONCLUSIONS</b>LDLT provides an excellent approach to addressing the problem of donor shortage in China even though the operation is complicated, uncompromising and difficult with respect to the safety of the donors and receptors. Despite early technical hurdles having been overcome, perfection of technique is still necessarily. At present, LDLT is a good choice for the patients with irreversible liver disease.</p>

Arthroscopically minimal invasion asisted percutaneous cannulated screw fixation of longitudinal fracture of the patella / 中国基层医药

Objective To evaluate the effectiveness of arthroscopically assisted percutaneous reduction and internal fixation with eannulated screws.Methods The fracture was reduced by closed manipulation or percutaneous leverage force by using the Kirschner wire.Then the patella was temporarily fixed by using a large size towel clamp or Kirschner wires.Under the guidance of knee arthroscopy,a micro-incision was made at the size of cannulated screw placement,the pilot holes were drilled at a proper depth,and the thread was configured.Two titanium screws were inserted parallelly.Results Following-up chekups for 4～24 months in 18 cases showed a satisfactory recovery of knee functions.According to the Bostman' standard,excellent effects were obtained in 16 cases and good effects in 2 cases.Conclusion Treatment of patellar fractures by percutaneous cannulated screw fixation under arrhroscope of- fered advantages of minimal invasion,accurate reduction,reliable fixation,and excellent recovery of joint functions.

Effect of GM-CSF on expansion and differentiation of CD34+ megakaryocyte progenitor cells from cord blood in vitro / 中国实验血液学杂志

To study the effect of GM-CSF on in vitro expansion of megakaryocyte progenitor cells from cord blood, CD34(+) cells isolated by magnetic cell sorting system (MACS) were cultured in serum-free medium containing TPO, IL-3, SCF and with or without various concentrations of GM-CSF (5, 20, 100 ng/ml). The numbers of MNC, proportion of CD34(+)CD41(+) cells and CFU-MK were measured at 6, 10 and 14 days. The results showed that the expansion of MNC and proportion of CD41(+) cells was accelerated distinctly by various concentrations of GM-CSF after 14 days, while 20 and 100 ng/ml GM-CSF exhibited higher expansion effect than that of 5 ng/ml. TPO + IL-3 + SCF with 5 ng/ml or 20 ng/ml GM-CSF could stimulate the formation of CFU-MK, while TPO + IL-3 + SCF with 100 ng/ml GM-CSF could inhibit it. It is concluded that GM-CSF can accelerate the expansion of megakaryocyte progenitor cells from CD34(+) cells in cord blood in the serum-free medium containing TPO + IL-3 + SCF.

Determination of ABO blood group genotypes with one tube PCR reaction / 中国实验血液学杂志

This study was aimed to establish one tube PCR reaction technique to determine ABO blood group genotypes. Salting-out method was adopted to extract genomic DNA; one tube polymerase chain reaction with GeneScan technique was used to identify ABO genotypes. The results showed that the ABO genotypes of 132 samples were in accordance with the phenotypes determined by serological technique. The frequencies of A, B and O were 0.205, 0.159 and 0.636 respectively. AA, AO, AB, BB, BO and OO genotypes were 8 (6.1%), 31 (23.5%), 7 (5.3%), 6 (4.5%), 23 (17.4%), and 57 (43.2%) respectively. It is concluded that one tube polymerase chain reaction with GeneScan technique can determine the genotypes of ABO blood group.

Processing and cryopreservation for 1963 units of human umbilical cord blood / 中国实验血液学杂志

The study was aimed to establish a standard procedure for human umbilical cord blood bank. The hematopoietic nucleated cells in cord blood were processed by using sedimentation and centrifugation method. After finishing CD34(+) cell counting, hematopoietic progenitor cell assay, microbial culture, infectious disease test and HLA typing, cord blood units were stored in the liquid nitrogen for further application. The results showed that nucleated cells of cord blood were (10.94 +/- 2.74) x 10(8) per unit; recovery rate of nucleated cells was (79.82 +/- 17.76)%. CD34(+) cells in cord blood were counted as (51.62 +/- 30.53) x 10(5) per unit. Eight units of cord blood were thawed after two years of cryopreservation, the recovery rate of nucleated cells, CD34(+) cells and CFU-GM were (91.4 +/- 6.0)%, (84.6 +/- 20.0)% and (85.8 +/- 14.9)% respectively. It is suggested that the methods and procedure reported for processing and cryopreservation of hematopoietic stem/progenitor cells in the human umbilical cord blood is effective.

Assessment on effect of short-term cryopreservation of cord blood hematopoietic cells / 中国实验血液学杂志

To study the effects of short-term cryopreservation of cord blood hematopoietic cells in liquid nitrogen, the viability and function of cord blood hematopoietic cells were investigated by using each of 8 samples cryopreserved for six months, one and two years after thawing respectively. Nucleated cells (NC) were detected by blood cell analyzer. CD34+ cells were analyzed by flow cytometry, CFU-GM were cultured and detected in vitro, the survival rate was determined by trypan blue staining. The results showed that the differences of recovery rate of NC, CD34+, CFU-GM were nonsignificant at three different cryopreserved times. In conclusion, the short-term storage in liquid nitrogen showed a good effect on cord blood hematopoietic cell without any significant change of activities and number of the cryopreserved hematopoietic cells.

Polymorphisms of six short tandem repeat loci in Zhejiang She ethnic population and Zhejiang Hans of China / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To investigate genetic polymorphism of six short tandem repeat (STR) loci (D3S1358,D16S539, TH01,TPOX, CSF1PO,D7S820) in Zhejiang She ethnic population and Han population.</p><p><b>METHODS</b>By use of AmpFlSTR Cofiler kit, 6 STR loci in 108 She samples and 102 Han samples were amplified. The PCR products were electrophoresed by ABI Prism 377 sequencer; the data were analyzed by Genescan software.</p><p><b>RESULTS</b>All genotype frequencies of the 6 STR loci in She and Han ethnic groups met Hardy-Weinberg equilibrium. In the She population, the heterozygosities (H) in D3S1358,D16S539,TH01,TPOX, CSF1PO and D7S820 were 0.8028, 0.9148, 0.7522, 0.6728, 0.9123,0.8338, the exclusion of paternity(EP) were 0.4067, 0.6057, 0.4437, 0.3200, 0.5250, 0.5358, discrimination power (DP) were 0.6690, 0.7841, 0.6447, 0.5382, 0.7298, 0.7296.The combined DP, PE and polymorphism information content were 0.9991,0.9805,0.9988 respectively. There were significant differences at D3S1358, D16S539 and TPOX loci, compared with Hans.</p><p><b>CONCLUSION</b>She population has its own STR allele distribution characteristic. The above data obtained from She population can be used not only in genetic researches and population investigation, but also in human identity and paternity testing.</p>