Somatostatin expression in rat islet of steroid diabetes mellitus

AIM: To investigate the effects of dexamethasone(DEX) on the pancreatic endocrine function when steroid diabetes mellitus(SDM) occurred. METHODS: 140 rats were randomly assigned to 4 groups according to the clinic doses of DEX administration, Radioimmunoassay and in situ hybridization were applied to evaluate islet hormone changes in pancreas and blood.RESULTS: Changes in fasting blood glucose(FBG) 、fasting serum insulin(FINS) caused by DEX were dose and time-dependent. Changes in somatostatin(SS)mRNA and protein in islet as well as FINS occurred earlier than that of FBG. CONCLUSION: The super-physiological DEX influenced the expression SS and insulin secretion in islet, which may play an important role in SDM.     

In vitro differentiation of exocrine pancreatic cells from mouse embryonic stem cells

AIM: To explore the effects of sodium butyrate, activin A and dexamthasone on inducing mouse embryonic stem (ES) cells to differentiate into exocrine pancreatic cells in vitro. METHODS: E14 mouse ES cells were cultured in suspension to form embryonic bodies (EBs). The EBs were cultured with differentiating medium containing different concentrations of sodium butyrate, and the spontaneously differentiated ES cells were used as control. Exocrine pancreatic genes such as amylase, chymotrypsinogen, elastase 1, elastase 2 and carboxypeptidase were detected by RT-PCR at different time points to determine the optimal concentration and exposure time of sodium butyrate. Furthermore, activin A or dexamthasone was also used to explore the effects on exocrine differentiation. After that, the combination of sodium butyrate, activin A and dexamthasone was used to promote the differentiation of exocrine pancreatic cells from ES cells. During the differentiation course, the gene expressions of amylase, chymotrypsinogen, elastase 1, elastase 2 and carboxypeptidase were detected by RT-PCR. Morphological changes were investigated by phase contrast microscopy. Amylase expression was examined by immunofluorescence staining. RESULTS: Exocrine pancreatic gene expressions such as amylase, chymotrypsinogen, elastase 1, elastase 2 and carboxypeptidase were detected in spontaneously differentiated EBs. A relatively lower concentration of sodium butyrate with a shorter exposure time significantly promoted those above gene expressions as compared to that of spontaneously differentiated EBs. Activin A and dexamethasone induced upregulation of exocrine gene expression. The combination of activin A, sodium butyrate and dexamethasone significantly enhanced the mRNA levels of amylase, chymotrypsinogen, elastase 1, elastase 2 and carboxypeptidase. Under the treatment of activin A, sodium butyrate and dexamethasone, differentiated cells were polygonal in shape with large, round, and center-situated nuclei. According to the observation of immunofluorescence staining, amylase was positive expressed at the final stage. CONCLUSION: These data indicate that exocrine pancreatic differentiation of ES cells is induced by sodium butyrate, activin A and dexamethasone. The combination of pancreatic inducing factors improves the differentiating efficiency.     

Biological characterics of human fetal cord blood mesenchymal stem cells

AIM: To investigate the biological characterics of human second-trimester fetal cord blood mesenchymal stem cells (MSC) and its application prospects in utero gene transfer/therapy (IUGT). METHODS: Nuclear cells separated from cord blood were cultured in DMEM medium. Surface antigens of the MSC were analyzed by the FACScan flow cytometry. Adipogenic and osteogenic mediums were used to assess the differentiation ability of the cells. Adenovirus vector deliver green fluorescent protein gene (Ad-GFP) was used to transfected the MSC and the expressing of GFP was detected by fluorescent microscope. The MSC were injected into the liver of newborn rat. The immunofluorescence analysis was conducted to determine the presence of double-positive CD105⁺/CD166⁺ cells in different organs of rats. MSC were subcutaneous injected into the human-nonobese diabetes/severe combined immunodeficiency disease (NOD/SCID) mice and carcinogenesises of the MSC in vivo were detected by pathological diagnosis. RESULTS: MSC could be separated from fetal cord blood. These cells were uniformly positive for CD29, CD44, CD59, CD105, CD166 and negative for CD34, CD45, CD80, CD86, HLA-DR. The cells had the abilities to differentiate into adipogenic and osteogenic cells in vitro, expressed the GFP at high levels (56.32%±3.28%). The MSC were located at different organs after injected into the newborn rats and didn't have carcinogenicity in vivo. CONCLUSION: Human second-trimester fetal cord blood MSC is an promising target cells in fetal IUGT.     

Embryonic stem cells transplant into subretinal space of C3B mouse

AIM:To study the differentiation of embryonic stem cells (ESC) at the subretinal space of C3B mouse whose retina are of normal structure. METHODS:Embryonic bodies (EB) were gained when embryonic stem cells were propagated 1 generation after anabiosis. The digested EB were transplanted into subretinal space of C3B mouse. The eyes were analysed by photo microscopy, electric microscopy and immune assay at 1st week, 3rd week and 2nd month after injection. RESULTS:The thickened retina appeared on the place of injection at 1st week. Teratoma formation was observed in 3 transplant recipients at 3rd week and 2nd month. The ratio of forming tumor was 25% in all eyes received cell transplant. Eye atrophy or scar at injection position was seen in other eyes. The nucleus of tumour cells had apparent strange type under electrical microscopy. The immune assay showed that part of the tumour were microtubule-associated protein 2 (MAP-2) positive;part of the tumour were glial fibrillary acid protein (GFAP) positive;a little cells were nestin positive. CONCLUSION:After transplantation into the subretinal space, ESC were not incorporated into retina or differentiated into retinal cells. The teratoma was formed in part of eyes. The security of application for ESC in eye was considerable.     

Purification and differentiation potentiality of fetal liver mesenchymal stem cells from mouse

AIM: To purify and investigate the differentiation potentials of fetal liver mesenchymal stem cells (flMSCs) from murine in vitro.METHODS: flMSCs from mouse fetuses at embryonic and fetal day (ED)13.5 or ED14.5 were isolated by adhering to plastic and passaged by modified method.Cell cycle and phenotype were analyzed by flow cytometry.The cell differentiation was induced by special induction media.The cells differentiated to adipose,cartilaginous and osteoid tissues were identified with oil red O,Toluid blue,alkaline phosphatease (ALP) and von Kossa’s staining.The cells differentiated to neural-like cells were detected by RT-PCR and immuno-staining.RESULTS: Fibroblast-like cells predominated in culture.(83.76±2.88)% of flMSCs stayed in the G₀/G₁ phases.Homogenous cells were positive for mesenchymal lineage markers CD44,CD29,but not for markers of hematopoietic cells CD45,CD11b.flMSCs were able to differentiate into adipogenic,chondrogenic,osteogenic and neurogenic cells.CONCLUSION: flMSCs can be purified by modified plastic-attachment method and have multiple differentiation,which is available to stem cell therapy for various diseases.     

Biological characteristics of long passaging rat mesenchymal stem cells

AIM:To investigate multi-potential of rat bone marrow mesenchymal stem cells (rBMMSC) and mutation inclination, the rBMMSC were long passaged in vitro. METHODS:Cellular cycles of different passages were assayed by FACSan flow cytometry and karyotypes of passage 6, passage 25 and passage 45 were compared by G-binding analysis. RESULTS:The early passages and long-term passages all showed strong proliferation; passage 6, passage 25 and passage 45 all showed normal karyotype. CONCLUSION:Long-term culture and passage of rBMMSC still remains strong proliferation. With this capability, the mutation inclination is not enhanced.     

Differentiation of rat marrow-derived mesenchymal stem cells into myoid cells in vitro

AIM:To study the differentiation of rat marrow-derived mesenchymal stem cells(MSCs) into myoid cells in vitro.METHODS:The MSCs of SD rat were cultured、passaged、induced and differentiated in vitro used by routine culture technique, and evaluated by FACScan flow cytometer, detected by immunohistochemistry and analyzed by Hitachi H-600 transmission electron microscope(TEM).RESULTS:FACScan shows that cells expressed the antigens of CD29 and CD44, not those of CD11b and CD45;cells show positive response of staining with desmin and myoglobin after processing by two compounds of 5-azacytidine and amphotericin B. There were stripform zone of myofilament without any organells beside the edge of membrane in myoid cell of induction and differentiation checked by TEM.CONCLUSION:The passaged cells were MSCs and the MSCs may have specific gene structures that can differentiate into myoid cells. The demethylation or hypomethylation may conduct by compounds of 5-azacytidine and amphotericin B, which could be involved in activating phenotype-specific genes to differentiate MSCs into myoid cells. There are good outlook on clinical treatment of illness of myatrophy using by MSCs.     

Protective role of carnosine on cardiac dysfunction in type 1 diabetes mellitus rat model

AIM:To explore the effects of carnosine (CAR) on cardiac dysfunction in type 1 diabetic mellitus rats and the underlying mechanism. METHODS:The SD rats were randomly divided into 4 groups:control (C) group, control+carnosine (C+CAR) group, diabetes mellitus (DM) group and diabetes mellitus+carnosine (DM+CAR) group (n=10). The rats were sacrificed after 12 weeks. The cardiac function was assessed by ventricular cannulation. The activity of superoxide dismutase (SOD) and the content of malondialdehyde (MDA) were assessed by ELISA. The mRNA levels of tumor necrosis factor α(TNF-α), interleukin-1β(IL-1β) and IL-6 were measured by real-time PCR. The distribution of connexin 43 (Cx43) was examined by immunofluorescence. The protein levels of Cx43 and protein kinase C (PKC) were determined by Western blot. RESULTS:Compared with the C group, the left ventricular end diastolic pressure (LVEDP) was increased whereas the left ventricular pressure maximum rise/fall velocity (±dp/dt_max) was decreased in the DM group (P<0.01). The activity of SOD decreased while the MDA increased in the left ventricular tissues (P<0.01). The mRNA levels of TNF-α, IL-1β and IL-6 were increased (P<0.01). The Cx43 distribution was irregular. The protein levels of phosphorylated Cx43 and PKCε were elevated (P<0.01). Compared with the DM group, the cardiac function of LVEDP and ±dp/dt_max in DM+CAR group was ameliorated (P<0.01), with increased SOD activity and decreased MDA content (P<0.05). The mRNA levels of TNF-α, IL-1β and IL-6 were reduced (P<0.01). The Cx43 distribution was improved and the protein levels of phosphorylated Cx43 and PKCε were decreased (P<0.01). CONCLUSION:CAR treatment can improve the cardiac function by its anti-oxidative and anti-inflammation effects and suppression of Cx43 abnormalities through PKCε in DM rats.     

Differentiation of mouse iPSCs into insulin-producing cells and experimental study on treating diabetes

AIM: To induce mouse induced pluripotent stem cells (iPSCs) to differentiate into insulin-producing cells (IPCs) by a new 3-step method, and to detect the efficiency and maturity for the treatment of diabetic mice. METHODS: We constructed iPSCs from mouse embryonic fibroblasts of male C57/C mouse by piggyBac transposon, then induced the iPSCs into IPCs by a 3-step method. The cell morphological change was traced by microscopy during the process of differentiation. The expression of mRNA and protein associated with islet β cell development was determined by real-time PCR and immunofluorescence staining. Flow cytometry was used to analysis the efficiency of differentiation. Insulin and C-peptide secretions of IPCs in response to glucose at high (25 mmol/L) or low (5.5 mmol/L) level were measured by ELISA. The IPCs were transplanted into the capsul of left kidney in the male C57/C diabetic mouse model. Blood glucose was continuously monitored for 28 day, serum insulin was tested by ELISA in different stages. The glucose tolerance test was performed on the 28th day, and the left kidney was excised. RESULTS: IPCs were obtained from mouse iPSCs by the 3-step method. The cells expressed the marker genes (Pdx1, Ngn3, Pax6 and Ins2) and proteins (Pdx1, Nkx6.1 and insulin) of β cells. The glucose stimulation induced the secretion of insulin and C-peptide. The efficiency of differentiation was 28% detected by flow cytometry. After transplantation of IPCs to the diabetic mice, the blood glucose was decreased to normal level on the 3rd day,and serum insulin level and the ability of regulating glucose were improved. IPCs were still alive after 28 d of transplantation by pathological observation. CONCLUSION: iPSCs is efficiently induced into IPCs by a 3-step method , and the induction time is shortened significantly. The hyperglycemia of diabetes mice is reversed after transplanting IPCs to same sex inbred strain mice.     

Isolation and identification of human fetal bone-derived mesenchymal stem cells and their differentiation to hepatocyte like cells

AIM: To separate and identify the mesenchymal stem cells (MSCs) from human fetal bone and to study their differentiation to hepatocyte like cells under the action of chemical induction.METHODS: The MSCs from human fetal bone were isolated and purified according to the different growth characteristic of attaching to the wall of cell culture flask.The cell cycle and surface markers of MSCs were identified using flow cytometry.The MSCs were pre-induced by adding DMSO,β-Me and 5-aza for 24 h,then adding the inductive medium of H-DMEM and rh-HGF to induce their differentiation to hepatocyte like cells (HLCs).HLCs were identified by the typical morphological change and the expression of special protein with the method of immunocytochemistry.RESULTS: The MSCs derived from human fetal bone expressed adhesion molecules CD29+,CD44+,but not antigens of hematopoietic CD34,CD45,and not antigens related to GVHD,such as HLA-DR,CD80 and CD86.Exposure of these cells to above-mentioned inductive agents resulted in obvious morphological change and an increase in expression of AFP and ALB.CONCLUSION: The results suggest the existence of plentiful MSCs in human fetal bone.MSCs derived from human fetal bone can easily differentiate to HLCs,and they have a lower immunogenic nature,which may provide the ideal source for tissue engineering (bioartificial liver) for cellular therapeutics.     

Supportive effects of human AGM region and fetal liver stromal cells on differentiation of murine embryonic stem cells into hematopoietic stem cells and in vivo hematopoietic reconstitution

AIM: To investigate the differentiation of murine embryonic stem cells (ESCs) into hematopoietic stem cells (HSCs) by the supportive effects of human aorta-gonad-mesonephros (AGM) region and fetal liver (FL) stromal cells.METHODS: E14 ESCs were induced into embryoid body (EB) first. Then the cells from EB were further co-cultured with human AGM region and FL stromal cells in non-contact system. On day 6, the cells derived from EB were collected for Sca-1⁺c-Kit⁺ cell analysis by flow cytometry, colony forming unit (CFU) assay and teratoma formation checking. BALB/c female mice conditioned with lethal dose of γ-ray irradiation were transplanted with EB cells from different culture systems. The survival rates, engraftment of donor cells, reconstitution of hematopoietic were monitored.RESULTS: Sca-1⁺c-Kit⁺ cells in EB cells co-cultured with human AGM region and FL stromal cells had the value of (21.96±2.54)%, and the total CFU was as (520±52)/10⁵ cells, which were statistically greater than those in EB cells only cultured with human AGM region stromal cells (P＜0.05). No teratoma was found in NOD-SCID mice after subcutaneous injection of EB cells co-cultured with human AGM region and FL stromal cells. In BALB/c female mice transplanted of EB cells co-cultured with human AGM region and FL stromal cells, the survival rate was 77.8%, and the peripheral blood cell count was obviously improved on day 14. PCR results showed the recipients all had sry gene copies from donor in bone marrow. The recipient mice transplanted with EB cells only cultured with human AGM region stromal cells all died within 15 days.CONCLUSION: Stromal cells from human AGM region and FL enhance the directed differentiation of ESCs into HSCs which can reconstruct hematopoiesis in vivo.     

Cell direct reprogramming：a new technique for treating diabetes

Diabetes is characterized by an absolute or relative deficiency in β-cell mass, which cannot be reversed with existing therapeutic strategies. The restoration of the endogenous islet β-cells can stabilize the level of blood glucose. The islet β-cells can be obtained from the directional differentiation of stem cells, but the process is complex and has the risk of teratomas generation. Cell direct reprogramming, one terminal differentiated cell can transdifferentiate into another kind of terminal differentiated cell, which is other than directional differentiation from stem cells. Direct reprogramming gives rise to the generation of islet β-cells from one terminal differentiated cell, may be preferable for diabetes therapy because of its unique advantage.     

Apoptotic mechanism of neuron-like cells differentiated from adult rat mesenchymal stem cells in vitro

AIM: To investigate the mechanism of apoptosis during the process of adult rat mesenchymal stem cells (MSCs) differentiating into neuron-like cells in vitro. METHODS: The MSCs were isolated primarily from adult rats bone marrow by density gradient and then expanded in medium as undifferentiated cells for 3-5 generations. The MSCs were divided into three groups at random. The control group was induced with β-mercaptoethanol (β-ME). Meanwhile, the U0126 group was given β-ME and U0126 (10 μmol/L) added at the beginning of induction. The PMA groups were treated with β-ME and different concentrations of PMA since pre-induction. The effects of U0126 and PMA on the activity of neuron-like cells were observed by MTT. The effects of U0126 and PMA on the expression of neuron specific marker nestin and expression of apoptosis-related protein caspase, Bcl-2, Bax in neuron-like cells were detected by using immunocytochemistry method. TUNEL technique was used to detect apoptosis index. RESULTS: Compared to control group, neuron viability, nestin and Bcl-2 increased and neuron apoptosis decreased in U0126 group (P<0.05). The activity of neuron-like cells, the expression of nestin and Bcl-2 in experiment group treated with 300 nmol/L PMA decreased (P<0.05), while Bax protein expression and apoptosis index increased (P<0.05). CONCLUSION: Both MAPK and PKC signal pathways may be involved in the differentiation of MSCs into neuron-like cells as well as the apoptotic process in differentiated neuron-like cells.     

Differentiation of rat bone marrow mesenchymal stem cells expressing hepatocyte growth factor into hepatocyte-like cells

AIM:To observe the hepatic differentiated efficiency of rat bone marrow mesenchymal stem cells (rMSCs) expressing hepatocyte growth factor (HGF) in three-dimensional microenvironment formed by hanging drop. METHODS:rMSCs were isolated and cultured in vitro, and flow cytometry was used to detect the expression of CD44, CD90, CD34 and CD45. Recombinant retrovirus carrying cDNA of human HGF (pLNCX2-hHGF) was constructed and infected with rMSCs (hHGF-rMSCs). hHGF expression in hHGF-rMSCs was detected by RT-PCR, immunofluorescence staining and ELISA. hHGF-rMSCs were cultured in hanging drop for 21 days. The expression of albumin (ALB),cytokeratin-18 (AFP) and alpha fetoprotein (CK-18) were detected by RT-qPCR and immunofluorescence staining in the 7th, 14th and 21st day, respectively. The secretion of albumin in cultured supernatant was measured by ELISA. RESULTS:CD44 and CD90 were highly expressed in the third generation of rMSCs, but CD34 and CD45 were hardly expressed. The expression of hHGF at mRNA and protein level were all detectable in the hHGF-rMSCs, and the secretion in the cultured supernatant was about (123.71±8.81) μg/L in a period of 21 days. In the hHGF-rMSCs, ALB, AFP and CK-18 were highly expressed at mRNA level from the 7th to the 21st day, and there were significant differences compared with rMSCs at the same time point (P<0.01). The results of immunofluorescence staining showed that the protein expression of AFP was negative on day 7 and 14, and positive on day 21; while the protein expression of ALB and CK-18 was positive on day 7 and lasted until day 21. ALB was positively observed in the culture supernatant of hHGF-rMSCs from 7th to 21st day measured by ELISA, and there was significant difference between the hHGF-rMSCs and rMSCs (P<0.01). CONCLUSION:hHGF transduced-rMSCs can be induced to differentiate into hepatocyte-like cells after cultured in hanging drop which provides a three-dimensional microenvironment. All these results might help to provide new seed cells for cell therapy of clinical liver diseases and in vitro bioartificial liver.     

Transplantation of neuron-like cells induced from rat bone marrow mesenchymal stem cells improves motor function in a MCAO rat model

AIM:To observe the amelioration of motor function in a media cerebral artery occlusion (MCAO) rat model after transplantation of neuron-like cells induced from rat bone marrow mesenchymal stem cells. METHODS:Transplant neuron-like cells derived from rat bone marrow mesenchymal stem cells, which were isolated, cultivated, predicated and induced in vitro, were introduced into infracted cerebral cortex. L-DMEM were injected in control group. Screen test, beam test, prehensile traction test and Morris maze test were conducted at 2 and 8 weeks alternately after transplantation. Rat brain tissues were stained with TTC and percentage of infracted volume was analyzed. RESULTS:There was significant difference between control group and test group in the time and length for rat finding aim and in the grade and time of screen test, beam test, Prehensile traction test (P<0.05). CONCLUSION:Transplantation of neuron-like cells induced from rat bone marrow mesenchymal stem cells improves the motor function of MCAO rats.     

Study on the biological characteristics of rat bone marrow mesenchymal stem cells

AIM: To investigate the basic biological characteristics of adult rat bone marrow mesenchymal stem cells(rBMMSCs), and compare to that of human BMMSCs (hBMMSCs). METHODS: rBMMSC and hBMMSCs were separated from bone marrow with the difference of adherence and Ficoll-Paque reagent, and expanded in culture medium in vitro, respectively. The proliferation and growth characteristics of the primary and different passage culture of rBMMSCs and hBMMSCs were analysed. The neural differentiation capacity of rBMMSCs with passages were observed. To detect the surface antigens of rBMMSCs, the labeled cells were analysed on a FACScan flow cytometer. The karyotype of rBMMSCs were detected by blocking cellular fission with colchicines. RESULTS: rBMMSCs and hBMMSCs have a strong self-renewal capacity. Approximately (4-8)×10¹² and (3-4)×10¹² cells were obtained after passage 15 in vitro, respectively. The ability of proliferation, CFU-Fs, and neural differentiation of rBMMSCs and hBMMSCs were decreased gradually with passages, but the ability of proliferation and CFU-Fs of rBMMSCs were higher than that of hBMMSCs at different passage. FACScan result showed rBMMSCs were uniformly positive for CD29 and CD44, and negative for CD11b, CD45, CD61, CD71, CD80, CD86,MHCⅠ and MHCⅡ. rBMMSCs had an normal karyotype, which had an average of 37.0±4.0 to 40.5±2.5 chromosomes. CONCLUSION: Adult rBMMSCs have strong self-renewal and neural differentiation capacity, and have an normal karyotype. So rBMMSCs can be used as the seed cells for tissue engineering.     

Effects of high-glucose on proliferation and apoptosis in endothelial progenitor cells of type 2 diabetes mellitus

AIM: This study aimed to observe the effects of high-glucose on proliferation and apoptosis of endothelial progenitor cells (EPCs) in type 2 diabetes mellitus patients,and tried to elucidate their possible role.METHODS: Various concentrations of glucose were added to the culture system of EPCs from 25 cases of type 2 diabetes mellitus patients (DM group) and 25 cases of healthy volunteers (control group).MTT assays were used to detect the proliferative rates.Annexin-V/PI stains were used to detect the apoptotic rates,and RT-PCR to detect the expression level of bcl-2 and bax.RESULTS: Proliferative activity of EPCs in both control group and DM group were attenuated when concentration of glucose was 33 mmol/L,while apoptotic rates increased.No significant change of proliferative rate and apoptotic rate of EPCs in DM group and control group in the presence of 5 mmol/L glucose was observed.The expression level of bax of EPCs in both DM group and control group increased while expression level of bcl-2 did not change much in the presence of 33 mmol/L glucose.CONCLUSION: High-glucose attenuates proliferative activity of EPCs and increases the apoptotic rate.Upregulation of bax may be its possible role.     

Transplantation of exogenous adipose tissue derived mesenchymal stem cells for treatment of acute myocardial infarction in rat

AIM: To establish a method of isolating,culturing the adipose tissue-derived mesenchymal stem cells in vitro and to investigate the possibility of exogenous transplanting the adipose tissue derived mesenchymal stem cells for treatment of rat acute myocardial infarction.METHODS: 18 male rats were separated randomly into 3 groups: sham surgery group (control,n=6),acute myocardial infarction control group (AMI,n=6) and myocardial infarction plus cell transplantation group (AMI+cell,n=6).The infarcted hearts were made by occlusion of left coronary artery.The mesenchymal stem cells were isolated from the rats’ peritoneum by using digestion methods and reproduced in vitro,then the cells were labeled with BrdU and implanted into the infarcted heart of the rats.Heart functions were measured 4 weeks after implantation.The hearts were also harvested for pathological and histoimmunochemical observations to determine the survival and location of the implanted cells.RESULTS: Plenty of mesenchymal stem cells were obtained from the adipose tissue of rats’ peritoneum.Compared with the AMI group,the left ventricular systolic pressure in the cell therapy group was increased significantly (P<0.01),the left ventricular end-diastolic pressure was decreased (P<0.01),and the ratio of the left ventricular pressure rise and decay (±dp/dt) was decreased (P<0.05).The number of blood vessels was increased at the boundary of infarction site by pathological observation.The labeled cells were founded in the infarcted myocardium and the blood vessel wall.CONCLUSION: The adipose tissue is a new optional stem cell source.The methods of exogenous transplantation of adipose tissue derived mesenchymal stem cells for treatment of AMI is effective and feasible.     

Effects of sodium deoxycholate on functional state of isolated pancreatic acinar cells of rat

AIM: To investigate the alteration of functional state of pancreatic acinar cells stimulated by sodium deoxycholate (SDOC), and to explore the possible signal pathway involved in the effects of SDOC. METHODS: Rat pancreatic acinar cells were isolated by collagenase digestion and were treated with varying concentration of SDOC or culture media respectively. At different time points (30 min, 1 h, 4 h, 10 h), cell viability was determined by MTT and supernatant of cells was collected to measurer the content of maloidialdehyde (MDA) and the activity of superoxide dismutase (SOD). Some cells were loaded with Fluo-3/AM, then exposed to varying doses of SDOC. ［Ca2+］i change of single pancreatic acinar cell in extracellular fluid with the absence or presence of Ca2+ was determined by laser scanning confocal microscopy. RESULTS: SDOC initiated cell damage in a time-and concentration-dependent manner (P<0.05). Egtazic acid (EGTA) at the concentration of 1 mmol/L decreased the cell mortality (P<0.05). SDOC did not induce a rise of ［Ca2+］i in the calcium-free extracellular fluid. Addition of extracellular calcium in the presence of SDOC resulted in a rapid and remarkable rise of ［Ca2+］i. The increase in ［Ca2+］i preceded the pathological and biological alteration of pancreatic acinar cells. The supernatant content of MDA increased (P<0.05) and the supernatant activity of SOD decreased in SDOC group(P<0.05). CONCLUSION: SDOC initiates cell damage in a time, concentration and calcium dependent manner. SDOC only induces the influx of Ca2+ from extracellular fluid. Calcium overload as an early pathogenetic event takes part in the damage of pancreatic acinar cells by the response of superoxidation. Calcium homeostasis disorder may be one of the causes or at least an important mediator of SDOC- induced pancreatic acinar cell damage.     

Electrophysiological characteristics of the membrane current of cardiomyocytes-like cells derived from rat bone marrow mesenchymal stem cells

AIM: To study the electrophysiological characteristics of the membrane currents of cardiomyocytes-like cells derived from rat bone marrow mesenchymal stem cells (MSCs).METHODS: MSCs were induced,cultured and identified according to the reference.At the fourth week after treatment with 5-azacytidine(5-aza),cardiomyocytes-like cells were detected for the membrane current with the whole-cell patch-clamp technique and compared with the undifferentiated MSCs.RESULTS: The undifferentiated MSCs only expressed potassium currents.MSCs were stained positive for troponin T after treatment with 5-aza,and two kinds of inward currents and three kinds of outward currents were expressed.They respectively were the fast inward sodium current (I_Na),the L-type calcium current (I_Ca),the transient outward potassium current (I_to),the ultra-rapid delayed rectifier potassium current (I_kur) and the slow delayed rectifier potassium current (I_ks).Compared with the undifferentiated MSCs,the potassium currents of cardiomyocytes-like cells derived from MSCs were mainly made up of I_kur and I_ks.CONCLUSION: After treatment with 5-azacytidine,MSCs are differentiated into cardiomyocytes-like cells,which express the current of I_Na,I_Ca,I_to,I_kur and I_ks.