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The expression and effects of proto-oncogene c-raf in spermatogonial stem cells in vitro
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Abstracts / Cell Biology International 32 (2008) S1eS67
cryopreservation of cord blood hematopoetic stem cells. The recovered cells’ viability was tested by typan blue staining, and the effect of EG group was best. The same result get from the recovery rate tested by MNC counted and receiver rate of CFU-GM. The result of the test of CFU-S showed that the effect of EG was better than DMSO (P < 0.05) and the diameter of CFU-S in EG group was the highest (P < 0.05), either about the WBC count in the peripheral blood of mouse (P < 0.05). These result suggest that the effects of protection were best when EG was composed of 1.5 M EG+ 0.25 M Suc + 1% Dex-40 in the vitrification cryopreservation of hematopoetic stem cells.
EFFECT OF EPO-ACTIVATED ASTROCYTE CONDITIONED MEDIUM ON DIFFERENTIATION OF NEURAL STEM CELL AND ITS PROTECTIVE EFFECT ON DIFFERENTIATED NEURAL STEM CELLS AFTER INJURY IN VITRO Jing Zhang, Hui Shen, Chun Lin Xia Department.of Anatomy & Cytoneurobiology Unit, Soochow University, Suzhou 215123, China Erythropoietin (EPO), a hematopoietic cytokine, has been shown to have developmental functions not only in erythrocyte but also in the nervous system. We previously found that EPO promoted differentiation of astrocytes, and astrocytes could promote neurogenesis. Thus, it is highly possible that neurotropic factors derived from EPO-treated astrocytes could influence neural stem cells (NSCs) differentiation into neuron. Therefore, in the present study we investigated whether EPO-activated astrocytes could secrete some factors to either promote the differentiation of NSCs or protect differentiated NSCs under injury condition in vitro. NSCs and astrocytes were separated from newborn rat cortex, and media were collected from astrocytes (ACM) and EPO activated astrocytes (EACM). The cells included three groups: the experiment group also called the EACM group (NSCs with EACM), the ACM group (NSCs with ACM) and the control group (NSCs with DMEM/F12 + 10% FBS). The expression of NF-200 in the NSCs showed strong positivity whereas the NSCs in control group scarcely exhibited morphological changes, and NF-200 immunocytochemical staining was nearly negative in corresponding period. The percentage of neuron positive in the EACM group was 59.18 2.77, while those in the ACM group and the control group were 23.58 1.21 and 16.98 0.91. As for EACM protective effects on differentiated NSCs after hydroxyl free-induced injuries generated by addition of FeSO4 and H2O2, the OD value was 0.381 0.027, while that in control group was only 0.270 0.013 (P < 0.05). The percentage of survived cell in experimental group (61.0 2.48%) was higher than control group (31.54 1.35 %) (P < 0.01). These results suggest that EPO activated astrocytes could secrete some factors to either promote the differentiation of neural stem cells or protect differentiated neural stem cells under injury condition in vitro.
THE EFFECT OF Notch1 SIGNAL TO THE EXPRESSION AND PHOSPHORYLATION OF TAU DURING THE DIFFERENTIATION OF EMBRYONIC STEM CELLS INTO NEURONS Ying Xing 1, Qiu Ying Zhang 1, Yi Teng Shen 1, Yuan Yuan Jiang 1, Xue Fei Han 1, Yan Xu 1,2, Wen Hai Yan 1,2 1 The Stem Cells Research Center, Zhengzhou University, China 2 Department of Pathophysiology, School of Basic Medicine, Zhengzhou University, China The Notch signaling pathway has been implicated in the regulation of cell-fate decisions such as differentiation of embryonic stem cells into neurons. In addition, The Notch signaling pathway is closely related to microtubule-associated protein Tau and its phosphorylation. We cultured mouse embryonic stem cells (ESCs) in vitro and transfected two plasmids, pSINsi-U6-Notch1 and PEGF-C1-Notch1, which respectively increased and decreased the expression of Notch1. Then we induced ESCs to differentiate into neural cells by Retinoic acid (RA). Respectively on days 1, 3, 5, 7 and 9 after induction, we examined the Tau protein and phosphorylation of tau at Serine 262 and Serine 396. Phosphokinase GSK-3b and phosphorylase PP2A were detected by immunocytochemistry. Western blotting was utilized to observe the expression of tau, GSK-3b, andPP2A, and the phosphorylation level of tau at Serine262 and
Serine 396 on day 5. We found that in ESCs transfected with pSINsi-U6Notch1 neuron-like cells differentiated from it increased regularly and formed a network structure. GSK-3b, PP2A and tau ser262 and tau ser396 were upregulated simultaneously from around day 3and on day 5, the expressions of tau, GSK-3b, PP2A, Serine262 and Serine 396 were more than the control group. In ESCs transfected with PEGF-C1- Notch1, neuron-like cells differentiated from it were not significantly changed on day 5, and the expressions of tau, GSK-3b PP2A; Serine262 and Serine 396 were lower than the control group. These findings suggest that the Notch signaling pathway promoted the differentiation of ESCs into neurons and at the same time enhanced the expression of tau but inhibiting its phosphorylation.
THE EXPRESSION AND EFFECTS OF PROTO-ONCOGENE C-RAF IN SPERMATOGONIAL STEM CELLS IN VITRO Si Fan Xu 1, Deng Ke Yan 2, Jin Lei Wang 2, Jia Xiang Chen 2 1 Chinese Minority Traditional Medical Center, College of Life and Environmental Science, Central University for Nationalities, Beijing, China 2 Medical College, Nan Chang University, Nan Chang, China Spermatogonial stem cells were separated by discontinuous Percoll gradient centrifugation from 9-day-old SD rat testes and purified by the different cells adhering speeds to dish. Spermatogonial stem cells were identified by immunohistochemistry of c-Kit protein expression. We then identified the homology between the amplified spermatogonial stem cells’ DNA and c-raf by sequencing and SeqMan. MTT assay was used to detect survival and proliferation of spermatogonial stem cells. TUNEL was used to assay the apoptosis index of spermatogonial stem cells and RT-PCR was utilized to observe the expressions of c-raf mRNA and caspase-3 mRNA in spermatogonial stem cells. The survival rates of cells separated by discontinuous Percoll gradient centrifugation and purified by the different cells adhering speeds to dish were 92.3 % and 91.5 % respectively. The percentage of spermatogonial stem cells expressing c-Kit was 90.1 %. Spermatogonial stem cells proliferation increased significantly in the DMEM containing 10 % NBS and decreased continuously in the DMEM without NBS. The highest proliferation rate of spermatogonial stem cells was seen in 120 h culture. The homology between the amplified spermatogonial stem cells’ DNA and the c-raf was 98 %. The survival rate of spermatogonial stem cells decreased significantly at culture 12 hours in 15 mmol/L c-raf AON group (P < 0.05). The expression of c-raf mRNA decreased and the expression of caspase-3 mRNA increased in c-raf AON group (P < 0.05). The apoptotic index of c-raf AON group (40.5 %) was much higher than those of the c-raf MON group (29.4 %) and control group (30.2 %) (P < 0.05). Our results show that discontinuous Percoll gradient centrifugation combined with the different cells adhering speeds to dish was an effective method for isolating and purifying spermatognial stem cells. C-kit could be used as a molecular marker in identifying the spermatogonial stem cells. The spermatogonial stem cells proliferated significantly at culture 120 hours in DMEM containing 10% NBS in vitro and the survival rate of spermatogonial stem cells decreased continuously in the DMEM without NBS. c-raf was expressed in the rat’s spermatogonial stem cells. The survival of spermatogonial stem cells could be prolonged by c-raf. C-raf might inhibit the apoptosis of spermatogonial stem cells by decreasing the expression of the capspase-3.
EXPERIMENTAL RESERCH ON THE CHEMOTHERAPEUTIC SENSITIVITY OF BRAIN TUMOR STEM CELLS Zhuan Yi Yang, Yong Wen Deng, Jia Sheng Fang, Yan Jin Wang, Feng Hua Chen, Ming Chu Li, Guo Ping Liu, Ling Feng Liu, Lei Wu, Ruo Kun Chen Department of Neurosurgery, Xiangya Hospital, Central South University, Changsha, Hunan 410008, P.R.China In order to explore the chemotherapeutic sensitivity of brain tumor stem cells (BTSCs), we obtained fresh specimens of astrocytoma and then cultivated them primarily into BTSCs and brain tumor cells (BTCs). By drug
Abstracts / Cell Biology International 32 (2008) S1eS67
cryopreservation of cord blood hematopoetic stem cells. The recovered cells’ viability was tested by typan blue staining, and the effect of EG group was best. The same result get from the recovery rate tested by MNC counted and receiver rate of CFU-GM. The result of the test of CFU-S showed that the effect of EG was better than DMSO (P < 0.05) and the diameter of CFU-S in EG group was the highest (P < 0.05), either about the WBC count in the peripheral blood of mouse (P < 0.05). These result suggest that the effects of protection were best when EG was composed of 1.5 M EG+ 0.25 M Suc + 1% Dex-40 in the vitrification cryopreservation of hematopoetic stem cells.
EFFECT OF EPO-ACTIVATED ASTROCYTE CONDITIONED MEDIUM ON DIFFERENTIATION OF NEURAL STEM CELL AND ITS PROTECTIVE EFFECT ON DIFFERENTIATED NEURAL STEM CELLS AFTER INJURY IN VITRO Jing Zhang, Hui Shen, Chun Lin Xia Department.of Anatomy & Cytoneurobiology Unit, Soochow University, Suzhou 215123, China Erythropoietin (EPO), a hematopoietic cytokine, has been shown to have developmental functions not only in erythrocyte but also in the nervous system. We previously found that EPO promoted differentiation of astrocytes, and astrocytes could promote neurogenesis. Thus, it is highly possible that neurotropic factors derived from EPO-treated astrocytes could influence neural stem cells (NSCs) differentiation into neuron. Therefore, in the present study we investigated whether EPO-activated astrocytes could secrete some factors to either promote the differentiation of NSCs or protect differentiated NSCs under injury condition in vitro. NSCs and astrocytes were separated from newborn rat cortex, and media were collected from astrocytes (ACM) and EPO activated astrocytes (EACM). The cells included three groups: the experiment group also called the EACM group (NSCs with EACM), the ACM group (NSCs with ACM) and the control group (NSCs with DMEM/F12 + 10% FBS). The expression of NF-200 in the NSCs showed strong positivity whereas the NSCs in control group scarcely exhibited morphological changes, and NF-200 immunocytochemical staining was nearly negative in corresponding period. The percentage of neuron positive in the EACM group was 59.18 2.77, while those in the ACM group and the control group were 23.58 1.21 and 16.98 0.91. As for EACM protective effects on differentiated NSCs after hydroxyl free-induced injuries generated by addition of FeSO4 and H2O2, the OD value was 0.381 0.027, while that in control group was only 0.270 0.013 (P < 0.05). The percentage of survived cell in experimental group (61.0 2.48%) was higher than control group (31.54 1.35 %) (P < 0.01). These results suggest that EPO activated astrocytes could secrete some factors to either promote the differentiation of neural stem cells or protect differentiated neural stem cells under injury condition in vitro.
THE EFFECT OF Notch1 SIGNAL TO THE EXPRESSION AND PHOSPHORYLATION OF TAU DURING THE DIFFERENTIATION OF EMBRYONIC STEM CELLS INTO NEURONS Ying Xing 1, Qiu Ying Zhang 1, Yi Teng Shen 1, Yuan Yuan Jiang 1, Xue Fei Han 1, Yan Xu 1,2, Wen Hai Yan 1,2 1 The Stem Cells Research Center, Zhengzhou University, China 2 Department of Pathophysiology, School of Basic Medicine, Zhengzhou University, China The Notch signaling pathway has been implicated in the regulation of cell-fate decisions such as differentiation of embryonic stem cells into neurons. In addition, The Notch signaling pathway is closely related to microtubule-associated protein Tau and its phosphorylation. We cultured mouse embryonic stem cells (ESCs) in vitro and transfected two plasmids, pSINsi-U6-Notch1 and PEGF-C1-Notch1, which respectively increased and decreased the expression of Notch1. Then we induced ESCs to differentiate into neural cells by Retinoic acid (RA). Respectively on days 1, 3, 5, 7 and 9 after induction, we examined the Tau protein and phosphorylation of tau at Serine 262 and Serine 396. Phosphokinase GSK-3b and phosphorylase PP2A were detected by immunocytochemistry. Western blotting was utilized to observe the expression of tau, GSK-3b, andPP2A, and the phosphorylation level of tau at Serine262 and
Serine 396 on day 5. We found that in ESCs transfected with pSINsi-U6Notch1 neuron-like cells differentiated from it increased regularly and formed a network structure. GSK-3b, PP2A and tau ser262 and tau ser396 were upregulated simultaneously from around day 3and on day 5, the expressions of tau, GSK-3b, PP2A, Serine262 and Serine 396 were more than the control group. In ESCs transfected with PEGF-C1- Notch1, neuron-like cells differentiated from it were not significantly changed on day 5, and the expressions of tau, GSK-3b PP2A; Serine262 and Serine 396 were lower than the control group. These findings suggest that the Notch signaling pathway promoted the differentiation of ESCs into neurons and at the same time enhanced the expression of tau but inhibiting its phosphorylation.
THE EXPRESSION AND EFFECTS OF PROTO-ONCOGENE C-RAF IN SPERMATOGONIAL STEM CELLS IN VITRO Si Fan Xu 1, Deng Ke Yan 2, Jin Lei Wang 2, Jia Xiang Chen 2 1 Chinese Minority Traditional Medical Center, College of Life and Environmental Science, Central University for Nationalities, Beijing, China 2 Medical College, Nan Chang University, Nan Chang, China Spermatogonial stem cells were separated by discontinuous Percoll gradient centrifugation from 9-day-old SD rat testes and purified by the different cells adhering speeds to dish. Spermatogonial stem cells were identified by immunohistochemistry of c-Kit protein expression. We then identified the homology between the amplified spermatogonial stem cells’ DNA and c-raf by sequencing and SeqMan. MTT assay was used to detect survival and proliferation of spermatogonial stem cells. TUNEL was used to assay the apoptosis index of spermatogonial stem cells and RT-PCR was utilized to observe the expressions of c-raf mRNA and caspase-3 mRNA in spermatogonial stem cells. The survival rates of cells separated by discontinuous Percoll gradient centrifugation and purified by the different cells adhering speeds to dish were 92.3 % and 91.5 % respectively. The percentage of spermatogonial stem cells expressing c-Kit was 90.1 %. Spermatogonial stem cells proliferation increased significantly in the DMEM containing 10 % NBS and decreased continuously in the DMEM without NBS. The highest proliferation rate of spermatogonial stem cells was seen in 120 h culture. The homology between the amplified spermatogonial stem cells’ DNA and the c-raf was 98 %. The survival rate of spermatogonial stem cells decreased significantly at culture 12 hours in 15 mmol/L c-raf AON group (P < 0.05). The expression of c-raf mRNA decreased and the expression of caspase-3 mRNA increased in c-raf AON group (P < 0.05). The apoptotic index of c-raf AON group (40.5 %) was much higher than those of the c-raf MON group (29.4 %) and control group (30.2 %) (P < 0.05). Our results show that discontinuous Percoll gradient centrifugation combined with the different cells adhering speeds to dish was an effective method for isolating and purifying spermatognial stem cells. C-kit could be used as a molecular marker in identifying the spermatogonial stem cells. The spermatogonial stem cells proliferated significantly at culture 120 hours in DMEM containing 10% NBS in vitro and the survival rate of spermatogonial stem cells decreased continuously in the DMEM without NBS. c-raf was expressed in the rat’s spermatogonial stem cells. The survival of spermatogonial stem cells could be prolonged by c-raf. C-raf might inhibit the apoptosis of spermatogonial stem cells by decreasing the expression of the capspase-3.
EXPERIMENTAL RESERCH ON THE CHEMOTHERAPEUTIC SENSITIVITY OF BRAIN TUMOR STEM CELLS Zhuan Yi Yang, Yong Wen Deng, Jia Sheng Fang, Yan Jin Wang, Feng Hua Chen, Ming Chu Li, Guo Ping Liu, Ling Feng Liu, Lei Wu, Ruo Kun Chen Department of Neurosurgery, Xiangya Hospital, Central South University, Changsha, Hunan 410008, P.R.China In order to explore the chemotherapeutic sensitivity of brain tumor stem cells (BTSCs), we obtained fresh specimens of astrocytoma and then cultivated them primarily into BTSCs and brain tumor cells (BTCs). By drug