OBJECTIVES: To observe the changes of the formation time of venous thrombus in rats, and to provide new ideas and methods for the estimation on thrombus formation time of the forensic cases died from thrombosis. METHODS: Totally 80 rats were randomly divided into 10 groups （0 h, 3 h, 6 h, 12 h, 1 d, 3 d, 1 week, 2 weeks, 3 weeks and 4 weeks after operation）. A vein thrombosis model was established by the "narrow" method. The processes of thrombosis, organization, recanalization and the features of change on hemosiderin and calcium salt were observed by HE stain, Perls stain and Von Kossa stain. The expression changes of CD61, α-SMA and CD34 were observed by immunohistochemical staining technique. RESULTS: Platelets adhered to the exposed blood vessel intima 3 h after operation, and platelet trabeculae were formed by the repeated accumulation of platelets 1 d after operation. The thrombus organization formed through the fibroblasts from vessel wall that grew into the interior of the thrombus 3 d after operation. Endothelial cells covered the surface of thrombus and then the new blood vessels were reformed, and the vessels were reconstructed. The expression of CD61 upregulated at the stages of the thrombus formation （3 h） and thrombus reformation （4 weeks）, and reached the peak 1 d after thrombus formation. The release of hemosiderin and the initial expression of α-SMA were detected 3 d later. Calcium deposit and expression of CD34 were observed 1 week later. CONCLUSIONS The hemosiderin, calcium salt, CD61, α-SMA and CD34 show time-dependent changing characteristics, which is expected to provide a reference for the estimation on thrombus formation time of the forensic cases died from thrombosis.
Animals ; Antigens, CD34/analysis* ; Hemosiderin/metabolism* ; Rats ; Venous Thrombosis/pathology*

Although widely applied in treating hematopoietic malignancies, transplantation of hematopoietic stem/progenitor cells (HSPCs) is impeded by HSPC shortage. Whether circulating HSPCs (cHSPCs) in steady-state blood could be used as an alternative source remains largely elusive. Here we develop a three-dimensional culture system (3DCS) including arginine, glycine, aspartate, and a series of factors. Fourteen-day culture of peripheral blood mononuclear cells (PBMNCs) in 3DCS led to 125- and 70-fold increase of the frequency and number of CD34⁺ cells. Further, 3DCS-expanded cHSPCs exhibited the similar reconstitution rate compared to CD34⁺ HSPCs in bone marrow. Mechanistically, 3DCS fabricated an immunomodulatory niche, secreting cytokines as TNF to support cHSPC survival and proliferation. Finally, 3DCS could also promote the expansion of cHSPCs in patients who failed in HSPC mobilization. Our 3DCS successfully expands rare cHSPCs, providing an alternative source for the HSPC therapy, particularly for the patients/donors who have failed in HSPC mobilization.
Antigens, CD34/metabolism* ; Hematopoietic Stem Cell Transplantation ; Hematopoietic Stem Cells ; Humans ; Leukocytes, Mononuclear/metabolism* ; Peptides/metabolism*

No abstract available.
Adult ; Antigens, CD34/metabolism ; Female ; Hemangiosarcoma/diagnosis/*pathology/surgery ; Humans ; Liver Neoplasms/diagnosis/*pathology/surgery

OBJECTIVETo investigate the mRNA expression profiles of megakaryocytes (MKs) from human cord blood CD34+ cells ex vivo expanded using Solexa technique.

METHODSCD34+ Cells were isolated using density gradient centrifugation and magnetic activated cell sorting. Cultures were stimulated with recombinant human thrombopoietin (100 ng/ml). After 12 days, the MKs fraction was separated from the non-MKs fraction using an anti-CD41 monoclonal antibody by immunomagnetic sorting. The mRNA expression of MKs and non-MKs was detected by Solexa sequencing.

RESULTSWe obtained 3 773 147 and 3 533 805 Tags from MKs and non-MKs, respectively. The amounts of unambiguous tags were 3 291 132 and 2 967 947 and those of distinct tags were 197 769 and 245 318. The expression of 1161 genes was up-regulated and that of 902 genes down-regulated. The expression of 2717 tags was up-regulated and that of 1519 tags down-regulated.

CONCLUSIONSMKs and non-MKs have remarkably different mRNA expression profiles. The differential gene-encoded products may be involved in cellular development, adhesion, apoptosis metabolism, intra- and intercellular signal transduction, and immune response. Further studies on this topic may clarify the expression mechanism, signal transduction, and regulation mechanisms.

Antigens, CD34 ; Cells, Cultured ; Fetal Blood ; cytology ; Humans ; Megakaryocytes ; cytology ; metabolism ; RNA, Messenger ; genetics ; Transcriptome

Vascular wall-resident stem cells (VW-SCs) play a critical role in maintaining normal vascular function and regulating vascular repair. Understanding the basic functional characteristics of the VW-SCs will facilitate the study of their regulation and potential therapeutic applications. The aim of this study was to establish a stable method for the isolation, culture, and validation of the CD34⁺ VW-SCs from mice, and to provide abundant and reliable cell sources for further study of the mechanisms involved in proliferation, migration and differentiation of the VW-SCs under various physiological and pathological conditions. The vascular wall cells of mouse aortic adventitia and mesenteric artery were obtained by the method of tissue block attachment and purified by magnetic microbead sorting and flow cytometry to obtain the CD34⁺ VW-SCs. Cell immunofluorescence staining was performed to detect the stem cell markers (CD34, Flk-1, c-kit, Sca-1), smooth muscle markers (SM22, SM MHC), endothelial marker (CD31), and intranuclear division proliferation-related protein (Ki-67). To verify the multipotency of the isolated CD34⁺ VW-SCs, endothelial differentiation medium EBM-2 and fibroblast differentiation medium FM-2 were used. After culture for 7 days and 3 days respectively, endothelial cell markers and fibroblast markers of the differentiated cells were evaluated by immunofluorescence staining and q-PCR. Furthermore, the intracellular Ca²⁺ release and extracellular Ca²⁺ entry signaling were evaluated by TILLvisION system in Fura-2/AM loaded cells. The results showed that: (1) High purity (more than 90%) CD34⁺ VW-SCs from aortic adventitia and mesenteric artery of mice were harvested by means of tissue block attachment method and magnetic microbead sorting; (2) CD34⁺ VW-SCs were able to differentiate into endothelial cells and fibroblasts in vitro; (3) Caffeine and ATP significantly activated intracellular Ca²⁺ release from endoplasmic reticulum of CD34⁺ VW-SCs. Store-operated Ca²⁺ entry (SOCE) was activated by using thapsigargin (TG) applied in Ca²⁺-free/Ca²⁺ reintroduction protocol. This study successfully established a stable and efficient method for isolation, culture and validation of the CD34⁺ VW-SCs from mice, which provides an ideal VW-SCs sources for the further study of cardiovascular diseases.
Mice ; Animals ; Endothelial Cells ; Cell Differentiation/physiology* ; Stem Cells ; Adventitia ; Fibroblasts ; Cells, Cultured ; Antigens, CD34/metabolism*

Adult ; Antigens, CD34 ; metabolism ; Humans ; Male ; Neoplasms, Fibrous Tissue ; metabolism ; pathology ; surgery ; Prostatectomy ; Prostatic Neoplasms ; metabolism ; pathology ; surgery ; Vimentin ; metabolism

OBJECTIVETo detect the expressions of transforming growth factor-beta1 (TGF-beta1), Desmin and CD34 in the penile corpus cavernosum of SD rats in different age groups.

METHODSWe randomly selected 10 SD rats in each of the 2-, 5- and 20-month age groups, harvested their penile corpus cavernosum tissues under ether anesthesia, and detected the mRNA and protein expressions of TGF-beta1, Desmin and CD34 by RT-PCR and immunohistochemistry.

RESULTSThe results of RT-PCR showed the mRNA expressions of TGF-beta1, Desmin and CD34 in the corpus cavernosum tissues, with significant differences between every two groups (P < 0.01). The TGF-beta1 protein was mainly expressed in the trabeculae and around the arteries of the corpus cavernosum for membrane and cytoplasm staining, the Desmin protein mainly in the membrane and cytoplasm for muscle tissue staining; and the CD34 protein mainly in the vascular and sinusoidal endothelia. The mRNA expression of TGF-beta1 was correlated positively (r = 0.944, P < 0.01) while those of Desmin and CD34 negatively with the age of the rats (r = -0.947, P < 0.01; r = -0.934, P < 0.01). And the mRNA expressions of both Desmin and CD34 had a significant correlation with that of TGF-beta1 (r = -0.888, P < 0.01; r = -0.887, P < 0.01).

CONCLUSIONWith the increase of age, the expression of TGF-beta1 is significantly up-regulated, while those of Desmin and CD34 significantly down-regulated in the corpus cavernosum tissues, and it is negatively correlated with the latter two. TGF-beta1 is an important influencing factor on ED.

Age Factors ; Animals ; Antigens, CD34 ; metabolism ; Desmin ; metabolism ; Male ; Penis ; metabolism ; Rats ; Rats, Sprague-Dawley ; Transforming Growth Factor beta1 ; metabolism

OBJECTIVE: To explore the extrinsic regulation mechanism of bone marrow microenvironment in leukemia cells, and investigate the promoting effect of osteoblast niche on the proliferation and self-renewal of leukemia stem cell by up-regulating the expression of interleukin-1 (IL-1) in leukemia cell. METHODS: The gene expression profiles on leukemia cells derived from AE9a mouse bone marrow endosteum and central bone marrow were determined by RNA sequencing and gene set enrichment analysis (GSEA). Quantitative real-time PCR (qRT-PCR) was used to detect the expression of IL-1 in AE9a mouse leukemia cells co-cultured with or without osteoblasts in vitro. In addition, qRT-PCR was also used to determine the expression of IL-1 in bone marrow mononuclear cell (BMMNC) from 43 patients with acute myeloid leukemia (AML). For leukemia cells co-cultured with osteoblasts or treated with IL-1β, colony forming ability of AE9a leukemia cells was determined by colony formation assay. RESULTS: In AE9a leukemia mouse, RNA-seq data and GSEA showed that the enrichment of the upregulated genes in leukemia cells located in endosteum fell into inflammatory response gene set, among them, IL-1α and IL-1β were significantly higher expressed in AE9a leukemia cells that located osteoblast niche (IL-1α: P<0.001, IL-1β:P<0.001). After AE9a leukemia cells were co-cultured with osteoblasts in vitro, the expression of IL-1α and IL-1β in leukemia cells were increased by 2.5 and 3.5 times respectively. In colony formation assay, the number of colonies was increased significantly after leukemia cells were co-cultured with osteoblasts (P<0.001). In addition, when AE9a leukemia cells were treated with IL-1β, the number of colonies was also increased significantly (P<0.01). In AML patients, BMMNC with high percentage of CD34 positive cells exhibited higher level of IL-1 expression. CONCLUSION Osteoblast niche can promote leukemia cell proliferation and self-renewal through up-regulating the expression of IL-1 in leukemia cells. In AML patients, the expression level of IL-1 was correlated to the percentage of CD34 positive cells in BMMNC.
Animals ; Antigens, CD34/metabolism* ; Bone Marrow/metabolism* ; Cell Proliferation ; Leukemia, Myeloid, Acute/metabolism* ; Mice ; Osteoblasts/metabolism* ; Stem Cells ; Tumor Microenvironment

OBJECTIVETo investigate the expression of CD133 in the bone marrow of patients with myelodysplastic syndrome (MDS) and explore its clinical significance.

METHODSThe expression of CD133 and CD34/CD38 in the bone marrow was detected using flow cytometry in 31 cases of refractory anemia with excess blasts (RAEB), 10 cases of refractory cytopenia with multilineage dysplasia (RCMD) and 11 cases of aplastic anemia (AA).

RESULTSThe percentage of CD133-expressing cells was 6.75% in patients with RAEB, significantly higher than that in patients with RCMD (1.41%) and AA (2.70%) (P<0.05); the percentage of CD133-positive cells were similar between the latter two patient groups (P>0.05). The percentage of CD34(+)/CD38- cells was similar in the 3 groups (P>0.05), all lower than 1%.

CONCLUSIONSAdvanced MDS patients are characterized by an increase of CD133-expressing cells, suggesting the value of CD133 in the diagnosis of RAEB. CD34(+)/CD38- cells do not show a significant value in the diagnosis of MDS.

AC133 Antigen ; Anemia, Aplastic ; metabolism ; Antigens, CD ; metabolism ; Antigens, CD34 ; metabolism ; Female ; Flow Cytometry ; Glycoproteins ; metabolism ; Humans ; Male ; Middle Aged ; Myelodysplastic Syndromes ; diagnosis ; metabolism ; Peptides ; metabolism

The Wilms' tumor gene (WT1) is a transcription factor involved in tumorigenesis, especially in leukemogenesis. However, the role of WT1 expression in nonmalignant hematopoietic cells remains unclear. Furthermore, due to alternative splicing at two sites: 17 amino acid residues of exon 5 (+17AA) and 3 amino acid residues (+KTS) between exons 9 and 10, WT1 gene has four main isoforms (17AA+/KTS+, 17AA+/KTS-, 17AA-/KTS+, 17AA-/KTS-, abbreviation: +/+, +/-, -/+, -/-). The isoforms probably existed in hematopoietic cells, which make the research more complex. The aim of study was to elucidate the expression and its isoforms of WT1 gene in different cell subsets of healthy bone marrow donors. The fluorescence RT-PCR detection system was established to measure the expressions of full-length WT1, WT1 (+17AA) and WT1 (+KTS) in CD34(+)CD38(-) (stem cell), CD34(+)CD38(+) (progenitor cell), CD15(+)CD11b(+) (granulocyte), CD33(+)CD14(+) (monocyte), CD20(+)CD5(-) (B-lymphocyte) and CD20(-)CD5(+) (T-lymphocyte) subsets from 18 normal human bone marrow samples. The results showed that WT1 expressed in CD34(+)CD38(-), CD34(+)CD38(+), CD15(+)CD11b(+) and CD33(+)CD14(+), but not in CD20(+)CD5(-) and CD20(-)CD5(+) subsets. The highest expression was in CD34(+)CD38(-), but decreased gradually in CD15(+)CD11b(+) and CD33(+)CD14(+) subsets. WT1 (+17AA), WT1 (+KTS) and WT1 (+/+) isoforms were predominant in CD34(+)CD38(-) and CD34(+)CD38(+) primitive subsets, while in CD15(+)CD11b(+) and CD33(+)CD14(+) the dominant isoforms were WT1 (-17AA), WT1 (-KTS) and WT1 (-/-). It is concluded that the expression of WT1 in normal bone marrow decreases gradually with cell differentiation. Hematopoietic cells may adjust the ratios of WT1 isoforms to inhibit or promote cell differentiation.
Antigens, CD34 ; analysis ; Bone Marrow Cells ; metabolism ; Cell Differentiation ; Gene Expression Regulation ; Humans ; Protein Isoforms ; metabolism ; WT1 Proteins ; metabolism