Bile acids inhibit ferroptosis sensitivity through activating farnesoid X receptor in gastric cancer cells.

BACKGROUND: Gastric cancer (GC) is associated with high mortality rates. Bile acids (BAs) reflux is a well-known risk factor for GC, but the specific mechanism remains unclear. During GC development in both humans and animals, BAs serve as signaling molecules that induce metabolic reprogramming. This confers additional cancer phenotypes, including ferroptosis sensitivity. Ferroptosis is a novel mode of cell death characterized by lipid peroxidation that contributes universally to malignant progression. However, it is not fully defined if BAs can influence GC progression by modulating ferroptosis. AIM: To reveal the mechanism of BAs regulation in ferroptosis of GC cells. METHODS: In this study, we treated GC cells with various stimuli and evaluated the effect of BAs on the sensitivity to ferroptosis. We used gain and loss of function assays to examine the impacts of farnesoid X receptor (FXR) and BTB and CNC homology 1 (BACH1) overexpression and knockdown to obtain further insights into the molecular mechanism involved. RESULTS: Our data suggested that BAs could reverse erastin-induced ferroptosis in GC cells. This effect correlated with increased glutathione (GSH) concentrations, a reduced GSH to oxidized GSH ratio, and higher GSH peroxidase 4 (GPX4) expression levels. Subsequently, we confirmed that BAs exerted these effects by activating FXR, which markedly increased the expression of GSH synthetase and GPX4. Notably, BACH1 was detected as an essential intermediate molecule in the promotion of GSH synthesis by BAs and FXR. Finally, our results suggested that FXR could significantly promote GC cell proliferation, which may be closely related to its anti-ferroptosis effect. CONCLUSION: This study revealed for the first time that BAs could inhibit ferroptosis sensitivity through the FXR-BACH1-GSH-GPX4 axis in GC cells. This work provided new insights into the mechanism associated with BA-mediated promotion of GC and may help identify potential therapeutic targets for GC patients with BAs reflux.

Photoinduced electron transfer and remarkable enhancement of magnetic susceptibility in bridging pyrazine complexes.

Three isostructural coordination polymers of the formula [MCl2(DMSO)2(pz)]n (pz = pyrazine, DMSO = dimethyl sulfoxide, M = Mn, Fe and Co for 1-3) were synthesized through the solvothermal reaction of MCl2, DMSO and pz. By introducing DMSO and chloride as electron donor ligands and pz as an accepter, a donor-metal-accepter (D-M-A) system was constructed. Radicals generated by photoinduced electron transfer (PET) from a donor to an accepter result in a dramatic change of vibronic absorption spectra in UV regions and remarkable enhancement of magnetic susceptibility. The mechanism of PET was also determined by PXRD, IR, EPR and XPS measurements. This is the first example of bridging pz complexes which exhibit both photochromism and photomagnetism in a PET process.

The "extracapsular cystic" sign in pancreatic serous cystic neoplasms: A clinicopathologic study of 177 patients with cystic pancreatic lesions.

OBJECTIVE: To assess a new imaging feature that we have named the extracapsular cystic sign which can make a constructive contribution towards differentiating serous cystic neoplasms (SCNs) from other pancreatic cystic lesions. MATERIAL AND METHODS: We retrospectively reviewed 177 CTs/MRIs of patients who underwent pancreatic resection of cystic lesions at two institutions from January 2011/2013, to September 2017. For each patient, demographic information, clinical presentation, especially imaging features were carefully investigated by two experienced abdominal radiologists, retrospectively. All statistical analyses were performed using SPSS V.23.0. RESULTS: Twenty-one lesions had extracapsular cystic signs which were newly discovered, 17 (28.3%) of 60 SCNs and 4 (3.4%) (mucinous cystic neoplasm = 1, walled-off necrosis = 2, retention cyst = 1) of 117 Non-SCNs were included, from which indicating that the extracapsular cystic sign was more often detected on SCNs. As for 21 lesions, 86% (n = 18) were females, and mean age at diagnosis was 51.2 years. 71% (n = 15) located in the pancreatic body and tail. Average size was 27.2 mm (23.7-53.4), mean (SD) ratio of biggest daughter cyst to mother cyst was 0.51[0.14] (p = 0.99), average (SD) angle between two of them was 105.5° [14.9] (p = 0.84). The average time interval between last imaging examination and surgery was 8.4 days. CONCLUSIONS: The new sign named the extracapsular cystic sign in SCNs may help differentiate SCNs from other pancreatic cystic lesions. Furthermore, this study supports an original diagnosis for SCNs when the sign of extracapsular cyst appears.

Novel single component tri-rare-earth emitting MOF for warm white light LEDs.

Single-phase white phosphors could overcome many drawbacks of traditional phosphors, and warm white light phosphors have been considered suitable for indoor illumination applications. Thus, synthesizing new single-phase warm white phosphors is significant for the production of LEDs. Based on the above considerations, a novel single component warm white light phosphor SmxTbyDy0.2-x-y-metal organic frameworks (MOFs) has been prepared successfully in this study. The compound Sm0.1Tb0.04Dy0.06-MOF shows ideal warm white light emission with CIE coordinates (0.333, 0.3522), a color rendering index (CRI) value of 86.7, a low correlated color temperature (CCT) value of 4444 K, and good heating/cooling circulation. In addition, the LED devices fabricated with this novel warm white light phosphor have excellent warm white light quality even at high temperatures, which is necessary for its practical application.

Nickel(0)-Catalyzed Hydroalkenylation of Imines with Styrene and Its Derivatives.

A nickel(0)-catalyzed hydroalkenylation of imines with styrene and its derivatives is described. A wide range of aromatic and aliphatic imines directly coupled with styrene and its derivatives, thus providing various synthetically useful allylic amines with up to 95 % yield. The reaction offers a new atom- and step-economical approach to allylic amines by using alkenes instead of alkenyl-metallic reagents. Experiments and DFT calculations showed that TsNH2 promotes the proton transfer from the coordinated olefin to the imine, accompanied by a new C-C bond formation.

Lymphadenitis associated with cat-scratch disease simulating a neoplasm: Imaging findings with histopathological associations.

The lymphadenitis associated with cat-scratch disease (CSD) is often confused with neoplasms by a number of radiologists and clinicians, and consequently, unnecessary invasive procedures or surgeries are performed. In the present study, the contrast-enhanced computed tomography (CT) and magnetic resonance imaging (MRI) findings of 10 patients (6 men and 4 women) with clinically and pathologically confirmed lymphadenitis associated with CSD were retrospectively analyzed (CT in 3 patients, MRI in 6 patients, and CT and MRI in 1 patient) at The Second Affiliated Hospital of Zhejiang University School of Medicine (Hangzhou, China) between January 2007 and November 2014. As a result, 17 enlarged lymph nodes were identified in 10 cases. The 5 nodes identified by CT scan exhibited relatively inhomogeneous isodensity to muscle, with patchy low density in the center. All 14 nodes identified by MRI scan exhibited homogeneous or heterogeneous isointensity to muscle or slightly increased intensity compared with that of muscle on T1-weighted images (T1WI), and homogeneous or heterogeneous hyperintensity on fat-suppressed T2WI. Following enhancement, all 17 enlarged lymph nodes associated with CSD demonstrated the following 3 different enhancement patterns: Moderate homogeneous enhancement (n=8), which was associated with histologically identified early disease stage; marked heterogeneous enhancement with no enhancement of the necrotic areas (n=4), and heterogeneous enhancement with progressively 'spoke-wheel-like' (defined as radiating enhancement from the center) enhancement of the patchy low-density area (n=1), which was associated with histologically identified intermediate disease stage; and astral low-density/hypointensity with marked enhancement (n=2) or a 'rose flower' sign (n=2), which was associated with histologically identified late disease stage. We hypothesized that the CT and MRI results of lymphadenitis in CSD may be associated with the pathological features. It may be suggested that the diagnosis of CSD may be formed when considering the characteristic CT and MRI features of astral low-density/hypointensity with marked enhancement or a 'rose flower' sign (defined as marginal petaloid enhancement) in the late disease stage, or the MRI results of homogeneous, moderate enhancement in the early disease stage, or the CT/MRI data of heterogeneous enhancement with non-enhancing area in the center in the intermediate disease stage, in solitary or multiple enlarged lymph nodes associated with general subcutaneous edema in the vicinity of the nodes on CT/MRI and with a history of cat exposure.

Contrast-enhanced multiple-phase imaging features of intrahepatic mass-forming cholangiocarcinoma and hepatocellular carcinoma with cirrhosis: A comparative study.

The intrahepatic mass-forming cholangiocarcinoma (IMCC) is frequently misdiagnosed as hepatocellular carcinoma (HCC) in patients with cirrhosis, by numerous radiologists and clinical doctors, which results in the incorrect therapeutic treatment. A retrospective case-control study was conducted, and the contrast-enhanced multiple-phase (CEMP) computed tomography (CT) and magnetic resonance imaging (MRI) findings of 22 pathologically confirmed IMCC patients and 22 HCC controls with underlying liver cirrhosis were analyzed at the present hospital, from January 2010 to December 2015. In addition, serum tests were conducted and clinical symptoms of patients evaluated. A statistical analysis revealed that the enhancement pattern, signal on MRI delayed phase (P<0.001), maximum diameter, capsule retraction, portal vein invasion, bile duct dilation and abdominal lymphadenectasis characteristics were different between IMCC and HCC patients with cirrhosis. On CEMP CT and MRI analysis, the most frequently occurring enhancement patterns of IMCC were progressive patterns (P=0.001 or P<0.001). Conversely, the most frequently occurring enhancement patterns present in HCC were the washout patterns (P<0.001). Therefore, the diagnosis of IMCC in cirrhotic patients should be verified with CEMP CT and MRI analysis for the future, to determine presence or absence of progressive and/or peripheral rim-like enhancement, a hyperintensive delayed phase with capsule retraction, portal vein invasion, bile duct dilation, abdominal lymphadenectasis and increased levels of CA199.

A Rapid and Facile Detection for Specific Small-Sized Amino Acids Based on Target-Triggered Destruction of Metal Organic Frameworks.

Most of the reported metal organic frameworks (MOFs)-based DNA sensors were developed by utilizing the different adsorption capacities of MOFs to different structural DNAs (for example, single-stranded DNAs (ssDNAs) and double-stranded DNAs (dsDNAs)) or ssDNAs with different lengths. Herein, we introduced another strategy for the design of MOFs-based biosensing platforms. We found that specific small-sized amino acids (for example, glycine and serine) could lead to the destruction of the MOFs formed by [Cu(mal)(bpy)]·2H2O], thus recovering the fluorescence of a fluorophore-labeled ssDNA that had been quenched by MOFs. The corresponding working mechanism was discussed. On the basis of this finding, a mix-and-detect fluorescence method was designed for the turn-on detection of specific small-sized amino acids. The feasibility of its use in real serum samples was also demonstrated. Besides biosensing applications, the discovery of amino acids-triggered destruction of MOFs can also enrich the building blocks of molecular logic gate. As an example, a biomolecular logic gate that performs OR logic operation was constructed using glycine and a DNA strand as inputs.

Nickel-Catalyzed Hydroacylation of Styrenes with Simple Aldehydes: Reaction Development and Mechanistic Insights.

The first nickel-catalyzed intermolecular hydroacylation reaction of alkenes with simple aldehydes has been developed. This reaction offers a new approach to the selective preparation of branched ketones in high yields (up to 99%) and branched selectivities (up to 99:1). Experimental data provide evidence for reversible formation of acyl-nickel-alkyl intermediate, and DFT calculations show that the aldehyde C-H bond transfer to a coordinated alkene without oxidative addition is involved. The origin of the reactivity and regioselectivity of this reaction was also investigated computationally, which are consistent with experimental observations.

Computational Prediction of One-Step Synthesis of Seven-membered Fused Rings by (5+2) Cycloaddition Utilising Cycloalkenes.

The (5+2) cycloaddition reaction utilising cycloalkenes is rare, although it is one of the most efficient methods of constructing seven-membered fused rings because of its high atom- and step-economy. In this study, we used quantum mechanical calculations to predict the plausibility of using the Rh-catalysed intermolecular (5+2) cycloaddition of 3-acyloxy-1,4-enynes and cycloalkenes to produce fused seven-membered carbocycles. The calculation results suggest a convenient, highly efficient and energetically practical approach. Strained cycloalkenes, such as cyclopropene, have been predicted to be active, and the desired bicyclic product should be favoured, accompanied by the formation of byproducts from rearrangement reactions. The energy barriers of the alkene insertion step were analysed by the distortion/interaction model to disclose the origins of the different reactivities of cycloalkenes with different ring sizes.

Dual responsive enzyme mimicking activity of AgX (X=Cl, Br, I) nanoparticles and its application for cancer cell detection.

Chitosan (CS) modified silver halide (AgX, X=Cl, Br, I) (CS-AgX) nanoparticles (NPs) were found to possess dual responsive enzyme mimetic activities. In the presence of H2O2, they were able to oxidize various colorimertic dyes, namely, peroxidase-like activity. Upon photoactivation, CS-AgX NPs could also oxidize the typical substrates in the absence of H2O2. Taking CS-AgI as an example, it was found that the photostimulated enzyme mimetics of CS-AgI NPs showed several unprecedented advantages over natural peroxidase or other existing alternatives based on nanomaterials, such as excellent enzyme-like activity over a broad pH range (3.0-7.0), the independence of hydrogen peroxide on activity, the easily regulated activity by light irradiation, and the good reutilization without significant loss of catalytic activity. The mechanism of the dual responsive enzyme-like activity of CS-AgI was investigated. On the basis of these findings, the photoactivated CS-AgI was designed to develop a facile, cheap, rapid, and highly sensitive colorimetric assay to detect cancer cells. The detection limit of the method for MDA-MB-231 was estimated to be as low as 100 cells, which was much lower than that reported by the method using peroxidase mimetics based on nanomaterials. We believe that CS-AgX NPs with dual responsive enzyme-mimicking activity, especially the excellent photostimulated enzyme-like activity, may find widely potential applications in biosensors.

Effect of carbodiimide cross-linking of decellularized porcine pulmonary artery valvular leaflets.

Decellularization provides low immunogenicity and is only slightly subject to calcification in tissue engineering. However, the mechanical properties of the tissues are weakened after decellularization. We adopted cross-linking agent 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) to treat decellularized porcine pulmonary artery valvular leaflets to improve their mechanical properties. Twenty porcine pulmonary artery valvular leaflets were divided into three groups: the fresh control group A, group B treated with trypsin and Triton X-100 to remove cells, and group C cross-linked with EDC after decellularization. All samples were evaluated the physical and mechanical properties and were then subcutaneously embedded in rabbits. These valvular leaflets were removed after 1, 2, or 4 weeks and checked for pathological changes. The cells of the valvular leaflets were completely removed. The thickness of the valvular leaflets was thinner in group B than in group A (P<0.01). In the subcutaneous embedding of the group B samples, there was mild immunological response after 1-2 weeks, and parts of the scaffolds were degraded. After 4 weeks, fibroblasts had grown into the scaffolds. In group C, there was an increase in the tensile strength and thermal shrinkage temperature in group C compared with group B (P<0.01). In subcutaneous embedding of the group C samples, there was a mild immunological response after 1-2 weeks. The fibroblasts had grown into the samples. The EDC-based cross-linking procedure can enhance the tensile strength of decellularized pulmonary artery valvular leaflets and both decrease the valvular leaflets' rejection and promote tissue regeneration in vivo.

Long-term results of surgical treatment of aortic and mitral regurgitation with enlarged left ventricle.

Mitral valve and aortic valve regurgitation associated with enlarged left ventricle remains difficult to manage and the long-term results following surgical treatment is uncertain. Between April 1988 and September 2000, 82 patients with aortic and mitral regurgitation associated with enlarged left ventricle underwent valve replacement at Anzhen Hospital. The valve disease was rheumatic in origin in 75 patients (91.5%) and congenital in 7 (8.5%). Twenty-eight patients were in New York heart Association Functional (NYHA) class II and 39 in class III and 15 in class IV. Echocardiogram showed severe aortic insufficiency associated with mild to moderate mitral regurgitation in 66 patients and severe mitral regurgitation associated with mild to moderate AI in 16 patients. The mean left ventricular diastole diameter (LVDD) was 77.8 ± 5.2 mm. Valve replacement was performed under hypothermic cardiopulmonary bypass (CPB). Early hospital mortality was 7.3%. Two weeks after surgery the echocardiogram showed a reduction of LVDD. Follow up was completed in 69 patients with mean of 13.5 years. 20 patients were in NYHA class I; 26 in Class II and 3 in Class III and 2 in class IV. The follow-up survival rate was 73.9%, and follow-up mortality was 26.1%. LVDD reduced from 77.8 ± 5.2 mm to 58.3 ± 4.5 mm (P < 0.001). In 24 patients, the LVDD was less than 50 mm. Double valve replacement and/or repair carried out an acceptable early and Long-term clinical outcomes in patients with MR and AI with associated LV great enlargement. Both LVDD and NYHA improved following surgical treatment in survival patients.

An in vivo model of in situ implantation using pulmonary valved conduit in large animals under off-pump condition.

BACKGROUND: The application of pulmonary valved conduit to reconstruct the continuity between right ventricles and pulmonary artery is one of the major surgeries. This study aimed to establish an in vivo model of in situ implantation using pulmonary valved conduit in large animals under off-pump condition to validate the long-term effects of artificial pulmonary valved conduit. METHODS: Domesticate juvenile male sheep and tissue-engineered porcine pulmonary valved conduit were used for the experiment: 30 sheep, weighing (15 ± 3) kg (range 13 to 17 kg) were randomly divided into two groups which were all operated under general anesthesia by off-pump surgery (group 1) and left thoracotomy (group 2). Two different off-pump surgical methods were used to perform cannulation in sheep pulmonary artery to replace part of sheep pulmonary artery with pulmonary valved conduit which will work together with sheep pulmonary artery and valves. During the experiments, animal survival, complication rates, operating time and blood loss were recorded to compare the results between groups and to establish a surgical method with minimal invasion, simplicity, safety, and high success rates. RESULTS: In group 1, a total of 15 cases of surgeries were performed, in which two sheep died; the operative mortality was 13.3% (2/15). In group 2, a total of 15 cases of surgeries were performed, and the surgical mortality rate was 0 (0/15). The operation time and blood loss in group 2 was significantly better than that in group 1. The postoperative echocardiograms showed that, after the surgeries by these two methods, the blood flows were normal, and the valves can open and close freely. Autopsy after 6 months showed that the inner wall and the valves of pulmonary valved conduit were smooth with no thrombus formation. CONCLUSION: These two off-pump methods are feasible and safe with fewer traumas; but the second method is better and particularly suitable for the establishment of a juvenile animal model.

A novel surgical procedure: scaffold-pulmonary autograft transplantation.

Mitral valve-related operations are easy to perform and show good results, but to prevent severe thromboembolism or a high ratio of prosthetic valve destruction by tissue, lifetime anticoagulant therapy is essential after the operation. Thus, identifying a new type of surgical procedure and prosthetic valve to cure mitral valve diseases is necessary. Pulmonary valve autograft transplantation (Ross II) with the "top hat" transplantation technique was first reported by Ross DN to cure mitral disease. Because the "top hat" procedure has some shortcomings, we designed the scaffold-pulmonary autograft transplantation procedure and performed animal experiments to confirm the feasibility and effectiveness of the procedure. A total of 13 minipigs, weighing 20-25 kg, were employed as experimental animals to undergo scaffold-pulmonary autograft valve transplantation in our surgical animal lab. The surgical procedure was performed under hypothermic general anaesthesia and extracorporeal circulation (or cardiopulmonary bypass, CPB). Briefly, the chest cave was opened through the left intercostal, the pulmonary valve autograft was harvested during on-pump beating heart, and the pulmonary valve autograft was mounted in a self-made pulmonary valve scaffold and transferred to the mitral valve annulus without removing the mitral instruments. Finally, the outflow tract of the right ventricle was re-established with a pig pulmonary homograft. After finishing data collection, all animals were executed 1 hour after removal from the CPB. For the 13 minipigs that underwent the operation, the CPB time was 182.4 ± 23.4 min. Two of the thirteen cases died of bleeding during the operation and of a post-operative pulmonary embolism, and the remaining eleven survived for one hour. The pressure of the left atrium did not increase significantly (P = 1.00), and the ultrasonic cardiograph (UCG) showed good function of the new mitral valves, with mean ejection fraction (EF) values of 63.6%. The mitral valve orifice areas were 1.10 ± 0.13 cm(2) (pre-operation) and 1.01 ± 0.08 cm(2) (post-operation) (P = 0.013). The function and structure of the new mitral valves were normal. We preliminarily consider scaffold-pulmonary autograft valve transplantation to be a new alternative to cure mitral valve disease, but advanced chronic animal experiments will be needed to confirm the long-term results of the operation. The results showed it could be a new alternative to cure mitral valve disease.

Decellularized porcine pulmonary arteries cross-linked by carbodiimide.

The physical properties of the tissues are weakened after decellularization, and the exposed collagen fibers are prone to thrombogenesis. Several studies have proven that the use of carbodiimide (EDC) as a cross-linking agent can improve the properties of decellularized xenogeneic scaffold materials. We adopted EDC for the treatment of porcine pulmonary arteries in an effort to improve the physical properties of these arteries following decellularization. Twenty porcine pulmonary arteries were randomly divided into 3 groups. The control group (group A) consisted of fresh porcine pulmonary arteries with no further processing; group B was treated with trypsin and the detergent Triton X-100 to remove cells; and group C was cross-linked with EDC after trypsin and Triton X-100 treatment, as in group B. The pulmonary arteries were assessed based on water content, thickness, tensile strength, and thermal shrinkage temperature, to evaluate the physical properties of all of the samples. The scaffolds were then subcutaneously embedded in rabbits. These constructs were removed after 4 weeks and checked. The cells and matrix components of the arterial walls were removed and the fibrous scaffolds were retained. In group B, the moisture content of the pulmonary arterial walls was increased; and the thickness of the walls and the tensile strength of the pulmonary arteries were decreased in comparison with group A. In subcutaneous embedding of the group B samples in rabbits, after 4 weeks, fibroblasts had grown into the scaffolds and regenerated the tissue. The water content was decreased in the pulmonary arterial walls, there was an increase in the tensile strength and the thermal shrinkage temperature in group C compared with group B. The EDC-based cross-linking procedure can enhance the tensile strength of decellularized pulmonary arteries and decrease scaffold rejection and degradation and promote tissue regeneration in vivo.

Expression of lymphocyte coding genes in peripheral blood and lymphocyte infiltration in cardiac tissues influenced by cyclosporin A in heterotopic heart transplantation model in rats.

UNLABELLED: To systematically compare the expression of coding genes with pathological changes of transplanted cardiac tissue and peripheral blood lymphocytes in an allo-heterotopic rat cardiac transplant model. Using SD rats as donors and Wistar rats as recipients, animals were divided into two groups, control and cyclosporine A intervention plus heart transplant groups. After transplant at 1, 3, 7, 10 and 12d, we assessed the ability of lymphocytes to infiltrate into cardiac tissues and levels of leukocyte coding genes in peripheral blood. Histopathological changes were monitored in cardiac tissue to determine the level of transplant rejection. RESULTS: (1) 24h after transplant peripheral blood lymphocytes' transcription and expression were temporarily reduced. (2) CD4(+) and CD8(+) lymphocytes infiltrate into cardiac tissue and Grade 1R pathological changes were observed 3d-7d after heart transplant. (3)Cyclosporine A was not able to completely block heart transplant rejection.(4) Although cyclosporine A was not able to effectively suppress CD4(+) T cell gene expression, it did suppress CD8(+) T cell gene transcription. (5) Cyclosporine A did not effectively reduce the rapid infiltration of CD4(+) or CD8(+) infiltration in 3d, but significantly reduced the degree of CD4(+) T cell infiltration in cardiac tissues between 3 and 7d. (6) Differential display (DD-PCR): Graft control group: there were differences in 2,3-bisphosphoglycerate, ribosomal protein S25, 12S ribosomal, gig18, MHC-III and ATPase H(+), which occurred 24h before CD4/CD8 surface protein expression. Cyclosporine A group: there were differences in thrombospondin-1, TCR, 2,3-bisphosphoglycerate, sodium channel beta-1, gig18 and TCR. In the cyclosporine A group 2,3-bisphosphoglycerate positive expression was observed 24h after the control group, which indicates that cyclosporine A slowed down the 2,3-bisphosphoglycerate transcription rate in peripheral lymphocytes and delayed its expression time. Cyclosporine A also suppressed gig18 transcription in peripheral lymphocytes. After 24h, sodium channel beta-1 was positively expressed in the cyclosporine A group. The relationship between molecular surface receptor expression and coding genes in cardiac tissue and peripheral blood after transplant indicates that early detection of acute rejection and anti-rejection drugs' curative effect can be assessed.

[Mesenchymal stem cells and endothelial progenitor cells obtained from rabbit bone marrow with differential adhesion methods and their biological characteristics].

This study was aimed to evaluate an effective and stable method for obtaining mesenchymal stem cells (MSC) and endothelial progenitor cells (EPC) from the rabbit bone marrow and to investigate the biological characteristics of MSC and EPC. Mononuclear cells were obtained from rabbit bone marrow using density gradient method, and were differentially adhered to the cell culture plate enclosed with fibronectin. Then, MSC and EPC were amplified with EGB-2MV medium. Trypan blue method was used to test the passage survival rate. Growth curve, MTT and DNA cycle were used to evaluate the proliferation ability of MSC and EPC. MSC were identified with induced differentiation into the osteoblasts and adipocytes, and their immune phenotype was detected by flow cytometry (FCM). EPC were characterized by the special digestion of Dil-ac-LDL, FITC-UEA-I, and the conjunction with CD133, VEGFR2/KDR and CD34, their purity was also calculated. The results indicated that the colony was obviously formed when the mononuclear cells were cultured for 24 hours and, 80% of the cells became long spindle and integrated at d 8. Cells, which were adhered for twice, were cultured with EGM-2MV medium, began to extend at d 3, and became strip-shaped and integrated for about 80% at d 8. Passage survival rates were more than 90% for both cells, and after passage 2 the growth curve was like "S". Optical density was changed obviously when the cells were cultured for 3 - 5 d, but there were no significant difference of cell cycles between MSC and EPC, which G0-G1 was (93.32 ± 1.65)% and (93.05 ± 1.95)% respectively. Positive rates were (99.7 ± 1.12)%, (99.1 ± 2.33)%, (4.8 ± 0.38)%, (6.8 ± 0.49)% and (0.4 ± 0.08)% for CD90, CD44, CD14, CD45 and CD79a respectively. MSC were identified by induced differentiation into osteoblasts and adipocytes. Positive rates of the EPC, which were adhered for twice and passaged 2, were (82.1 ± 3.4)% for fluorescent staining of Dil-ac-LDL and FITC-UEA-I, and (74.2 ± 3.2)%, (64.7 ± 4.3)% and (43.5 ± 1.5)% for CD133, VEGFR2/KDR and CD34 respectively. It is concluded that high-purity MSC can be obtained with density gradient and differential adhesion method, and high-proliferation EPC can be cultured with EGM-2MV medium in cell plates enclosed with fibronectin, so they may become the optimal seed cells for tissue engineering study.

Expressional time phase of leukocyte molecules induced by allogenic cardiac antigen and cyclosporin A in rats' in vitro model.

UNLABELLED: The immunosuppressive agent cyclosporin A has been proven to reduce the rejection rate and prolong the survival time of transplanted hearts. But some reports showed that cyclosporine A did not completely suppress the rejection. We performed in vitro studies to model a time course to observe the effect of cyclosporin A. METHODS: The experiment was divided into a control group (group I), an antigen group (group II), a cyclosporin A group (group III) and an antigen + cyclosporin A group (group IV). After transplantation, at 2 h, 6 h, 12 h, 24 h, 48 h and 72 h, leukocyte molecules were monitored. RESULTS: The expression of IL-2R peaked at 12 h in group II and at 6 h in group III. There was a gradual decline in the expression of the P59 gene in group I, positive expression at 2 h and between 12 h and 24 h in group II, in group IV, there was a decrease at 48 h. The expression of the CD4 gene was lowest at 2 h in group I and at 6 h in group II. CD4 expression then quickly increased to a maximum at 48 h in group III, at 2 h in group IV. There was a minimal expression was reached at 12 h in group I and IV and at 6 h in group III in the expression of the CD8 gene. CONCLUSIONS: Alloantigen induced lymphocytes to release IL-2R and P59 and stimulated the induction of the CD4 gene' transcription for 6 h. Cyclosporin A stimulated the release of IL-2R for 2 h. These results provide an in vitro basis for describing the time phases of rejection inhibited by cyclosporin A.

[Primary culture and biological characteristics of cardiac fibroblasts of adult mice].

AIM: To establish a set of reliable methods of isolation, culture and characterization of cardiac fibroblasts from adult mice. METHODS: Fibroblasts were isolated from adult mice and cultured using combined trypsin-type II collagenase digestion method or tissue block culture method. The cell growth of cardiac fibroblasts was observed under an inverted phase contrast microscope. Cell viability of the different cell passages was evaluated by Trypan blue staining. The proliferation of cardiac fibroblasts was analyzed by growth curve, MTT assay and DNA cell cycle analysis, respectively, to observe the effects of different culture media on the growth of cardiac fibroblasts. The expressions of vimentin, fibronectin and discoidin domain receptor 2 (DDR2) on the cardiac fibroblasts were observed by immunofluorescence staining. RESULTS: After 1 d culture with the enzyme digestion method, the adherent cells were ellipse observed under an inverted phase contrast microscope and after 3 d, cells were spindle and grew quickly. But only after 4 d isolation and culture by the block culture method, it was observed that cells grew from the border of tissues and after 7 d, the cells started to amplify gradually. The cell viability rate of both culture methods were more than 97%. Growth curve of the third passage presented a "S" shape by both methods. MTT assay showed the optimal cell proliferation value on day 3 to day 5. The ratio of G0-G1 phase and S+G2+M phase were respectively 62.61% and 30.87% by enzyme digestion method, and 69.24% and 28.05% by block culture method. Log phase was found in cardiac fibroblasts cultured in HG/DMED supplemented with 10% fetal bovine serum. The cell colonies like a swirl were observed at the third passage by HE staining. Under a fluorescent microscope, the cells were highly positive for the expressions of vimentin, fibronectin and DDR2 which were the classical phenotype of the fibroblasts. CONCLUSION: The cardiac fibroblasts could be effectively obtained by both enzyme digestion and block culture methods. Compare to the block culture method, the cells isolated by the combined enzyme digestion method expressed the higher levels of fibroblasts markers such as vimentin, fibronectin and DDR2.