A glass micropipette vacuum technique of cerebrospinal fluid sampling in C57BL/6 mice / 生理学报

The purpose of this study was to establish a suitable method for extracting cerebrospinal fluid (CSF) from C57BL/6 mice. A patch clamp electrode puller was used to draw a glass micropipette, and a brain stereotaxic device was used to fix the mouse's head at an angle of 135° from the body. Under a stereoscopic microscope, the skin and muscle tissue on the back of the mouse's head were separated, and the dura mater at the cerebellomedullary cistern was exposed. The glass micropipette (with an angle of 20° to 30° from the dura mater) was used to puncture at a point 1 mm inboard of Y-shaped dorsal vertebral artery for CSF sampling. After the first extraction, the glass micropipette was connected with a 1 mL sterile syringe to form a negative pressure device for the second extraction. The results showed that the successful rate of CSF extraction was 83.33% (30/36). Average CSF extraction amount was (7.16 ± 0.43) μL per mouse. In addition, C57BL/6 mice were given intranasally ferric ammonium citrate (FAC) to establish a model of brain iron accumulation, and the CSF extraction technique established in the present study was used for sampling. The results showed that iron content in the CSF from the normal saline control group was not detected, while the iron content in the CSF from FAC-treated group was (76.24 ± 38.53) μmol/L, and the difference was significant. These results suggest that glass micropipette vacuum technique of CSF sampling established in the present study has the advantages of simplicity, high success rate, large extraction volume, and low bleeding rate, and is suitable for the research on C57BL/6 mouse neurological disease models.

GBA mutations and Parkinson's disease / 生理学报

Parkinson's disease (PD) is a common neurodegenerative disease characterized by the degeneration of dopaminergic neurons in the substantia nigra and the intraneuronal Lewy bodies in this area. Genetic mutations in PD pathogenesis have been explored and better understood in recent years. GBA variants are now considered to be the single largest risk factor for PD. Gaucher disease (GD) is a lysosomal storage disorder disease and an inherited deficiency of lysosomal glucocerebrosidase (GCase) arising from mutations in the gene GBA. A group of patients with GD exhibit parkinsonian symptoms, meanwhile, GBA mutations are more frequently observed in patients with PD. These lines of evidence suggest a close relationship between GBA mutations and PD. GBA mutations are associated with an earlier onset age and a distinct cognitive decline in PD. GCase loss-of-function caused by GBA mutations interferes with the degradation of α-synuclein, and α-synuclein pathology in turn inhibits normal GCase function in PD, which forms a vicious cycle. However, the exact mechanisms for this bidirectional pathogenic loop have not to be fully elucidated. In this review, we summarize the current understandings on the potential link between GBA mutations and PD pathogenesis, which may show novel insights into PD etiology and therapeutics.

Clinical significance of distribution of ABO blood group in patients with deep sternal wound infection after cardiac surgery / 中国感染控制杂志

Objective To explore the clinical significance of distribution of ABO blood group in patients with deep sternal wound infection(DSWI)after cardiac surgery.Methods Clinical data of 84 patients with DSWI after cardiac surgery in the department of cardiothoracic surgery in General Hospital of China Aviation of China Medical University in 2012-2014 were analyzed retrospectively, according to ABO blood group, patients were divided into 4 groups:A blood group, B blood group, AB blood group, and O blood group, according to whether the blood group was A group, they were divided into A blood group and non-A blood group.Distribution of ABO blood group in DSWI patients was analyzed, risk factors, clinical manifestations, and etiological characteristics of DSWI patients with different ABO blood groups were compared.Results Among patients with DSWI, A blood group and non-A blood group were 33 cases(39.3％)and 51 cases(60.7％)respectively(B, O, and AB blood group were 16 cases[19.1％], 29 cases[34.5％], and 6 cases[7.1％]respectively);the proportion of A blood group in DSWI patients was higher than that of the healthy population, but the difference was not statistically significant(P=0.055). Distribution of baseline characteristics and incidences of various clinical manifestations among DSWI patients of different blood groups were not statistically significant(all P＞0.05).However, compared with non-A blood group or other ABO blood groups, DSWI patients with A blood group had higher incidence of elevated white blood cell count, difference was statistically significant(P＜0.05), positive detection rate of gram-positive bacteria in A blood group was also higher, difference was statistically significant(P＜0.05).In addition, only 3 strains of Pseudomonas aeruginosa was detected only in B blood group, while gram-negative bacteria were not detected in AB blood group. Conclusion ABO blood group may play a role in the pathogenesis of DSWI after cardiac surgery, which may be associated with a specific bacterial infection.

Preparation and characterization of a gene-activated matrix mimicking extracellular matrix / 药学学报

This study aims to establish a novel gene-activated matrix that mimics the structure and function of extracellular matrix (ECM-m-GAM). The structure, mechanical property and release profile were also characterized. Firstly, the liposome/DNA lipoplex (LPD) was modified with cell penetrating peptide TAT. The obtained TAT-LPD was then mixed with RGD grafting hyaluronic acid solution. After addition of the matrix metalloproteinase (MMPs) sensitive crosslinker (HS-MMP-SH), hyaluronic acid was crosslinked and TAT-LPD was encapsulated in the subsequently formed hydrogel. As a result, the cell adhesion factor RGD, MMPs sensitive substrate and the efficient gene transfer vector TAT-LPD were all integrated in the hyaluronic acid hydrogel, which was named as ECM-m-GAM. The release profile of DNA from ECM-m-GAM in different release medium was evaluated with PicoGreen kits. The results suggested that the mean diameter of the spherical TAT-LPD was (263.0 ±4.30) nm. TAT-LPD was successfully encapsulated in ECM-m-GAM, which had the typical porous network structure of hydrogels. The mechanical strength of GAM was enhanced with the increasing of hyaluronic acid content. When the content was 4%, the elastic modulus of GAM reached 1 600 Pa. The highly elastic GAM may be suitable for implantation and tissue regeneration. The DNA release showed significant MMPs sensitive property. Especially, the released DNA still existed in form of nanoparticles. Bone marrow mesenchymal stem cells (BMSCs) were successfully transfected with GAM and the green fluorescent protein was expressed. The results have laid a solid foundation for future study of the cell transfection and tissue regeneration.

Current Use of Oral Anticoagulants and Prognostic Analysis in Patients with Atrial Fibrillation Undergoing Coronary Stenting / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>It is currently believed that triple oral antithrombotic therapy in patients with atrial fibrillation (AF) after percutaneous coronary intervention (PCI) should be recommended if there are no contraindications. However, selecting triple therapy for AF patients undergoing PCI is still challenging when bleeding risk is considered. This study aimed to investigate the current use of oral anticoagulants (Vitamin K antagonists [VKA]) and perform prognostic analysis in real-world patients with AF undergoing coronary stenting.</p><p><b>METHODS</b>A total of 276 consecutive coronary artery disease (CAD) patients with or without AF undergoing coronary stenting were retrospectively evaluated and analyzed. The univariate and multivariate analyses were conducted to explore the current use of VKA and prognosis of patients with AF undergoing coronary stenting. The primary end-point was composite of all-cause death, nonfatal recurrent myocardial infarction, stroke, serious bleeding events, unplanned repeat revascularization, and worsening heart failure at 12-month follow-up after coronary stenting.</p><p><b>RESULTS</b>AF patients undergoing coronary stenting have more clinical concomitant diseases. Only 9.0% AF patients after coronary stenting received triple antithrombotic therapy (VKA, aspirin, and clopidogrel) at discharge. AF was independently associated with increased risk of the 12-month composite end-points (relative risk = 5.732, 95% confidence interval 1.786-18.396, P = 0.003).</p><p><b>CONCLUSIONS</b>In real-life AF patients undergoing coronary stenting, guideline-recommended VKA was less used. AF patients had adjusted worse prognosis during 12-month follow-up after discharge. It is of utmost importance to improve the current status of oral anticoagulants use.</p>

Association of 5-lipoxygenase expression and clinicopathological factors in colorectal carcinoma / 中华胃肠外科杂志

<p><b>OBJECTIVE</b>To examine the association of 5-lipoxygenase (5-LOX) expression with clinicopathological factors in colorectal cancer.</p><p><b>METHODS</b>Immunohistochemical stain was used to detect the 5-LOX expression in 52 resected specimens of colorectal cancer. The association between 5-LOX expression and clinicopathological factors was examined.</p><p><b>RESULTS</b>The positive rate of 5-LOX expression in 52 specimens of colorectal carcinoma was 73.1% (38/52). In 41 colorectal cancer specimens with lymph node metastasis, the positive rate of 5-LOX expression was higher than that in the specimens without metastasis (87.8% vs. 18.2%, P<0.05). The positive rate of 5-LOX expression in the specimens with deep infiltration (T3 and T4) was higher than that in the specimens with superficial infiltration (T1 and T2) (81.1% vs. 53.3%, P<0.05). The positive rate of 5-LOX expression in TNM stage III and IIII cancer was higher than that in stage I and II (79.5% vs. 53.8%, P<0.05). The positive rate of 5-LOX expression in cancers of poor differentiation and non-differentiation adenocarcinoma was higher than that of well and moderately differentiated cancer (100% vs. 50.0%, P<0.05). There were no significant differences of 5-LOX expression with tumor size,vascular invasion and peritoneal dissemination.</p><p><b>CONCLUSION</b>5-LOX expression in colorectal carcinoma is closely associated with lymph node metastasis, infiltration depth, differentiation degree and TNM stage.</p>

Quantitative analysis of dendritic cell subsets in bone marrow of patients with acute myeloid leukemia / 中国实验血液学杂志

In order to study the quantity of dendritic cell (DC) subsets of bone marrow in patients with acute myeloid leukemia (AML), the bone marrow aspirate were collected from 77 newly diagnosed AML patients and from 30 healthy persons. The quantity of DC subsets (myeloid dendritic cells, mDC and plasmacytoid dendritic cells, pDC) were detected by flow cytometry and analysed by 3-color and 4-color cytometric gate. Based on the conventional 3-color panel, mDC were identified by Lin-HLA-DR+CD11c+ and pDC were identified by Lin-HLA-DR+CD123+. Based on the 4-color panel, mDC were identified by Lin-HLA-DR+CD11c+ BDCA-1+ and pDC were identified by Lin-HLA-DR+CD123+BDCA-2+. The results showed that a reduction of mDC was found in 74.0% (57/77) and 58.4% (45/77) patients, a reduction of pDC was found in 90.9% (70/77) and 46.8% (36/77) patients respectively by 3-color and 4-color cytometric analysis. Meanwhile an expansion of mDC was showed in 19.5% (15/77) and 22.1% (17/77) patients, an expansion of pDC was showed in 1.3% (1/77) and 27.3% (21/77) patients respectively by 3-color and 4-color cytometric analysis. In subtypes of AML-M2, AML-M3 or AML-M4/5, 81.4%, 100% and 42.1% patients showed mDC decrease and 88.4%, 100% and 89.5% patients showed pDC decrease respectively by 4-color cytometric analysis. It is concluded that the 4-color cytometric gate is better method for detection of mDC and pDC from bone marrow of newly diagnosed AML patients as compared with 3-color cytometric gate, the majority of AML patients showed reduction of mDC and pDC. The percentages of patients with mDC normal or mDC increase in AML-M4/5 subtypes are more than that in AML-M2/3 subtypes, while the pDC does not show difference between AML subtypes.

Identification and enumeration of dendritic cells in peripheral blood and bone marrow of healthy individuals / 中国实验血液学杂志

The study was aimed to analyse and enumerate the dendritic cells (DC) subsets in peripheral blood and bone marrow (BM) of healthy individuals in China by using 2 panel 4-color flow cytometry (FCM). The percentage and absolute number of Lin-HLA-DR+CD11chiBDCA1+ myeloid DC (mDC) and Lin-HLA-DR+CD123hiBDCA2+ plasmacytoid DC (pDC) were detected in 35 normal BM (NBM) and 29 normal peripheral blood (NPB), the results were compared with the Lin-HLA-DR+CD11chimDC and Lin-HLA-DR+CD123hi pDC obtained by 3-color FCM. The results indicated that both absolute count of DC subset and relative count of pDC in BM were decreased along with increase of age, the absolute count of DC subset in male BM was higher than that in femoral BM (p<0.05). The DC subsets in NBM and NPB were different whatever by 3 or 4-color cytometric analysis, there were more mDCs than pDCs in PB and the ratio of mDC to pDC was 2.70 and 2.31 respectively. In contrast, pDCs predominated in BM, the ratio of mDC to pDC in BM was 0.90 and 0.71 respectively. The quantity of DC subsets significantly correlated to both frequency (mDC r=0.86; pDC r=0.96, p<0.05) and absolute number (mDC r=0.95; pDC r=0.98, p<0.05) between 3 and 4-color cytometric analysis. The quantity of DC subsets in PB and BM were significantly different, counted by 3 and 4-color cytometric analysis except pDC in PB (p<0.001). The quantity of DC subsets were much higher by 3-color than that by 4-color analysis. Since some Lin-HLA-DR+CD11chimDC and Lin-HLA-DR+CD123hi pDC were BDCA1- and BDCA-2dim/- respectively, that more were in BM than in PB. It is concluded that the DC absolute enumeration is correlated with sample type, gender, age and total nucleated cells (p<0.05). 4-color antibody combination may help to identify the real DC subsets in BM. DC subsets in NBM and NPB are different, that more mDC are in PB whereas more pDC in BM.

Expression of 5 genes in CD7 positive acute myeloid leukemia stem/progenitor cells from bone marrow / 中国实验血液学杂志

This study was aimed to investigate abca5, mdr-1, kdr, dapk and irf-1 expressions in leukemia stem/progenitor cells (LSC) from CD7 positive acute myeloid leukemia, the expression of these 5 genes in mononuclear cells (MNC) from 15 normal bone marrow (NBM) and 16 AML patients bone marrow (AML BM) specimen were detected by real-time quantitative PCR (RQ-PCR). CD34(+)CD38(+) progenitor cells and CD34(+)CD38(-)Lin(-) stem cells were sorted by flow cytometry (FCM) from the MNCs of 10 NBM and 21 AML BM specimen. These 5 gene expressions in the sorted cells were detected by small amount cell RQ-PCR. The results showed that these 5 genes above mentioned all expressed in NBM-MNC, in which the expression levels of irf-1 and dapk were highest with the relative expression levels 4.08 and 3.86, the expression levels of abca 5 and mdr-1 were in the middle with the relative expression 0.49 and 0.84 respectively, the kdr expression was lowest with the relative expression level 0.02. In CD34(+)CD38(+) progenitor cells, the expression level of kdr increased dramatically (p < 0.05) while irf-1 and dapk dramatically decreased (p < 0.05). There was no obvious change of expression in the rest 2 genes. In CD34(+)CD38(-) stem cells the expression level of these 5 genes all increased nearly 2 times as much as that in CD34(+)CD38(+) progenitor cells, but kdr increased 3 times as much, and the increase of kdr and irf-1 expressions was of statistical significance (p < 0.05). Compared with the NBM, expression levels of 5 genes in AML-MNC decreased, and out of them abca 5, mdr-1, kdr and dapk were decreased most remarkably (p < 0.05). Comparison between AML CD34(+)CD38(+) cells and AML MNC showed that the expression level of irf-1 and dapk were decreased dramatically (p < 0.05) while the rest 3 genes increased their expression with statistical significance (p < 0.05). The expression levels of these 5 genes were higher in CD34(+)CD38(-) cells than those in CD34(+)CD38(+) stem cells, and the increase of kdr and irf-1 expressions showed statistical difference (p < 0.05). These 5 genes expression levels were all higher than those in CD34(+)CD38(+) cells whether in AML CD34(+)CD38(-)CD7(+) cells or CD34(+)CD38(-)CD7(-) cells. The increase of kdr expression in CD34(+)CD38(-)CD7(+) cells as well as kdr and irf-1 expressions in CD34(+)CD38(-)CD7(-) cells were all of statistical significance (p < 0.05). In conclusion the expression level of kdr in NBM was highest in stem cells while dapk and irf-1 were highest in differentiated cells. The expression levels of these 5 genes in CD34(+)CD38(-)Lin(-) stem cells were higher than those in CD34(+)CD38(+) progenitor cells. The gene expressions in AML CD34(+)CD38(-)CD7(+) cells and CD34(+)CD38(-)CD7(-) cells are in accordance with the characteristics of stem cells.

Establishment of a Huntington’s Disease in vitro Drug Screening Cell Model / 中国生物工程杂志

To develop a Huntington’s disease(HD) cell model in vitro to screen drugs targeting the aggregation of polyQ,different length of CAG repeat fragments were amplified by random primer PCR, identified by DNA sequencing and were fused to the N-terminus of CAT in the pCAR system respectively which had been constructed and identified before. Recombinant plasmids were transformed into and induced to express in the host E.coli. SDS-PAGE and chloramphenicol resistance test were done to determine the solubility of the polyQ and chloramphenicol resistance levels of the fusions. With different length of CAG repeat fragments cloned and expressed in the CAT-fusion protein reporting system, it is found that when the length of the fragments increased over 40, their encoding polyQ expressed as insoluble protein and chloramphenicol resistance levels are lower, while under 40, the polyQ expressed as soluble ones and chloramphenicol resistance levels are higher. A in vitro HD model that could minimize the pathological process of the HD thus has been developed. With which by measure the recombinant bacteria’s resistance to chloramphenicol, the polyQ’ solubility and folding state in vitro by quality and quantity could be determined. Thus this model can be used to screen drugs or bioactivity materials that can inhibit aggregation of the polyQ, which thereby shedding new light on the prevent, diagnosis and therapy of HD.

Effects of Progesterone on Brain Damage in Hypoxia Mouse / 实用儿科临床杂志

Objective To observe the changes of water, potassium and sodium in the hypoxic cerebral tissues in mice and the effects of progesterone on the tissues and investigate the neuroprotective role of progesterone (PROG)in the cerebral anoxia. Methods Thirty - two male mice were divided into control group, simple hypoxia group, lower dosage group and higher dosage group. Progesterone was injected intraperitoneally in the dosage of 4 mg/kg or 8 mg/kg respectively 30 min before hypoxia in the last two groups. The contents of water, potassium and sodium in brain tissues in mice were evaluated at 24 hours after cerebral anoxia. Results The water, potassium and sodium contents in simple hypoxia group were significantly higher than those in control group(P0.05)in higher dosage group(8 mg/kg PROG)compared with those of simple hypoxia group. Conclusions At 24 hours after cerebral anoxia, there are significant increases of water, potassium and sodium in brain tissues. Progesterone may pro-duce neuroprotective role by inbibiting the rise of them.