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1.
目的:建立免疫磁珠法,分离纯化小鼠肝脏Kupffer细胞并进行功能鉴定。方法:采用原位肝脏酶灌注、制备肝非实质细胞悬液、加入PE标记的大鼠抗小鼠F4/80一抗,然后结合山羊抗大鼠IgG Dynabeads免疫磁珠分选Kupffer细胞,流式细胞仪检测其纯度并检测其吞噬功能。结果:在经过胶原酶Ⅳ型消化、免疫磁分选,获得小鼠肝Kupffer细胞产量约为(8.05±0.34)×106/鼠肝,细胞纯度为(96.6±0.7)%,细胞活力大于90%。结论:应用免疫磁珠法能有效地分离纯化小鼠肝脏Kupffer细胞,为体外进一步研究提供了高纯度和活力的Kupffer细胞群。  相似文献   

2.
目的 建立稳定高效分离SD大鼠肝细胞与Kupffer细胞的实验流程,研究SD大鼠Kupffer细胞与肝实质细胞共同培养Kupffer细胞对肝实质细胞生长、形态功能的影响,为体外原代培养肝细胞的应用及进行相关研究提供参考数据.方法 (1)原位2步Ⅳ型胶原灌注法消化SD大鼠的肝脏;(2)低速离心法分离肝实质细胞;(3)Percoll液密度梯度离心法分离肝Kupffer细胞;(4)单独培养肝实质细胞;以61比例贴壁共培养肝实质细胞与Kupffer细胞;(5)光镜分别观察单独培养与共同培养情况下肝实质细胞的生存和形态;(6)全自动生化仪检测单独与共同培养情况下培养上清白蛋白和糖水平并进行比较.结果 每只大鼠可分离的肝细胞产量平均为(2.03±0.26)×108,细胞活力为88%~95%,平均(91.7±2.11)%.Kupffer细胞产量平均为(3.19±0.20)×107,纯度为85%~92%.平均(88.5±1.83)%.192 h原代贴壁培养观察,单独培养组肝细胞的生长、增殖迅速,并向正常肝细胞的形态演变.2周后细胞开始死亡,培养至21 d时细胞全部死亡.结论 (1)通过本实验研究成功地建立了以肝下腔静脉为流人道的Ⅳ型胶原酶二步灌流法分离肝实质细胞以及联合Pereoll液密度梯度离心法分离Kupffer细胞的实验室操作程序.该操作程序稳定高效实用.(2)按61的比例在体外进行肝实质细胞与Kupffer细胞192 h贴壁共同培养实验,实验结果提示,肝实质细胞和Kuffer细胞在适宜的培养条件下可进行共培养;这两种细胞简单的同步贴壁共同培养,肝细胞的生长、白蛋白合成功能和糖代谢会受到影响,提示在共培养体系中,要实现肝实质细胞租Kupffer细胞良好生长和两者功能的协调发挥,尚需进一步建立更适宜的共同培养系统.  相似文献   

3.
目的 建立稳定高效分离SD大鼠肝细胞与Kupffer细胞的实验流程,研究SD大鼠Kupffer细胞与肝实质细胞共同培养Kupffer细胞对肝实质细胞生长、形态功能的影响,为体外原代培养肝细胞的应用及进行相关研究提供参考数据.方法 (1)原位2步Ⅳ型胶原灌注法消化SD大鼠的肝脏;(2)低速离心法分离肝实质细胞;(3)Percoll液密度梯度离心法分离肝Kupffer细胞;(4)单独培养肝实质细胞;以6:1比例贴壁共培养肝实质细胞与Kupffer细胞;(5)光镜分别观察单独培养与共同培养情况下肝实质细胞的生存和形态;(6)全自动生化仪检测单独与共同培养情况下培养上清白蛋白和糖水平并进行比较.结果 每只大鼠可分离的肝细胞产量平均为(2.03±0.26)×108,细胞活力为88%~95%,平均(91.7±2.11)%.Kupffer细胞产量平均为(3.19±0.20)×107,纯度为85%~92%.平均(88.5±1.83)%.192 h原代贴壁培养观察,单独培养组肝细胞的生长、增殖迅速,并向正常肝细胞的形态演变.2周后细胞开始死亡,培养至21 d时细胞全部死亡.结论 (1)通过本实验研究成功地建立了以肝下腔静脉为流人道的Ⅳ型胶原酶二步灌流法分离肝实质细胞以及联合Pereoll液密度梯度离心法分离Kupffer细胞的实验室操作程序.该操作程序稳定高效实用.(2)按6:1的比例在体外进行肝实质细胞与Kupffer细胞192 h贴壁共同培养实验,实验结果提示,肝实质细胞和Kuffer细胞在适宜的培养条件下可进行共培养;这两种细胞简单的同步贴壁共同培养,肝细胞的生长、白蛋白合成功能和糖代谢会受到影响,提示在共培养体系中,要实现肝实质细胞租Kupffer细胞良好生长和两者功能的协调发挥,尚需进一步建立更适宜的共同培养系统.  相似文献   

4.
大鼠肝星形细胞、枯否细胞的同步分离培养与鉴定   总被引:1,自引:0,他引:1  
目的:建立经济、简便的同步分离培养肝星形细胞及枯否细胞的方法。方法:应用胶原酶和蛋白酶对大鼠肝脏进行原位灌流、消化,获得大鼠肝脏非实质细胞,经18%(W/V)的Nycodenz密度梯度离心,一步分离肝星形细胞及枯否细胞;肝星形细胞处在界面,枯否细胞则处在界面下;台盼蓝染色鉴定细胞活力;Desmin免疫细胞化学染色鉴定肝星形细胞;溶菌酶免疫细胞化学染色鉴定枯否细胞。结果:该方法分离的肝星形细胞和枯否细胞的纯度达95%左右。肝星形细胞得率(3.0±0.5)×107/肝,枯否细胞得率(4.0±0.5)×106/肝。结论:应用Nycodenz一步密度梯度离心法可以很好的同时分离肝星形细胞和枯否细胞。  相似文献   

5.
目的 :分离培养大鼠肝星形细胞 ,为深入研究肝纤维化的发生机制奠定基础。方法 :采用IV型胶原酶、链酶蛋白酶消化肝脏 ,密度梯度离心分离HSC ,台盼蓝染色排斥法鉴定细胞活率 ,Desmin免疫细胞化学法鉴定HSC纯度。结果 :每只成年大鼠肝脏的HSC得率为 (2 33± 0 1 5)× 1 0 7个 ,细胞存活率在 95 %以上。免疫细胞化学显示 ,desmin阳性细胞为 (90 3± 2 8) % ,传代培养后阳性细胞在 98%以上。结论 :建立了稳定、高产的HSC分离方法 ,细胞得率、活率、纯度方面达到了国内外文献报道的水平  相似文献   

6.
张晓勇  周春祥 《安徽医学》2014,(10):1437-1439
目的探讨青蒿琥酯对大鼠肝巨噬(Kupffer)细胞分泌致炎细胞因子的影响。方法 CCl4皮下注射SD大鼠进行肝纤维化造模,Percoll密度梯度离心法分离纯化大鼠肝Kupffer细胞,ELISA检测不同浓度青蒿琥酯处理LPS刺激的大鼠肝Kupffer细胞分泌致炎细胞因子TNF-α、TGF-β1和IL-6的变化。结果造模12周后,模型组大鼠出现明显的肝纤维化,Percoll密度梯度离心纯化后的大鼠肝Kupffer细胞在LPS刺激后TNF-α、TGF-β1和IL-6的表达量在模型组较对照组显著增加(P<0.05)。0μmol/L、125μmol/L、175μmol/L、225μmol/L 4个浓度的青蒿琥酯处理后,模型组较对照组致炎细胞因子TNF-α、TGF-β1和IL-6的表达量显著下调,并且175μmol/L为最佳抑制浓度(P<0.05)。结论青蒿琥酯可以抑制大鼠肝Kupffer细胞分泌致炎细胞因子  相似文献   

7.
目的建立一种稳定、高效、同时分离非酒精性脂肪性肝炎(nonalcohlic steatohepatitis,NASH)大鼠肝细胞(hepatocytes,HCs)和库普弗细胞(Kupffer cells,KCs)的新方法。方法采用离体循环灌注Ⅳ型胶原酶,低速离心获取HCs;差速离心、密度梯度离心和选择性贴壁获得KCs。用Typan blue染色和流式细胞术(FCM)分别进行活性和纯度鉴定。结果每只大鼠获得纯化的HCs[(4.0~4.5)×108]、KCs[(1.5~2.0)×107],经Typanblue染色活力均在95%以上,FCM检测纯度均在90%以上。结论此实验建立同时分离NASH大鼠HCs和KCs的方法高效、稳定,细胞数量和纯度都符合进一步实验的要求。  相似文献   

8.
成年大鼠胰岛分离纯化的实验研究   总被引:2,自引:0,他引:2       下载免费PDF全文
为探讨成年大鼠胰岛分离、纯化的方法 ,为成人胰岛细胞的分离纯化奠定基础 ,采用胰管内注射胶原酶水浴孵化以及不连续密度梯度法进行成年Lewis大鼠胰岛分离纯化 ,并对获得的胰岛细胞通过体外葡萄糖刺激胰岛素分泌实验进行功能活性检测。分离后获得 (93 8± 2 1 8)个胰岛细胞 ,染色观察胰岛细胞形态完好。纯化后获得 (80 2±1 81 )个胰岛细胞 ,平均纯度为 (68± 1 1 ) % ,纯化后细胞回收率为 (85 .9± 5 .9) % ,纯化后细胞形态完好。体外葡萄糖刺激胰岛素分泌实验 ,低糖情况下胰岛素分泌量为 (2 .5 3± 0 .1 8)mIU/L ,高糖情况下为 (5 .41± 0 .3 1 )mIU/L ,两者相比有极显著性差异 (P =0 .0 0 1 )。低糖刺激下C肽分泌量为 (7.5 1± 1 .0 9)ng/L ,高糖情况下为 (8.5 6± 1 .0 6)ng/L ,具有极显著性差异 (P =0 .0 0 6)。提示 :采用胰管内胶原酶灌注水浴孵化进行成年大鼠胰岛分离及不连续密度梯度法纯化可获得较多的〔(80 2± 1 81 )个〕形态及功能完好的胰岛细胞 ,为临床进行成人胰岛分离纯化打下了基础  相似文献   

9.
目的:对大鼠Kupffer细胞分离方法进行优化。方法:采用D-Hanks液和Ⅳ型胶原酶液两步原位灌注法结合离体消化,差速离心法和Percoll分离液密度梯度离心法分离并选择性贴壁纯化Kupffer细胞。采用328 nm波长自发荧光法、CD68和CD163免疫荧光染色法及吞噬实验鉴定Kupffer细胞。结果:Kupffer细胞获得量为9.8×10~6个/鼠肝,其中活细胞率占93%以上,证实细胞为Kupffer细胞且吞噬功能正常。结论:该方法花费时间短,相对简便、经济,且细胞数量、功能活性和纯度等均达到实验要求。  相似文献   

10.
目的枯否细胞(Kupffer cells,KC)是肝内巨噬细胞群,活化的KC参与多种生物、病理学反应过程。然而,KC分离、培养技术进展缓慢。为研究KC致炎、抗炎活化机制,旨在建立经济、简便、有效、稳定分离培养SD大鼠肝KC的方法。方法选用健康雄性SD大鼠,采用0.05%IV型胶原酶灌注肝,经25%和50%percoll密度梯度离心,提取肝KC。荧光显微镜下观察细胞自发荧光情况,锥虫蓝拒染实验鉴定细胞活力,倒置显微镜下观察KC培养过程中细胞形态变化,吞噬墨水、latex珠实验以及免疫细胞化学染色法鉴定细胞纯度。结果大鼠肝KC得率为(3.22±0.31)×107(n=24),细胞活力为(92.35±0.13)%,细胞纯度均在(94.62±0.24)%。结论该方法简单、可行,为进一步研究KC功能和作用机制奠定基础。  相似文献   

11.
Objective To observe blood pressure change with age in salt-sensitive teenagers whose salt sensitivity were determined by repeated testing.Methods Salt sensitivity was determined through intravenous infusion of normal saline combined with volume-depletion by oral diuretic furosemide in 55 teenagers. After five years, salt sensitivity was re-examined and subject blood pressure was followed up. Blood pressure changes in salt-sensitive teenagers were compared to that of non-salt sensitive teenagers over five years.Results After 5 years, the repetition rate of salt sensitivity determined by intravenous saline loading is 92.7%. In teenagers with salt sensitivity on the baseline, both the systolic blood pressure increments and increment rates were much higher than non-salt sensitive teenagers (12.7±12.1 mmHg vs. 2.8±5.2 mmHg, P< 0.01; 12.2%± 12.0% vs. 2.5% ±4.4%, P< 0.001,respectively). There was a similar trend for diastolic blood pressure (8.4 ± 6.4 mmHg vs. 3.7 ± 6.4 mmHg, P = 0.052; 13.2% ±10.6 % vs. 6.8%± 10.1%, P = 0.053, respectively).Conclusions Salt sensitivity determined by intravenous saline loading showed good reproducibility. Blood pressure increments with age were much higher in salt-sensitive teenagers than non-salt sensitive teenagers, especially in terms of systolic blood pressure.  相似文献   

12.
Objective: To evaluatel the value of D-dimers in patients with acute aortic dissection (AAD). Methods: This study consisted of 16 patients with AAD and 27 non-AAD patients. Serum D-dimets were measured by Sta-Liatest D-DI immunoturbidimetric assay. Results: D-dimer level was higher (P < 0.001) in patients with AAD(7.91 ± 5.52 μg/ml) than that in non- AAD group(1.57±1.24 μg/ml). D-dimer was positive (>0.4 μg/ml) in all patients with AAD and in 10 control group patients (37%). Among patients with acute AAD, D-dimers tended to be higher in Stanford A than in Stanford B (8.67 ± 4.31 μg/ml vs. 3.24±1.27 μg/ml, P <0.01). D-dimer values tended to be higher in more extended disease(3.84 ± 1.65 μg/ml, 8.57 ± 3.58 μg/ml and 11.87 ± 5.69 μg/ml in thoracic aorta, thoracic and abdominal aorta, thoracic and abdominal aorta and iliacal arteries, respectively, P < 0.05 for both 8.57 ± 3.58 and 11.87 ± 5.69 vs. 3.84 ± 1.65 ). Including the control group into the analysis, we found a sensitivity of 100%, a negative predictive value of 100%, and a specificity of 66% and a positive predictive value of 64% for D-dimer in diagnosis of AAD in our patients with suspected AAD. Conclusion: D-dimer was elevated in patients with AAD. A negative D-dimer test result could be useful in excluding AAD.  相似文献   

13.
Objective: To set up a simple and reliable rat model of combined liver-kidney transplantation. Methods: SD rats served as both donors and recipients. 4℃ sodium lactate Ringer's was infused from portal veins to donated livers,and from abdominal aorta to donated kidneys, respectively. Anastomosis of the portal vein and the inferior vena cava (IVC) inferior to the right kidney between the graft and the recipient was performed by a double cuff method, then the superior hepatic vena cava with suture. A patch of donated renal artery was anastomosed to the recipient abdominal aorta. The urethra and bile duct were reconstructed with a simple inside bracket. Results: Among 65 cases of combined liver-kidney transplantation, the success rate in the late 40 cases was 77.5%. The function of the grafted liver and kidney remained normal. Conclusion: This rat model of combined liver-kidney transplantation can be established in common laboratory conditions with high success rate and meet the needs of renal transplantation experiment.  相似文献   

14.
Increasing maternal age is the only etiological factor unequivocally linked to Down's syndrome in humans. The occurrence rate of newborns with Down's syndrome is about 1/220 in women over 35 years old. However, the occurrence rate in embryos fertilized in vitro, of the elder woman is unclear. Using FISH we screened the number of chromosome 21 in preimplanted embryos of 5 elderly women (average age, 38.4 years) to study the feasibility and necessity of screening trisomy 21 in embryos in patients over 35 years old at the in vitro fertilization (IVF) center.  相似文献   

15.
Real-time three-dimensional echocardiography (RT3DE)is a new ultrasound technique that enables dynamic threedimensional visualization and quantification of the heart in real time. Investigation of feasibility and methodology of RT3DE in determining left ventricular (LV) and right ventricular (RV) volumes, RT3DE was performed in 35 normal adults using Philips SONOS 7500 system with a 2-4 MHz matrix array transducer. The 60°×60° "pyramid" volume database was obtained and analyzed on a TomTec echo workstation. Both LV and RV volumes were calculated with four 3DE methods (i.e. apical 2, 4, 8, and 16-plane) through manually tracing ventricular endocardial borders in end diastole and end systole. Stroke volumes were then calculated. LV volume was also measured by 2DE Simpson's rule using GE VIVID 7 ultrasound machine.  相似文献   

16.
A clinical guideline for the therapeutic interventions of integrative medicine may be defined as a written document which states a series of recommendations on therapeutic interventions of integrative medicine for a special disease or condition. The guideline may provide assistance to medical professionals in making clinical decisions aimed at improving the clinical outcome of patients and reducing the costs of medical care(~'4~. Recommendations issued by a guideline should be based on the best available evidence in both Western and Chinese medicine. For fulfilling this purpose, the development of clinical guidelines for therapeutic interventions in the field of integrative medicine should follow scientific principles and undergo a rigorous processes.  相似文献   

17.
Objective:To probe into the influence of changes of ovarian hormones on the pathogenesis of the specific sub-type premenstrual syndrome(PMS)and reveal partial microcosmic mechanisms of adverse flow of liver-qi.Methods:Estradiol(E2)and progesterone(P)levels in serum were determined at different phases of menstrual cycle by radioimmunoassay.Results:In the group of PMS with adverse flow of liver-qi.the secretive peak value Of E2 and P at the follicular phase significantly decreased,and the secretive peak value at the luteal phase did not come into being.Conclusions:Low E2 and P secretive peak at the follicular phase and absence of secretive peak at the luteal phase is one of the microcosmic mechanisms of PMS with adverse flow of liver-qi.One of the pathophysiologic mechanisms of specific sub-type PMS is probably the continuous low level of E2and P.  相似文献   

18.
Journal of Nanjing Medical University (English Edition) JNMU, sponsored by Nanjing Medical University, was established in 1987. It is a bimonthly comprehensive English medical journal published locally and abroad.Since 2007, Journal of Nanjing Medical University (English Edition )was granted Elsevier the full publishing and distribution rights worldwide for the Electronic Edition, excluding the People's Republic of China.  相似文献   

19.
目的:评价使用安心颗粒对急诊经皮冠状动脉介入术(PPCI)术后生活质量的影响.方法:将160例接受PPCI的急性ST段抬高型心肌梗死患者随机分为安心颗粒组(术前顿服安心颗粒8.8g,术后安心颗粒4.4 g/次,每日2次)和对照组(仅接受基础药物治疗).所有患者均服用阿司匹林、氯吡格雷和阿托伐他汀.分别在入院时、出院前1d、出院后180 d时,应用心肌梗死多维度量表(MIDAS)、中文版SF-36评价量表对患者生活质量评分.并观察术后30 d以内的出血并发症、血小板减少症发生情况.结果:入院时和出院前1d,两组患者的心肌梗死MIDAS、SF-36量表评分比较无差异(P>0.05);出院后180 d时,与对照组比较,安心颗粒组MIDAS、SF-36评分明显减低(P<0.05);组内与入院时比较,两组出院前1d、出院后180 d时,MIDAS、SF-36评分均降低(P<0.05).两组患者在随访期间均无大量出血、少量出血、重度和极重度血小板减少症发生,安心颗粒组有4例、对照组有7例发生不明显出血(P>0.05).两组发生轻度血小板减少症的患者数比较无差异(P>0.05).结论:PPCI使用安心颗粒,能改善急性ST段抬高型心肌梗死患者的生活质量,且不增加出血风险.  相似文献   

20.
Objective:To investigate the influences of urapidil and nicardipine on rabbit sinus function,atrio-ventricular node function and hemodynamics.Methods:Thirty-two Angora's rabbits were selected and randomly divided into four groups.U1 group:urapidil 0.25 mg/kg;U2 group:urapidil 0.5 mg/kg;N1 group:nicardipine 10 μg/kg;N2 group:nicardipine 20 μg/kg.All these medicine were administrated within 30 seconds.Measurements were taken before and after the administration of urapidil or nicardipine for the following data:mean blood pressure(MAP),heart rate(HR),sino-atrial conduction time(SACT),maximal sinoatrial recovery time(SNRTmax)corrected sinus node recovery time(CSNRT),index of sinus node recovery time(SNRTI),Wenckebach A-V conduction frequency (WB),and P-R interval.Results:Significant MAP and HR changes were identified in all of the four groups before and after administration of both urapidil and nicardipine.No significant changes could be found in the rest of the parameters.Intergroup analysis showed that SACT and CSNRT of N1 and N2 groups were shorter than those of the U2 group(P<0.01);the MAP decreased(P<0.01)and the HR increased drastically(P<0.01).Conclusions:Neither urapidil(0.25 mg/kg,0.5 mg/kg)nor nicardipine(10μg/kg,20μg/kg)has any significant influence on rabbit sinus function or rabbit atrio-ventricular node function.Nicardipine could be a better choice than urapidil for parafunctional sinus node patients.  相似文献   

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