Experimental study on inducing bone mesenchymal stem cells to differentiate into cardiomyocytes in vitro

AIM: To study the differentiation of rat bone mesenchymal stem cells (MSCs) into cardiomyocytes in vitro. METHODS: MSCs were isolated and purified from the bone marrow of rats by density gradient centrifugation and adhering to the plastic culture. The third passage MSCs were treated by 5-azacytidine (5-aza). The induced cells were evaluated by immunocytochemistry staining and RT-PCR analysis. RESULTS: After being induced by 5-aza, some MSCs became bigger and longer. The connection of the cells were formed on day 14.The direction of the cells arraying was similar gradually. The induced cells were stained positively for desmin, α-actin and troponin I. RT-PCR showed that these cells expressed β myosin heavy chain. CONCLUSION: 5-aza can induce MSCs to differentiate into cardiomyocytes in vitro.     

Changes of cardiac function,RAGE expression and calcium dysregulation in type 2 diabetic rats

AIM: To investigate the changes of cardiac structure and function in rats with type 2 diabetic mellitus (T2DM), and to explore the mechanisms underlying diabetic cardiomyopathy. METHODS: The cardiac structure and function were measured by echocardiography in Zucker diabetic fatty (ZDF) rats and their control Zucker lean (ZL) rats. The size of the cardiomyocytes was determined by wheat germ agglutinin staining. The protein expression of atrial natriuretic peptide (ANP), β-myosin heavy chain (β-MHC), receptor for advanced glycation end products (RAGE), L-type cal-cium channel α1C subunit (Ca_V1.2) and Orai1 was assessed by Western blot. RESULTS: Compared with the ZL control rats, the thickness of left ventricular wall, ejection fraction (EF), fractional shortening (FS) and the sizes of cardiomyocytes were significantly increased, and diastolic function was decreased in the ZDF rats (P<0.05). The protein expression of β-MHC, ANP, RAGE and Orai1 was increased, while the expression of Ca_V1.2 was decreased in ZDF rats (P<0.05). CONCLUSION: T2DM rats show the prominent features including cardiomyocyte hypertrophy, ventricular hypertrophy and compensatory enhancement of cardiac function, and the Ca²⁺ handling and increase in RAGE expression may play important roles in the processes.     

南京不同类型梅花品种香气成分的比较研究

1 材料与方法 采用活体植株动态顶空套袋采集法和TCT／GC／MS（热脱附／气相色谱／质谱联用）技术分析南京梅花山真梅种系直枝梅类宫粉型‘玉露宫粉’、玉蝶型‘宇治里’、黄香型‘黄金鹤’、朱砂型‘姬千鸟’、绿萼型‘变绿萼’、洒金型‘复瓣晚跳’及垂枝梅类白碧垂枝型‘双碧垂枝’的香气组成。2002年3月513采样，将采样后的吸附管套上聚四氟乙烯套，放在干燥器中低温保存。样品分析时间为2002年4月27日。同时采集和分析空气作对照。采用Xcalibur 1．2版本软件及NIST98谱图库进行梅花香气成分的检索，兼顾挥发物出峰的保留时间鉴定。     

Adrenomedullin gene transfection enhances the therapeutic effects of transplantation of bone marrow mesenchymal stem cells on cardiac function and ventricle remodeling in acute myocardial infarction rats

AIM: To investigate adrenomedullin gene transfection enhances the therapeutic effects of homogeneous transplantation of bone marrow mesenchymal stem cells (MSCs) on cardiac function and ventricle remodeling in acute myocardial infarction rats. METHODS: MSCs were isolated and expanded using the preplating method. The infection efficiency of adenovirus vector to MSCs was tested by X-gal staining. Ad-ADM expression in MSCs and its secretion in culture medium were measured by ELISA. The left anterior descending branch of rats was ligated to establish a myocardial infarction model. The MSCs were labeled by DAPI, and were directly implanted into the acute infarct site via focal injection. Four weeks later, cardiac function was evaluated using physiological recorder. Hearts were harvested and sliced to be analyzed by immunohistochemistry (factor Ⅷ and ADM) and the DAPI-labeled cells were identified. Sirius red staining was used to identify interstitial collagen on slides. Analysis of collagen type I and III was performed using a polarized filter on sections stained for collagen with Sirius red, and the ratio of collagen type I and III were detected. RESULTS: With X-gal staining, MSCs were effectively transfected by adenovirus in vitro. The transfection efficiency showed the dose-effect relationship with multiplicities of infection (MOI). When MOI was 150, the infection efficiency was 95.4%. The expression of ADM was traced in culture medium and expressed in the time-dependent manner. A maximum production of ADM was observed at 7 d after infection ［(26.53±1.42) ng/L vs (1.34±0.08) ng/L, P<0.05］, and ADM secretion reduced to normal level at 15 d ［(2.20±1.44) ng/L vs (1.52±0.33) ng/L, P>0.05］. DAPI-labeled MSCs transplantation was found in the hearts of the recipients. Immunohistochemical studies demonstrated that intense immunostaining for ADM was higher in Ad-ADM plus MSCs group, compared to other groups. Compared with control, MSCs transplantation significantly increased capillary density in infarct area (P<0.01). A combination of Ad-ADM trensfection and MSCs transplantation demonstrated a further increase in capillary density compared with Ad-ADM or MSCs alone. MSCs transplantation decreased the ratio of collagen type I and III, obviously improved the left ventricular functions. Furthermore the combination treatment resulted in further decrease in the ratio of collagen type I and III, and significantly improved the left ventricular functions. CONCLUSION: Ad-ADM transfection enhances the angiogenic potency of MSCs transplantation and decreases the ratio of collagen type I and III through increasing ADM expression in infarct area, thus contributes to reverse the ventricular remodeling and improves the cardiac function.     

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.     

Effect of leonurine on expression of miR-1 in rats with myocardial fibrosis induced by isoproterenol

AIM: To investigate effect of leonurine on the expression of microRNA-1 (miR-1) in rats with myocardial fibrosis induced by isoproterenol (ISO). METHODS: SD rats (n=10) were used as normal control group, and 80 rats were given ISO by intraperitoneal injection daily for 2 weeks to establish the model of myocardial fibrosis. The model rats were randomly divided into 5 groups:model group, low-dose (7.5 mg·kg^-1·d^-1) leonurine group, middle-dose (15 mg·kg^-1·d^-1) leonurine group, high-dose (30 mg·kg^-1·d^-1) leonurine group and p38 mitogen-activated protein kinase (p38 MAPK) inhibitor (0.3 mg·kg^-1·d^-1) group. After the treatment for 2 weeks, the ultrastructure of left ventricular myocardial tissues was observed under electron microscope. Masson staining was used to detect collagen fibrosis, and the expression of collagen I and collagen Ⅲ was determined by the method of immunohistochemistry. The contents of endothelin-1 (ET-1) and angiotensin Ⅱ (Ang Ⅱ) were measured by ELISA. The expression of miR-1 and ET-1 mRNA was detected by real-time PCR, and the protein expression of p38 MAPK, β-myosin heavy chain (MHC) and α-MHC was determined by Western blot. RESULTS: Compared with model group, the ultrastructure of left ventricular myocardial tissues in high-dose leonurine group was attenuated, and the expression of miR-1 and the protein expression of α-MHC in left ventricular myocardial tissues of high-dose leonurine group were increased (P<0.05). Collagen volume fraction, collagen I, collagen Ⅲ, the ratio of collagen Ⅰ/collagen Ⅲ, the contents of ET-1 and Ang Ⅱ, the mRNA expression of ET-1, and the protein expression of p38 MAPK and β-MHC in high-dose leonurine group were lower than those in model group (P<0.05). CONCLUSION: Leonurine attenuates myocardial fibrosis in the rats induced by ISO, and it is potentially associated with affecting the expression of miR-1, and inhibiting ET-1/p38 MAPK signaling pathway.     

Effect of livin-modified BM-MSCs transplantation on cardiac function following acute myocardial infarction in a rat model

AIM: To study the effect of livin gene-modified bone marrow mesenchymal stem cells(BM-MSCs) transplantation on the cardiac function following acute myocardial infarction in a rat model and the expression of livin, caspase-3, caspase-7 and caspase-9 in the livin gene-modified BM-MSCs. METHODS: The MSCs were obtained by the whole bone marrow culture method, and the apoptosis of the MSCs after infection with adenovirus vector carrying enhanced green fluorescent protein(EGFP) gene and livin recombinant vector(rAd-livin) were detected by flow cytometry. The expression of livin, caspase-3, caspase-7 and caspase-9 was detected by Western blot. After permanent left anterior descending artery occlusion, the rats were randomized to receive intramyocardial injection of DMEM without cells(vehicle group), or containing MSCs(MSCs group), MSCs(EGFP)(rAd-control/MSCs group) or MSCs(livin)(rAd-livin/MSCs group). Left ventricular systolic pressure(LVSP), left ventricular end-diastolic pressure(LVEDP), the maximum increased rate of left ventricular pressure(-dp/dt_max) and the maximum decline rate of left ventricular pressure(+dp/dt_max) were recorded for evaluating the cardiac functions. RESULTS: The apoptosis of rAd-livin/MSCs was significantly decreased as compared with MSCs and rAd-control/MSCs(P<0.05). Meanwhile, the expression of caspase-3, caspase-7 and caspase-9 was significantly downregulated as compared with the other 2 groups(P<0.05). The cardiac function in rAd-livin/MSCs group was significantly improved as compared with DMEM group, and those in the other 2 groups got the similar results, but the function in rAd-livin/MSCs group was better improved. Meanwhile, the number of surviving cells in rAd-livin/MSCs group was significantly improved as compared with the other 2 groups. CONCLUSION: The apoptosis of MSCs is decreased after rAd-livin transfection, and the expression of caspase-3, caspase-7 and caspase-9 is also significantly downregulated while the expression of livin is significantly upregulated. Transplantation of livin-modified BM-MSCs by lentiviral vector results in better prognosis for treating myocardial infarction by enhancing cell survival.     

Effect of mesenchymal stem cells transfected with human heme oxygenase-1(HO-1)gene on myocardial apoptosis and angiogenesis

AIM: To investigate the effects of mesenchymal stem cells(MSCs)transfected with human heme oxygenase-1(HO-1)gene on myocardial apoptosis and angiogenesis. METHODS: MSCs were acquired from the bone marrow of adult rats. The cells were isolated, purified, cultured, and transfected with Adv-HO-1 in vitro before transplantation. At 1 h after left coronary artery ligation, Adv-HO-1-MSCs or MSCs were directly injected into the border of cardiac infarction in rats. Western blotting analysis was used to measure HO-1, and Bax protein expression in the border of cardiac infarction. ELISA was used to measure the expressions of VEGF and bFGF in the border of cardiac infarction. At 4 weeks after transplantation, the heart functions in survival rats were examined by the Buxco system. The rats were killed, then the myocardial infarct size was measured with Masson’s trichrome, and the expression of CD34 in myocardial infarction area was detected by immunohistochemical method. RESULTS: HO-1-MSCs exhibited increased HO-1 expression. The expression of HO-1, VEGF and bFGF in the border of cardiac infarction in the rats treated with HO-1-MSCs were higher than those in the rats treated with MSCs and PBS(P<0.01). However, the expression of apoptotic protein Bax was significantly lower than that in the rats treated with MSCs and PBS(P<0.01). The number of capillary vessels in the border of cardiac infarction in the rats treated with HO-1-MSCs was significantly higher than that in the rats treated with MSCs and PBS. The cardiac function in the rats treated with HO-1-MSCs was better than that in the rats treated with MSCs and PBS(P<0.01). CONCLUSION: The favorable effect on heart function appears to be a combined outcome of HO-1 and paracrine factors released by MSCs.     

Effect of SCF and G-CSF pretreatment on the proliferation and the differentiation of bone mesenchymal stem cells

AIM: To investigate the effect of pretreatment of stem cell factor (SCF) and granulocyte colony-stimulating factor (G-CSF) on the proliferation and the differentiation of mesenchymal stem cells (MSCs) into cardiomyogenic cells. METHODS: The MSCs, isolated primarily from bone marrow, and purified by passage culture, were obtained from the adult rats of four groups: the rats were pretreated by 5 daily injections of SCF; the rats were pretreated with G-CSF; the rats were pretreated with SCF and G-CSF; the rats were treated without any intervention. The 4th passage of MSCs was labeled by DAPI and cellular cycle analysis was conducted by flow cytometry before co-culture. The neonatal rat cardiomyocytes cultured for 3 days were co-cultured with DAPI-MSCs. The percentage of the differentiation of MSCs into cardiomyogenic cells during the five co-culture days was analyzed. The morphologic changes of MSCs and the proteins expression of cardiac myosin heavy chain (MHC) and troponin T (TnT) were recorded respectively with digital microscope camera system and immunofluorescence technique. The percentage of the differentiation of MSCs into cardiomyogenic cells was also calculated. RESULTS: The percentage of MSCs in G₀/G₁ phase in SCF/G-CSF group was significantly lower than that in SCF group, G-CSF group and the control group. The percentage of MHC protein-positive MSCs in SCF/G-CSF group was markedly higher than that in SCF group, G-CSF group and the control group, and that in SCF group and G-CSF group was significantly higher than control group. The percentage of TnT protein-positive MSCs in SCF/G-CSF group, SCF group and G-CSF group was significantly higher than that in control group.CONCLUSION: SCF and G-CSF show the ability to stimulate the proliferation of MSCs and induce MSCs to differentiate into cardiomyocytes. The combination of using SCF and G-CSF is more effective than using only SCF or G-CSF.     

Changes in the expression of endothelin system in rats with chronic heart failing

AIM: To study the changes of endothelin system during chronic heart failure (CHF) and imply the relationship between endothelin system and the course of CHF by observing the mRNA expression of endothelin receptors (ET_AR and ET_BR) and PreproET₁ in early stage and later stage of CHF caused by left coronary artery ligation. METHODS: The mRNA expression of ET_A, ET_B receptors and PreproET₁ were detected by RT-PCR technique. The plasma concentrations of ET₁ and ANP were determined by RIA method. RESULTS: The plasma concentrations of ET₁ and ANP, and the mRNA expression of ET receptors and PreproET₁ in the lefe ventricle increased significantly in early stage (myocardial infarction 10 d). While the plasma concentrations of ET₁ and ANP in later stage (myocardial infarction 70 d) were higher than those in the early stage. However, the mRNA expression of ET_AR, ET_BR and PreproET₁ decreased significantly. The mRNA expression of ET_AR in myocardial infarction (MI) 70 d rats had no difference with those in sham-operated rats and the mRNA expression of ET_BR and PreproET₁ in MI 70 d rats was lower significantly than those in MI 10 d rats, but significantly higher than those in sham-operated rats. CONCLUSION: The changes of ET receptors and PreproET₁ mRNA expression are involved in the cardiac function modulation during the different stages in chronic heart failure.     

Isolation and differentiation of stem cells derived from human placenta into cardiomyocytes

AIM: To isolate the stem cells from human placenta and induce them into cardiomyocytes. METHODS: Stem cells were isolated from human placenta and characterized by morphologic analysis. The “hanging drop” methods were used to inducte stem cells into cardiomyocytes. The expressions of atrial natriuretic factor(Anf) and β-myosin heavy chain(β-MHC) genes were analyzed by RT-PCR. RESULTS: Stem cells derived from human placenta were self-renewed and differentiated into cardiomyocytes in vitro. RT-PCR showed that Anf and β-MHC genes were expressed in the “beating cells”.CONCLUSION: Human placenta is an abundant source of stem cells.     

Effect of rosuvastatin combined with irbesartan on remodeling of myocardial hypertrophy in rats

AIM: To evaluate the effect of rosuvastatin combined with irbesartan on the remodeling of myocardial hypertrophy in the rats. METHODS: Male SD rats(n=50) were randomly divided into control group, model group, rosuvastatin group, irbesartan group and combination group. The model of myocardial hypertrophy was established by subcutaneous injection of isoproterenol at dose of 2.5 mg/kg for 14 d. From the first day of modeling, the rats in control group and model group received intragastrical saline, and the rats in rosuvastatin group, irbesartan group and combination group were treated with rosuvastatin(4 mg·kg^-1·d^-1), irbesartan(15 mg·kg^-1·d^-1) and rosuvastatin(4 mg·kg^-1·d^-1)+ irbesartan(15 mg·kg^-1·d^-1), respectively. The interventions continued for 4 weeks. After the interventions, the cardiac mass index and left ventricular mass index of the SD rats were measured. Besides, the degree of myocardial hypertrophy was observed with HE staining. The mRNA expression of hypertrophy-related factors, such as ANF, β-MHC and AT1R was determined by RT-PCR, and the protein expression of AT1R was determined by Western blot. RESULTS: Compared with control group, the cardiac mass index, left ventricular mass index, as well as the mRNA expression of ANF and β-MHC in model group were significantly increased(P<0.05). Compared with model group, the above factors in rosuvastatin group and irbesartan group were decreased(P<0.05), and the factors in combination group were lower than those in rosuvastatin group and irbesartan group(P<0.05). In addition, the expression of AT1R at mRNA and protein levels in rosuvastatin group and irbesartan group was lower than that in model group(P<0.05), while the expression AT1R at mRNA and protein levels in combination group was lower than that in rosuvastatin group and irbesartan group(P<0.05). CONCLUSION: Rosuvastatin and irbesartan are equally effective drugs to resist the formation of myocardial hypertrophy by decreasing the expression of AT1R. Moreover, combination of the 2 drugs is more effective to improve the degree of myocardial hypertrophy than the 2 drugs alone.     

Inhibitory effect of aerobic exercise on left ventricular remodeling and sympathetic neural remodeling in rats with heart failure after myocardial infarction

AIM: To investigate the effects of long-term aerobic exercise on the heart and sympathetic neural remodeling (structure and function remodeling) in heart failure rats induced by myocardial infarction. METHODS: Heart failure model after myocardial infarction was performed by ligating anterior descending coronary artery in the Wistar rats. Four weeks after operation, the rats were randomly divided into sham operation sedentary (S) group, heart failure sedentary (H) group and heart failure exercise (HE) group. The animals in HE group underwent 10-week treadmill running, while those in S group and H group were sustained in a resting state. The cardiac structure and function including left ventricular internal diameter at diastole (LVIDd), left ventricular internal diameter at systole (LVIDs), left ventricular anterior wall diameter at diastole (LVAWDd), left ventricular anterior wall diameter at systole (LVAWDs), left ventricular posterior wall diameter at diastole (LVPWDd) and left ventricular posterior wall diameter at systole (LVPWDs), and cardiac function parameters including fractional shortening (FS) and left ventricular ejection fraction (LVEF) were measured by echocardiography. The myocardium was collected for histopathological observation with Masson staining, and the collagen volume fraction (CVF) was determined. The concentrations of norepinephrine (NE) in the myocardium and plasma were measured by high-pressure liquid chromatography. The frequency domain analysis was applied for determining the heart rate variability (HRV) via subcutaneous recording electrode involving total power (TP), normalized low power (LFn), normalized high power (HFn) and LF/HF ratio. The mRNA expression of collagen type I (Col-I), collagen type III (Col-III), atrial natriuretic factor (ANF), α-myosin heavy chain (α-MHC), β-myosin heavy chain (β-MHC), sarcoplasmic endoplasmic reticulum Ca²⁺-ATPase (SERCA2a) was detected by real-time PCR. The protein levels of nerve growth factor (NGF) and its receptor (TrkA), and tyrosine hydroxylase (TH) were measured by Western blotting. RESULTS: (1) Compared with S group, body weight (BW), LVIDd, FS, LVEF, TP, HFn, the mRNA expression of α-MHC and SERCA2a, and the protein levels of NGF, TrkA and TH decreased (P<0.05). Left ventricular weight (LVW), left ventricular mass index (LVMI), LVAWDd, LVAWDs, LVPWDd, LVPWDs, CVF, plasma and myocardial NE content, LFn, LF/HF, and the mRNA expression of ANF, β-MHC, Col-I and Col-III increased (P<0.05) in H group. (2) Compared with H group, LVW, LVMI, LVIDd, FS, LVEF, TP, HFn, the mRNA expression of α-MHC and SERCA2a, and the protein levels of NGF, TrkA and TH were raised (P<0.05), while CVF, plasma and myocardial NE content, LFn, LF/HF, and the mRNA expression of ANF, β-MHC, Col-I and Col-III decreased (P<0.05) in HE group. CONCLUSION: Long-term aerobic exercise training leads to inhibition of heart and sympathetic neural remodeling and improvement of cardiac function and autonomic modulation in the rats after myocardial infarction.     

Preliminary investigation on combining autologous marrow stromal cells transplantation with granulocyte colony stimulating factor for improvement of rabbit cardiac performance after actue myocardiac infarction

AIM: To test the hypothesis that autologous marrow stromal cells (MSCs) transplantation combined with granulocyte colony stimulating factor (G-CSF) can enhance cardiac function of ischemic hearts in vivo.METHODS: In order to achieve a safe and persistent effect,we explored the potential of autologous MSCs transplantation.Acute myocardial infarction induced by occlusion of left anterior descending artery,autologous MSCs labeled with BrdU bromodeoxyuridine in vitro were administered intramyocardially into the infarct area of the same donor rabbits and G-CSF was administrated by subcutaneous injection.Four weeks later,the transplanted labeled MSCs were detected by laser scanning confocal microscopy and the cardiac functions were examined by echocardiogram and multichannel physiologic recorder.Myocardial infarct size was measured from mid-transverse sections stained with Massons trichrome.RESULTS: After 4 weeks,transplanted MSCs were demonstrated myogenic differentiation with the expression of α-sarcomeric actin and connexin 43 located in intercalated disk.MSCs combined with G-CSF transplantation improved the left ventricular contractility and reduced myocardial infarct size markedly compared to that without G-CSF tratment.CONCLUSION: Our finding indicates that autologous MSCs combined with G-CSF transplantation may represent a promising therapeutic strategy on ischemic heart disease.     

Inhibition of Rac1 protects cardiac functions in STZ-induced diabetic cardiomyopathy through decreasing pho-p38 MAPK

AIM: To investigate the effects of Rac1 inhibition on the size of cardiomyocytes and left ventricular functions in diabetic cardiomyopathy. METHODS: Type 1 diabetes was induced in 8-week old C57 mice by streptozotocin (STZ, ip injection). Diabetic mice were treated with NSC23766, a specific inhibitor of Rac1 (STZ+NSC group, n=15) or without treatment (STZ group, n=15). Nondiabetic mice were used to serve as empty control (Con group, n=10) or NSC23766 control (NSC group, n=10). The survival rate, LVHW/BW and left ventricular functions were detected at the end of 8 weeks after induction of diabetes. The expression of ANP, BNP, β-MHC and pho-p38 MAPK in the cardiac tissues were analyzed by real-time RT-PCR and Western blotting, respectively. Hematoxylin and Eosin staining (HE) was used to measure the cell size in the cardiac tissues. RESULTS: The functions of left ventricle were significantly impaired in the diabetic animals with decreased survival rate and increased LVHW/BW, which was accompanied by significant increases in ANP, BNP and β-MHC, and elevated pho-p38 MAPK expression in diabetic hearts. The increased survival rate, improved left ventricular functions and decreased LVHW/BW were observed in the diabetic animals treated with NSC. The mRNA expression of ANP, BNP, β-MHC and pho-p38 MAPK was significantly attenuated in the diabetic hearts by NSC treatment. The size of the cardiomyocytes, which increased in diabetic hearts, was decreased in the NSC-treated diabetic cardiac tissue. CONCLUSION: Rac1 inhibition protects left ventricular functions and attenuates hypertrophy, which is associated with the decrease in pho-p38 MAPK expression in diabetic heart. These data suggest that inhibition of Rac1 might be beneficial to diabetic cardiomyopathy.     

Protective effects of bone marrow stromal cells on ischemia/reperfusion hippocampal slices

AIM:To study the protective effect of bone marrow stromal cells (MSCs) on ischemia /reperfusion hippocampal slices.METHODS:Ischemia/reperfusion models of hippocampal slices from newborn rats were established. MSCs obtained from adult bone marrow were cultured, isolated and purified. Cell death was assessed using propidium iodide fluorescence. And brain-derived neurotrophic factor (BDNF) expression in MSCs was determined by immunocytochemistry and Western blot.RESULTS:Maximal dead cells appeared in hippocampal slices 3 to 7 days after reperfusion. When the slices were co-cultured with MSCs, only a few cells were dead. The protective effect of MSCs on the slices was diminished significantly when anti-BDNF antibody was added to the medium. The protein of BDNF was faintly expressed in MSCs under normal conditions. When MSCs were co-cultured with ischemia /reperfusion hippocampal slices, the expression of BDNF in MSCs was increased gradually especially when co-cultured for 3 to 7 days. However, MSCs co-cultured with normal hippocampal slices expressed BDNF at a lower level at any times of co-culture.CONCLUSIONS:In an in vitro model of simulated ischemia, MSCs reduce cell death. Ischemia/reperfusion hippocampal slices co-cultured with MSCs promote the expression of BDNF in MSCs, which in turn protect the ischemic neurons.     

Genes expression and electrophysiological characterization of ES cell-derived cardiomyocytes

AIM: To study the expression of αMHC, Anf, MLC2v genes and characterization of electrophysiology of cardiomyocytes derived from murine transgenic PαMHC-EGFP embryonic stem cells in vitro. METHODS: At 0 d, 3 d, 5 d, 7 d, 10 d, 14 d, gene expression profiles of αMHC, Anf, MLC2v were made by RT-PCR and electrophysiology profiles were made by patch clamp analysis in cardiomyocytes. RESULTS: EGFP positive clusters of cells were first observed as early as 7 d and 8 d of development. αMHC gene expressed at 7 days of EB, and was enhanced at 10 d and 14 d (P<0.05). Anf and MLC2v gene expressed in cardiomyocytes at 10 d and 14 d. Patch clamp experiments showed that ES cell-derived cardiomyocytes in early stage comprised the different cardiac subtypes. 73.5% (n=34) of EGFP positive beating cardiomyocytes displayed atrial-like ［APD90=（78.4±3.1）ms］, 20.5% of those cells displayed pacemaker-like ［APD90=（112.6±5.5）ms, n=7］, only one cell displayed (APD90=200 ms) ventricular-like. CONCLUSION: αMHC, Anf, MLC2v genes expressed in the cardiomyocytes during early stages of development. Therefore, αMHC, Anf, MLC2v could be used as marker genes for developing chamber myocardial cells in vitro.     

Long-term insulin treatment improves postischemic cardiac structure and function via increasing serum BNP levels

AIM：To investigate the influence of long-term insulin treatment on postischemic cardiac structural and functional changes, and to further explore the underlying mechanisms. METHODS：Adult male SD rats were randomly divided into 4 groups (8~10 rats per group): sham group, myocardial infarction (MI) + saline (1 mL·kg-1·d-1, hypodermic injection for 4 weeks) group, MI + insulin (2 U·kg-1·d-1, hypodermic injection for 4 weeks) group and MI + insulin (2 U·kg-1·d-1, hypodermic injection for 4 weeks) + wortmannin ［a phosphatidylinositol 3-kinase (PI3K) inhibitor; 15 μg·kg-1·d-1, intraperitoneal injection 15 min before each insulin treatment］ group. The rats in the latter 3 groups were subject to ligation of the left anterior descending coronary artery, while those in sham group underwent the same surgical procedures without tying the sutures. The cardiac structural and functional changes were observed by echocardiogram, heart catheterization and microscopy with HE and Masson trichrome staining. Blood glucose was determined by Roche blood glucose meter, and the serum levels of insulin and brain natriuretic peptide (BNP) were detected by ELISA. The protein expression and phosphorylation of PI3K, Akt, glycogen synthase kinase 3β (GSK3β) and p38 mitogen-activated protein kinase (p38 MAPK) in myocardial tissues were detected by Western blotting. The mRNA expression of BNP, β-myosin heavy chain (β-MHC) and atrial natriuretic peptide (ANP) in myocardial tissues was determined by real-time fluorescence quantitative PCR. RESULTS：At the end of the 4th week, MI rats receiving long-term insulin treatment showed decreased ratio of heart length/heart weight, smaller systolic left ventricle cavity, thicker systolic interventricular septum, and increased cardiac ejection fraction, left ventricular development pressure and instantaneous first derivate of left ventricle pressure (P<0.05 vs MI + saline group). Moreover, insulin treatment significantly increased the phosphorylation of PI3K and Akt and the serum level of BNP, and inhibited the phosphorylation of p38 MAPK (P<0.05 vs MI + saline group), but did not change the mRNA expression of BNP in myocardial tissues. The effects of insulin on BNP were not blocked by wortmannin (P>0.05 vs MI + insulin group). CONCLUSION：Insulin improves postischemic cardiac structure and function by increasing serum BNP levels possibly independent of PI3K-Akt signaling pathway.