OBJECTIVE To establish a preparation system for megakaryocytes(MKs)derived from human embryonic stem cells(hESCs)and MK microparticles(MKMPs),and to assess the pro-angio-genic efficiency of these microparticles.METHODS ①hESCs were induced to mesodermal progenitor cells via monolayer culture with the first-stage induction medium for 2 days before the cells were induced to hemogenic endothelial/hematopoietic progenitor cells by culturing with the second-stage induction medium for another 3 days.Then,the cells were dissociated into single cells,seeded into the third-stage induction medium,and cultured using the suspension method for 8 days to obtain MKs.The specific characters of differentiated cells were identified through morphological observation and flow cytometry before stage-specific marker proteins in different periods were analyzed[hESCs:TRA-1-60,sialyl glycolipid stage-specific embryonic antigen4(SSEA4)];mesodermal progenitor cells:brachyury;hemogenic endothelial/hematopoietic progenitor cells:CD34,CD43;MKs:CD41a,CD42b),and immu-nofluorescence staining[β1-tubulin,von Willebrand factor(VWF)],[friend leukemia integration 1(FLI1),CD42].② MKMP collection and verification:MKMPs were collected via differential centrifugation.The concentration and size of these MKMPs were determined by nanoparticle tracking analysis(NTA),and both the morphology and ultrastructure were examined by transmission electron microscopy(TEM).Besides,the MKMPs-specific proteins[CD41,tumor susceptibility gene 101(TSG101)and CD9]were detected by Western blotting analysis.③ Biological function of MKMPs:MKMPs were stained with CD41a-PE antibodies and co-cultured with human umbilical veinvascular endothelial cells(HUVECs)labeled by CD34-APC for 3 h.Live-cell immunofluorescence was employed to find out whether HUVECs could absorb MKMPs.To find out whether MKMPs could affect the role of HUVECs in angio-genesis and cell migration,platelet microvesicles(PMPs)were used as positive controls.The experi-mental groups were added with different concentrations of microparticles(1,5,10 and 20 mg·L-1)while the control group was given no microparticles(0 mg·L-1).The number of nodes that formed the lumen after 5 h of incubation in Matrigel was counted,and the size of healing of the scratch area was analyzed after 6 h.To elucidate the mechanism through which MKMPs impacted angiogenesis,ELISA was used out to quantitatively detect the concentration of proteins in microparticles.RESULTS ① A three-stage differentiation cultural system was established to develop hESCs into MKs.Flow cytometry revealed progressive loss of pluripotency markers SSEA4 and TRA-1-60,while the mesodermal progenitor marker brachyury peaked at d 2.Subsequently,hemogenic endothelial/hematopoietic progenitor markers CD34 and CD43 emerged at d 5,followed by megakaryocytic markers CD41a and CD42b at d 13.Immunofluorescent images further demonstrated that MKs expressed specific proteins CD42,β1-tubulin,von VWF and FLI1 at d 13.②Microparticles were collected via differential centrifuga-tion.Transmission electron microscopy revealed that their substructure exhibited a typical double-layered membrane.Nanoparticle tracking analysis indicated that the size was(164.3±14.0)nm.The result of WB demonstrated that the microparticles expressed specific markers,including TSG101,CD9 and CD41.③ MKMPs were absorbed after being co-cultured with HUVECs for 3 h and enhanced the ability of HUVECs to form tubes and migrate.Notably,the treatment of 5 mg·L-1 MKMPs was more effective than 5 mg·L-1 PMPs treatment.The results of ELISA showed that the content of VEGF from MKMPs was higher than from PMPs,which may be the key factor in regulating endothelial biological function.CONCLUSION MKs derived from hESCs can generate functional microparticles which can promote angiogenesis.

OBJECTIVE To establish a preparation system for megakaryocytes(MKs)derived from human embryonic stem cells(hESCs)and MK microparticles(MKMPs),and to assess the pro-angio-genic efficiency of these microparticles.METHODS ①hESCs were induced to mesodermal progenitor cells via monolayer culture with the first-stage induction medium for 2 days before the cells were induced to hemogenic endothelial/hematopoietic progenitor cells by culturing with the second-stage induction medium for another 3 days.Then,the cells were dissociated into single cells,seeded into the third-stage induction medium,and cultured using the suspension method for 8 days to obtain MKs.The specific characters of differentiated cells were identified through morphological observation and flow cytometry before stage-specific marker proteins in different periods were analyzed[hESCs:TRA-1-60,sialyl glycolipid stage-specific embryonic antigen4(SSEA4)];mesodermal progenitor cells:brachyury;hemogenic endothelial/hematopoietic progenitor cells:CD34,CD43;MKs:CD41a,CD42b),and immu-nofluorescence staining[β1-tubulin,von Willebrand factor(VWF)],[friend leukemia integration 1(FLI1),CD42].② MKMP collection and verification:MKMPs were collected via differential centrifugation.The concentration and size of these MKMPs were determined by nanoparticle tracking analysis(NTA),and both the morphology and ultrastructure were examined by transmission electron microscopy(TEM).Besides,the MKMPs-specific proteins[CD41,tumor susceptibility gene 101(TSG101)and CD9]were detected by Western blotting analysis.③ Biological function of MKMPs:MKMPs were stained with CD41a-PE antibodies and co-cultured with human umbilical veinvascular endothelial cells(HUVECs)labeled by CD34-APC for 3 h.Live-cell immunofluorescence was employed to find out whether HUVECs could absorb MKMPs.To find out whether MKMPs could affect the role of HUVECs in angio-genesis and cell migration,platelet microvesicles(PMPs)were used as positive controls.The experi-mental groups were added with different concentrations of microparticles(1,5,10 and 20 mg·L-1)while the control group was given no microparticles(0 mg·L-1).The number of nodes that formed the lumen after 5 h of incubation in Matrigel was counted,and the size of healing of the scratch area was analyzed after 6 h.To elucidate the mechanism through which MKMPs impacted angiogenesis,ELISA was used out to quantitatively detect the concentration of proteins in microparticles.RESULTS ① A three-stage differentiation cultural system was established to develop hESCs into MKs.Flow cytometry revealed progressive loss of pluripotency markers SSEA4 and TRA-1-60,while the mesodermal progenitor marker brachyury peaked at d 2.Subsequently,hemogenic endothelial/hematopoietic progenitor markers CD34 and CD43 emerged at d 5,followed by megakaryocytic markers CD41a and CD42b at d 13.Immunofluorescent images further demonstrated that MKs expressed specific proteins CD42,β1-tubulin,von VWF and FLI1 at d 13.②Microparticles were collected via differential centrifuga-tion.Transmission electron microscopy revealed that their substructure exhibited a typical double-layered membrane.Nanoparticle tracking analysis indicated that the size was(164.3±14.0)nm.The result of WB demonstrated that the microparticles expressed specific markers,including TSG101,CD9 and CD41.③ MKMPs were absorbed after being co-cultured with HUVECs for 3 h and enhanced the ability of HUVECs to form tubes and migrate.Notably,the treatment of 5 mg·L-1 MKMPs was more effective than 5 mg·L-1 PMPs treatment.The results of ELISA showed that the content of VEGF from MKMPs was higher than from PMPs,which may be the key factor in regulating endothelial biological function.CONCLUSION MKs derived from hESCs can generate functional microparticles which can promote angiogenesis.

The application of Raman spectroscopy in the field of laboratory medicine is making continuous progress and development. The biosensor platform based on Raman spectroscopy provides a new means for accurate molecular diagnosis of diseases. In particular, as a fast and non-destructive detection method, surface-enhanced Raman scattering has the advantages of simple sample preparation, little interference from water and real-time detection, and shows great application potential in the field of medical examination. At the same time, with the integration of SERS and other technologies, including electrochemistry, new nano-materials, microfluidic, biochip, DNA nano-machine, artificial intelligence and machine learning, it will play a more and more important role in the field of medical laboratory. With the deepening of SERS research and the cross-integration between multiple disciplines, it will be widely used in biomedical detection and is expected to become an important technology platform for the next generation of precision diagnosis.

Objective:To observe the effect of high expression of polypyrimidine tract-binding protein-associated splicing factor (PSF) on low concentration of 4-hydroxynonenal (4-HNE) induced human retinal microvascular endothelial cells (HRMECs), and explore the possible mechanism.Methods:The HRMECs cultured in vitro were divided into 4-HNE treated group, PSF overexpression group combined with 4-HNE group (PSF+4-HNE group), PSF overexpression+ML385 treatment combined with 4-HNE group (PSF+ML385+4-HNE group), and 4-HNE induced PSF overexpression group with LY294002 pretreatment (LY294002+4-HNE+PSF group). Cell culture medium containing 10 μmmol/L 4-HNE was added into 4-HNE treatment group, PSF+4-HNE group, PSF+ML385+4-HNE group for 12 hours to stimulate oxidative stress. 1.0 μg of pcDNA-PSF eukaryotic expression plasmid were transfected into PSF+4-HNE group and PSF+ML385+4-HNE group to achieve the overexpression of PSF. Also cells were pretreated with ML385 (5 μmol/L) for 48 hours in the PSF+ML385+4-HNE group, meanwhile within the LY294002+4-HNE+PSF group, after pretreatment with LY294002, cells were treated with plasmid transfection and 4-HNE induction. Transwell detects the migration ability of PSF to HRMECs. The effect of PSF on the lumen formation of HRMECs was detected by using Matrigel in vitro three-dimensional molding method. Flow cytometer was used to detect the effect of PSF overexpression on reactive oxygen (ROS) level in HRMECs. Protein immunoblotting was used to detect the relative expression of PSF, nuclear factor E2 related factor 2 (Nrf2), heme oxygenase-1 (HO-1) protein, and phosphoserine threonine protein kinase (pAkt) protein. The comparison between the two groups was performed using a t-test. Results:The number of live cells, migrating cells, and intact lumen formation in the 4-HNE treatment group and the PSF+4-HNE group were 1.70±0.06, 0.80±0.13, 24.00±0.58, 10.00±0.67, and 725.00±5.77, 318.7±12.13, respectively. There were significant differences in the number of live cells, migrating cells, and intact lumen formation between the two groups ( t=12.311, 15.643, 17.346; P<0.001). The results of flow cytometry showed that the ROS levels in the 4-HNE treatment group, PSF+4-HNE group, and PSF+ML385+4-HNE group were 816.70±16.67, 416.70±15.44, and 783.30±17.41, respectively. There were statistically significant differences between the two groups ( t=16.311, 14.833, 18.442; P<0.001). Western blot analysis showed that the relative expression levels of pAkt, Nrf2, and HO-1 proteins in HRMECs in the 4-HNE treatment group, PSF+4-HNE group and LY294002+4-HNE+PSF group were 0.08±0.01, 0.57±0.04, 0.35±0.09, 0.17±0.03, 1.10±0.06, 0.08±0.11 and 0.80±0.14, 2.50±0.07, 0.50±0.05, respectively. Compared with the PSF+4-HNE group, the relative expression of pAkt, Nrf2, and HO-1 proteins in the LY294002+4-HNE+PSF group decreased significantly, with significant differences ( t=17.342, 16.813, 18.794; P<0.001). Conclusion:PSF upregulates the expression of HO-1 by activating the phosphatidylinositol 3 kinase/Akt pathway and inhibits cell proliferation, migration, and lumen formation induced by low concentrations of 4-HNE.

OBJECTIVE： To determine and compare the contents of catalpol and aucubin in different parts （root， stem， leaf and flower） of wild Centranthera grandiflora， and to provide reference for the selection of medicinal parts and source development. METHODS： HPLC method was used to determine the contents of catalpol and aucubin in root， stem， leaf and flower of wild C. grandiflora， and the contents of different parts were analyzed comparatively. The determination of catalpol was performed on Agilent TC-C18 column with mobile phase consisted of methanol-0.1％ phosphoric acid （1 ∶ 99， V/V） at the flow rate of 1 mL/min； the detection wavelength was set at 210 nm， and sample size was 20 μL. The column temperature was 35 ℃； the determination of aucubin was performed on SPHERI-5RP-C18 column with mobile phase consisted of acetonitrile-water （3 ∶ 97， V/V） at the flow rate of 1 mL/min； the detection wavelength was set at 205 nm， and sample size was 20 μL； the column temperature was 25 ℃. RESULTS： The linear range of catalpol and aucubin were 0.061 5-3.321 and 0.000 36-0.216 mg/mL （all r＝0.999 9）. The limits of detection were 0.016 and 0.007 μg/mL. The limits of quantitation were 0.052 and 0.023 μg/mL. RSDs of precision， stability （24 h） and reproducibility tests were all lower than 2.00％ （n＝6）. The average recoveries were 99.34％ and 99.61％， and RSDs were 1.06％ and 1.12％， respectively （n＝6）. The average content of catalpol in root， stem， leaf and flower wild C. grandiflora were 1.609， 3.030， 11.095 and 1.921 mg/g， respectively. The contents of aucubin in different parts were 0.441， 0.020， 0.005 and 0.006 mg/g，respectively. CONCLUSIONS：The established HPLC method meets the requirements of quantitative analysis. Catalpol is mainly distributed in the leaves of wild C. grandiflora， and aucubin is mainly distributed in the roots of wild C. grandiflora. The experimental conclusion provides a reference for the reasonable selection of different medicinal parts as raw materials to develop medicine with different efficacy.

Objective To compare the effects of two kinds of transparent dressings for patients with skin graft-versus-host disease (GVHD) after allogeneic hematopoietic stem cell transplantation (Allo-HSCT) in PICC catheter maintenance.Methods Retrospective analysis was conducted among 86 patients who had complicated skin GVHD after Allo-HSCT and used PICC catheter. We selected 42 patients who used dressings of 10 cm×12 cm from March 2012 to January 2014 as the control group,and selected 44 patients who used dressings of 6 cm×7 cm from March 2014 to January 2016 as the observation group. During the maintenance of PICC catheters,the maintenance effect of catheters in two groups were analyzed.Results There was no significant difference between two groups in the effect of catheter maintatence,catheter-related infection,and the dressing replacement interval (P>0.05). While the medical adhesive related skin injury of the observation group was significantly lower than that of the control group (P<0.05).Conclusions During the maintenance of PICC catheters,the transparent dressing of 6 cm×7 cm is much safer,more comfortable with less costs. It is especially suitable for the patients with skin GVHD after Allo-HSCT.

Objective To investigate the prevalence and influencing factors of dysmenorrhea among female college students in Changchun city, so as to provide scientific basis for health promotion and effective preventive measurement. Methods Non-randomized convenience sampling and face to face interview were used to collect information from female college students aged between 17 and 25 years in 14 universities in Changchun. Chi-square test and logistic regression model were used to analyze influencing factors of dysmenorrhea. Results The average age of 1 071 subjects was 21.21 ± 1.83 years. The prevalence of dysmenorrhea was 86.55%. The proportion of mild dysmenorrhea among the subjects was 62.56%, followed by 33.01% with moderate dysmenorrhea and 4.43% with severe dysmenorrhea; 80.76% of subjects paid attention to keep warm in the daily life. Normal BMI, sleeping before 23 o'clock or between 23 to 24 o'clock, taking exercise frequently or everyday might be the protective factors of dysmenorrhea, and the OR values (95% CI) were respectively as 0.60 (0.37-0.97), 0.56 (0.37-0.84), 0.42 (0.22-0.78) and 0.63(0.42-0.97). Tension and the family history of dysmenorrhea might be the risk factors, and the OR values (95%CI) were respectively 1.63 (1.10-2.41), 4.84 (2.80-8.35). Conclusion The prevalence of dysmenorrhea is high among female college students. Lacking exercise, BMI less than 18.5 kg/m2, staying up late, tension and the family history of dysmenorrhea may be the influencing factors of dysmenorrhea among female college students.

Objective To observe the prevention effect of frozen fresh aloe mouthwash on oral cavity mucous membrane inflammation in patients with hematopoietic stem cell transplantation, so as to find an economic and effective method for prevention the oral mucosa inflammation.Methods A total of 42 patients were admitted in our hospital from January 2013 to January 2014.They were divided into the experimental group (20 cases) and the control group (22 cases).The control group was given the conventional oral intervention measures.On the basis of it, the experimental group was given frozen fresh aloe mouthwash, with fresh Curacao aloe vera 30 to 40 gram, peeled its thorn, and put in the 250 ml boiled water (100℃).After cooling down, it was stored for 6 to 8 hours in the 2 -4 ℃ refrigerator.The degree of oral mucositis of the two groups was compared.Results In the experimental group, eleven patients had 0 degree of oral mucositis, six patients hadⅠdegree of oral mucositis, two patients had Ⅱdegree of oral mucositis, one patient had Ⅲ degree of oral mucositis.Those numbers of patients in the control group were five, three, ten, four, respectively.There was a significant difference between two groups (U=112.5,P<0.01).Conclusions Frozen fresh aloe mouthwash can prevent oral mucositis among patients with hematopoietic stem cell transplantation.It is easy for patients to accept, and it is economic and effective.

OBJECTIVETo investigate the post-transcriptional regulation of dual-specificity phosphatase-1 (DUSP1) by the RNA- binding protein HuR in heat shock.

METHODSThe recombinant plasmids carrying wild-type (WT) HuR or its mutants at threonine 118 were constructed and transiently transfected into NIH 3T3 cells via liposome, and the changes in the expressions of DUSP1 mRNA and protein were detected by quantitative real-time PCR and Western blotting, respectively.

RESULTSHeat shock caused significantly enhanced phosphorylation of HuR at the residue T118. In 3T3 cells transfected with the plasmids carrying wild-type HuR for its over-expression showed significantly up-regulated DUSP1 mRNA and protein expressions at 24 h after transfection. Over-expression of HuR(T118A) down-regulated DUSP1 mRNA and protein expressions in cells challenged with heat shock, while HuR(T118E) over-expression significantly increased DISP1 expression at both mRNA and protein levels. After heat shock, HuR(WT) translocated from the cell nucleus to the cytoplasm to form particles. HuR(T118E) was diffusely distributed in the cytoplasm before heat shock and formed particles after heat shock. HuR(T118A) did not undergo such translocation in response to heat shock challenge.

CONCLUSIONHuR regulates DUSP1 mRNA and protein expression at the post-transcriptional level to increase its expression after heat shock by enhancing the phosphorylation HuR T118.

Animals ; Cell Nucleus ; Cytoplasm ; Dual Specificity Phosphatase 1 ; genetics ; metabolism ; ELAV Proteins ; metabolism ; Gene Expression Regulation ; Heat-Shock Response ; Hot Temperature ; Mice ; NIH 3T3 Cells ; Phosphorylation ; RNA, Messenger ; Real-Time Polymerase Chain Reaction ; Transfection ; Up-Regulation

Background and purpose:Aberrant DNA methylation that leads to the inactivation of tumor suppressor genes plays important roles in development and progression of breast cancer. Clinically, related gene methylation is considered to be a promising biomarker for tumor diagnosis and prognosis. This study aimed to investigate the methylation status ofSox17 gene in breast cancer tissue and its corresponding plasma circulating DNA, as well as to investigate its value in breast cancer early diagnosis and prognosis.Methods:TheSox17 gene promoter methylation status was detected by MSP in 86 cases of breast cancer, 36 normal breast tissues and its paired plasma DNA, the results were analyzed with corresponding clinical and pathological features.Results:The frequency ofSox17 gene methylation rate among 86 breast cancer tissues was 77.9%(67/86), and was 61.6%(53/86)in plasma circulating DNA, however, noSox17 gene methylation was found in normal breast tissues.Sox17 gene promoter methylation in plasma circulating DNA was signiifcantly associated with the methylation status in tumor tissues (r=0.502,P=0.000). In breast cancer tissue specimens,Sox17 methylation status was significantly correlated with tumor stage (χ2=6.18,P=0.041) and lymph node metastasis (χ2=13.54,P=0.001);Sox17 gene methylation rate was signiifcantly correlated with tumor stage (χ2=27.06,P=0.000), tumor size (χ2=9.65,P=0.007) and lymph node metastasis (χ2=20.80,P=0.000) in plasma samples, and there was no signiifcant difference ofSox17 gene methylation between patient age, histological grade and ER, PR, HER-2/neu status.Conclusion:Sox17 gene promoter methylation plays an important role in the carcinogenesis and development of breast cancer, and may be associated with the prognosis of breast cancer. Furthermore, methylatedSox17 gene may be a useful tumor biomarker in plasma circulating DNA for breast cancer detection and disease monitoring.