Isolation and identification of exosomes from mouse primary peritoneal macrophages / 中国生物制品学杂志

@#Objective To isolate,purify and identify exosomes secreted by mouse primary peritoneal macrophages.Methods Five male C57BL/6 mice were intraperitoneally injected with 3% mercaptoacetate broth respectively,and the primary peritoneal macrophages were obtained by lavage,and then the purity was analyzed by flow cytometry.The exosomes of mouse primary peritoneal macrophages were extracted by ExoQuick TC exosome kit,which were measured for the protein content with BCA kit,observed for the morphology by transmission electron microscopy,detected for the particle size and distribution with nanoparticle tracking analyzer,and determined for the expression of exosome-specific markers(CD9,CD63 and TSG101) by Western blot.Results About 5 × 10~6 peritoneal macrophages with the purity of(99.17±0.65)%were obtained from each mouse.Approximately 869 μg of exosomal protein was extracted from 5 mL of mouse primary peritoneal macrophage culture supernatant.The exosomes of mouse primary peritoneal macrophages were typical tea saucerlike vesicles with strong refraction under electron microscopy,and highly expressed the exosome-specific markers TSG101,CD63 and CD9.The particle size distribution was concentrated between 100 and 200 nm,with an average particle size of175.2 nm.Conclusion Intraperitoneal injection of mercaptoacetate broth can improve the yield of mouse primary peritoneal macrophages.ExoQuick TC.exosome kit can extract sufficient amount of exosomes with high purity from mouse primary peritoneal macrophages.

A Non-woven Path: Electrospun Poly(lactic acid) Scaffolds for Kidney Tissue Engineering

Chronic kidney disease is a major global health problem affecting millions of people; kidney tissue engineering provides an opportunity to better understand this disease, and has the capacity to provide a cure. Two-dimensional cell culture and decellularised tissue have been the main focus of this research thus far, but despite promising results these methods are not without their shortcomings. Polymer fabrication techniques such as electrospinning have the potential to provide a non-woven path for kidney tissue engineering. In this experiment we isolated rat primary kidney cells which were seeded on electrospun poly(lactic acid) scaffolds. Our results showed that the scaffolds were capable of sustaining a multipopulation of kidney cells, determined by the presence of: aquaporin-1 (proximal tubules), aquaporin-2 (collecting ducts), synaptopodin (glomerular epithelia) and von Willebrand factor (glomerular endothelia cells), viability of cells appeared to be unaffected by fibre diameter. The ability of electrospun polymer scaffold to act as a conveyor for kidney cells makes them an ideal candidate within kidney tissue engineering; the non-woven path provides benefits over decellularised tissue by offering a high morphological control as well as providing superior mechanical properties with degradation over a tuneable time frame.

Comparison of different special staining techniques of chondrocytes and their application values / 中国比较医学杂志

Objective To compare the advantages and values of several special staining methods of chondrocytes . Methods Twelve 7-day old healthy C57BL/6J mice were killed to obtain the cartilage tissue of the knee joint in order to isolate the chondrycytes .Type II collagen was used to assess the chondrocytes .Then the chondrocyte climbing slices were prepared.The materials were fixed, and HE staining, Safranin O-fast green staining, SA-β-gal staining and immunohistochemical staining of Type II collagen were performed and compared .Results HE staining showed clear morphology of the chondrocytes .The cell nuclei were stained blue and the cytoplasm was pink .Safranin O-fast green staining showed that the nuclei were pink and the cytoplasm green .SA-β-gal staining showed that the aging cells were green while the young cells were colorless .Immunohistochemical staining of type II collagen showed the distribution of type II collagen and they were stained brown while the cell nuclei were blue .Conclusions HE staining and safranin O-fast green staining can provide more information than the other staining techniques .SA-β-gal staining is useful in the analysis of aging chondrocytes .Immunohistochemical of type II collagen can be used to study type II collagen .

Molecular mechanism of apoptosis induced by valproate in primary cells from acute lymphoblastic leukemia in vitro / 白血病·淋巴瘤

Objective To investigate the relation between demethylation effect and apoptosis of valproate (VPA) in primary acute lymphoblastic leukemia (ALL) cells in vitro.Methods 10 cases of ALL patients was choosed to acquire leukemia cells.Cell growth curve were assessed by the MTT assay,the apoptosist of primary ALL was analyzed with DNA Ladder and Annexin-V-FITC/PI by flow cytometry.The expression methylation level of p15 was detected by hn-MSPCR,and p15mRNA were detected by RT-PCR.All dates was analyzed by SPSS16.0.Results The 50 ％ inhibition rate of VPA were 1.898 mmol/L to primary ALL cells assayed by MTT respectively.DNA ladder showed the apoptosis of primary ALL cells increased by adding VPA dose.Annexin-V-FITC/PI tests showed that the apoptosis percentage of primary ALL cells were (0.44±0.04) ％ in control group,(5.80±0.65) ％ in 1.0 mmol/L VPA group,(48.46±2.49) ％ in 2.0 mmol/L VPA group,(76.45±2.98) ％ in 4.0 mmol/L VPA group,the apoptosis percentage increased significantly (P ＜ 0.05).The demethylation of p15 INK4B gene decreased by adding VPA dose,the expression of p15 mRNA expression increased significantly compared with control group by RT-PCR (P ＜ 0.05).Conclusion It is found that VPA could induce demethylation of p15 INK4B gene,which could upregulate the p15 mRNA expression,due to the apoptosis of primary ALL cells.