Objective To improve disadvantages such as asynchrony during embryoid bodies(EBs)preparation,and to establish a novel method to prepare EBs by using attached mouse embryonic stem Cells.Methods Mouse R1 embryonic stem(ES)cells were trypsinized to a single cell suspension when reaching a sub-confluent state of 70 %～80 %,then 1?106 ES cells were plated into 100 mm tissue culture dishes.After being cultured in medium containing low concentration leukemia inhibitory factor(LIF,1 ?g/L)for 3 days,EBs were collected and suspended in EBs culture medium for further development.Morphology studies were performed on suspended EBs and their cryosections,then immunofluorescent staining of ?-fetoprotein(AFP),platelet endothelial cell adhesion molecule-1(PECAM-1),and neurofilament 68 KD(NF-68)were performed on suspended and attached EBs.Results EBs obtained by this method were homogeneous in size,synchronous in developmental stage;typical in structure and could well display the developmental process from simple EBs to mature cystic EBs.Immunofluorescent staining showed that AFP,PECAM-1 and NF-68 were positive.Conclusion Compared with present methods of preparing EBs,this novel method have advantages of simple manipulation,high efficiency of EBs formation and obtaining EBs at synchronous developmental stage,furthermore,these EBs have typical structures and the potential to differentiate into derivatives of all three embryonic germ layers.Thus this method can be used as an ideal tool for studies on early embryonal development,ES cell differentiation and so on.

BACKGROUND:It has been found that cel ular repressor of E1A-stimulated genes (CREG) is a lysosomal protein binding directly to the mannose-6-phosphate (M6P)/insulin-like growth factor II receptor (IGFIIR) and depends on the interaction with M6P receptors for efficient delivery to lysosomes OBJECTIVE:To study the interactions between the exogenous CREG protein and cathepsins and M6P/IGFIIR and to confirm the effect of CREG protein on expression and distribution of M6P/IGFIIR. METHODS:Double-stained immunofluorescence and coimmunoprecipitation were applied to observe the interactions between the exogenous CREG protein and cathepsin B, cathepsin L and M6P/IGFIIR. Using gain-of-function and loss-of-function approaches, the effect of CREG on expression and distribution of M6P/IGFIIR were studied by western blot assay and immunofluorescence staining. RESULTS AND CONCLUSION:Double-stained immunofluorescence and coimmunoprecipitation analyses confirmed the direct interactions between the exogenous CREG protein and cathepsin B, cathepsin L and M6P/IGFIIR. It was verified that CREG plays a critical role not in the expression but in the distribution of M6P/IGFIIR using gain-of-function and loss-of-function approaches. These findings provide evidence that exogenous CREG protein is located in lysosomes and has interactions with cathepsins and M6P/IGFIIR, also CREG plays a critical role in the distribution of M6P/IGFIIR.

ObjectiveTo investigate the effect of ulinastatin on perioperative renal function in patients undergoing orthotopic liver transplantation.MethodsSixty ASA Ⅱ or Ⅲ patients of both sexes aged 35-64 yr weighing 50-75 kg with normal blood urea nitrogen (BUN) and creatinine (Cr) before operation undergoing orthotopic liver transplantation were randomly divided into 2 groups ( n ＝ 30 each):control group (group C) and ulinastatin group ( group U).Anesthesia was induced with midazolam,fentanyl,etomidate and vecuronium and maintained with isoflurane inhalation,propofol TCI,continuous remifentanil infusion and intermittent iv boluses of fentanyl and vecuronium.The patients were tracheally intubated and mechanically ventilated.PET CO2 was maintained at 30-35 mm Hg.Ulinastatin 400 000 IU in normal saline 20 ml was infused iv after induction of anesthesia.Ulinastatin 200 000 IU was then infused every 4 h until 48 h after operation.Urine volume and the amount of furosemide administered were recorded before anhepatic phase,and during anhepatic and neohepatic phase.Venous blood samples and urine were collected before induction of anesthesia (T1),at 15 min of anhepatic phase ( T2 ),at 15min of neohepatic phase (T3),at the end of operation (T4) and 48 h after operation (T5) for determination of serum concentrations of BUN,Cr and creatinine clearance rate and urinary N-acetyl-beta-D-glucosaminidase (NAG)activity and microalbumin concentration.ResultsCompared with group C,ulinastatin significantly decreased the amount of furosemide administered and increased urine volume during anhepatic and neohepatic phase,decreased serum Cr concentration,increased creatinine clearance rate at T2.5,decreased urinary NAG activity and microalbumin concentration at T4.5 and serum BUN concentration at T3-s.ConclusionUlinastatin has protective effect on rehal function during perioperative period in patients undergoing orthotopic liver transplantation.

Objective To explore medical equipment support in actual combat training to improve mobile medical unit in medical service support.Methods Combined with the characteristics of actual combat training,the problems and difficulty of medical equipment support were discussed in actual combat training.Results Some measures were put forward for medical equipment support such as cherishing of medical serviceman on equipment,equipment quality control and maintenance beforeutilization,effective protection and fixation,minimization of influence of instable power supply and plan preparation for loading and transport.Conclusion The study on medical equipment support in actual combat training contributes to enhancing medical support ability of mobile medical unit.

Objective To investigate whether chemokine CC motif 2 (CCL2) is involved in the high residual platelet response,and the mechanism of CCL2 being involved in the regulation of platelets.Methods Forty patients with ST elevation myocardial infarction (STEMI) were admitted.P2Y12 reaction unit (PRU) was detected by VerifyNow.Forty patients were divided into high platelet reactivity group (high reactivity group,n=24) and normal platelet reactivity group (normal reactivity group,n=16) according to the results of PRU detection.Plasma CCL2 concentration of the STEMI patients was examined by ELISA.The expressions of CCL2 and CCR2 in the platelets were detected by Western blotting.After CCL2 stimulation,the kinases of which phosphorylation was changed in the platelets were screened by ARY003B protein chips.The phosphorylation of p38MAPK and HSP27 in the platelets was tested by Western blotting after CCL2 stimulation in the presence or absence of CCR2 antagonist (RS 102895) or p38MAPK signal pathway inhibitor (SB 203580).Results The plasma CCL2 concentration of high reactivity group was markedly higher than that of normal reactivity group.Moreover,compared with normal reactivity group,the expressions of CCL2 and CCR2 in the platelets of high reactivity group significantly increased.After the platelets were stimulated by CCL2,the phosphorylation of p38α and HSP27 enhanced in the platelets by protein chips screening.When RS 102895 or SB 203580 was treated before CCL2 stimulation,the phosphorylation of p38MAPK and HSP27 decreased.Conclusions CCL2 participates in high residual platelet response in an autocrine/paracrine way.CCL2/CCR2 might affect the function ofplatelets through p38MAPKHSP27 signal pathway.

Objective: To explore the effect and possible mechanisms of intermittent alkaline on rat vascular smooth muscle cells (VSMCs) calcification induced by high phosphorus. Methods: VSMCs were isolated from rat thoracic aorta and cultured in vitro. The fourth generation VSMCs were randomly divided into control group, high phosphorus+ pH7.4, high phosphorus+ pH7.5, high phosphorus+ pH7.6 and high phosphorus+ pH7.7 group with random number table. The control group was cultured in DMEM with 10% fetal bovine serum. Other groups were cultured in DMEM with 10 mmol/L β-glycerophosphate and alkalized by 7.4% NaHCO₃ to adjust the pH respectively. After the intervention of 4 hours, the control group was replaced with the normal medium containing 10% fetal bovine serum, the other 4 groups were replaced with high phosphorus based on the pH value of the culture medium, and then replaced the culture medium every other day. After 4 days intervention, the mRNA and protein expression of L type calcium channel β₃ subunit(LTCC β₃) and Runt related transcription factor 2 (Runx2) were detected by RT-PCR and Western blot. After 4 days intervention, the level of VSMC calcium ion was detected by Fluo-3/AM. After 14 days intervention, alkaline phosphatase (ALP) activity was measured by enzyme linked immunosorbent assay (ELISA) and the calcification was observed by measuring calcium content. Results: (1) Compared with control group, the gene and protein expressions of LTCC β₃ were higher in high phosphorus+ pH7.4 group (0.49±0.03 vs. 0.23±0.02 and 0.45±0.03 vs. 0.26±0.02 respectively, all P<0.05). Compared with high phosphorus+ pH7.4 group, the mRNA(0.86±0.05) and protein(0.62±0.04) expressions of LTCC β₃ were higher in high phosphorus+ pH7.5 group (P<0.05). Compared with high phosphorus+ pH7.5 group, the mRNA(0.99±0.05) and protein(0.80±0.03) expressions of LTCC β₃ were higher in high phosphorus+ pH7.5 group (all P<0.05). Compared with high phosphorus+ pH7.6 group, the mRNA(1.16±0.05) and protein(0.93±0.03) expressions of LTCC β₃ were higher in high phosphorus+ pH7.7 group (all P<0.05). (2) Compared with control group, calcium ion influx were higher in high phosphorus+ pH7.4 group (124.61±6.06 vs. 75.68±7.82, P<0.05). Compared with high phosphorus+ pH7.4 group, calcium ion influx was higher in high phosphorus+ pH7.5 group(210.85±9.75, P<0.05). Compared with high phosphorus+ pH7.5 group, calcium ion influx was higher in high phosphorus+ pH7.6 group(298.44±11.42, P<0.05). Compared with high phosphorus+ pH7.6 group, calcium ion influx was higher in high phosphorus+ pH7.7 group(401.13±11.41, P<0.05). (3) Compared with control group, the mRNA and protein expressions of Runx2 and ALP were higher in high phosphorus+ pH7.4 group (0.60±0.04 vs. 0.34±0.03, 0.42±0.04 vs. 0.21±0.02, 67.2±4.3 vs. 23.2±2.3 respectively, all P<0.05). Compared with high phosphorus+ pH7.4 group, the mRNA(0.76±0.05) and protein(0.68±0.03) expressions of Runx2 and ALP(102.1±5.4) were higher in high phosphorus+ pH7.5 group (all P<0.05). Compared with high phosphorus+ pH7.5 group, the mRNA(0.90±0.05) and protein(0.90±0.05) expressions of Runx2 and ALP(139.3±4.9) were higher in high phosphorus+ pH7.6 group (all P<0.05). Compared with high phosphorus+ pH7.6 group, the mRNA(1.11±0.05) and protein(1.08±0.06) expressions of Runx2 and ALP(197.0±6.7) were higher in high phosphorus+ pH7.7 group (all P<0.05). (4) Compared with control group, the calcium content were higher in high phosphorus+ pH7.4 group ((75.4±4.3)mg/g pro vs.(25.2±2.1)mg/g pro, P<0.05). Compared with high phosphorus+ pH7.4 group, the calcium content were higher in high phosphorus+ pH7.5 group ((100.8±5.7) mg/g pro, P<0.05). Compared with high phosphorus+ pH7.5 group, the calcium content were higher in high phosphorus+ pH7.6 group ((143.5±6.1) mg/g pro, P<0.05). Compared with high phosphorus+ pH7.6 group, the calcium content were higher in high phosphorus+ pH7.7 group ((205.1±8.2) mg/g pro, P<0.05). Conclusion Intermittent alkaline stimulation can promote high phosphorus induced rat VSMCs calcification possibly through upregulating LTCC β₃ subunit gene and protein expression, increasing calcium ion influx and enhancing VSMCs phenotypic transformation.

Objective To study epigenetic diversity of Astragalus mongolicus by methylation-sensitive amplification polymorphism(MSAP)technique,and to establish and optimize the response system of MSAP technique applicable to Astragalus Mongolicus.Methods Astragalus membranaceus(Fisch.)Bge.var.mongholicus(Bge.)Hsiao was used as material.The single factor and orthogonal design method were used to optimize the key influencing factors of the reaction system of Astragalus Mongolian methylation-sensitive amplification polymorphism(MSAP)technique,and the best reaction system of Astragalus Mongolian MSAP was established.Results 0.5 μL each of EcoR I and Msp I/Hpa II were added to the 20 μL double-enzyme digestion reaction system,and the digestion was completed in a water bath at 37℃for 6 h to complete the digestion;the optimal ligation system was 25 μL,including 10 μL digestion product,0.2 μL T4 DNA Ligase,1 μL EcoR I-adapter,1 μL Hpa II/Msp I-adapter,2.5 μL 10×T4 Buffer;25 μL optimal pre-amplification reaction system,including 2.5 μL 10×PCR buffer,0.5 μL dNTP,2.0 μL ligation product,0.3 μL Taq DNA polymerase,1.0 μL upstream and downstream primers;25 μL optimal selective amplification system,including 2 μL 20-fold diluted pre-amplification product,1.0 μL 10×PCR buffer,1.0 μL dNTP,0.2 μL Taq DNA polymerase,0.8 μL primers.Conclusion The optimized MSAP system was detected by polyacrylamide gel electrophoresis,which showed that the polymorphism was good,the system was stable,the bands were clear and repeatable,which laid the foundation for the subsequent MSAP analysis of Astragalus mongolica.

ObjectiveTo establish a droplet digital PCR (ddPCR) method for detecting hepatitis B virus (HBV) covalently closed circular DNA (cccDNA). MethodsHBV cccDNA standard substance was constructed, and HBV cccDNA primers and probes were designed based on the structural differences between HBV cccDNA and relaxed circular DNA (rcDNA). HBV plasmid was amplified to obtain HBV cccDNA standard substance, and a ddPCR detection method was established with the standard substance after gradient dilution as the template for HBV cccDNA detection; the limit of detection and repeatability of this method were analyzed. Liver tissue samples were collected from 20 patients who attended Beijing YouAn Hospital, Capital Medical University, from June 2017 to October 2020, all of whom were diagnosed with HBV infection, and DNA of the samples was extracted and digested with plasmid-safe ATP-dependent DNA enzyme to obtain HBV cccDNA template; the ddPCR detection method was evaluated in clinical samples and was compared with the quantitative real-time PCR (qPCR) detection method. The chi-square test was used for comparison of categorical data between the two groups. ResultsThe HBV cccDNA detection method based on ddPCR was established, which accurately detected HBV cccDNA in standard substance after gradient dilution, with a limit of detection of 1 copy/μl, and the coefficients of variation of 1×103, 1×102, and 1×101 copies/μl standard substances were 441%, 3.98%, and 5.09%, respectively. HBV cccDNA was detected in the samples of 20 patients with HBV infection; the ddPCR detection method detected HBV cccDNA in 17 patients, with a positive rate of 85%, while the qPCR detection method detected HBV cccDNA in 11 patients, with a positive rate of 55%, and there was a significant difference between the two methods (χ2=4.286, P=0038). ConclusionThe established ddPCR method for detecting HBV cccDNA has a low limit of detection and good repeatability, which provides an effective tool for further clinical detection.

【Objective】 A framework to support nurses and midwives making the clinical decision and providing the written instruction for blood transfusion has been developed and implemented in the United Kingdom as a response to the changing needs of the patient and in recognition that blood transfusion services to patients could be improved by using the untapped knowledge and expertise of experienced nurses and midwives.Special education and training program for this role development are provided jointly by the national blood and nurse management authority, higher education institutions and transfusion societies.The British government has issued and implemented a compulsory professional indemnity which cover nurses and midwives as well.The development and implementation of the framework, policies and procedures for this role development is based on the regulatory compliance and the collaboration of, and beneficial to the multiple stakeholders, with the gaps left by doctors being fillled, work load of doctors reduced, nurses and midwives achieving professional development, hospitals performing more efficiently, and most importantly, the patients having a better transfusion services.At present, there is no similar policy or program for nurses and midwives in China.Therefore, this paper introduces the policy framework and implementation for this role development in UK, which would be a valuable reference for the role development and extension of nurses and the organization, education and training for transfusion professional teams as well in China in the near future.

Objective:To establish a rapid and accurate method for the detection of Klebsiella pneumoniae carbapenemase (KPC) carbapenemase gene based on recombinase aided amplification (RAA)-CRISPR-Cas13a (CRISPR-Cas13a) technology. Methods:Twenty-five clinical isolates of carbapenem-resistant Klebsiella pneumoniae (CRKP) and five carbapenem-sensitive Klebsiella pneumoniae (CSKP) strains preserved in 2020-2021 in Beijing Chuiyangliu Hospital were randomly collected, and the total DNA samples of the strains was extracted. RAA primers specific for KPC DNA and CRISPR RNA (crRNA) were designed to establish a rapid and accurate method for the detection of KPC carbapenemase gene based on RAA-CRISPR-Cas13a technology. The method was evaluated by plasmids and clinical sample strains, and the detection was also performed by Quantitative real-time PCR (qPCR) method to compare the detection rate and consistency of the two methods. Results:The RAA-CRISPR-Cas13a method can detect KPC plasmids and samples with a sensitivity of 1 copy/μl, which is higher than that of qPCR (10 1 copies/μl). Among the 30 clinical strains (including 25 CRKP strains and 5 CSKP strains), 23 strains were detected to carry KPC gene by both RAA-CRISPR-Cas13a method and qPCR method, and 7 strains were not detected with KPC gene. The detection rate of KPC gene in the 25 CRKP strains was 92% (23/25). The positive coincidence rate of the two methods was 100% (23/23). Conclusions:This study combined RAA amplification technology with CRISPR-Cas13a technology to establish a rapid and accurate method for detecting KPC carbapenemase gene. The method is useful for accurate screening of KPC carbapenemase-producing strains. It has a wide application prospect in drug resistance monitoring and infection control.