Rabbit olfactory stem cells. Isolation protocol and characterization

PURPOSE: To describe a new technique for isolation of a mesenchymal stem cells (MSCs) population from the olfactory mucosa in rabbits. METHODS: Olfactory stem cells (OSCs) were retrieved from under the cribriform plate of the Ethmoid bone. Several assays were accomplished to characterize the cell population and attest its viability in vitro. The cells were submitted to flow cytometry with the antibodies CD34, CD45, CD73, CD79, CD90 and CD105 and also they were induced to differentiate in three lineages. Functional evaluation involved analysis of in vitro growth behavior, colony forming unit like fibroblasts (CFU-f) and cryopreservation response. Further transduction with Green Fluorescent Protein (GFP) was also performed. RESULTS: The OSCs showed mesenchymal features, as positive response to CD34, CD73 and CD90 antibodies and plasticity. Additionally, these cells have high proliferated rate, and they could be cultured through many passages and kept the ability to proliferate and differentiate after cryopreservation. The positive response to the transduction signalizes the possibility of cellular tracking in vivo. This is a desirable feature in case those cells are used for pre-clinical trials. CONCLUSION: The cells harvested were mesenchymal stem cells and the technique described is therefore efficient for rabbit olfactory stem cells isolation.

Expression of CD90 and P75NTR stem cell markers in ameloblastomas: a possible role in their biological behavior

Abstract Multicystic and unicystic ameloblastomas are benign odontogenic tumors that present distinct biological behavior. The investigation of stem cells has become an important branch of tumor biology, with several studies addressing the possible role of these cells in tumor growth, angiogenesis, progression, infiltration and invasiveness. This study evaluated the immunohistochemical expression of CD90(Thy-1) and P75NTR stem cell markers in multicystic and unicystic ameloblastomas. Seventeen (17) samples of ameloblastomas (multicystic, n = 10; unicystic, n = 7) were submitted to immunohistochemical reactions and graded semi-quantitatively. The Kolmogorov-Smirnov test was used to verify possible differences in CD90 and P75NTR expressions between multicystic and unicystic ameloblastomas (p < 0.05). CD90 immunostaining was observed in all multicystic ameloblastoma specimens (n = 10), in the cytoplasm of the fibroblasts and vascular endothelial cells of the tumor stroma, near the neoplastic odontogenic epithelia. The staining of stromal CD90 was significantly higher in multicystic than in unicystic ameloblastomas (p = 0.003). Nuclear P75NTR immunostaining was observed in all ameloblastoma specimens. A significant difference was seen in the epithelial staining of P75NTR between multicystic and unicystic types (p = 0.007). The increased expression of CD90 and P75NTR found in multicystic ameloblastomas suggests a behavioral biological difference between multicystic and unicystic ameloblastomas, as well as a difference in ameloblastoma development.

Isolation, culture, and identification of human spermatogonial stem cells / 中华男科学杂志

<p><b>OBJECTIVE</b>To isolate, identify and culture human spermatogonial stem cells (SSC) and then obtain purified and enriched human SSCs for research and application.</p><p><b>METHODS</b>We detected the expression of CD90 in the human testis using the immunofluorescence technique and isolated human testicular spermatogenic cells by two-step enzymatic digestion, followed by differential plating and magnetic-activated cell sorting (MACS) with CD90 as an SSC marker. Then we identified the isolated CD90-positive spermatogenic cells by RT-PCR and immunocytochemistry, and meanwhile cocultured them with Sertoli cells in SG medium in vitro.</p><p><b>RESULTS</b>The isolated CD90-positive cells showed a relatively homogeneous characteristic in size and morphology and expressed the genes specific for human SSCs, with high expressions (90.5%) of GFRA1, GPR125, and UCHL1. After coculture with Sertoli cells in the SG medium for 2 weeks, the isolated CD90-positive cells maintained a good activity.</p><p><b>CONCLUSION</b>CD90 can be regarded as a speci- fic marker for human SSCs and used to obtain highly enriched human SSCs by differential plating and MACS. Furthermore, the isolated human SSCs can be cultured in SG medium in vitro.</p>

Presença de marcadores de células-tronco em linhagens celulares humanas de câncer de mama: possível valor em diagnóstico, prognóstico e terapía / Analysis of stem cells markers in human breast cancer cell lines

O câncer de mama é a doença maligna que mais acomete as mulheres no mundo. Apesar dos inúmeros tratamentos, o óbito se deve principalmente à doença metastática que pode se desenvolver a partir do tumor primário. Esta progressão tumoral decorre da dificuldade de se estabelecer um prognóstico mais preciso. Atualmente, a teoria de células iniciadoras de tumor vem sendo estudada para tentar explicar a biologia do câncer e descrever novos alvos para prognósticos e terapias. O carcinoma mamário foi o primeiro tumor sólido para o qual foi identificada uma subpopulação celular, definida como CD44+/CD24-, apresentando as características de células iniciadoras tumorais. Embora este fenótipo venha sendo muito utilizado para descrever as células iniciadoras tumorais de mama, muitos trabalhos tem questionado a relevância clínica desses marcadores, enfatizando que outros marcadores devem ser identificados. Assim, o objetivo deste trabalho é analisar e caracterizar marcadores de células-tronco que possam estar relacionados com o grau de malignidade no modelo de câncer de mama. Inicialmente, analisou-se a expressão de 10 marcadores de células-tronco em diferentes linhagens de câncer de mama que apresentam graus crescentes de malignidade. O CD90 foi selecionado devido à alta expressão desse marcador na linhagem mais agressiva Hs578T. Para a caracterização deste marcador, realizou-se ensaios funcionais, através do silenciamento do CD90 na linhagem tumorigênica Hs579T e sua superexpressão na linhagem não-tumorigênica MCF10A. As linhagens celulares geradas foram caracterizadas quanto ao crescimento celular, potencial invasivo e metastático. Foi possível observar que houve uma alteração da morfologia nas linhagens transformadas com o CD90 e, também, um maior tempo de dobramento na linhagem Hs578T-CD90- e um menor na MCF10A-CD90+. Além disso, a linhagem MCF10-CD90+ foi capaz de crescer independentemente de EGF. Através da análise da via EGF, foi possível observar que houve um aumento da expressão da forma fosforilada do receptor e dos fatores Erk, c-Jun, e Jnk na linhagem MCF10A-CD90+ e uma diminuição dos mesmos na linhagem Hs578T-CD90-. A análise da atividade do elemento responsivo do fator de transcrição AP1 comprovou que a via de EGF é funcional na linhagem MCF10-CD90+. Também foram analisados os marcadores de transição epitélio-mesenquimal, verificando-se aumento da expressão dos marcadores mesenquimais na linhagem MCF10A-CD90+ e diminuição na linhagem Hs578T-CD90-. Os ensaios in vitro de invasão mostraram que as células MCF10-CD90+ são capazes de migrar e invadir e as células Hs578T-CD90- apresentam diminuição significativa da habilidade de migração e invasão. Além disso, os ensaios de metástase in vitro e in vivo, mostraram que a superexpressão de CD90 levou à malignização das células MCF10A. Por outro lado, a linhagem Hs578T-CD90- apresentou menor potencial metastático in vitro. Portanto, neste trabalho, pela primeira vez, o CD 90 foi caracterizado funcionalmente como um marcador envolvido na transformação maligna do carcinoma mamário, contribuindo, assim, para melhor entendimento da biologia do câncer de mama e para que se possa desenvolver novas ferramentas de diagnóstico/prognóstico e novos protocolos clínicos e terapêuticos

Breast cancer is the malignant disease which affects the highest number of women in the world. In spite of the numerous treatments available, death is primarily due to the metastatic disease that may develop from the primary tumor. This tumor progression occurs because of the difficulty in establishing an accurate diagnosis/prognosis. Currently, the tumor initiating cells theory is being applied in an attempt to explain cancer biology and to unveil new diagnostic and therapeutic targets. Mammary carcinoma was the first solid tumor in which a cellular subpopulation, defined as CD44+/CD24-, was associated with tumor initiating cells. Although this phenotype has been widely used to describe breast tumor initiating cells, several studies have questioned the clinical relevance of these markers, emphasizing that additional markers should be identified. The objective of the present study is to analyze and characterize stem cell markers that may be related to malignancy stages in the breast cancer model. Initially, the expression of 10 stem cell markers was analyzed in different breast cancer cell lines displaying different malignancy grades. CD90 was selected due to its high expression levels in the most aggressive cell line, namely: Hs578T. In order to further characterize this marker, a functional study was performed in which CD90 was silenced in the Hs578T tumorigenic cell line and overexpressed in the non-tumorigenic MCF10A cell line. The resulting cell lines were characterized relative to growth rate and invasive and metastatic potential. A change in morphology readily was observed in the cell lines overexpressing CD90. In addition, the Hs578T-CD90-cell line presented an increased doubling time (DT), while the MCF10A-CD90+ cell line displayed a lower DT.. Furthermore, MC10-CD90+ cells were able to grow in the absence of EGF. Analysis of components of the EGF pathwayrevealed increased expression levels of the phosphorylated form of Erk, c-Jun and Jnk receptors in the MCF10-CD90+ cell line, while Hs578T-CD90- cells presented decreased expression of the same factors and receptors. Analysis of the activity of the AP1 responsive element allowed confirmation that the EGF pathway is functional in the MCF10-CD90+. . Epithelial-mesenquimal transition markers presented increased expression levels in the MCF10A-CD90+ cell line, accompanied by decreased expression levels in Hs578T-CD90- cells. In vitro invasion assays showed that MCF10A-CD90+ cells are capable of migrating and invading, while Hs578T-CD90- cells presented a significant decrease in their ability to migrate and invade. Additionally, in vitro and in vivo metastasis assays showed that malignization ensued upon overexpression of CD90 in MCF10A cells and a lower tendency to form metastasis in vitro was observed for the Hs578T-CD90- cell line. Therefore, the present study presents, for the first time in the literature, the functional characterization of CD90 as a genetic marker involved in the malignant transformation of mammary carcinoma, leading to a better understanding of the breast cancer biology, which may, in turn, lead to the development of new clinical and therapeutic protocols

Immunophenotyping of leukemic stem cells and chromosome karyotype characteristics in Uyghur leukemia pediatric patients / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To study the immunophenotype and chromosome karyotype characteristics of leukemic stem cells (LSC) in Uyghur leukemia pediatric patients.</p><p><b>METHODS</b>The morphological features of LSC in culture in vitro was observed by flow cytometry. The immunophenotype was assessed by detective flow cytometry. The chromosome karyotype was analyzed by R-banding technique.</p><p><b>RESULTS</b>The LSC showed suspended floating colonies growing in the culture medium, and grew well and proliferated constantly in culture over 8 months. Among the 13 children with AML, there were 10 CD34(+)CD38(-)CD123(+) and CD33(+) cases, 10 CD44(+) cases, 10 CD96(+) cases, and 5 CD90(+) cases. Among the 13 children with B-ALL, there were 6 CD34(+)CD20(-)CD19(+) cases, 7 CD9(+) cases, and 5 CD123(+) cases. Among the 9 children with acute T lymphoblastic leukemia (T-ALL), there were 5 CD34(+)CD7(-) and CD90(+) cases, and 4 CD123(+) cases. Among the 13 cases of AML, 5 cases showed chromosome translocation t(15;17), one case chromosome translocation t(8;21), and 7 cases showed no chromosome karyotype abnormality. Among the 22 ALL cases, there were chromosome translocation t(12;21) in 1 case, t(9;22) in 3 case, hyperdiploid in 2 cases, and 16 cases without karyotype abnormalities. Twenty-nine children received induction remission therapy. Among them, 12 died, including 9 CD96(-)positive cases and 3 CD96(-)negative cases, with a statistically significant difference (P < 0.05).</p><p><b>CONCLUSIONS</b>The LSC of Uyghur leukemia pediatric patients in Xinjiang express CD9 and CD19 in ALL, and express CD123 and CD90 simultaneously in ALL and AML. The expression of CD96 is one of factors of poor prognosis.</p>

Expression of CD90/EpCAM/CD24 in hepatocellular carcinoma cell lines at various stages of differentiation / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To confirm the malignant phenotype of hepatocarcinoma cell (HCC) lines at various stages of differentiation (MHCC97L, MHCC97H and HCCLM3) and to explore their expression levels of cancer stem cell (CSC) markers.</p><p><b>METHODS</b>The invasive and proliferative properties of each HCC line were assessed by transwell assay and the Cell Counting Kit-8 (CCK-8) colorimetric assay. Sensitivity to chemotherapy was assessed by treatment with oxaliplatin and determination of the half inhibitory concentration (IC50). The expression of CD90, EpCAM and CD24 was measured by flow cytometry.</p><p><b>RESULTS</b>The number of cells that migrated through the invasion assay membrane were significantly different between the three HCC lines: HCCLM3 (30.57 +/- 8.95) more than MHCC97H (21.33 +/- 4.17) more than HCC97L (9.33 +/- 3.85), P less than 0.01. The IC50 was significantly different between the three HCC lines: HCCLM3 (36.57 +/- 6.95) mumol/L more than MHCC97H (26.35+/-3.88) mumol/L more than MHCC97L (17.68 +/- 3.25) mumol/L. The CSC marker with the highest expression on all three HCC lines was CD90 (HCCLM3: 0.92% +/- 0.21%, MHCC97H: 1.98% +/- 0.23%, and MHCC97L: 2.55% +/- 0.34%), followed by EpCAM (2.11% +/- 0.32%, 3.23% +/- 0.18%, and 4.38% +/-0.49%, respectively), and CD24 as the lowest (0.68% +/- 0.37%, 1.22% +/- 0.26%, and 1.36% +/- 0.24%, respectively).</p><p><b>CONCLUSION</b>Higher expression of CSC markers on HCC lines is associated with a stronger invasive ability and higher sensitivity to chemotherapy.</p>

Expression of neonatal Fc receptor on human nephritis and rat nephritis models / 中华病理学杂志

<p><b>OBJECTIVE</b>To study the expression of neonatal Fc receptor in podocytes in human nephritis and immune-induced rat nephritis models: anti-Thy1.1 nephritis and Heymann nephritis.</p><p><b>METHODS</b>Thirty-nine cases of renal biopsies were enrolled from September 2009 to February 2010, including 8 cases of minimal change disease, 4 cases of focal segmental glomerulosclerosis, 9 cases of membranous nephropathy, 12 cases of IgA nephropathy and 6 cases of lupus nephritis. Five normal kidney tissue samples adjacent to renal clear-cell carcinoma were served as normal controls. Laser capture microdissection and real-time RT-PCR were used to assess the expression level of FcRn mRNA in glomeruli of various glomerulonephritides, and immunohistochemistry (IHC) of FcRn by SuperVision method was performed. In addition, rat models of mesangial proliferative nephritis (anti-Thy1.1 nephritis) and passive membranous nephropathy (Heymann nephritis) were established and FcRn was examined in renal tissues by IHC.</p><p><b>RESULTS</b>The FcRn mRNA level in lupus nephritis was statistically higher than that of normal controls (P < 0.05). FcRn protein expression by IHC was seen in lupus nephritis (6/6), membranous nephropathy (6/9) and IgA nephropathy (7/12), significantly higher than that of normal controls (0/5), P < 0.05. Minimal change disease and focal segmental glomerular sclerosis showed minimal or none expression of FcRn (1/8, 0/4 respectively) and not statistically difference from that of normal controls. Furthermore, FcRn expression in podocytes was detected in rat anti-Thy1.1 (3/5) and Heymann nephritis models (2/7) but was not detected in normal controls.</p><p><b>CONCLUSIONS</b>Expression of FcRn in podocytes was up-regulated in immune-induced human nephritis and rat nephritis models of anti-Thy1.1 nephritis and Heymann nephritis. FcRn may play a role in the development of immune-induced glomerulonephritis.</p>

FTY720 attenuates rat anti-Thy-1 mesangial proliferative glomerulonephritis by inhibition of transforming growth factor β1-connective tissue growth factor pathway / 中华病理学杂志

<p><b>OBJECTIVE</b>To investigate whether FTY720 inhibits rat mesangial proliferation and extracellular matrix expansion through suppression of transforming growth factor β1-connective tissue growth factor (TGFβ1-CTGF) pathway, and to explore experimental evidence for its effect on mesangial proliferative glomerulonephritis.</p><p><b>METHODS</b>A rat model of anti-Thy-1 mesangial proliferative glomerulonephritis was established and FTY720 intervention was performed. Periphery blood lymphocyte count, urine protein excretion, glomerular mesangial proliferation, protein and gene expression of TGFβ1 and CTGF and extracellular matrix protein including fibronectin, laminin and collagen IV in isolated glomeruli were documented at 1, 3 and 7 days after injection of anti-Thy-1 antibody.</p><p><b>RESULTS</b>The model group developed proteinuria at 1, 3 and 7 days after injection of anti-Thy-1 antibody, which were significantly higher [(27.9 ± 7.3), (63.5 ± 18.8) and (52.4 ± 15.4)mg/d, respectively] than those in the control group [(8.4 ± 2.4), (8.4 ± 2.1) and (10.4 ± 3.2) mg/d; respectively, P < 0.01]. FTY720 intervention group showed significantly decreased proteinuria at 3 and 7 days after injection [(31.4 ± 7.0), (25.5 ± 7.7) mg/d, respectively] than model group (P < 0.01), although higher than the control group (P < 0.01). After intervention for 3 and 7 days, FTY720 significantly down-regulated both TGFβ1 and CTGF gene and protein expression in cultured glomeruli, and suppressed the production of glomerular extracellular matrix protein secretion, leading to attenuated mesangial cell proliferation and extracellular matrix expansion in rat anti-Thy-1 mesangial proliferative glomerulonephritis.</p><p><b>CONCLUSION</b>FTY720 significantly attenuates mesangial proliferation and extracellular matrix expansion through inhibition of TGFβ1-CTGF pathway in rat, and thus ameliorates the development of anti-Thy-1 mesangial proliferative glomerulonephritis.</p>

Role of transcription factor special AT-rich binding protein 2 in the osteoblasts differentiation of bone marrow stromal cells / 中华口腔医学杂志

<p><b>OBJECTIVE</b>To investigate the role of transcription factor special AT-rich binding protein 2 (SATB2) in the osteoblasts differentiation of bone marrow stromal cells (BMSC) in vitro.</p><p><b>METHODS</b>Rats bone marrow stromal cells were isolated by Percoll sedimentation and the cells were placed and allowed to attach for three times. After passages, expression plasmid pBABE-hygro-satb2 was constructed, then transfected into BMSC. BMSCs were inoculated in conditioned medium and osteogenic factors were detected by western blotting and reverse transcription polymerase chain reaction (RT-PCR).</p><p><b>RESULTS</b>The morphological observation of BMSC showed either spindle or polygonal pattern. The cellular phenotypic marker of the third passage was CD29 positive and CD34 negative. The growth curve possessed "S" pattern. The intensity of calfilication in BMSC was higher in SATB2 transfection group (IA value 125974 ± 241) than that in the control groups (IA value 178486 ± 406). Moreover, cell migration rate increased in SATB2 transfection group [width of scratch (0.72 ± 0.01) mm] compared with control group [width of scratch (0.83 ± 0.03) mm]. In addition, the mRNA expression of osteogenic factors runt-related transcription factor 2, Osterix, activating transcription factor 4, integrin-binding sialoprotein were upregulated.</p><p><b>CONCLUSIONS</b>Cells cultured with this method have general biological characteristics and osteogenic differentiation potential in vitro. SATB2 can promote osteoblasts differentiation of BMSC.</p>

Rapid Isolation of Adipose Tissue-Derived Stem Cells by the Storage of Lipoaspirates

PURPOSE: This study examined a rapid isolation method decreasing the time and cost of the clinical application of adipose tissue-derived stem cells (ASCs). MATERIALS AND METHODS: Aliquots (10 g) of the lipoaspirates were stored at 4degrees C without supplying oxygen or nutrients. At the indicated time points, the yield of mononuclear cells was evaluated and the stem cell population was counted by colony forming unit-fibroblast assays. Cell surface markers, stem cell-related transcription factors, and differentiation potentials of ASCs were analyzed. RESULTS: When the lipoaspirates were stored at 4degrees C, the total yield of mononuclear cells decreased, but the stem cell population was enriched. These ASCs expressed CD44, CD73, CD90, CD105, and HLA-ABC but not CD14, CD31, CD34, CD45, CD117, CD133, and HLA-DR. The number of ASCs increased 1x1014 fold for 120 days. ASCs differentiated into osteoblasts, adipocytes, muscle cells, or neuronal cells. CONCLUSION: ASCs isolated from lipoaspirates and stored for 24 hours at 4degrees C have similar properties to ASCs isolated from fresh lipoaspirates. Our results suggest that ASCs can be isolated with high frequency by optimal storage at 4degrees C for 24 hours, and those ASCs are highly proliferative and multipotent, similar to ASCs isolated from fresh lipoaspirates. These ASCs can be useful for clinical application because they are time- and cost-efficient, and these cells maintain their stemness for a long time, like ASCs isolated from fresh lipoaspirates.

Expression of THY1 gene in epithelial ovarian cancer / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To detect the expession of THY1 in ovarian serous cystadenocarcinoma tissues.</p><p><b>METHODS</b>Immunohistochemistry was performed to detect the expression of THY1 gene in formalin-fixed, paraffin-embedded specimens of normal ovaries (n = 25), ovarian serous cystadenoma (n = 25), and serous cystadenocarcinoma (n = 53). The correlation of THY1 expression with clinicopathological parameters was statistically analyzed.</p><p><b>RESULTS</b>The positive expression rates of THY1 protein in normal ovaries, ovarian serous cystadenomas and ovarian serous cystadenocarcinomas were 60.0% (15/25), 72.0% (18/25) and 34.0% (18/53), respectively. The values of IOD of THY1 protein expression were 288,449.2 +/- 60,087.3, 271,655.6 +/- 66,588.7 and 252,087.6 +/- 45,559.4, respectively. The expression of THY1 protein was significantly down-regulated in ovarian serous cystadenocarcinoma tissues compared with that in normal ovarian tissues and ovarian serous cystadenoma tissues (P < 0.05). THY1 expression was negatively correlated with surgical-pathological staging, histological differentiation and lymph node involvement (P < 0.05).</p><p><b>CONCLUSION</b>The decreased level of THY1 expression may be related with the occurrence and development of ovarian serous cystadenocarcinoma.</p>

Significance of expression of THY1 protein in epithelial ovarian cancer / 中华肿瘤杂志

<p><b>OBJECTIVE</b>The purpose of this study was to investigate the clinical significance of THY1 protein expression in epithelial ovarian cancer.</p><p><b>METHODS</b>Immunohistochemistry (IHC) and terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) staining were used to detect the protein expression of THY1, Ki67 and cell apoptosis in 76 epithelial ovarian cancers by tissue microarray. The correlation between THY1 expression and patients' clinical features was analyzed.</p><p><b>RESULTS</b>Of the 76 epithelial ovarian cancer samples, 64 were informative for IHC and TUNEL assays and 42 (65.6%) among them showed down-regulated/loss expression of THY1 protein. A significant positive correlation of THY1 protein expression with clinical stage and distant metastasis was observed in this ovarian cancer cohort (P < 0.05). The more advanced the tumor stage, the more frequency of loss expression of THY1 protein. In addition, the mean positive rate of Ki67 staining in tumors with down-regulated/loss expression of THY1 was 33.7% +/- 3.5%, significantly higher than that in the tumors with normal expression of THY1 (17.3% +/- 6.1%, P = 0.0027). However, no significant correlation was observed between THY1 protein expression and tumor cell apoptosis as well as patients' survival in this series (P > 0.05).</p><p><b>CONCLUSION</b>Down-regulated/loss expression of THY1 protein in epithelial ovarian cancer is significantly correlated with cancer cell proliferation and metastasis in the epithelial ovarian cancer, and it may be used as one of the new molecular biomarkers to predict the disease progression in patients.</p>

Construction of THY1 eukaryotic expression plasmid and its effects on growth of ovarian cancer SKOV3 cells / 生物医学工程学杂志

This study was designed to construct THY1 eukaryotic expression plasmid and assess its effects on epithelial ovarian cancer cell line SKOV3. The gene fragment coding for THY1 was inserted into pcDNA3.1(+) for constructing the recombinant plasmid pcDNA3.1(+)-THY1. The eukaryotic expression plasmid was analyzed by PCR, restriction endonucleases digestion and DNA sequencing. The recombinant plasmid pcDNA3.1(+)-THY1 was transfected into SKOV3 cells by liposome protocol. The experimental cells were classified into three groups: SKOV3-THY1, SKOV3-Null and SKOV3. The pcDNA3.1(+)-THY1 has been transfected into SKOV3 cells by RT-PCR and immunohistochemistry. The cell inhibitory rate of SKOV3-THY1 (56.6% at the fifth day) was higher than that of SKOV3-Null (12.5%), there was significant difference between them (P<0.05). The ratios of G1 phase of SKOV3 cells after transfection were increased and the ratios of S phase were decreased significantly. There was significant difference between SKOV3-THY1 and SKOV3-Null or SKOV3 (P<0.05), but there was no significant difference between SKOV3-Null and SKOV3 (P>0.05). We have constructed the recombinant plasmid pcDNA3.1(+)-THY1 sucessfully. THY1 transfection can inhibit the growth of SKOV3 cells in vitro.

Isolation and characterization of human rheumatoid arthritis fibroblast-like synoviocytes / 南方医科大学学报

<p><b>OBJECTIVE</b>To isolate and characterize human rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLSs).</p><p><b>METHODS</b>The synovial membrane tissues were obtained from 4 RA patients, 1 chondroma patient and 1 healthy subject and FLS were isolated by means of tissue culture. The cell morphology was observed by phase-contrast microscope and the cell surface markers were detected by flow cytometry.</p><p><b>RESULTS</b>The FLSs were successfully cultured from the synovial membrane tissues with good cell homogeneity after the third passage. The FLSs of the 3rd to 7th passages were stable and proliferated actively, followed by slow proliferation and aging since the 8th passage. Flow cytometry showed that the 4th-passage FLSs from the RA patients contained 99.04% CD90(+) cells, 2.73% CD3(+) cells, 0.29% CD3(-)CD19(+) cells, 2.81% CD3(-)CD16(+)CD56(+) cells, 5.89% CD14(+) cells, and 54.17% CD55(+) cells. The presence of interleukin-1 receptor type I (IL-1RI, 158.63-/+20.32 pg/ml) and IL-1beta (4.67-/+0.82 pg/ml) were detected in the cell culture supernatant of the 4th-passage FLSs from the RA patients by enzyme-linked immunosorbent assay ELISA.</p><p><b>CONCLUSION</b>FLSs from RA patients can be effectively culture by means of tissue culture, and the cultured FLSs show high expressions of CD90, IL-1RI and IL-1beta.</p>

Growth inhibitory effects of THY1 gene on epithelial ovarian cancer SKOV3 cells / 南方医科大学学报

<p><b>OBJECTIVE</b>To construct THY1 eukaryotic expression plasmid and study its effects on the growth of epithelial ovarian cancer cell line SKOV3.</p><p><b>METHODS</b>THY1 gene fragment was obtained from normal human ovarian tissue using RT-PCR, and inserted into the eukaryotic expression plasmid pcDNA3.1(+) to construct the recombinant plasmid pcDNA3.1(+)-THY1, which was transformed into E. coli JM109 followed by selection of the positive clones containing the target inserts. The eukaryotic expression plasmid was analyzed by PCR, restriction endonucleases digestion and DNA sequencing. SKOV3 cells divided into SKOV3-THY1, SKOV-3-Null and SKOV3 groups were transfected via liposome with the recombinant plasmid pcDNA3.1(+)-THY1, empty plasmid, or not transfected, respectively. The expression of THY1 mRNA and its protein were examined by RT-PCR and Western blot methods. The cell growth and apoptosis were evaluated by MTT assay and flow cytometry.</p><p><b>RESULTS</b>The gene fragment of exogenous THY1 was correctly inserted into the eukaryotic expression plasmid pcDNA3.1(+) as verified by PCR, restriction endonucleases digestion and DNA sequencing, and recombinant expression plasmid pcDNA3.1(+)-THY1 transfection resulted in stable expression in SKOV3 cells as shown by RT-PCR and Western blotting. The cell growth inhibition rate of SKOV3-THY1 group (56.6% at the fifth day) was significantly higher than that of the SKOV3-Null group (12.5%, P<0.05), and the cell apoptosis rate in SKOV3-THY1 group (31.8%) was significantly higher than those in SKOV3-Null group (10.5%) and SKOV3 group (9.8%, P<0.05), but the apoptosis rate between the latter two groups was similar (P>0.05).</p><p><b>CONCLUSIONS</b>The recombinant plasmid pcDNA3.1(+)-THY1 can be expressed stably in human ovarian cancer cell line SKOV3. THY1 transfection can inhibit the growth of SKOV3 cells in vitro, suggesting the important role of THY1 gene in pathogenesis and development of ovarian cancer.</p>

Nephritic model induced by anti-Thy1.1 monoclonal antibody and its application to study on Chinese materia medica / 中国中药杂志

The anti-Thy1.1 glomerulonephritis (GN) model induced by anti-Thy1.1 monoclonal antibody (mAb) is a widely used animal model for human mesangial proliferative glomerulonephritis (MsPGN), which is characterized by significant proteinuria and acute or progressive mesangial injury following the complement-mediated mesangiolysis and glomerular inflammatory cell infiltration. In this review, it has been discussed that the pathogenesis of reversible anti-Thy1.1 GN or irreversible anti-Thy1.1 GN induced by mAb 1-22-3 injection, the mechanisms governing inflammatory cells infiltration and several injurious cytokines in glomeruli, and some of the processes involved in the resolution of mesangial lesion such as mesangial cell proliferation and matrix expansion. Using these models, it has been reported to examine the effects of Chinese materia medica, including multi-glycoside of Tripterygium wilfordii Hook. f. (GTW) and Sairei-to on mesangial damage and proteinuria, and then to clarify the mechanism of these herbs at molecular level by examining the effects on various injurious factors.

Biologic characteristics of rat bone marrow mesenchymal stem cells cultured in vitro / 中华病理学杂志

<p><b>OBJECTIVE</b>To investigate biological characteristics of rat bone marrow mesenchymal stem cells (MSC) cultured in vitro and to explore their potential applications.</p><p><b>METHODS</b>MSC were isolated from rat bone marrow by density gradient centrifugation and were induced to differentiation. Flow cytometry was used to characterize their surface antigen expression, cell cycle status and cell growth parameters. Telomerase activity was determined by TRAP-ELISA assay.</p><p><b>RESULTS</b>Fusiform MSC became larger and flattener with increasing passages of culture. After the fourth passage, the MSC showed an immunophenotype of CD29 (94.75% +/- 3.68%), CD71 (95.43% +/- 2.23%), and CD90 (98.08% +/- 3.88%). After the seventh passage, MSC with such immunophenotype decreased with CD29: 50.00% +/- 3.35%, CD71: 50.70% +/- 2.43%, and CD90: 48.60% +/- 2.83%. Cells with such immunoprofile completely disappeared after passage 9. Overall, MSC grew faster during the first 5 passages. The number of MSC in S and G(2)/M phases were 38.36% +/- 2.01% and those in G(0)/G(1) phase were 61.64% +/- 2.13% after 3 passages. The cell growth decreased after passage 7. Percentage of MSCs in S and G(2)/M phases was 10.83% +/- 1.63% and that in G(0)/G(1) was 89.17% +/- 1.96% after passage 12, after which the cells failed to further divide. After passage 9, MSCs lost their ability to differentiate to Von Kossa and oil red O positive staining cells. In addition, telomerase activity of MSC also gradually decreased with the prolonged passages, from the original 52.7% +/- 0.78% to no telomerase activity.</p><p><b>CONCLUSION</b>The biological and immunophenotypical characteristics of cultured MSC showed obvious alterations with increasing numbers of passage of culture.</p>

Expression of cell surface antigens during the differentiation of osteoblast by human bone marrow-derived mesenchymal stem cells / 中国医学科学院学报

<p><b>OBJECTIVE</b>To explore the phenotypic changes of some cell surface antigens in the process that the bone marrow-derived mesenchymal stem cells (MSCs) differentiate into osteoblast lineage under certain conditions.</p><p><b>METHODS</b>The mononuclear cells were isolated from human bone marrow and cultured in the medium in the presence (experimental group) or absence (control group 1) of dexamethasone, ascorbic acid and beta-glycerophosphate, or in the alpha-MEM only (control group 2). The expressions of CD45, CD34, CD117, CD90, and HLA-DR were examined by flow cytometry on culture day 7, 12, and 17.</p><p><b>RESULTS</b>Human bone marrow MSCs can differentiate into mature cells with the characteristics of osteoblasts in vitro. The expression of CD45 was low in the experimental group and in the control groups on culture day 7, and became negative from day 12 in all groups. The expressions of CD34 and CD117 were negative at all time points. The expression of CD90 increased on day 12 in all groups, and was more obvious in the control groups. The expression of HLA-DR was gradually elevated along with the differentiation and maturation of osteoblasts in the experimental group, but dropped in the control groups in the later time points.</p><p><b>CONCLUSIONS</b>MSCs can differentiate into mature osteoblasts after induction. The expression of cell surface antigens during differentiation has characteristic changes, which may be key markers in the early stage of osteoblasts differentiation.</p>

Correlation of changes in circulating bone marrow stem cells in pregnant rabbits after acute myocardial infarction with serum estradiol / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the changes in circulating bone marrow stem cells in pregnant rabbits after AMI (AMI) and their relationship with estradiol.</p><p><b>METHODS</b>Three groups of rabbits were used, namely pregnancy and AMI group, AMI group without pregnancy, and sham operation group with pregnancy. The ratio of CD90(+) cells in the peripheral blood was determined with flow cytometry in all the rabbits, and serum estradiol level measured. Four weeks after AMI, hemodynamic measurements were carried out. The morphological changes of the myocardial tissues were examined with ImageJ 1.31.</p><p><b>RESULTS AND CONCLUSION</b>Four weeks after AMI, the two pregnancy groups showed a higher Left ventricular end systolic pressure(LVESP) and+dp/dtmax, lower left ventricular end-diastolic pressure (LVEDP) and -dp/dtmax and high levels of CD90(+) cells in peripheral blood than AMI group without pregnancy (P<0.01). The ratio of circulating CD90(+) cells increased gradually with gestational age and peaked at the end stage of pregnancy. After delivery the circulating CD90+ cell ratio decreased sharply, showing a significant correlation with serum estradiol level (r=0.725, P<0.01). Four weeks after AMI, the pregnancy group had smaller myocardial infarction (MI) volume than the non-pregnant group (22.17+/-6.34% vs 38.86+/-5.97%, P<0.05). Circulating bone marrow stem cells increased during pregnancy with gestational age and peaked at the end stage of pregnancy. Ten days after delivery, the stem cells resumed basically the normal level. The proportion of circulating bone marrow stem cells was significantly correlated with the level of serum estradiol during pregnancy, and mobilization of the bone marrow stem cells induced by acute ischemic event in pregnant rabbits was advanced. 4 weeks after AMI, the pregnant rabbits showed better heart contraction and diastolic function than the non-pregnant ones.</p>

The biological behavior of hepatocarcinoma stem cells in rats / 中华肝脏病杂志

<p><b>OBJECTIVES</b>To study the biological behavior of hepatocarcinoma stem cells in rats.</p><p><b>METHODS</b>Primary liver carcinomas were induced in rats using diethylnitrosamine. Tumor cells from 8 rats were separated according to rats oval cell (OVC) markers CD34, c-Kit, Thy-1, AFP, CK7, CK8, CK14, CK18, CK19 and GGT and then they were separately injected into the livers of nude mice. The tumors grown from the different subpopulation of OVC markers in the nude mice livers (10 OVC markers negative or positive cells) were weighted 1 month after the inoculations. The hepatocarcinoma cell subpopulations with higher ability in causing tumor growths were further studied in vitro. The cell cycles and DNA content of those subpopulation cells were investigated using flow cytometry.</p><p><b>RESULTS</b>(1) Subpopulation cells with CK7(-), Thy-1(+) and AFP(+) markers had a higher ability in causing tumors in nude mice; (2) Subpopulation cells, exhibiting characters of TSC, had a low growth rate in vitro.</p><p><b>CONCLUSIONS</b>(1) Different subpopulations of hepatocarcinoma cells had different abilities in causing tumors in rats. Some subpopulation cells, such as CK7(-), Thy-1(+) and AFP(+) cells, have characteristics of tumor stem cells. (2) The hepatocarcinoma stem cells may have a low growth rate in vitro.</p>