Expression of EphA2 and ephrin-A1 in endometrial endometrioid adenocarcinoma and their relationship with angiogenesis

AIM: To investigate the expression of erythropoietin-producing hepatocellular receptor A2 (EphA2) and its ligand ephrin-A1 in endometrial endometrioid adenocarcinoma (EEA), and to analyze their relationship with angiogenesis of the tumor. METHODS: The CD34-stained microvessel density (MVD) and the expression of ephA2 and ephrin-A1 were detected by immunohistochemical assay in 56 cases of EEA, 20 cases of endometrial hyperplasia, 30 cases of normal proliferative endometrium and 30 cases of normal secretory endometrium. The correlations among the expression of EphA2 and ephrin-A1, MVD and clinicopathological features were analyzed. RESULTS: MVD and the expression of EphA2 and ephrin-A1 in EEA were significantly higher than those in the tissues from endometrial hyperplasia and normal endometrium (P<0.05). They were related to FIGO stage, histological differentiation, depth of myometrial invasion, lymphovascular invasion and progesterone receptor expression (P<0.05). A significant positive correlation between MVD and the expression of EphA2 and ephrin-A1 was observed by Spearman rank correlation test (r=0.476, P<0.05; r=0.501, P<0.05). CONCLUSION: Overexpression of EphA2 and its ligand ephrin-A1 in EEA may be involved in the angiogenesis and progesterone resistance.     

Downregulation of beta tropomyosin protein expression in colon adenocarcinoma

AIM: To investigate the cancer associated proteins and sensitive biomakers for early diagnosis in colon adenocarcinoma by using proteomic technique.METHODS: Two-dimensional gel electrophoresis was used to define patterns of protein expression in adenocarcinoma tissue from 8 patients with matched normal colonic mucosa. Proteins expressed differently of a 2-fold change were cut and analyzed by MALDI-TOF/TOF mass spectrometry. RESULTS: Two-dimensional protein maps of adenocarcinoma and normal colonic mucosa were gained successfully. Gel-analysis software identified an average of 3 289 spots in adenocarcinoma while 3 066 in normal mucosa and statistical filtering yielded 31 spots of a 2-fold change, 18 of which were identified by using mass spectrometry, including cytokeratin 8, cytokeratin 10, S100A6, beta tropomyosin (TMβ), protein disulfide isomerase, etc. Functional analysis revealed that these proteins were associated with adenocarcinoma cellular oncogenesia, proliferation, differentiation, metastasis and so on. The results of Western blotting validated that the expression level of TM β in colon adenocarcinoma was much lower than that in matched normal colonic mucosa.CONCLUSION: Proteomic analysis can identify the proteins with variance of colon adenocarcinoma versus normal colonic mucosa. Downregulation of TM β might serve as a new biomarker of colon adenocarcinoma.     

Proliferation and apoptosis of neuroendocrine cells in ovarian epithelial tumors

AIM: The purpose of this study was to observe the morphological features of neuroendocrine cells (NECs)， their proliferation and apoptosis in ovarian epithelial tumors, and to discuss their biological and clinical significance. METHODS: 79 specimens of ovarian epithelial tumor samples were collected, of them 20 benign, 18 boderline, 41 milignant tumors, and 22 normal ovaries were investigated immunohistochemically. Chromogranin A was used to detect NECs and their proliferation and apoptosis were examined by double-label staining of chromogranin A and Ki67 or TUNEL. RESULTS: The positive rate of CgA, distribution and staining intensity in ovarian epithelial tumors were higher than those in normal ovary. NECs showed various shapes with neuronoid protuberances stretching to the neighboring cells or basement membrane. Occasionally, they might touch together. No TUNEL positive coexpression in all NECs was observed by double-label staining, but some NECs were coexpressed with Ki67. CONCLUSION: NECs of ovarian epithelial tumors like cancer cells showed a proliferation, but no apoptosis. Their secretion might promote their neighboring non-NECs to proliferate and prevent them from apoptosis.     

Clinical significance of KLF15 expression in human lung adenocarcinoma

AIM: To explore the clinical significance of Krüpple-like factor 15 (KLF15) protein expression in the patients with lung adenocarcinoma for exploring the therapeutic and prognositic biomarkers of lung cancer. METHODS: Four cases of lung adenocarcinoma tissues and matched adjacent tissues were collected from our hospital, and the expression of KLF15 protein in these tissues was analyzed by Western blot. At the same time, 72 cases of archived paraffin-embedded samples and clinical data of the patients with lung adenocarcinoma were also collected. The KLF15 protein expression in the archived paraffin-embedded lung adenocarcinoma samples was detected by immunohistochemical staining. The correlations between KLF15 protein expression and clinical characteristics of the patients including prognosis were also analyzed. In addition, the KLF15 protein was up-regulated in A549 cells, and then the effects of KLF15 protein on the viability of the cells were measured by CCK-8 assay. RESULTS: The protein expression of KLF15 in the 4 cases of lung adenocarcinoma tissues was significantly lower than that in matched paracancerous tissues. Fifty-three cases of lung adenocarcinoma specimens showed low expression or no expression of KLF15 protein in total 72 cases (73.6%). The 5-year survival rate of the patients with high expression of KLF15 protein in their specimens was higher than that of the patients with the low expression of KLF15 protein (P<0.01), and the expression of KLF15 protein was significantly correlated with the pathological staging (P<0.01) and T stage (P<0.01) of the patients with lung adenocarcinoma. Furthermore, the low expression of KLF15 protein was an important poor prognostic indicator of the patients. Up-regulation of KLF15 protein in the A549 cells significantly inhibited the growth of the cells. CONCLUSION: KLF15 inhibits the growth of lung adenocarcinoma cells. It could be used as a therapeutic target and a prognostic biomarker for the patients with lung adenocarcinoma.     

Expression of osteopontin in endometrial carcinoma and cervical cancer

AIM: To study the expression and significance of osteopontin (OPN) in endometrial carcinoma and cervical cancer. METHODS: Immunohistochemical S-P assay was used to detect the expression of OPN in paraffin-embedded sections of 30 cases of endometrial carcinoma, 20 cases of cervical cancer and 30 cases of normal control tissues. The relationship between OPN expression and clinical-pathological characteristics was evaluated. RESULTS: The positive immunostaining rates of OPN in endometrial carcinoma (70%) and cervical cancers (55%) were significantly higher than that in the normal secretive and proliforative endometrium (50% and 10%) and normal cervical epithelium (10%), respectively (P<0.01). The positive immunostaining rate of OPN in squamous cell carcinoma and adenocarcinoma of the cervix was 53.3% and 60%, respectively, there was no significant difference between the two groups (P>0.05). The positive immunostaining rate of OPN in stage Ⅲ and stage Ⅱ and G3 and G2 of endometrial carcinoma was significantly higher than that in stage Ⅰ and type G1 of endometrial carcinoma. The positive immunostaining rate of OPN in stage Ⅱb and type G3 of cervical cancer was significantly higher than that in stage Ⅰa and type G1 of cervical cancer. CONCLUSION: OPN is significantly highly expressed in both endometrial carcinoma and cervical cancer, and its expression is closely related to the stage and grading of these malignant tumors.     

Clinical significance and prognostic value of β-catenin expression in colonic adenocarcinoma

AIM: To observe the expression of β-catenin in colonic adenocarcinoma and to investigate its clinical significance and prognostic value. METHODS: The integrated clinical and follow-up data of 52 patients, who were undergone radical operation, were retrospectively analyzed from June 2000 to June 2004 in our hospital. The paraffin-embedded tissues of 52 colonic adenocarcinoma specimens and 20 cases of normal paraneoplastic colonic mucous tissues were detected for β-catenin expression by immunohistochemical method. The relationships between β-catenin and clinical variables and prognostic value were statistically analyzed. RESULTS: β-catenin was normally expressed in all 20 normal cases. In the cases of colonic adenocarcinoma, the abnormal expression rate of β-catenin was 71%. The abnormal expression of β-catenin did not correlated with gender, age, histological differentiation and blood CEA (P>0.05), but it was correlated with lymph node metastasis and clinical stage (P<0.05). The abnormal expression of β-catenin were also correlated with the postoperative survive time (P<0.05). CONCLUSION: The abnormal expression of β-catenin is correlated with the lymph node metastasis and clinical stage, and the abnormal expression is an important adverse prognostic factor for survival in the patients with colonic adenocarcinoma. β-catenin may be a molecular prognostic marker in the patients with colonic adenocarcinoma.     

Differentiation of bone marrow mesenchymal stem cells into cardiomyocyte-like cells in vitro via cardiotrophin 1

AIM: To investigate the effects of cardiotrophin 1 (CT-1) on differentiation of swine bone marrow mesenchymal stem cells (MSCs) into cardiomyocyte-like cells in vitro.METHODS: MSCs were isolated and proliferated from Tibet miniswine. Adipogenic and osteogenic potentials were identified. MSCs were divided into 4 groups for induction: untreated group, 5-azacytidine (5-Aza) group,CT-1 group and 5-Aza combined with CT-1 group. After induction for 4 weeks, the expression of cardiac cell markers including α-actin and cardiac troponin-T (cTnT) was estimated by immunofluorescence staining. Finally, the rates of red fluorescence positive-staining cells were calculated. RESULTS: The expression of α-actin in the 4 groups by red fluorescence staining was as follows: the differentiation rate of cardiomyocyte-like cells in combination group was 29.90%±4.76%, significantly higher than that in 5-Aza group (17.73%±2.34%, P<0.01), CT-1 group (6.63%±0.55%, P<0.01) and untreated group (1.62%±0.09%, P<0.01). The differentiation rate in 5-Aza group was significantly higher than that in CT-1 group (P<0.01) and untreated group (P<0.05). The differentiation rate in CT-1 group was significantly higher than that in untreated group (P<0.01). The expression of cTnT in the 4 groups was as follows: the differentiation rate of cardiomyocyte-like cells in combination group was 36.50%±4.09%, significantly higher than that in 5-Aza group (14.37%±1.65%, P<0.01), CT-1 group (7.50%±0.61%, P<0.01) and untreated group (1.12%±0.23%, P<0.01). The differentiation rate in 5-Aza group was significantly higher than that in CT-1 group (P<0.01) and untreated group (P<0.01). The differentiation rate in CT-1 group was significantly higher than that in untreated group (P<0.01).CONCLUSION: Appropriate concentrations of 5-Aza (10 μmol/L) and CT-1 (0.1 μg/L) induce swine bone marrow MSCs to differentiate into cardiomyocyte-like cells in vitro. CT-1 combined with 5-Aza significantly increases the differentiation rate.     

Toll-like receptor 4 expression on mast cells in human gingival tissues with chronic periodontitis

AIM: To observe the expression of Toll-like receptor 4 (TLR4) on mast cells in human gingival tissues with chronic periodontitis. METHODS: A total of 68 volunteers, including 23 cases of mild chronic periodontitis, 25 cases of severe chronic periodontitis and 20 healthy controls, were involved in this study, and their gingival specimens were taken and fixed in 4% neutral formalin. The histological changes of gingival tissues were observed by HE staining. The expression of TLR4 in gingival tissues was detected by immunohistochemical staining, and TLR4 expression on mast cells was detected by immunofluorescence double staining. RESULTS: The expression of TLR4 in gingival tissues and on mast cells in chronic periodontitis groups was significantly higher than that in normal control group (P<005), and that in severe chronic periodontitis group was significantly higher than that in mild chronic periodontitis group (P<005). CONCLUSION: The expression of TLR4 in gingival tissues and on mast cells is increased with the severity of chronic periodontitis, suggesting that TLR4, especially TLR4 on mast cells, may play an important role in human chronic periodontitis.     

Mortalin expression in cervical squamous-cell carcinoma and its effect on tumor metastasis

AIM: To investigate the significance of mortalin expression in clinical pathology of cervical squamous-cell carcinoma. METHODS: Immunofluorescence staining was used to detect the location of mortalin in human cervical squamous-cell carcinoma SiHa cells. The protein expression of mortalin was detected in 59 cases of normal cervical epithelial tissues and 93 cases of cervical squamous-cell carcinoma tissues by immunohistochemical staining, and its correlation with clinicopathological features of cervical squamous-cell carcinoma was also analyzed. MTT assay was used to evaluate the optimal concentration and dosing time of mortalin inhibitor MKT-077. After the protein expression of mortalin in SiHa cells was inhibited, wound-healing and migration assays were performed. The protein expression of epithelial-mesenchymal transition (EMT)-related molecules was determined by Western blot. RESULTS: Immunofluorescence staining showed that mortalin was located in the cytoplasm of SiHa cells. The positive rate and strongly positive rate of mortalin in the cervical squamous-cell carcinoma patients were 88.7% (55/62) and 61.3% (38/62), respectively, and they were significantly higher than those in normal cervical epithelial tissues (23.7% and 5.1%, P < 0.01). Additionally, mortalin expression was statistically correlated with the histological grade, clinical stage and lymph node metastasis. After inhibiting the expression of mortalin in the SiHa cells by MKT-077, the results of wound-healing and migration assays showed that the migration ability of SiHa cells was down-regulated. The protein expression of E-cadherin was up-regulated, and vimentin and Snail were significantly down-regulated. CONCLUSION: Mortalin over-expression is an effective biomarker for prediction of malignant potential and poor prognosis of cervical squamous-cell carcinoma.     

Differentiated expression of myosin VI in diverse human carcinomas

AIM: To investigate the expression profile of myosin VI in various human carcinomas and adjacent normal tissues. METHODS: A piece of cancer profiling array containing 154 matched cDNA pairs from 19 tumors and the adjacent normal tissues and 10 diverse tumor cell lines were separately hybridized with radiolabeled probes for myosin VI and housekeeping gene ubiquitin. Immunohistochemistry (IHC) was used to confirm the expression profile of myosin VI in 40 cases of ovarian adjacent normal tissues, 8 cases of cystadenoma, 16 cases of borderline tumors and 52 cases of endometrioid carcinoma by tissue microarray. RESULTS: Myosin VI was expressed in all the tissues and cell lines. The expression of myosin VI was significantly higher in ovarian and colon cancer tissues than that in matched normal tissues. The results of IHC confirmed that myosin VI expression rates were 100% (52/52), 81.3% (13/16) and 10.4% (5/48) in the ovarain carcinoma, boderline tumor and nomal ovarian epithelium or cystadenoma, respectively. The expression of myosin VI protein was significantly higher and stronger in ovarain carcinoma than that in the borderline tumor, benign tumor or normal ovary tissues. A significant correlation was also found between the expression of myosin VI and the tumor grade of ovarain carcinoma. CONCLUSION: Differentiated expression of myosin VI is found in diverse malignant tumor tissues and cell lines, suggesting myosin VI plays an important role in the tumor development and progression.     

Significance of CK19 expression in detecting lymph node micrometastases in patients with laryngeal carcinoma

AIM: To evaluate the significance of cytokeratin19 (CK19) expression in diagnosing micrometastases in lymph nodes in patients with laryngeal carcinoma. METHODS: Forty cases of laryngeal carcinoma together with 163 lymph nodes were studied by the staining with hematoxylin and eosin(HE)and immunostaining with antibody against cytokeratin 19 (CK19) on histological sections of the primary tumor and regional lymph nodes. RESULTS: In all cases, CK19 was positively expressed in the primary tumor. Among 163 lymph nodes, metastases were confirmed by HE in 23 lymph nodes, and in 42 lymph nodes staining with immunohistochemistry, micrometastases were found in 19 lymph nodes. Micrometastases in the lymphonodes were significantly related to the T staging. CONCLUSION: Immunohistochemical staining is a valuable method for the detection of node micrometastases in patients with laryngeal carcinoma.     

Biological characteristics of chronic myelogenous leukemia bone marrow derived mesenchymal stem cells

AIM: To study the biology characteristics of mesenthymal stem cells (MSCs) derived from chronic myelogenous leukemia(CML) and normal adult bone marrow.METHODS: Mononuclear cells from chronic myelogenous leukemia (n=19) and normal adult (n=8) bone marrow were obtained, cultured in expanded medium with low serum concentration.Cell morphology, cell cycle, immunophenotype and in vitro differentiation capacity were investigated.The differentiations of osteocytes and adipocytes were detected by von Kossa staining and Oil-red O staining.The chimeric oncogene BCR/ABL and Ph chromosome, two hall marks of CML, were detected in CML derived MSCs, normal adult MSCs, CML derived hematopoietic cells and K562 cells.RESULTS: CML and normal adult derived MSCs showed similar characteristics in cell morphology, phenotype and growth pattern.A typital fibrablast like morphology was observed.Under suitable conditions, CML and normal adult MSCs had the similar ability to differentiate into adipocytes and osteoblasts in vitro.Moreover, CML and normal adult MSCs did not express BCR/ABL gene products and Ph chromosome was not observed.CONCLUSIONS: We isolated and cultured a population of cells with characteristics of multipotent stem cells from CML bone marrow.There were similar biologic characteristics and differentiation ability between normal adult and CML bone marrow-derived MSCs.     

Influence of all-trans retinoic acid on proliferation and differentiation of neural stem cells from new born rat striatum

AIM: To study the effect and mechanism of all-trans retinoic acid (atRA) on neural stem cell (NSCs) proliferation and differentiation from new born Sprague-Dawley rat striatum. METHODS: NSCs were isolated from the brains of new born Sprague-Dawley rat striatum, and the features of cells were characterized by immunofluorescence staining. The effects of different culture medium on cell cycle distribution and proliferation of NSCs were determined by flow cytometry (FCM). The effects of atRA on differentiation of NSCs were determined by immunofluorescence staining and classified count of differentiated cells. RESULTS: FCM assay indicated that atRA inhibited the proliferation of NSCs. The percentage of cells in G0/G1 phase in atRA treatment group was significantly higher than that in control, and the proliferation index (PI) was significantly low. The percentage of neurons differentiated from NSCs in atRA group was 2.5 times of the control group after induced by adding 10% FCS in culture medium. CONCLUSION: atRA counteracts the effects of bFGF on the promotion of mitosis and inhibition of differentiation of NSCs. atRA also promotes NSCs to differentiate into neurons in vitro.     

Effect of interfering TGF-β receptor Ⅱ expression on viability,differentiation and apoptosis of NB4 cells

AIM: To evaluate the effect of interfering TGF-β receptor Ⅱ (TβRⅡ) expression on the viability and differentiation of human acute promyelocytic leukemia NB4 cells induced by all-trans retinoic acid (ATRA) and their apoptosis induced by arsenic trioxide (ATO). METHODS: The technique of lentivirus-mediated RNA interference was used to obtain stable NB4 cells with TβRⅡ knockdown, named TβRⅡ-shRNA NB4 cells. CCK-8 assay was used to detect the viability of TβRⅡ-shRNA NB4 cells. The expression level of CD11b was analyzed by flow cytometry, and Wright-Giemsa staining was used to detect the effects of ATRA on the differentiation of TβRⅡ-shRNA NB4 cells. Double staining (Annexin V-FITC/PI) and AO/EB staining were used to detect the effects of ATO on the apoptosis of TβRⅡ-shRNA NB4 cells. RESULTS: The viability of TβRⅡ-shRNA NB4 cells was significantly higher than that of NB4 parental cells. The differentiation was induced in TβRⅡ-shRNA NB4 cells and NB4 parent cells by treatment with ATRA at different concentration (0.01, 0.02, 0.04, 0.08, 0.1 μmol/L) for 96 h. The differentiation rate of TβRⅡ-shRNA NB4 cells was lower than that of NB4 parental cells in a dose-dependent manner. ATO induced apoptosis of TβRⅡ-shRNA NB4 cells and NB4 parent cells at different concentrations (2, 4 and 8 μmol/L) for 24 h. The apoptotic rate of TβRⅡ-shRNA NB4 cells was lower than that of NB4 parental cells dose-dependently. At the concentration of 8 μmol/L for 24 h, the apoptotic rates in TβRⅡ-shRNA NB4 cells and NB4 cells were (49.15±2.05)% and (66.85±2.41)%, respectively (P<0.01). CONCLUSION: Down-regulation of TβRⅡ increases the viability of NB4 cells, inhibits NB4 cell differentiation induced by ATRA, and also inhibits apoptosis induced by ATO.     

Expression of GLUT4 in endometrium of rats with polycystic ovary syndrome

AIM:To explore the expression of glucose transporter 4 (GLUT4) in the endometrium of rats with polycystic ovarian syndrom (PCOS) and evaluate the relationship between GLUT4 expression and insulin resistance (IR). METHODS:54 female SD rats of 85 days were randomized to control group (n=20), PCOS model group (n=17) and metformin treatment group (n=17). The rats in the latter two groups were induced by Poretsky’s method for PCOS model, followed by placebo or metformin, respectively. After 14 days of treatment, the rats were sacrificed and the expression of GLUT4 in endometrium was detected by Elivision^TM Plus two steps immunohistochemical staining. RESULTS:The expression of GLUT4 and insulin receptor(INS-R) proteins of endometrial glandulan epitheliu in PCOS rats were significantly lower (P<0.01,P<0.05) than those in control group, however, the expression of insulin(INS) protein in PCOS rats was higher than that in control group (P<0.01). The expression of GLUT4 in the treatment group increased (P<0.01), but was still lower than that in control group (P<0.01). However, compared with PCOS group, the expression of INS protein was decreased (P<0.05), but was still higher than that in control group (P<0.05). There was no GLUT4 expression in interstitial cells in endometrium, and the changes of the expressions of INS and INS-R proteins in those cells were similar with those in glandulan epitheliu. CONCLUSION:The decrease in GLUT4 expression of endometrium in PCOS rats is related with endometrial insulin resistance.     

Expression relevance of GATA-1 and miR-451a in erythroid differentiation of K562 cells under hypoxia

AIM To investigate the expression relevance of GATA binding protein-1 (GATA-1) and microR?NA-451a (miR-451a) in erythroid differentiation of human chronic myeloid leukemia K562 cells under hypoxia. METHODS The K562 cells were divided into 2 groups: normoxia group and hypoxia (1% O₂) group, and 40 μmol/L hemin chloride was used to induce K562 cell differentiation for 48 and 72 h. The mRNA expression of γ-globin was detected by RT-qPCR, hemoglobin production was observed by benzidine staining, and flow cytometry was used to detect CD235a expression for verifying erythroid differentiation model. The protein expression of GATA-1 during K562 cell differentiation under normoxia and hypoxia was determined by Western blot. RT-qPCR was used to detect the mRNA expression of GATA-1 and the expression level of miR-451a, and their correlation was analysis. The K562 cells were infected by lentivirus for over-expression or knock-down of GATA-1. Meanwhile, the morphological changes of the cells in the above groups were analyzed by Wright-Giemsa staining method to clarify the erythroid differentiation of K562 cells. The expression miR-451a was detected by RT-qPCR after GATA-1 over-expression or knock-down. REULTS: Under normoxia and hypoxia conditions, the expression levels of γ?-globin and CD235a and the positive rate of benzidine staining at 48 and 72 h were significantly higher than those at 0 h (P<0.05).At 72 h, the expression levels of γ?-globin and CD235a and the benzidine staining positive rate in hypoxia group were significantly higher than normoxia group (P<0.05). The expression of GATA-1 mRNA and miR-451a under hypoxia showed an upward trend during the erythroid differentiation of K562 cells, and was significantly higher than that in normoxia group at 72 h (P<0.05). Correlation analysis showed that the mRNA expression of GATA-1 was positively correlated with miR-451a expression under hypoxia (P<0.01). After over-expression of GATA-1 under hypoxia, the expression of γ-globin and CD235a, the positive rate of benzidine staining, and the cell counts of size augmentation, nuclear deflection and nuclear shrinkage at 72 h were significantly higher than those in negative control group (P<0.05). After knock-down of GATA-1 under hypoxia, the expression of γ-globin and CD235a, the benzidine staining positive rate, and the cell counts of size augmentation, nuclear deflection and nuclear shrinkage at 72 h were significantly lower than those in negative control group (P<0.05). Compared with negative control group under hypoxia, the expression of miR-451a was significantly increased after GATA-1 over-expression (P<0.05), while the expression of miR-451a was significantly decreased after GATA-1 knock-down (P<0.05). CONCLUSION Hypoxia increases the expression of GATA-1 and then up-regulates miR-451a to promote erythroid differentiation of K562 cells.     

In vitro hematopoietic stem cells from embryonic stem cells reconstruct hematopoiesis in mice

AIM: To investigate the potential of hematopoietic stem cells (HSCs) derived from mouse embryonic stem cells (ESCs) to reconstruct hematopoiesis in vivo. METHODS: Using a three-step method, a mice embryonic stem cell line, E14.1 was induced into hematopoietic stem cells. The cell markers with CD34+/ Sca-1+ were identified by flow cytometry analysis, then HSCs (1×109 cells/L) from third-step were injected into SCID mice for observing teratoma formation. To validate function of HSCs, colonogenic cell assay was conducted and the hematopoiesis in lethally irradiated mice was reconstituted. RESULTS: The method of three-step differentiation, combined to use more hematopoietic stimulating factor promoted the E14.1 cell differentiation into HSCs with highest percent of CD34+/Sca-1+ cells (as high as 58.64%±4.20%) with more CFU-E, CFU-GM and CFU-GEMM populations. The cells showed the character of hematopoietic progenitors by Wright-Giemsa staining. Positive selected CD34+/Sca-1+ cells by magnetic sorting from third-step differentiation were transplanted into 7 lethally irradiated female mice while predominant hematopoietic reconstitution were observed in 10 d after transplantation, with 71.4% (5/7) successful engraftment rate. Three recipients showed that the cell population of the peripheral blood leukocytes, red blood cells and hemoglobin approached to normal index at 40 d after transplantation, but followed relative slow renew in platelet count. Survival rate in transplant group was 43%, compared to 100% mortality in control mice. Karyotyping assays confirmed the female mice with XY. CONCLUSION: The three-step differentiation and the culture conditions described here support the differentiation of mouse ESCs into HSCs. HSCs derived from mouse ESCs can reconstruct hematopoiesis.     

Acylation stimulating protein induces preadipocyte differentiation

AIM: To study the role of acylation stimulating protein (ASP) in the differentiation of 3T3-F442A preadipocytes. METHODS: Differentiation of 3T3-F442A preadipocytes was induced by ASP. The morphological changes were observed by Oil-Red O staining and the differentiation rate was compared. TG synthesis and TG mass in these cells were also assayed. DNA synthesis was measured by ［3H］-TdR incorporation. A typical differentiation inducer, insulin, was used as a positive control to compare these results. RESULTS: (1) 3T3-F442A preadipocytes were induced to differentiate by ASP alone. Fat droplets were clearly visible in the cytoplasm of 3T3-F442A cells. The differentiation rate was high (90%), but no significant difference was observed, compared with that in insulin group (95%). (2) In ASP group, TG synthesis and TG mass were significantly increased, both of them were higher than that in control group (P<0.05), but there was no significant difference, compared with insulin group (P>0.05). CONCLUSION: As a new adipocytes hormone, ASP plays an important biological role in the differentiation of preadipocytes.