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1.
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   

2.
大鼠肝星状细胞的分离培养鉴定   总被引:1,自引:1,他引:1  
目的改良分离培养大鼠肝星状细胞的简便方法。方法经过门静脉插管、推注肝素稀释液、肝脏灌流、胶原酶离体循环消化、过滤、离心分离等步骤,分离肝星状细胞,省去了链霉蛋白酶E消化过程,采用台盼蓝染色排斥法鉴定细胞活率,desmin免疫细胞化学染色法鉴定HSCs纯度。结果每只鼠肝HSCs的得率为0.5~1.0×107,纯度为(92.2±2.0)%,活率为(97.8±0.3)%,数量及活率均符合实验要求。结论该方法简便、廉价、实用,值得进一步探索。  相似文献   

3.
目的:探讨一种简便、高产的大鼠肝星状细胞(HSC)分离方法,为研究肝细胞相关实验提供细胞原材料。方法:应用悬浮缓冲液校正Nycodenz密度梯度分离液,使其浓度为11.3%(w/v),采用单层一步梯度离心法分离HSC。采用台盼蓝染色排斥法鉴定细胞活率,desmin免疫细胞化学染色法鉴定HSCs纯度。结果:肝星状细胞得率每只大鼠约(4.0±0.5)×10^7个,细胞活力达到90%,细胞纯度达到95%。结论:本法简便、高产,是一种理想的分离肝星状细胞的方法,值得推广。  相似文献   

4.
大鼠肝星状细胞分离与培养方法的实验研究   总被引:2,自引:0,他引:2  
目的:探讨一种经济易行的分离与培养大鼠肝星状细胞的方法,为研究肝纤维化的发病机制提供良好条件。方法:采用离体循环酶液灌注法对成年大鼠的肝脏进行消化,经两种密度的Nycodenze密度梯度液高速离心(20000r/min)和细胞悬液低速离心(40×g)去杂质后得到肝星状细胞。台盼蓝拒染实验鉴定细胞活率,desmin和GFAP免疫细胞化学双抗单染色法鉴定肝星状细胞的纯度。结果:肝星状细胞的得率为(3.6±0.1)×10^7/肝,肝星状细胞存活率为(92.8±0.8)%,对培养24h的肝星状细胞行desmin和GFAP共同鉴定DAB显色,细胞阳性率为(96.2±0.5)%。结论:本改良法在保证大鼠肝星状细胞产量的前提下提高了细胞纯度,有利于对原代大鼠肝星状细胞及肝纤维化的进一步研究。  相似文献   

5.
目的建立一种经济、稳定可靠的HSC分离方法,为体外研究提供细胞模型.方法 Hanks液在体灌洗大鼠肝脏,离体后用Ⅳ胶原酶、链蛋白酶、DNaseI消化肝脏,12%Nycodenz连续梯度液分离大鼠HSC,计数细胞得率,0.2%台盼蓝染色计算细胞活率.自发荧光、Desmin、α-SMA免疫荧光染色对HSC进行鉴定和纯度分析.结果分离HSC得率(3.7±0.6)×107/只大鼠,细胞活率>90%,分离第1天自发荧光和第3天desmin染色阳性细胞>90%.HSC随着体外培养形态明显改变,分离培养7 d后α-SMA阳性细胞>90%,培养14 d或传代后>95%.结论肝脏离体后消化能达到在体消化同样的效果,应用Nycodenz密度分离介质可获得满意的HSC得率和纯度.  相似文献   

6.
目的建立一种可以同时分离大鼠肝星状细胞(HSC)和枯否细胞(KC)的方法。方法选用SD大鼠,经胶原酶和蛋白酶灌注肝脏后,用18%Nycodenz密度梯度离心,分离大鼠HSC和KC,HSC处在界面,KC处在界面下。常规台盼蓝染色行细胞活力鉴定;荧光显微镜下观察自发荧光确定HSC的纯度;KC免疫细胞化学检测。结果成功分离大鼠肝星状细胞和枯否细胞,HSC得率为2×107/肝,KC得率为(3~6)×106/肝,活性>90%,纯度>90%。结论该方法建立的HSC和KC同时分离培养的方法,简单、易行、可靠,细胞纯度高,同时获得的细胞可保持良好的生物学性状。  相似文献   

7.
分离肝星形细胞的简便方法   总被引:15,自引:4,他引:11  
目的 建立一种简便而可靠的肝星形细胞 (HSC)的分离方法。 方法 雄性 SD大鼠 ,常规门脉穿刺插管 ,蠕动泵灌注不含钙 D- Hanks液后游离肝脏 ,先后用含链霉蛋白酶 E、 型胶原酶消化液 37℃体外灌注消化和振荡消化 ,细胞悬液过滤后 ,经 1 1 % Nycodenz密度梯度离心 (2 730 r/ min× 1 5 m in)分离 HSC,锥虫蓝染色排斥法鉴定细胞活率 ,以 1× 1 0 6 m L- 1 密度混悬后接种在培养瓶。结蛋白免疫细胞化学染色鉴定 HSC纯度。 结果 成功分离 HSC,细胞得率 (1 .2~ 2 .3)× 1 0 6 g- 1 肝组织 ,活率 >90 % ,纯度 >90 % ,可用于实验研究。 结论 链霉蛋白酶E和 型胶原酶体外灌注消化及 1 1 % Nycodenz密度梯度离心是分离大鼠 HSC简便、可靠的方法  相似文献   

8.
目的:建立一种合理、经济、便捷的大鼠原代肝星状细胞(hepatic stellate cells,HSC)分离方法。方法:运用改良肝脏原位灌注D-Hanks后,改用Ⅳ型胶原酶和DNA酶Ⅰ离体灌注,10%、16%Optiprep密度梯度离心分离大鼠肝星状细胞,台盼蓝排斥试验鉴定细胞活力,α-SMA免疫细胞化学法鉴定细胞纯度。结果:我们采用Optiprep密度梯度离心法成功分离出大鼠HSC,且活力、纯度均达到研究要求,HSC得率约为2×107/肝,细胞存活率为95%以上,纯度为90%以上。结论:本方法分离大鼠HSC合理、经济、便捷。  相似文献   

9.
目的在肝脏二步酶灌注法分离大鼠肝星状细胞的基础上,探讨一种简单易行且结果稳定的分离方法。方法采用大鼠肝脏二步酶灌注法对成年大鼠的肝脏进行消化,经密度梯度离心去杂质后得到肝星状细胞。台盼蓝拒染实验鉴定细胞活率,结蛋白(Desmin)免疫荧光鉴定肝星状细胞的纯度。结果肝星状细胞的得率为4.0×107以上,肝星状细胞纯度为96%左右,肝星状细胞存活率为(95.0±1.2)%。结论本实验采用的大鼠肝星状细胞的分离方法,结果稳定,细胞纯度较高,有利于进一步的生物学研究。  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.
Objective To measure Derpl and Blot5 allergen levels in asthmatics' homes in Hongkong.Methods Seventy houses were enrolled for a mite indoor environment study. Dust samples were obtained from two sites of each patients' house: bed and floor. Derpl and Blot5 levels were quantified by a two-site monoclonal antibody-based ELISA technique.Results The levels of Derpl allergens found in bed (geometric mean (GM) 3.43 μg/g of dust; 95%CI, 1.89-4.96 μg/g)and on the floor (GM 1.12 μg/g of dust; 95%CI, 0.71-1.53 μg/g) indicated significant differences (P=0.005). However, the levels of Blot5 allergens found in bed (GM 19.00 μg/g of dust; 95%CI, 0.89-38.90 μg/g) and on the floor (GM 6.14 μg/g of dust; 95%CI, 0.40-11.90 μg/g) showed no statistically significant difference. In addition, in regards to the exposure index for Derpl and Blot5 allergens found in bed and on the floor, 17.6% in bed and 8.6% on the floor had levels of Blot5 ≥ 10 μg/g of dust, higher than those obtained for Derp1 (7.2% and 0% in bed and on the floor respectively, P< 0.05); higher percentages in bed and on the floor (25.0% and 35.7%) were observed for levels of Blot5 =0 μg/g of dust as compared with Derpl in bed and on the floor (4.3% and 14.5% respectively, P< 0.05).Conclusions Derpl and Blot5 are the major allergens found in this regional study, Blot5 is a more potent allergen in Hongkong, probably reflecting the high level of exposure to Blomia tropicalis (Bt). Bt and Dermatophagoides pteronyssinus (Dp) allergens should be included for precise diagnosis and effective immuno-therapeutic treatment of mite allergy in Hongkong.  相似文献   

20.
ANTI-PROLIFERATION EFFECT OF ORIDONIN ON HL-60 CELLS AND ITS MECHANISM   总被引:4,自引:0,他引:4  
Objcetive To investigate the anti-proliferation effect of oridonin on leukemic HL-60 cells and its mechanism. Methods HL-60 cells in vitro in culture medium were given different concentrations of oridonin. The inhibitory rate of cells were measured by microculture tetrazolium (MTT) assay, cell apoptotic rate was detected by flow cytometry (FCM), morphology of cell apoptosis was observed by hoechst 33258 fluorescence staining, and the activity of telomerase was detected using telomere repeat amplification protocol (TRAP) PCR-ELISA before and after apoptosis occurred. Results Oridonin could decrease telomerase activity, inhibit growth of HL-60 cells, and cause apoptosis significantly. The suppression was both in time- and dose-dependent manner. Marked morphological changes of cell apoptosis including condensation of chromatin and nuclear fragmentation were observed clearly by hoechst 33258 fluorescence staining especially after cells were treated 48-60 hours by oridonin. Conclusions Oridonin has apparent anti-proliferation and apoptotic effects on HL-60 cells in vitro, decreasing telomerase activity of HL-60 cells may be one of its most important mechanisms. These results will provide strong laboratory evidence of oridonin for clinical treatment of acute leukemia.  相似文献   

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