Comparison of pretreatment techniques for single-cell RNA sequencing of airway aspirate specimens / 中华微生物学和免疫学杂志

Objective:To investigate the appropriate pretreatment methods for single cell RNA sequencing of airway aspirate cells.Methods:Four fresh airway aspirate specimens were collected from four patients with acute respiratory tract infections. These specimens were digested with airway aspirate digester and prepared into single cell suspension. The cells were used for library construction directly (DE), or fixed with 10×Genomics Chromium Next GEM Single Cell Fixed RNA Sample Preparation Kit and then mixed to construct the library (DF), or cryopreserved, thawed, fixed (FF) before mixed to construct the library. All three methods were treated with oil emulsion using 10 4 cells and subjected to single-cell sequencing using the 10×Genomics platform. The number of obtained cells, data quality, annotated cell types and expression of marker genes were analyzed. Differences in the expression of highly variable genes (HVGs) of the same cell subsets obtained by the three pretreatment methods were compared using Pearson correlation. Expression of the differentially expressed genes in the same cell subpopulation obtained by different pretreatment methods was also compared. The correlation of the expression of differentially expressed genes between the same cell subsets obtained by the three pretreatment methods was analyzed by Pearson correlation. Results:The median numbers of single cells obtained using DE, FF and DF methods were 2 733, 1 140 and 5 897 ( P>0.05). The unique molecular identifiers were higher than 500. The median numbers of genes obtained using the three methods were 801, 887 and 1 259 ( P>0.05). The cells with novelty score over 0.8 accounted for 99%, 87% and 93%, respectively. There were nine cell subsets obtained by the three methods, including squamous cells, secretory cells, ciliated cells, T cells, B cells, macrophages, plasma cells and neutrophils. DF and FF methods could obtain more basal cells with specific high expression of keratin 5 than DE method. The differentially expressed and highly variable genes in the same cell subsets obtained by the three pretreatment methods showed high consistency in their expression with a significant correlation ( P<0.001). Conclusions:Under the same sequencing data volume, the quality of data obtained from fixed airway aspirate single-cell suspensions using the method of probe hybridization and transcriptome sequencing was comparable to that obtained directly from fresh cells. This method was more suitable for the pretreatment of clinical samples used for single-cell RNA sequencing.

Effects of Gentiopicroside on the Apoptosis of Human Pancreatic Cancer Cells PANC- 1 and IL- 6/JAK2/STAT3 Signaling Pathway / 中国药房

OBJECTIVE：To study the effects of gentiopicroside on the apo ptosis o f human pancreatic cancer cells PANC- 1，and to explore its mechanism from the perspective of IL- 6/JAK2/STAT3 signaling pathway. METHODS ：Using PANC- 1 cells as model ， the proliferation inhibition rate of cells was tested by MTT assay after treated with 0（negative contro ），2，4，8，16，32，64，128 mg/L gentiopicroside for 72 h and IC 50 were calculated. The cells were divided into negative control group ，gemcitabine group （positive control，4 mg/L）and gentiopicroside low-concentration ，medium-concentration and high-concentration groups （15，30，60 mg/L）. After cultured for 1，3，5，7 d，Trypan blue exclusion staining was used to count the survival cell ，and the growth of cells was investigated. After cultured for 72 h，colony formation assay was used to observe colony formation rate of cells ；the apoptotic rate of cells was detected by Hoechst 33258 staining；the mRNA and protein expressions of IL- 6，JAK2，STAT3 in cells were detected by RT-PCR and Western blotting assay. RESULTS ：4-28 mg/L gentiopicroside could inhibit the proliferation of cells （P＜0.05 or P＜ 0.01），in concentration dependent trend ；IC50 was 9.54 mg/L. Compared with negative control group ，survival cell count （cultured from 3，5，7 d），mRNA and protein expressions of IL- 6，JAK2 and STAT 3 in cells were decreased significantly in gemcitabine group ， gentiopicroside medium-concentration and high-concentration groups （P＜0.05 or P＜0.01），while the apoptotic rate was increased significantly （P＜0.01）. The colony formation rate of cellswere decreased significantly in gemcitabine group and gentiopicroside high-concentration group （P＜0.01）. mail：hb.gz@163.com Compared with gemcitabine group ，there was no statistical significance in above indexes of gentiopicroside high- 6716008。 concentration group （P＞0.05）. CONC LUSIONS：30，60 mg/L gentiopicroside could inhibit the proliferation and induce apoptosis of PANC- 1 cells，and 60 mg/L gentiopicroside is similar to gemcitabine in the effects. Its mechanism may be related to inhibiting the activation of IL- 6/JAK2/STAT3 signaling pathway.

Relationship between clinical features and peripheral blood test indicators and curative effect in patients with acquired hemophagocytic syndrome / 白血病·淋巴瘤

Objective To explore relationship between clinical features and peripheral blood test indicators and curative effect in adult patients with acquired hemophagocytic syndrome (HPS). Methods A total of 61 adult patients with acquired HPS who were admitted to the First Affiliated Hospital with Nanjing Medical University and the Affiliated Jiangning Hospital of Nanjing Medical University from April 2014 to March 2017 were enrolled, including 38 males and 23 females, with a median age of 48 years (17-86 years). The retrospective analyses of their clinical data and laboratory examination results were made in this study. Results There was no significant difference in the therapeutic effective rate of 61 HPS patients caused by different inducements after treatment (P ＝0.184). The prothrombin time (PT) before treatment was higher than that after treatment [(12.90±1.97) s vs. (12.35±1.78) s, P＝ 0.046]; the level of lactate dehydrogenase (LDH) before treatment was higher than that after treatment (median: 476 U/L vs. 231 U/L, P ＝ 0.000); the level of D-dimer (D-D) before treatment was higher than that after treatment (median: 1.46 mg/L vs. 0.51 mg/L, P ＝ 0.007); the level of aspartate aminotransferase (AST) before treatment was higher than that after treatment (median: 54.9 U/L vs. 26.0 U/L, P＝ 0.000); the level of serum calcium before treatment was lower than that after treatment [(2.07±0.20) mmol/L vs. (2.18±0.23) mmol/L, P ＝ 0.043]. The peripheral blood platelet counts (Plt) in the effective group (32 cases) before treatment was higher than that in the ineffective group (29 cases) (median: 104.0×109/L vs. 63.5×109/L, P ＝0.007), the level of albumin (ALB) in the effective group was higher than that in the ineffective group [(35.50 ±6.17) mmol/L vs. (31.93 ±6.54) mmol/L, P ＝ 0.033], the level of serum calcium in the effective group was higher than those in the ineffective group [(2.18±0.18) mmol/L vs. (2.08±0.20) mmol/L, P ＝ 0.047], the level of prothrombin time (PT) in the effective group was lower than that in the ineffective group [(12.40±1.76) s vs. (13.43±2.06) s, P ＝ 0.041], and the level of LDH in the effective group was lower than that in the ineffective group (median: 415.0 U/L vs. 593.5 U/L, P＝ 0.032). Conclusion The lower expressions of Plt, ALB and serum calcium, and the higher expressions of PT and LDH may indicate the poor prognosis of adult acquired HPS, and there fore these patients need to be paid attention.

Analysis of prognosis of acute myeloid leukemia patients based on genetic mutations / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To correlate the clinical features of patients with acute myeloid leukemia (AML) with mutations of FLT3-ITD, NPM1, CEBPA, c-KIT, DNMT3A and ND4 genes as well as chromosomal aberrations.</p><p><b>METHODS</b>Somatic mutations of aforementioned genes in 412 newly diagnosed AML patients were detected with PCR and direct sequencing. All patients were also subjected to R-banding chromosomal analysis. The results were correlated with the clinical features and prognosis of the patients.</p><p><b>RESULTS</b>The mutation rates of FLT3-ITD, NPM1, CEBPA, c-KIT, DNMT3A and ND4 were 9.0% (26/289), 19.1% (50/262), 18.9% (34/180), 3.4% (7/208), 6.6% (9/137) and 6.9% (4/58), respectively. Patients with poor prognosis based on genetic mutations had lower blood platelet count than those with intermediate and good prognosis (P=0.001 and P=0.001, respectively). None of the three groups attained median overall survival (OS) (P> 0.05). The complete remission (CR) was similar among the three groups (P> 0.05). For patients with different prognosis based on cytogenetic findings, white blood cell count in those with intermediate prognosis was higher than those with good and poor prognosis (P< 0.001 and P=0.004, respectively), while the blood platelet count of the intermediate group was higher than that of the group with good prognosis (P=0.018). No significant difference was found among the three groups in terms of hemoglobin level (P> 0.05). The group with poor prognosis has attained shorter OS compared with those with good and intermediate prognosis (P< 0.001 and P=0.003, respectively). However, the CR rate of the group with good prognosis was higher than that of the intermediate group (P=0.001). For the group with intermediate prognosis, presence of genetic mutations did not correlate with the clinic characteristics such as white blood cell count, blood platelet count, hemoglobin level, OS and CR rate (P> 0.05 for all comparisons).</p><p><b>CONCLUSION</b>Genetic mutations combined with cytogenetic analysis can facilitate the prognosis and personalized treatment for patients with AML.</p>

Study on mechanism of protective effect of sevoflurane pretreatment on myocardial ischemia-reperfusion injury in rats / 重庆医学

Objective To explore the role of non-receptor tyrosine kinase(c-Src)in sevoflurane pretreatment for relieving myocardial ischemia-reperfusion injury.Methods By using the random number table,the healthy male Wistar rats were randomly divided into 5 groups (n=10):sham operation group (Ⅰ),ischemia-reperfusion group(Ⅱ),sevoflurane pretreatment group(Ⅲ), sevoflurane pretreatment plus dimethyl sulfoxide(DMSO,Ⅳ)and sevoflurane pretreatment plus c-Src specific inhibitor SU6656 group(Ⅴ)groups.The group Ⅲ,Ⅳ and Ⅴ were performed the sevoflurane aftertreatment before reperfusion;the group Ⅴ was in-jected by SU6656 at 5 min before reperfusion;the group Ⅳ was given the equal volume DMSO.The arterial blood sample in each group was collected at 120 min after reperfusion for detecting serum LDH level and CK-MB activity.Rats were killed for taking the heart and separating the left ventricle to calculate the area of myocardial infarctio;the expression levels of Src,phosphorylated Src (p-Src),CAT and SOD in myocardial tissue were detected in each group.Results Compared with the groupⅠ,the level of serum CK-MB and LDH activity,myocardial infarct area and p-Src/Src,CAT,SOD in the other 4 groups were increased significantly (P <0.05);comparing with the group Ⅲ,the serum CK-MB and LDH activity,myocardial infarct area and SOD,CAT,in the group Ⅱ,Ⅳ and Ⅴ were increased,however the level of p-Src/Src was decreased significantly (P <0.05).Conclusion The c-Src-reactive ox-ygen signaling pathway might mediate the role of sevoflurane pretreatment for reducing myocardial ischemia-reperfusion injury in rat.

Effects of furin inhibitor on metastasis of human breast cancer MCF-7 cells / 中国病理生理杂志

[ ABSTRACT] AIM:To investigate the mechanism underlying breast cancer metastasis and to provide theoretical da-ta for studying the pathogenesis of breast cancer onset and development.METHODS: Human breast cancer MCF-7 cells were treated with different concentrations of furin inhibitorα1-PDX for 48 h.Wound healing assay and Transwell assay were applied to detect the migration and invasion abilities of the MCF-7 cells.The expression of cell migration-associated proteins, including membrane-type 1 matrix metalloproteinase ( MT1-MMP) , vascular endothelial growth factor ( VEGF)-C and VEGF-D, was determined by Western blotting.The protein levels of MMP2 and MMP9 in the supernatant were measured by ELISA. RESULTS:Compared with control group, 200 nmol/L of furin inhibitor exerted significant inhibitory effects on the cell mi-gration (P<0.05).The expression of cell migration-associated proteins MT1-MMP, VEGF-C and VEGF-D was significantly inhibited after treated withα1-PDX ( P<0.05 ) .Significant inhibitory effects of α1-PDX on the expression of MMP9 and MMP2 (P<0.05) in the supernatant were observed.CONCLUSION:Furin inhibitor suppresses the metastasis of MCF-7 cells via down-regulating the expression of MMPs and VEGFs.

Triazole Schiff base derivative induces cannibalism of SMMC-7721 cells in vitro / 解剖学报

ObjectiveTo a nalyze the morphologic features of SMMC-7721 cannibalistic cells that induced by triazole Schiff base derivative(LH-37) in vitro. Methods The SMMC-7721 cells (1×10~4/ml)were cultured in the medium containing of 1×10~(-5) mol/L LH-37 for 24h,48h.The character of cells was detected by Papanicolaou and Wright′s Staining. Immunohistochemical method was used to observe the cleaved Caspase-3 positive cells. The ultrastructure of cannibalism cells was observed by JEM 100CX-II transmission electronic microscope. Results Microscopic analysis demonstrated the complete internalization of one cell within another. We noted that some cannibalistic cells in small aggregates appeared to be inside of large vacuoles, suggesting that they were internalized within a neighboring cell. The proportion of cannibalistic cells were increased after SMMC-7721 cells were cultured in the presence of LH-37 for 48 hours. The proportion of the cannibalistic cells in control and LH-37 group was 0.47% and 5.23% respectively . Many internalized cells were positive for cleaved caspase-3 staining . Ultrastructural analysis of engulfed cells from 24 hours exhibited evidence of live-cell internalization consistent with cannibalism, The most common fate for internalized cells was death after treatment with LH-37 for 48 hours, as evidenced by nuclear degradation and the eventual disappearance of some cells within the enveloping cell . Conclusion The data presented indicate that LH-37 can lead to an increase of cannibalism in human hepatocarcinoma cell in vitro.

Autophagic death induced by matrine in BEL-7402 cells / 中草药

Objective To study the effects of matrine on inducing autophagy and autophagic death in BEL-7402 cells. Methods BEL-7402 cells were cultured in RPMI1640 alone or exposed to different concentration of matrine. Cell growth inhibition was assessed by MTT assay. Apoptosis was evaluated by flow cytometry. Autophagosome marker LC3 was examined by immunofluorescence microscopy. The ultrastructure change of cells was analyzed by transmission electron microscope. Results The cell proliferation was inhibited by matrine in a dose dependent manner. Its IC50 value was 1.1 mg/mL at 48 h. Treatment with 0.6-1.6 mg/mL matrine for 12 h induced apoptosis of BEL-7402 cells and the apoptosis rate reached (44.88?0.78)% at concentration of 1.2 mg/mL. The autophagy positive cells were greatly increased after 1.2 mg/mL matrine treated in BEL-7402 cells and the number of cells reached (63.16?0.29)%. Morphological features of typical autophagic death were apparent in the cytoplasm at the ultrastructural level. Conclusion Matrine could induce autophagy and autophagic death of BEL-7402 cells in vitro.

Effects of triazole schiff base derivative on the differentiation and apoptosis of human hepatocarcinoma cells / 中国药理学通报

Aim To study the effect of 3-pyridin-3-yl-4-[(4-hydroxy-3-methoxy-benzylidene)amino]-5-me-thylsulfanyl-4H-[1,2,4] triazole (LH-37) on induction of differentiation and apoptosis of hepatocarcinoma BEL-7402 cells. Methods BEL-7402 cells were cultured in RPMI 1640 and treated by LH-37 at different doses. The proliferation of the cells and the inhibition effect of LH-37 on the cell proliferation were examined by MTT assay. The reverse transcriptase-polymerase chain reaction (RT-PCR) was used to detect the changes of alpha fetoprotein(?-FP)mRNA and albumin(Alb). The concentration of ?-FP in the cells was detected by ELISA assay. Cell morphology was observed by fluorescence microscope techniques. The protein expressions of active caspase-3 in BEL-7402 cells were observed by immunohistochemical staining. Western blot was used to assay caspase-3 and caspase-9. Colorimetric method was used to assay activity of superoxide dismutase (SOD) and catalase (CAT). The apoptotic cells were assayed by flow cytometry (FCM) with annexin V-FITC conjugated and propidium iodide (PI) staining. Results The cell proliferation was inhibited by LH-37 at 10 ?mol?L-1~1 mmol?L-1 in dose dependent manners. After treated with 10 ?mol?L-1 or 1.0 ?mol?L-1 LH-37, the mRNA and protein expression of ?-FP were significantly reduced but mRNA expression of Alb was significantly increased. Treatment of BEL-7402 cells with different LH-37 concentrations for 48 hours increased the percentage of active caspase-3 positive cells and protein expression of caspase-3 and caspase-9. More apoptosis features of cells were observed in LH-37 ( 100 ?mol?L-1) treatment groups than in control group. LH-37 markedly promoted the viability of SOD and decreased CAT in BEL-7402 cells. Counclusion LH-37 might inhibit proliferation and induce differentiation and apoptosis of human hepatocarcinoma BEL-7402 cells, which might be related to the cytotoxicity of the intracellular hydrogen peroxide (H2O2).