Kidney transplantation is an ideal treatment for patients with end-stage renal disease. Circulating alloantibodies against donor human leukocyte antigens and blood group antigens can impair allografts, shorten allograft survival, and limit access to kidney transplantation. Furthermore, the presence of donor specific antibodies is associated with increased incidence of antibody-mediated rejection and decreased graft survival following transplantation. Plasmapheresis, an extracorporeal therapy directed at removing plasma proteins that has been found to minimize the effects of perioperative sensitization in kidney transplantation. Plasmapheresis enables transplantation across the barrier of ABO blood group incompatibility. In addition, it is also an important approach for the treatment of antibody-mediated rejection. Therefore, studying the application of plasmapheresis in perioperative period of kidney transplantation is expected to increase the chance of transplantation and improve the outcomes following transplantation. This article introduces the application of plasmapheresis in the perioperative period of kidney transplantation.
The incidence of cardiovascular disease remains high, and surgery is an important measure for the treatment of cardiovascular disease. However, cardiovascular surgery is complicated and difficult, and it is one of the departments with the highest rate of allogeneic blood transfusion. Allogeneic blood transfusion significantly increases the complications and mortality of patients, while autologous blood transfusion can effectively reduce allogeneic blood transfusion and adverse reactions. Autologous plateletpheresis technology is a popular autotransfusion method in recent years. This article reviews the autologous plateletpheresis technology and its clinical application in cardiovascular surgery.
Objective To explore the proper dosage of establishment of stable hepatic oval cells （HOC） prolif-eration model by using 2-acetaminofluorene （2-AAF） combined with two-third partial hepatectomy （2/3 PH） surgery， and to explore isolated and cultured method of HOC in vitro. Methods The 174 Wistar rats were randomly divided into 4 experimental groups （each group enrolled 30 rats）， saline group （n=30）， and untreated group （n=24）. Rats of 4 experi-mental groups were underwent gavage of 5， 10， 15， and 20 mg/（kg ? d） 2-AAF， corresponding to the groups from No.1 to No.4 group. Rats of saline group received saline gavage and rats of untreated group didn’t received any treatment. A standard 2/3 PH surgery was performed on the 5th day after gavage， then the same gavage method was still administrated as preoperation untill rats were sacrificed. The liver tissues of 6 selected rats were adopted and identified by HE staining and immunohistochemical staining on 4， 8， 12， and 16 days after PH for observation of the proliferation of HOC in every group， on 4 days， levels of alanine aminotransferase （ALT） and aspartate aminotransferase （AST） were tested in addition. HOC were isolated and purified by collagenase perfusion method and percoll gradient centrifugation. Results The surv-ival rates of untreated group，saline group，No.1 group，No.2 group，No.3 group，and No.4 group were 100% （24/24），93% （28/30），93% （28/30），90% （27/30），90% （27/30），and 80% （24/30） respectively. Compared with the saline group and untreated group， the levels of serum ALT and AST increased significantly in No.2， No.3， and No.4 group on the 4th day after PH （P＜0.05）. The results of HE staining showed that No.2， No.3， and No.4 group were observed visibly different level of damage at liver tissue， and the proliferation level of HOC were most obviously in No.3 and No.4 group. The results of immunohistochemical staining revealed that proliferation cells were positively expressed oval cell marker-6 （OV-6）. The number of OV-6 positive cells were increased significantly with the increase of dosage of 2-AAF between 4 days and 12 days after operation， and proliferation levels were related with dosages of 2-AAF （P＜0.05）. In all cultured cells， 80% of cells were OV-6 positive cells after isolation and culture by using collagenase perfusion method and percoll gradient centrifugation. Conclusions The methods of gavage of 2-AAF at 15 mg/（kg ? d） combined with 2/3 PH surgery can establish the HOC proliferation model on the 12th day， as well as the rats have lower mortality and better tolerance， especially. The collagenase perfusion method and percoll gradient centrifugation can be used to isolate HOC effectively.
【Abstract】 Objective To improve the method of obtaining purified and viable human hair foll icle stem cells (HFSCs)from bulge cells (BCs). Methods Firstly, the BCs were isolated from human hair foll icles by microdissection. Secondly, the CD200+ HFSCs were selected from BCs using magnetic cell sorting method. The viabil ity of these purified HFSCs was detected under l ight microscope. The purification rate was analyzed by flow cytometry. The pre- and post-purification cells were compared by immunofluorescence staining. Results The adherent BCs displayed a typical cobblestone morphology on day 6. The BCs expressed K19 bly. The viabil ity rate of pre-purification cells was 95.0% ± 0.6% while that of post- purification cells was 94.2% ± 1.0%. There was no significant difference (P lt; 0.05). By flow cytometry and immunofluorescence staining examination, the CD200+ cell rate was 8.31% before cell sorting purification while that was 82.31% after cell sorting purification. Con clu sion Highly purified and viable HFSCs could be obtained by micromanipulation and magnetic cell sorting assay.
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.
ObjectiveTo compare the effectiveness of flexible fixation and rigid fixation in the treatment of ankle pronation-external rotation fractures with distal tibiofibular syndesmosis.MethodsA retrospective analysis was made on the clinical data of 50 patients with ankle pronation-external rotation fractures and distal tibiofibular syndesmosis treated between January 2013 and December 2015. Suture-button fixation was used in 23 patients (flexible fixation group) and cortical screw fixation in 27 patients (rigid fixation group). There was no significant difference in age, gender, weight, side, fracture type, and time from trauma to surgery between 2 groups (P>0.05). The operation time, medial clear space (MCS), tibiofibular clear space (TFCS), tibiofibular overlap (TFO), American Orthopaedic Foot and Ankle Society (AOFAS) score, and Foot and Ankle Disability Index (FADI) score were compared between 2 groups.ResultsThe operation time was (83.0±9.1) minutes in the flexible fixation group and was (79.6±13.1) minutes in the rigid fixation group, showing no significant difference (t=1.052, P=0.265). All patients achieved healing of incision by first intention. The patients were followed up 12-20 months (mean, 14 months). The X-ray films showed good healing of fracture in 2 groups. There was no screw fracture, delayed union or nounion. The fracture healing time was (12.1±2.5) months in the flexible fixation group and was (11.3±3.2) months in the rigid fixation group, showing no significant difference between 2 groups (t=1.024, P=0.192). Reduction loss occurred after removal of screw in 2 cases of the rigid fixation group. At last follow-up, there was no significant difference in MCS, TFCS, TFO, AOFAS score and FADI score between 2 groups (P>0.05).ConclusionSuture-button fixation has similar effectiveness to screw fixation in ankle function and imaging findings, and flexible fixation has lower risk of reduction loss of distal tibiofibular syndesmosis than rigid fixation.
ObjectiveTo investigate the preparation and osteogenic properties of poly (L-lactic acid)(PLLA)/lecithin porous scaffolds with open pore structure.MethodsPLLA/lecithin porous scaffolds with different lecithin contents (0, 5%, 10%, 20%, 30%, 40%, 50%) were prepared by thermally induced phase separation (groups A, B, C, D, E, F, and G, respectively). Scanning electron microscopy (SEM) was used to observe the surface morphology of the scaffolds. Wide-angle X-ray diffraction (XRD) and differential scanning calorimetry (DSC) were used to detect the crystallinity of the scaffolds. The water uptake ability of the scaffolds was measured. The cell growth and viability of bone marrow mesenchymal stem cells (BMSCs) of mouse on each scaffold was assessed by cell counting kit 8 (CCK-8) method. The osteogenic differentiation ability of BMSCs on each scaffold was evaluated by alkaline phosphatase (ALP) activity. Finally, a critical-size rat calvarial bone defect model was used to evaluate the osteogenesis of the scaffolds in vivo. Micro-CT was used to reconstruct the three-dimensional model of the defect area, and the bone volume and bone mineral density were quantitatively analyzed.ResultsSEM results showed that the lecithin could slightly reduce the pore size; when lecithin content was 50%, platelet-like structure could be observed on the scaffolds. Wide angle XRD and DSC showed that the crystallinity of scaffolds gradually decreased with the increase of lecithin content. The water uptake ability test showed that the hydrophilicity of scaffolds increased with the increase of lecithin content. CCK-8 assay showed that cell activity gradually increased with the increase of culture time. After 7 days of culture, the absorbance (A) value of groups C, D, E, and F were significantly higher than that of groups A, B, and G (P<0.05), but no significant difference was found among groups C, D, E, and F (P>0.05). After 14 days of osteogenic induction, with the increase of lecithin content, there was a significant difference in ALP activity of each group. The ALP activity in groups D, E, F, and G were significantly higher than that in groups A, B, and C (P<0.05).In vivo, the results of Micro-CT examination and bone volume and bone mineral density showed that the scaffolds with 30% lecithin had the best repairing effect.ConclusionPrepared by thermally induced phase separation, the cytocompatibility, osteogenic differentiation, and bone repair ability of the PLLA/lecithin porous scaffold is obviously better than that of pure PLLA scaffold. PLLA/lecithin porous scaffold with suitable lecithin content is a promising scaffold material for bone tissue engineering.
Objective To explore the isolating methods of rat submandibular gland cell for primary culture. Methods Rat submandibular gland cell were isolated by direct isolation and pancreatin digestion respectively, and then were cultured and subcultured on DMEM. The shape and structure of cultured cells were observed with phase contrast microscope. The cell survival rate was detected by using trypan blue elimination test. The vital force of culture cells was estimated with MTT colorimetric method. The cultured cell secretion function was evaluated by assay of amylase activity. Results By direct isolatin, the cell survival rate was 70% and the cell vital force was 0.16±0.014. By pancreatin digestion, the cell survival rate was 85% and the cell vital force was 0.45±0.13; the cells had good shape and attached well. The Ck8.13 and keratin antibodies were epithelium specific and α-SMA antibodies were myoepithelium specific. The cells were stained positively with CK8.13, keratin and α-SMA antibodies. Conclusion The method of pancreatin digestion for the isolation of submandibular gland cell is better than that of idrect isolation.
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.
To separate the overlapped protein spots in two-dimensional gel electrophoresis (2-DE) images, we proposed an auto-separating algorithm based on valley characteristics. Firstly, the marker-controlled watershed algorithm was used to detect the initial outlines of the object regions. Secondly, medial axis transform and hierarchical branch pruning method were applied to the main skeletons of the object regions, and each main skeleton was fitted into line segments to describe the overlap directions. Then, the 3-dimensional model of the object region was scanned on the normal planes of the line segments to find the valley locations. And finally, a validation model was adopted to construct separation lines. The experiments on 2 real scanned 2-DE images showed that the true overlap separate (TOSs) were 78.95% and 85.71%, respectively. The results indicated that the proposed algorithm was better than the existing algorithms and could be used in engineering practice.