2.Construction of exogenous recombinant eukaryotic expression vector pcDNA3.1-hBMP-7 and transfection into rabbit bone marrow stromal cells
Zhen XUE ; Songcen Lü ; Liyuan NIU ; Jindong ZHAO ; Yashan GUO ; Gang AN ; Ying WU
Chinese Journal of Tissue Engineering Research 2009;13(40):7985-7990
BACKGROUND:Under the in vitro conditions of cell harvesting, culture, and transplantation, whether bone marrow stromal cells (BMSCs) can be effectively applied in local gene therapy remains unclear.OBJECTIVE: To construct a recombinant eukaryotic expression plasmid carrying human bone morphogenetic protein-7 (hBMP-7) gene, and to expect to enhance osteoinductive properties of rabbit BMSCs transfected.DESIGN, TIME AND SETTING: A cell-genomics in vitro observation was performed at the Laboratory of Scientific Research, Second Affiliated Hospital of Harbin Medical University between July 2006 and July 2007.MATERIALS: Human healthy fresh placental tissue was provided by the Department of Gynaecology and Obstetrics, Second Affiliated Hospital of Harbin Medical University. Written informed consent was obtained from the women. One healthy male New Zealand rabbit was provided by the Laboratory Animal Center, Harbin Medical University.METHODS: hBMP-7 gene was cloned from human placental tissue to construct a recombinant eukaryotic expression plasmid carrying hBMP-7 gene by conjugating with eukaryotic expression vector pcDNA3.1. BMSCs were isolated from rabbit bone marrow and cultured in vitro. Then they were divided into 3 groups: pcDNA3.1-hBMP-7-transfected, pcDNA3.1 -transfected, and untransfected. 5×106 BMSCs were inoculated into a 60 mm3 flask containing antibiotic-free medium 1 day prior to transfection.MAIN OUTCOME MEASURES: RT-PCR and immunohistochemistry were employed to detect hBMP-7 expression in BMSCs, alkaline phosphatase activity, hydroxypreline content, and osteocalcin production in each group. RESULTS: After 72-hour transfection, a 1.3 kb fragment was seen in the pcDNA3.1-hBMP-7-transfected group, showing brown granules in the endochylema, but not seen in the pcDNA3.14ransfected and untransfected groups. ALP activity in the pcDNA3.1-hBMP-7-transfected group significantly increased at 2 days after transfection, peeked at 8 days, and still increased at 10 days. At each time point, alkaline phosphatase activity, hydroxyproline content, and osteocalcin production were significantly higher in the pcDNA3.1-hBMP-7-transfected group than in the pcDNA3.1 -transfected and untransfected groups (P<0.05 or P<0.01).CONCLUSION: Recombinant eukaryotic expression vector pcDNA3.1- BMP-7 was constructed successfully. Results indicated that hBMP-7 was expressed in BMSCs sufficiently and was involved in inducing differentiation of BMSCs into osteoblasts. The method would provide substantial basement for hBMP-7 gene therapy.
3.Effects of calcitonin gene-related peptide on repolarization process in isolated guinea-pig atrial myocardium at the physiological temperature.
Rong XU ; Bei-Ying LIU ; Wei-Zhen NIU
Acta Physiologica Sinica 2002;54(2):154-158
The purpose of this study was to investigate the effects of calcitonin gene-related peptide (CGRP) on the repolarization process in isolated guinea-pig atrial cells and to determine the contribution of K(+) channels to the CGRP-induced changes in action potential using conventional microelectrode method at the physiological temperature. We found that: (1) CGRP (16 nmol/L) antagonized the influences of potassium channel blockers, 4-AP and BaCl2, on action potential; (2) CGRP (16 nmol/L) increased the amplitude and maximum depolarizing velocity of slow action potential and shortened the conducting time in guinea pig atrial myocardium at extracellular K(+) concentration of 18.5 mmol/L; (3) CGRP (16 nmol/L) alleviated triggered activity induced by superfusion with solution containing CsCl and no potassium ion; and (4) the effects of CGRP on the configuration of action potential were temperature-dependent. At the temperature of 36.5+/-0.5 degrees C, CGRP (5, 16, and 50 nmol/L) increased the amplitude of the action potential and shortened APD(20), APD(50) and APD(90). The CGRP effects on APD(20) and APD(50) were dose-dependent and reversible. On the contrary, CGRP prolonged APD(20), APD(50) and APD(90) at the temperature of 25.5+/-2.1 degrees C. The present study suggests that CGRP possesses multiple effects on various ionic channels. Among them the effects on potassium currents are major determinants in the changes in action potential induced by CGRP under physiological temperature. It is necessary to further study the influences of CGRP on different types of potassium channels.
Action Potentials
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drug effects
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physiology
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Animals
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Body Temperature
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Calcitonin Gene-Related Peptide
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pharmacology
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Cells, Cultured
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Female
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Guinea Pigs
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Heart Atria
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cytology
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drug effects
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Male
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Potassium Channels
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physiology
4.A comparison of calcitonin gene-related peptide effects on coronary flow and cardiac conduction system in the guinea pig
Wei-Zhen NIU ; Yan-Li GAO ; Ping LIU ; Bei-Ying LIU ; Gang YE
Acta Physiologica Sinica 2000;52(3):259-262
The purpose of this study was to examine the effects of calcitonin gene-related peptide (CGRP) on coronary flow and various parts of cardiac conduction system (CCS) in the isolated guinea pig heart by recording the cardiac electrogram and His bundle activity simultaneously. The coronary flow (CF) was measured and the right atrium was paced at incremental frequencies periodically. Perfusing heart with CGRP at the concentrations of 3~30 nmol/L increased CF strikingly and dose-dependently. CGRP (30 nmol/L) caused a moderate and significant increase in the spontaneous heart rate, but failed to influence the atrioventricular nodal and His bundle-Purkinje fiber conduction time and the longest atrial pacing cycle length inducing 3∶2 Wenckebach and 2∶1 AV nodal conduction. The results indicate that CGRP in the guinea pig heart might act mainly on coronary vessels and then the sinus node, but did not affect the conductivity and refractoriness of the AV conduction system.
5.Applicability Study on QAMS for Multi-component Content Determination of Lonicerae Japonicae Caulis
Wei TIAN ; Ya-Qin ZHEN ; Xin-Guo WANG ; Yu-Rou TIAN ; Jun-Shan LI ; Li-Ying NIU
Chinese Journal of Information on Traditional Chinese Medicine 2018;25(11):77-82
Objective To establish a QAMS method for content determination of six compositions (chlorogenic acid, caffeic acid, cryptochlorogenin acid, isochlorogenic acid A, isochlorogenic acid C and loganin) from Lonicerae Japonicae Caulis; To verify the feasibility and applicability of this method in quality control of Lonicerae Japonicae Caulis. Methods Chlorogenic acid was set as internal reference substance. The HPLC analysis was performed on a Waters Symmetry C18 column (4.6 mm × 250 mm, 5 μm) with a mobile phase consisted of acetonitrile and 0.4% phosphoric acid solution in gradient elution manner at a flow rate of 1 mL/min. The column temperature was maintained at 35 ℃, and the detection wavelength was set at 327 nm for chlorogenic acid, caffeic acid, cryptochlorogenin acid, isochlorogenic acid A, isochlorogenic acid C and 236 nm for loganin. Results The relative correction factors of caffeic acid, cryptochlorogenin acid, isochlorogenic acid A, isochlorogenic acid C and loganin were established; there was no obvious difference between calculated value of QAMS and measured value of external standard method. Conclusion The quality control mode of QAMS can be used for multi-index synchronization quality evaluation of the six compositions from Lonicerae Japonicae Caulis.
6.Advance in Rehabilitation Effects for Individualized Exercise Prescription on Cardiovascular Disease (review)
Shuang ZHANG ; Ying CHEN ; Xi WANG ; Zhen YU ; Li-Niu PAN ; Wei-Hong ZHANG
Chinese Journal of Rehabilitation Theory and Practice 2019;25(1):60-63
Currently, individualized exercise prescription plays a vital role in the cardiac rehabilitation of patients with chronic cardiovascular diseases. Many cardiopulmonary exercise tests proved that individualized exercise prescription can lower blood pressure and glucose of patients with cardiovascular diseases, improve cardiopulmonary function, and improve exercise endurance and quality of life. At the same time, this paper also summarized that the individualized exercise prescription should be formulated in compliance with the principle (individuality, effectiveness, safety, professionalization, comprehensiveness and permanence), exercise intensity evaluation method (from previous heart rate, fatigue grading methods into cardiopulmonary exercise test) and the contents of the individualized exercise prescription (with a focus on the exercise intensity formulation).
7.Vertebral bone marrow infiltration of acute leukemia: diffusion-weighted imaging study
Jinliang NIU ; Congcong LIANG ; Junfeng LI ; Jun WANG ; Wenjin LI ; Ying ZHENG ; Jie ZHENG ; Zhizhen SONG ; Xiaojun LI ; Yi XU ; Junping ZHEN
Chinese Journal of Radiology 2011;45(9):807-811
Objective Using single direction dispersion breathless DWI, to analyze the value of DWI for vertebral bone marrow infiltration in patients with acute leukemia (AL). MethodsForty-two patients with AL and 15 healthy volunteers received vertebral sagittal DWI with single shot spin-echo echoplan imaging (SS-SE-EPI) sequence( b value = 0,650 s/mm2) at a GE Signa Excite 1. 5 T scanner with 8 channels body coil. DWI for all patients were performed from three directions, including from superior to inferior (S/I), from anterior to posterior (A/P) and from right to left (R/L). The apparent diffusion coefficient (ADC) value was measured on ADC map from each direction using GE-Function tool DWI software. Forty two patients consisted of 13 onset with untreated patients and 29 treated patients (7 nonremission,8 complete remission and 14 consolidation therapy). The ADC values among the three diffusion directions were compared. Analysis of variance and t test were used to compare the ADC values in different AL stages, Pearson correlation analysis was used to analyze the correlation between ADC values and the percentage of bone marrow progenitor cells. Results The ADC values from S/I, A/P and R/L of 362 vertebras in the 57 subjects are (0. 758 ±0. 009) × 10-3 mm2/s, (0. 732 ±0. 009) × 10 -3 mm2/s and (0. 758 ±0. 009) × 10 -3 mm2/s, respectively. There is no statistical significance( F = 2. 958, P > 0. 05 ).The ADC values from S/I of 94 vertebras in 15 healthy volunteers is (0. 697 ± 0. 122) × 10 -3 mm2/s, of 85 vertebras in 13 untreated AL patients is (0. 592 ±0. 071 ) × 10-3mm2/s. There is statistical significance between them ( t = 2. 568, P < 0. 05 ) ; The ADC value of 183 vertebras in 29 treated AL patients [ ( 0. 796 ±0. 225 ) × 10-3mm2/s]is higher than that in untreated patients with statistical significance (t = -1. 332,P <0. 05). One hundred and forty vertebras in patients with complete remission and consolidation therapy were [ (0. 786 ±0. 184) × 10-3 mm2/s],and 43 vertebras in patients with non-remission(NR) [ (0. 804 ±0. 327 ) × 10 - 3 mm2/s], there was not statistical significance between them ( t = - 0. 160, P > 0. 05 ). The ADC values from S/I direction of untreated patients showed significant negative correlation with the proportion of the blast cell in the bone marrow ( median value 26. 4%. Min 7.9%, Max 48. 2% ) ( r =- 0. 524, P < 0. 05 ). ConclusionsDWI of vertebral bone marrow is isotropy. ADC value is a non-invasive and quantitative index for evaluating the pathogenetic condition of AL.
8.Rapid quantification of periodontal bacteria using an impedic immunosensor
Zhen-Hua PEI ; Zhong-Ying NIU ; Sheng-Gen SHI ; Chu-Hua TANG
Chinese Journal of Stomatology 2013;48(z1):87-90
Objective To develop an electrochemical impedance immunosensor based on polypyrrole modified microelectrodes for periodontal bacteria rapid quantification.Methods Mcirofluidic chip with embedded IDAM was prepared by conventional photolithography process.Then polypyrrole structure was deposited on microelectrodes through electropolymerization method.Polypyrrole was biofuncationalized with mouse anti-Porphyromonas gingivalis gingipain K IgG antibody using N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide-hydrochloride (EDC) chemistry.The linear relationship between the impedance and bacterial concentration,the impedance characteristic and the feasibility of the developed impedic immunosensor in quantifing Porphyromonas gingivalis was investigated respectively.Results The polypyrrole membrane structure was observed on work electrodes,and the lowest detection limit of the immunosensor was 107 cells/L in pure culture.In the concentration range of 107-1012 cells/L,a linear relationship was found between the normalized impedance change and the logarithmic value of the cell concentration.The total detection time from sampling to measurement was less than 1 h.Conclusions The immunosensor developed in the present study offered some insight into chair-side quantifing periodontal bacteria.
9.Comparison of exosome extracting methods from human umbilical cord mesenchymal stem cells
Ying GUO ; Xiu-Wei WANG ; Yu-Hu NIU ; Li WANG ; Nan ZHOU ; Bai-Yi LI ; Zhen-Dong WANG ; Pin ZHANG ; Ya-Jie GAO ; Bo NIU
Chinese Journal of Tissue Engineering Research 2018;22(9):1382-1388
BACKGROUND: Cell-free stem cell therapy has been an issue of concern, but there is no conclusion on how to extract high-quality exosomes. OBJECTIVE: To extract exosomes from human umbilical cord mesenchymal stem cells by using three different methods, and then to screen the optimal method. METHODS: Exosomes were extracted from human umbilical cord mesenchymal stem cells by using the Total Exosome Isolation test kit, Exo Quick test kit and differential ultracentrifugation method, respectively. Then, transmission electron microscopy was used for morphological observations, BCA was utilized to quantify the protein, and western blot assay was applied to detect surface markers CD9, CD81 and CD63. RESULTS AND CONCLUSION: Extraction of exosomes was completed by all the three methods, and round or oval membranous vesicles were observed under the transmission electron microscope. The protein content and purity of exosomes was highest in the differential ultracentrifugation group, followed by the Exobiology Quick kit group, and lowest in the Total Exosome Isolation kit group, and there were significant differences among the three groups (P < 0.05). Under the same protein concentration, surface specific markers, CD81, CD63 and CD9, were expressed highest in the differential ultracentrifugation group, followed by the Exobiology Quick kit group, and lowest in the Total Exosome Isolation kit group. The operating time was significantly lower in the Exobiology Quick kit group compared with the other two groups (P < 0.05). To conclude, despite a longer operating time, the differential ultracentrifugation method is a rational method to extract enough exosomes with relative high purity.
10.The study on relationship of body mass index and blood pressure in children and adolescents of Beijing.
Wen-juan WANG ; Ke-an WANG ; Chun-ming CHEN ; Ruo-xiang CAO ; Ya-min BAI ; Lin-mao MA ; Zhen-ying REN ; Zhen-hua NIU ; Quan GAO
Chinese Journal of Epidemiology 2004;25(2):109-112
OBJECTIVETo identify the relationship of body mass index (BMI) and blood pressure in 7 - 15 years children and adolescents of Beijing so as to provide scientific basis for early prevention of hypertension and to provide evidence for verification on the category criterion of overweight and obesity in children and adolescents of China, recommended by the Working Group on Obesity in China (WGOC) to sensitively distinguish the blood pressure in normal weight, overweight and obesity populations.
METHODSA cross-sectional survey on epidemiological characteristics of obesity with stratified cluster sampling method carried out in Beijing in April and May, 2000. 5155 students aged 6 - 15 years were selecte das research subjects. The category criterion of overweight and obesity in children and adolescents of China was recommended by WGOC, the diagnostic criterion of hypertension in children was recommended by CDC in the USA. Statistics analysis system (SAS 8.1) including partial person correlation analysis, t-test, chi(2) test and logistic multi-factors regression analysis was used to analyses the data from 4982 subjects aged 7 - 15 years.
RESULTS(1) after the age and gender were adjusted, the BMI positive correlation with systolic blood pressure (SBP) and diastolic blood pressure (DBP) was found independent in 7 - 15 years children and adolescents (P < 0.0001) and the partial relation coefficients(r) between BMI and SBP and DBP were 0.323 87 and 0.245 88 respectively. (2) the means of SBP and DBP in obesity group were significantly higher then overweight, while overweight was significantly higher then normal weight group (P < 0.0001). (3) the prevalence rates of hyper-SBP, hyper-DBP and hypertension were significantly different (P < 0.0001). When compared with the normal weight group, the relation risk (RR) for hypertension in overweight group and obesity group were 2.96 and 4.85 respectively. The prevalence rates of hypertension in overweight and obesity group were 19.70% and 24.22% respectively. (4) the results of logistic multi-factors regression analysis showed that both age and weight were effecting on hyper-SBP, hyper-DBP and hypertension (P < 0.0001). After age was adjusted, the RR for hypertension was 2.62, and their confidence interval (CI) was 2.36 - 2.91 in obesity or overweight, between overweight and normal weight.
CONCLUSION(1) the BMI positive correlation with SBP and DBP was found independent in 7 - 15 years children and adolescents of Beijing, and the risk for hypertension maybe increased when these people with overweight and obesity, it is very important for hypertension prevention and control that overweight and obesity prevention and control in children and adolescents. (2) the sensitivity of the category criterion of overweight and obesity in children and adolescents of China, recommended by WGOC have been verified on distinguish the blood pressure in normal weight, overweight and obesity populations.
Adolescent ; Age Factors ; Blood Pressure ; Body Mass Index ; Child ; China ; Cross-Sectional Studies ; Female ; Humans ; Hypertension ; etiology ; Logistic Models ; Male ; Risk Factors ; Sex Factors