Objective To study a simple and practical method of isolation, culture and identification of hepatic oval cells from adult rat. Methods Wistar adult rats were fed by 2-acetaminofluorere （AAF） and were stimulated by partial hepatectomy to activate the proliferation of hepatic oval cells. After operation 12 days, the livers were resected for isolating oval cells. Hepatic tissue was digested by 0.10% collagenase Ⅳ and the obtained heterogeneous liver cells were then isolated and purified by density gradient centrifugation. The expressions of albumin and CK19 mRNA in hepatic oval cells were analyzed by immuno-fluorescence and RT-PCR. Results The survival rate of the newly isolated oval cells was more than 90%. The hepatic stem cells were shown by immuno-fluorescence of stem cell’s antigen c-kit. The expressions of mRNA CK19 and albumin of the oval cell were also detected by PCR. The proliferation activity of the newly isolated oval cells was significantly high and they could be induced to differentiate into both hepatic and bile ductal cells by some growth factors. Conclusion The successful development of the simple and feasible isolation and purification procedure as well as the identification method for hepatic oval cells may provide a fundamental for further studies about bionomics of the hepatic stem cell and the relation between stem cells and hepatic carcinoma.
Objective To establish a method to isolate the CD4+CD25+ regulatory T cells (Tregs) and to identify the purity and function of these cells. Methods The peripheral blood (8 mL) were collected from the great saphenous vein of 10 rhesus monkeys (4 females and 6 males, aged 4-5 years, and weighing 5-8 kg). The mononuclear cells were isolated with density gradient centrifugation. CD4+ T cells were separated by the Magnetic cell sorting (MACS) negative selection and MACS positive selection. The cell yield rate, the cell viability, and the cell purity were compared between MACS negative selection and MACS positive selection. In CD4+ MACS negative selection, the anti-biotin MicroBeads and biotin-antibody cocktai in CD4+CD25+ Tregs isolation kit non-human primate were used, and in MACS positive selection, the anti-APC MicroBeads in CD4+CD25+ Tregs isolation kit non-human primate and CD4-APC were used. The CD4+ T cells separated by positive selection were selected to obtain CD4+CD25 Tregs with CD25 MicroBeads. The purity, activity, the FoxP3 level, and the suppressive function to concanavalin A (ConA) activated autologous CD4+CD24- effective T cells (Teffs) of CD4+CD25+ Tregs were detected by flow cytometry. Results After CD4+ T cells were separated by MACS negative selection and MACS positive selection, the cell viabilities were all up to 95%, showing no significant difference (P gt; 0.05). The cell yield rate and purity of CD4+ T cells by positive selection were significantly higher than those of CD4+ T cells by negative selection (P lt; 0.05). CD4+CD25+ Tregs can be successfully isolated by MACS double positive selection. The classifying purity was 76.2% ± 8.6%; the cell activity was 93.3% ± 4.7%; and the level of FoxP3 was 74.2% ± 6.9%. The CD4+CD25+ Tregs had suppressive effect on ConA activated autologous CD4+CD25- Teffs. Conclusion MACS double positive selection can be used to isolate high-purity CD4+CD25+ Tregs from the peripheral blood of rhesus monkeys and the process does not influence the activity of CD4+CD25+ Tregs.
Objective To make a comparative study on the effects of whole bone marrow culture method and density gradient centrifugation method in isolating hBMSCs. Methods hBMSCs were obtained from healthy adult volunteers and isolated by whole bone marrow culture method and density gradient centrifugation method. Primary cell morphology was observed using inverted phase contrast microscope and the cells in the 2nd passage were stained with HE after being cultured for 7 days. And then, the generation time of the primary, 2nd and 3rd passage hBMSCs was comparedbetween two methods and the surface markers were detected by flow cytometer. In addition, the ALP expression inosteoinductive hBMSCs were evaluated by ALP activity kit at 3, 6 and 9 days and ALP staining was used for osteoinductivehBMSCs with Kaplow method at 9 days. Results Primary cells isolated with whole bone marrow culture method showedaggregation growth while cells isolated with density gradient centrifugation method showed diffusion growth. HE stainingshowed no significant difference in the morphology of the 2nd passage cells between these two methods. The generationtime of primary cells isolated by whole bone marrow culture method (15.36 ± 1.67) days was significantly shorter than that of cells isolated by density gradient centrifugation method [(18.57 ± 1.05) days] (P lt; 0.01), while the generation time of the 2nd and 3rd passage cells showed no statistically significant differences between these methods (P gt; 0.05). The concent of positive surface markers (CD29, CD44, CD71, CD105, CD166) and negative surface marker CD34 in the 2nd cells showed no significant difference between these two isolation methods (P gt; 0.05); however, negative markers CD14 and CD45 showed significant difference (P lt; 0.01). The ALP expression in osteoinductive cells showed no statistical significant (P gt; 0.05) at 3, 6 and 9 days; and the ALP staining positive cell ratio of whole bone marrow culture method was basically in accordance with that of density gradient centrifugation method at 9 days. Conclusion hBMSCs could be isolated by whole bone marrow culture method, and the cell isolation effects of whole bone marrow culture method are equivalent with density gradient centrifugation method.
Objective To find a feasible method that can fast isolate seed cells, keratinocyte stem cell and fibroblasts, for composite tissue engineered skin. Methods The foreskin could be attained from posthectomy, the subcutaneous tissue was removed completely, and the full-thick skin was cut into pieces, 2 mm×2 mm in size, then the pieces were submerged into a centrifuge tube containing collagenase Ⅰ in a oscillator. After 3-hour digestion at 37℃, the dermis was dissolved completely with all the fibroblasts in the digestion solution and the epidermis could be separated easily.With more than 10minute digestion in trypsin at 37℃, the epidermal cells could be harvested. Then flowcytometry and FITCimmunofluorescence for cytokeratin 19 of epidermal cells and FITC-immunofluorescence for vimentin of fibroblast were conducted to identify keratinocyte stem cells in the epidermal cells and fibroblasts in the digestion solution. Moreover, epidermal cells and fibroblasts were cultured in vitro for 7 days to investigate their biological behavior. Results Using collagenase Ⅰ combined with trypsin, epidermal cells andfibroblasts could be isolated at one time within 3 hours. Up to 17% cells demonstrated cytokeratin 19 positive in the epidermal cells, with fibroblast vimentin positive. The amount of fibroblast could be enlarged to more than 100 times within 6 days, but the putative keratinocyte stem cells were difficult for subculture. Conclusion Seed cells for composite tissue engineered skin could be harvested fast at one time, that made it possible to reconstruct composite tissue-engineered skin in vitro.
Objective To study the effect of soybean trypsin inhibitor (STI) on the rat islets’ yield and function during the process of isolation and purification. Methods The rats were divided into experiment group and control group according to whether STI was put into the collagenase. STI (2.0 mg/ml) was put into the collagenase digestive juice of the experiment group and none to the control group. For both of two groups, islets were isolated by situ perfusion collagenase into the rat pancreas and they were purified by un-continuous gradient centrifugation with Ficoll-400. The quantities of the obtained rat islets before and after purification were recorded, and the morphosis and function of the purified rat islets were tested, then their vivo function were observed after islets plantation. Results After digest and before purification, there was no obvious deviation of the obtained islets quantity between two groups 〔(624±38.2) IEQ vs (586±37.7) IEQ, P＞0.05〕; After purification, there were significant deviation in the islets quantity 〔(408±28.3) IEQ vs (189±27.1) IEQ, P＜0.05〕 and purity quotient 〔（93±2.4）% vs （75±2.1）%, P＜0.05〕. For two groups, there was no obvious deviation of the obtained islets in insulin stimulation and secretion experiment as well as their vivo function experiment. Conclusion The ultimate yield and purity quotient of the rat islets can be obviously improved by using collagenase digestive juice with SIT in situ perfusion on the rat pancreas, and it has no obvious effect on the islets function.
Objective To explore a simple, effective and stable method for the isolation and purification of Kupffer cells from rat liver, enabling further study on the structure and function of these cells in vitro. Methods After laparotomy, a catheter was inserted into the portal vein and secured with artery clamp. Then, the rat liver was perfused and digested with solution Ⅰ and solution Ⅱ containing 0.05% collagenase Ⅳ respectively. The cell suspension was centrifuged with isopycnic sedimentation in a two-step Percoll gradient to harvest Kupffer cells. The isolated Kupffer cells were purified by selective adherence after 30 min of cultivation, and identified by evaluation of phagocytosis of India ink and peroxidase staining with DAB through light and electron microscopy. Results It was verified that the viability of isolated Kupfffer cells was more than 90% through Trypan blue staining. Those Kupffer cells could attach to plastic quickly and phagocytose ink, and had the appearance of “fried eggs” in positive peroxidase staining with a purity of 95%. Under the light microscopy, the appearance of newly isolated Kupffer cells was round with uniform shape and size. After two days of culture, Kupffer cells appeared to distend with irregular or stellate shape. The typical features were observed in the transmission electron micrographs. There were numerous pseudopods and occasional cup-like indentations in the cell membrane of Kupffer cells. The cytoplasm contained numerous types of lysosomes and other phagocytotic vesicles. Conclusion The method for isolating and culturing Kupffer cells in this study is effective and stable, and the biological characters are preserved in the cultured cells.
【Abstract】ObjectiveTo develop a method of adult porcine pancreatic islet isolation.MethodsThe tails of adult porcine pancreas were perfused through the pancreatic duct with 0.1% cold collagenase(type Ⅺ) and incubated at 38.5 ℃.The digested tissue was dispersed in 4 ℃ Hanks balanced salt solution(HBSS).The tissue suspensions were filtered through a 600 μm mesh.The residual tissue was resuspended in cold HBSS,and put in the Ricordi’s chamber and shaken for 5 minutes,then filtered again.The isolated islets were divided into three groups: control group(n=14),Pefabloc(trypsin inhibitor,n=8) group and FOY(trypsin inhibitor,n=5) group.The collagenase solution of the Pefabloc and FOY group was supplemented with 1.0 mmol/L Pefabloc and FOY respectively. ResultsThe islet yields of the Pefabloc group and FOY group 〔(11 848±3 530) islet/g pancreas and (14 496±3 693) islet/g pancreas〕 were significantly higher than that of the control group 〔(8 505±3 349) islet/g pancreas〕,P＜0.05.The activity of pancreatic protein enzyme in digestive fluid after digestion in control group was higher than the activity of pancreatic duct before injection and Pefabloc group(P＜0.01),which the control group, pancreatic duct before injection and Pefabloc group were (114.7±50.0) BAEEU,(4.0±1.8) BAEEU and (5.5±2.7) BAEEU,respectively.The pancreatic duct before injection and Pefabloc group showed no significant difference in statistics. In control group,when the harvest of islet was more than 8 000/g,the activity of pancreatic protoin enzyme was less than that with the harvest of islet below 8 000/g 〔(78.3±26.7) BAEEU vs (137.5±48.4) BAEEU,P＜0.05〕.Islet after purification in control group,Pefabloc group and FOY group showed good insulin secretion ability for different concentration of glucose.ConclusionA higher porcine pancreatic islet yield can be obtained by this method of pancreatic islet isolation and prophylactic administration of trypsin inhibitors consistently produce excellent islet yields.
ObjectiveTo evaluate the efficacy of the epicardial circumferential left atrial ablation (CLAA) with pulmonary vein isolation (PVI) in curing atrial fibrillation (AF). MethodsThirty experimental pigs, weight from 60-78 kg, were divided into 3 groups with the method of random sampling:a group of AF (AF group, n=10), a group of PVI (PVI group, n=10), and a group of CLAA and PVI (CLAA+PVI group, n=10). AF mode was induced by rapid atrial pacing. After AF was induced, no ablation was performed for pigs in the AF group, PVI was performed for pigs in the PVI group with bipolar radiofrequency ablation clamp, and the CLAA+PVI group underwent CLAA after accepted PVI with bipolar radiofrequency ablation clamp. After ablation, we applied electrovert on AF pigs to recover to sinus rhythm, then we tested the vulnerability and lasting time of AF in all 3 groups. ResultsAll pigs developed a stable and sustained AF by rapid left atrial pacing. The pigs of the PVI group and the CLAA+PVI group successfully underwent ablation with the beating heart. Isolated PVI terminated AF in 3 of 20 pigs, and CLAA with PVI terminated AF in 5 of 8 pigs (15% vs. 62.5%, P=0.022). After all pigs recovered to the sinus rhythm, compared with the AF group (10/10), the incidence of sustained AF by burst pacing was statistically decreased in the PVI group (3/10, P=0.003) and the CLAA+PVI group (0/10, P<0.001). There was no statistical difference between the PVI group and the CLAA+PVI group (P=0.211). There was a statistical decreasing of AF duration in the PVI group (P=0.003) and the CLAA+PVI group (P<0.001) compared with the AF group and there was a statistical decreasing of AF duration in the CLAA+PVI group compared to that of the PVI group (P=0.008). ConclusionCompared with isolated PVI, CLAA+PVI may effectually stop the lasting of AF, restrain the recurrance of AF, and improve the treatment effect of AF.
Carbapenemase producing Enterobacteriaceae (CPE) has emerged as a significant global public health challenge and placing infected patients at risk of potentially untreatable infections. When resistance to carbapenems occurs, there are often few alternative treatments available. Numerous international guidelines have performed systematic and evidence review to identify new strategies to prevent the entry and spread of CPE in healthcare settings. Several key strategies have been shown to be highly effective. Firstly a new strategy that is proven to be effective is the early identification of the CPE carrier patients through active surveillance cultures. While waiting for the screening results, suspected CPE carriers will be put on preemptive isolation in single room and healthcare worker will at the same time practice contact precautions. The active surveillance culture and prompt preemptive isolation will limit the entry and spread of CPE from getting into hospital. Secondly, it is of utmost importance to incorporate enforcement of the basic infection prevention and control best practices in the hospital including, full compliance to hand hygiene, appropriate use of personal protective equipment, execute antibiotic stewardship program to control abuse of antibiotics, effective environmental cleaning and decontamination, staff education and feedback, as well as surveillance of healthcare-associated infections. Such a holistic approach has been shown to be effective in inhibiting CPE from gaining foothold in the hospital.
ObjectiveTo explore the effect of using a stent graft to treat a Stanford type A aortic dissection with the ascending aorta in the cavity.MethodA retrospective review was made of the clinical data of a patient with Stanford type A aortic dissection admitted to Zhangye People’s Hospital Affiliated to Hexi University in December 2016.ResultsAfter the patient underwent general anesthesia aortic dissection and stent graft treatment, the dissection fracture completely disappeared. After 2 years of follow-up, the patient’s pseudocavity hematoma was completely absorbed. The operative time was 30 min and the blood loss was about 5 mL. There were no complications such as avulsion of dissection, internal leakage, cerebral infarction, myocardial infarction, nervous system, and other complications occurred.ConclusionFor Stanford type A aortic dissection with a tear located in the ascending aorta, intracavitary treatment with coated stent is feasible for ascending aortic dissection with good vascular conditions and tear location through accurate preoperative assessment.