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We developed a high-efficiency microfluidic chip for extracting exosomes from human plasma. We collected peripheral blood from normal human, designed and fabricated a microfluidic chip based on nanoporous membrane and agarose gel electrophoresis to isolate exosomes. The extracted exosomes were characterized by transmission electron microscopy, nanosight and Western blotting, the morphology, concentration and particle size of exosomes were identified and analyzed. Meanwhile, we used ultracentrifugation and microfluidic chip to isolate exosomes separately. The particle size and concentration of the exosomes extracted by two methods were compared and analyzed, and their respective extraction efficiency was discussed. Finally, the expression level of miRNA-21 in exosomes was analyzed by RT-PCR. The microfluidic chip isolated (in 1 hour) high-purity exosomes with size ranging from 30-200 nm directly from human plasma, allowing downstream exosomal miRNA analysis. By comparing with ultracentrifugation, the isolation yield of microfluidic chip was 3.80 times higher than ultracentrifugation when the volume of plasma sample less than 100 μL. The optimized parameters for exosome isolation by gel electrophoresis microfluidic chip were: voltage: 100 V; concentration of agarose gel: 1.0%; flow rate of injection pump: 0.1 mL/h. The gel electrophoresis microfluidic chips could rapidly and efficiently isolate the exosomes, showing great potential in the research of exosomes and cancer biomarkers.
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Humans , Exosomes , MicroRNAs/genetics , Microfluidics , Plasma , UltracentrifugationABSTRACT
Lung cancer is a malignant tumor with high incidence and mortality in the world, which seriously threatens people's safety and health. Early diagnosis of lung cancer is the key part in the process of prevention and treatment of lung cancer. It can improve the survival of patients with lung cancer. Exosomes are closely related to the invasion and metastasis process of tumor, it plays an important role in the development of lung cancer. Biomarkers based on exosomes have become a powerful diagnostic tool of lung cancer. Exosomes are lipid bilayer vesicles with uniform size and diameter of 30 nm-200 nm secreted by cells. Exosomes contain different types of nucleic acids and proteins. These nucleic acids and proteins are derived from their parent cells (including parent cancer cells), which have a wide range of physiological functions, including immune regulation, intercellular communication and other physiological activities. Biomacromolecules in exosomes, such as single-stranded RNA, long noncoding RNA, microRNA, protein and lipids, which can provide valuable genetic information for early clinical diagnosis of lung cancer. This review described the origin, structural characteristics, extraction methods, biological characteristics of exosomes and the relationships of exosomes in the early diagnosis of lung cancer. .
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Objective To establish a rapid and sensitive liquid chromatograph-mass spectrometry (LC-MS/MS) method for the determination of thiabendazole in honeysuckle flowers. Methods The acetic ether was selected as extraction solvent. The mass spectrometer analysis was conducted in the positive ionization electrospray mode using SIM. The transitions m/z 202→175 was used to quantify thiabendazole. Results The satisfactory linearity was obtained in the range of 0.1×10-5-2×10-5mg for thiabendazole (r=0.999 5), and the limit of detection (LOD) of 10.0 ng/ml and the mean recovery of 93.70% were obtained by this LC-MS/MS method. Conclusions The method of LC-MS/MS is sensitive, simple and accurate, and it proved to be suitable for the determination of thiabendazole in Honeysuckle flowers.
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Objective:To study the effect of atractylodes lactideⅠ,Ⅱand Ⅲ on the expression of cytokines by inducing M1 type macrophage model. Methods:Apoptotic macrophages were induced by sodium thioglycolate, and then the rat peritoneal inflammatory macrophages were purified by a differential adherence method. The expression of macrophage marker antigen (ED1) was identified by flow cytometry macrophages. Tumor necrosis factor ( TNF-α) , proinflammatory cytokines ( IL-1β) and interleukin-6 ( IL-6 ) were measured by ELISA in vitro. The levels of expression of arginase 1, inducible nitric oxide synthase (iNOS), inflammatory cytokines (IL-1β) chemokine (CCL22) and transforming growth factor (TGF-β) in inflammatory macrophages were determined by RT-PCR. Results:As for the purification of cultured rat inflammatory macrophage expressing ED1, atractylenolideⅠand Ⅲ could reduce the ex-pression levels of iNOS and IL-1βand increase the expression levels of arginase1 CCL22 and TGF-β. The release of inflammatory fac-tor IL-1β decreased, and the release levels of TGF-β and chemokines CCL22 were promoted(P<0. 05). Atropine lactoneⅠ and Ⅲ had significant inhibitory effects on TNF-α, IL-1β and IL-6 in macrophages(P<0. 05). Conclusion: Atractylodes lactone Ⅰand Ⅲ with anti-inflammatory activity can promote the expression of cytokines in inflammatory macrophages, while the change induced by at-ractylaxanthin Ⅱ is not obvious.
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Objective:To establish a method for determining osthole in Baicao Fuyanqing suppositories by HPLC. Methods:The de-termination was carried out on a DL-Cl8column (4.6 mm ×150 mm, 5 μm). The mobile phase consisted of acetonitrile-water (65 ∶35) with the detection wavelength at 321nm and the flow rate of 1. 00 ml·min-1 . Results:The linear range of osthole was 2. 0-80. 0 μg· ml-1(r=0. 999 1). The average recovery of osthole was 97. 5%(RSD=1. 95%,n=6). Conclusion:The method is simple, rapid and accurate, which can be used for the determination of osthole in Baicao Fuyanqing suppositories.
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Objective:To establish a rapid and sensitive method for the determination of carbendazim in honeysuckle flowers by LC-MS/MS. Methods:The sample was extracted by acetic ether. The mass spectrometer was operated in the positive ionization elec-trospray ( ESI) mode using selection ion monitoring ( SIM) . The transition m/z 192→160 was used to quantify carbendazim. Results:The method had a satisfactory linearity within the range of 50-5 000 ng·ml-1 for carbendazim (r=0. 999 4), the limit of detection (LOD) was 2. 0 ng·ml-1, and the mean recovery was 93. 2%. Conclusion:The method of LC-MS/MS is sensitive, simple and ac-curate,which is proved to be suitable for the determination of carbendazim in honeysuckle flowers.
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Objective:To establish an HPLC method to determine the content of rosmarinic acid in Wuling capsules. Methods:The chromatographic column was Linksil-ODS (250 mm × 4. 6 mm,5 μm) with a gradient elution mode at the flow rate of 0. 8 ml· min-1,and the mobile phase was acetonitrile-water (32∶68) including 0. 5% acetic acid. The detection wavelength was 319 nm, the column temperature was 25 ℃ and the injection volume was 20 μl. Results: The linear range was 2-80 μg·ml-1 and the standard curve showed good linear regression within the range(r=0. 999 8). And the average recovery was 97. 07﹪ (n=6) with the inter-day precision (RSD) of 1. 35 %. Conclusion:The method is accurate and rapid with good reproducibility in the determination of ros-marinic acid in Wuling capsules, which can be used to control the quality of rosmarinic acid in Wuling capsules.
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Objective: To establish a method for the determination of atractylenolide Ⅰ in Atractylodes macrocephala by HPCE. Methods:The separation conditions of HPCE were as follows:50 mmol·L-1 borate buffer was used as the running buffer, the UV de-tection was set at 210 nm, the separation voltage was 20 kV, the column temperature was 25℃ and the pressure injection was 50 mbar × 5 sec. Results:The lower detection limit was 0. 5 μg·ml-1 . The concentration of atractylenolide Ⅰ showed a linear plot within the range of 2-100 μg·ml-1(r=0. 996 0). The average recovery was 97. 1%, and the intra-and inter-day RSD was 1. 3% and 2. 5%, respectively. Conclusion:The established method is simple, sensitive and economic. The method is suitable for the quality control of atractylodes macrocephala.
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Objective Inflammation plays a critical role in the presence , development and maintenance of atrial fibrillation ( AF) , but it remains unclear what factors induce inflammation in AF patients , especially in those with valvular heart disease ( VHD) . The aim of this paper was to investigate the role of the shedding of Syndecan -4 in left atrial inflammation in patients with valvular atrial fibrillation. Methods Sixty VHD patients scheduled for valvuloplasty or valve replacement surgery were divided into three groups of equal number:sinus rhythm (SR), paroxysmal atrial fibrillation (PaAF), and persistent atrial fibrillation (PeAF).Another 10 pa-tients with congenital heart disease but no valve damage and atrial fibrillation were included in a control group .Baseline clinical data were recorded and tissues were obtained from the left atrial appendage during operation .The expressions of iNOS , HMGB1, and Syn-decan-4 in the left atrium were detected by Western blot , and the pathological changes of the left atrial tissue observed by HE staining . Results Western blot analysis was performed to detect expression levels of proteins .The iNOS level was significantly higher in pa-tients from the paroxysmal AF group (1.61 ±0.10) and persistent AF group (1.67 ±0.08) than those from sinus group (1.06 ± 0.11) and control group (1.02 ±0.12), as was the protein level of HMGB1 (0.63 ±0.05, 0.95 ±0.10, 0.45 ±0.07 and 0.46 ± 0.06 in paroxysmal AF group, persistent AF group, sinus group and controlgroup respectively ).Inflammatory cell infiltration in-creased, while syndecan 4 was down-regulated in AF groups.All these comparisons were significant (P<0.05). Conclusion The decreased expression of Syndecan-4 and enhanced inflammatory response in the left atrial tissue indicate that the shedding of Synde-can-4 may play a role in the presence and development of inflamma-tion in the left atrium .
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Objective:To establish an HPLC method to simultaneously determine the content of protocatechualdehyde, rosmarinic acid and salvianolic acid A in Salvia miltiorrhiza Bunge. Methods: The chromatographic column was a Linksil-ODS ( 250 mm × 4. 6 mm,5 μm) column,the mobile phase was 1% acetic acid water solution-acetonitrile (7∶3) with the flow rate of 0. 8 ml·min-1 . The detection wavelength was 280nm , the column temperature was 30℃ and the injection volume was 20 μl. Results: The linear range of protocatechualdehyde, rosmarinicacid and salvianolic acid A was 3-300 μg·ml-1(r>0. 999 0), and the recovery was within the range of 95. 22%-99. 68% with RSD below 2% (n=5). Conclusion:The method is accurate, rapid and reproducible, and can be used to control the quality of Salvia miltiorrhiza Bunge.
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The qualitative and quantitative analysis of active constituents in Fructus Psoraleae is presented by high-performance liquid chromatography (HPLC) coupled with different detections.Extracts of Fructus Psoraleae were examined by HPLC with ion trap mass spectrometry (IT-MS) and 18 major compounds of coumarins,benzofuran glycosides,flavonoids,and meroterpene were identified.The determination of four major constituents including bavachin,isobavachalcone,bavachinin,and bakuchiol was accomplished by HPLC with UV,MS,and electrochemical detection (ECD).These methods were evaluated for a number of validation characteristics (repeatability,LOD,calibration range,and recovery).ECD obtained a high sensitivity for analysis of the four components; MS provided a high selectivity and sensitivity for determination of bavachin,isobavachalcone,and bavachinin in negative-ion mode.After optimization of the methods,separation,identification.and quantification of the four components in Fructus Psoraleae were comprehensively tested by HPLC with UV,MS,and ECD.
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Relying on unique wild Chinese crude drug, animal and plant resources of Wudang Mountain and Shennongjia, biological pharmacy technology and talent advantages, Shiyan area has a definite competitive advantage in the development of biomedicine industry. This paper analyzed the current situation of biomedical industry and put forward corresponding strategies in Shiyan area based on SWOT.
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Objective To discuss the health education modes in order to promote the effect of health education for TURP(transurethral resection of prostate)patients and their family members.Methods A total of 380 cases after TURP were divided into the observation group and the control group at random with 190 cases in each group.The discharge instruction for the patients and their family members in the observation group was in the form of health education prescription,while the discharge instruction for the control group was in the form of oral instruction.The awareness rate of disease-related knowledge and incidence of complication were compared between the two groups. Results The awareness rate of disease-related knowledge in the observation group was higher,and incidence of complications was lower than that of the control group.Conclusions The application of health education prescription in the discharge instruction for the patients after TURP can guarantee the effectiveness of health education,simultaneously decrease the incurrence of complications and improve the life quality of patients.
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OBJECTIVE:To establish a HPLC method for the determination of yohimbine in corynanthe yohimbe K.schum.METHODS:The analysis was performed on C18 column with column under room temperature.The mobile phase consisted of methanol-phosphoric acid buffer-triethylamine(30∶ 70∶ 0.5,pH5.0)with a flow rate of 1.0mL? min-1.The wavelength was set at 270nm.RESULTS:There was a good linear relationship when the sample size of yohimbine was over a range of 10～ 1 000? g? mL-1(r=0.999 8),with recovery at 96.5% and precision at 1.23%.CONCLUSION:This method was simple and reliable,and suitable for the determination of content of yohimbine.
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OBJECTIVE:To analysis and identify the liposoluble constituents extracted with supercritical CO 2 fluid ex-traction(SFE)from atractylodes macrocephala by gas chromatography-mass spectrometry(GC/MS).METHODS:Volatile oil was extracted from atractylodes macrocephala,to analsis and identify the chemical components in liposoluble constituents with GC/MS,DB-5MS capillary column was the chromatographic column and helium was carrier gas,the flow rate was 2.0ml/min,and the column temperature was raised from 70℃ to 250℃ at an increasing rate of 10℃/min.EI ion sources were adopted in mass spectra.RESULTS:A total of 19 chromatographic peaks were separated and identified,most of which were unsatu-rated fatty acids and esters,accounting for 96.2% of the total peak area.CONCLUSION:Using GC/MS,the chemical com-position of the liposoluble constituents in atractylodes macrocephala can be analyzed,which provides references for the further exploitation and utilization of atractylodes macrocephala.