Changes of cardiac function, RAGE expression and calcium dysregula-tion in type 2 diabetic rats / 中国病理生理杂志

AIM:To investigate the changes of cardiac structure and function in rats with type 2 diabetic melli-tus(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(CaV1.2)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 cardiomyo-cytes 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 hyper-trophy and compensatory enhancement of cardiac function, and the Ca2+handling and increase in RAGE expression may play important roles in the processes.

L-type calcium channel involved in heterogeneity of arterial constriction in different organs of rats / 中国药理学通报

Aim To study whether there was arterial heterogeneity and association with L-type calcium channel (LCC) in different parts of arteries in re-sponse to certain vasoconstrictor. Methods The aor-ta, renal arteries and coronary arteries were dissected from rats. Arterial ring contractions induced by pheny-lephrine (Phe), 5-hydroxyl tryptamine (5-HT) or U46619 in concentration-dependent manner were meas-ured using the Multi Myograph system and the response to nifedipne was observed. Results (1) Phe had no obvious effect on the tension of coronary artery,but in-duced concentration-dependent vasoconstriction in aor-ta and renal artery,and pEC50of aorta was significantly higher than that of renal artery (P<0.05). The inhi-bition rate of nifedipine on the aortic contractile re-sponses was significantly higher than that of renal arter-y (P<0.05). (2) The contraction induced by 5-HT on aorta was not obvious, but was significant on renal artery and coronary artery. The inhibitory rate of nife-dipine on coronary artery vasoconstriction was signifi-cantly higher than that of renal artery (P <0.05). (3) U46619 could induce aorta,renal artery and coro-nary artery concentration- dependent contraction, but the Emaxof them were both higher than that of renal ar-tery (P<0.05). And the pEC50of aorta was the lar-gest (P<0.05). Nifedipine significantly inhibited the contraction of aorta, renal artery and coronary artery induced by U46619 with the greatest inhibitory rate on the coronary artery vasoconstriction and minimal inhibi-tion on aortic vasoconstriction. Conclusions The re-sponse to certain vasoconstrictor is different among aor-ta, renal artery and coronary artery in rats, and the contraction mediated by L-type calcium channel is also different.

Effect of impaired glucose tolerance on cardiac dysfunction in a rat model of prediabetes / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>The effect of impaired glucose tolerance (IGT) on cardiac function during the chronic prediabetes state is complicated and plays an important role in clinical outcome. However, the molecular mechanisms are not fully understood. This study was designed to observe cardiac dysfunction in prediabetic rats with IGT and to determine whether glucose metabolic abnormalities, inflammation and apoptosis are linked to it.</p><p><b>METHODS</b>The IGT rat models were induced by streptozocin, and the heart functions were assessed by echocardiography. Myocardial glucose metabolism was analyzed by glycogen periodic acid-Schiff staining, and the pro-apoptotic effect of IGT was evaluated by TUNEL staining. Additionally, caspase-3 activation, macrophage migration inhibitory factor (MIF) and G-protein coupled receptor kinase 2 (GRK2) were detected by Western blotting in cardiac tissue lysates.</p><p><b>RESULTS</b>Area-under-the-curve of blood glucose in rats injected with streptozotocin was higher than that in controls, increased by 16.28%, 38.60% and 38.61% at 2, 4 and 6 weeks respectively (F = 15.370, P = 0.003). Abnormal cardiac functions and apoptotic cardiomyocytes were observed in the IGT rats, the ejection fraction (EF) being (68.59 ± 6.62)% in IGT rats vs. (81.07 ± 4.59)% in controls (t = 4.020, P = 0.002). There was more glucose which was converted to glycogen in the myocardial tissues of IGT rats, especially in cardiac perivascular tissues. Compared to controls, the cleaved caspase-3, MIF and GRK2 were expressed at higher levels in the myocardial tissues of IGT rats.</p><p><b>CONCLUSIONS</b>IGT in the prediabetes period resulted in cardiac dysfunction linked to abnormal glycogen storage and apoptosis. Additionally, MIF and GRK2 may be involved in the pathogenesis of cardiac dysfunction in prediabetes and their regulation may contribute to the design of novel diagnostic and therapeutic strategies for those who have potential risks for diabetic cardiovascular complications.</p>

Effect of decellularization on tissue composition and immunogenicity of porcine and human aortic valves / 南方医科大学学报

<p><b>OBJECTIVE</b>To study the changes of tissue composition and immunogenicity of porcine and human aortic valves after decellularization.</p><p><b>METHODS</b>Three cryopreserved human aortic valves and 4 porcine valves were decellularized with trypsin, and the leaflet tissue was homogenized for SDS-PAGE protein electrophoresis and U-937 migration assay.</p><p><b>RESULTS</b>Trypsin effectively removed the cells from the valve. SDS-PAGE demonstrated an obvious difference in the tissue composition between porcine and human valves. Although decellularization significantly diminished the differences between the valves, decellularized procine aortic valve stilled contained more protein components (between 26 000 and 43 000) than human valve. U-937 migration assay showed an obvious decrease of cell migration in the valves by decellularization (from 832.7×10(3) to 152.4∓31.1×10(3) for porcine valves, P<0.01, and from 644.9×10(3) to 91.2×10(3) for the human valves, P<0.01). Decellularized porcine valves induced a significantly greater cell migration than decellularized human valves (P<0.05).</p><p><b>CONCLUSION</b>Decellularization with trypsin can effectively decrease the immunogenicity of human or porcine heart valve, but can not completely eliminate the antigen, and decellularized porcine valve still retain strong immunogenicity.</p>

Effect of ginsenoside Rg1 on the microenvironment dependent differentiation of human bone marrow mesenchymal stem cell to vaso-endothelioid formative cells in vitro / 中国中西医结合杂志

<p><b>OBJECTIVE</b>To investigate the effect of ginsenoside Rg1 on the microenvironment dependent differentiation of human mesenchymal stem cells (hMSCs) to vaso-endothelioid cells (VECs) in vitro.</p><p><b>METHODS</b>The in vitro differentiation of hMSCs to VECs were established adopting the in vivo environment simulated semi-permeable membrane separated non-contact co-culturing method. The mRNA expressions of endothelial markers, such as platelet endothelial adhesive factor-1 (CD31), vascular hemophillia factor (vWF) and vascular endothelial cadherin (VE-cadherin) were analyzed by RT-PCR; the protein expressions of CD31 and vascular endothelial adhesive factor-1 (VCAM1) were detected by fluorescence immunohistochemistry; structural identification for the endothelial characteristics of differentiated hMSCs were made under electron microscopy; and the percentage of CD31 expression in differentiated hMSCs was determined by flow cytometry to explore the effect of ginsenoside Rg1 on the differentiation.</p><p><b>RESULTS</b>The bone marrow mesenchymal stem cells co-cultured with mature endothelial membrane showed a microenvironment dependent capacity for differentiating to endothelium, with the morphological changes revealed starting from the 2nd week, showing cell body contraction, polygonal-shaped change; and at the 3rd week, the markedly speedily cell proliferation with elliptic or slabstone-like change of cells. High levels of classic endothelial cell markers, such as mRNA expressions of CD31, vWF, VE-cadherin, and protein expressions of CD31 and VCAM1, were shown; the typical weibel-palade body of endothelial cell was found in the differentiated cells. Moreover, percentage of CD31 expression in the differentiated hMSCs was increased after Rg1 treatment dose-dependently.</p><p><b>CONCLUSION</b>Under the microenvironment of co-culture, hMSCs could differentiate into cells presenting the characteristics of endothelial cell in aspects of the morphology and ultrastructure of cells, as well as the gene and protein expressions of cell markers; ginsenoside Rg1 can promote the microenvironment dependent differentiation of hMSCs to VECs system in vitro.</p>

High glucose up-regulates GRK2 gene expression in H9C2 cardiomyoblasts in vitro / 南方医科大学学报

<p><b>OBJECTIVE</b>To study the effect of high glucose on GRK2 gene expression in H9C2 cardiomyoblasts in vitro.</p><p><b>METHODS</b>H9C2 cardiomyoblasts were cultured for 72 h in the presence of 0, 5.5, 12.5, 25 or 33 mmol/L glucose (with the osmotic pressure adjusted with monnitol). Semi-quantitative detection of GRK2 gene expression in H9C2 cardiomyoblasts was carried out using RT-PCR and phosph-Akt (Ser473) protein level was measured by Western blotting.</p><p><b>RESULTS</b>Glucose in the culture medium (5.5 to 33 mmol/L) concentration-dependently increased the mRNA expression of GRK2 concentration and decreased phosphorylation Akt (ser473) level in in H9C2 cardiomyoblasts.</p><p><b>CONCLUSION</b>Increased GRK2 gene expression may play an important role in cardiac dysfunction in diabetes.</p>

Effect of ketamine on transient outward potassium current of isolated human atrial myocytes / 药学学报

The effects of ketamine on transient outward potassium current (I(to)) of isolated human atrial myocytes were investigated to understand the mechanism of part of its effects by whole-cell patch-clamp. Atrial myocytes were enzymatically isolated from specimens of human atrial appendage obtained from patients under going cardiac valve displacing. Ito is recorded in voltage-clamp modes using the patch-clamp technique at room temperature. Currents signals were recorded by an Axopatch 200B amplifier with the Digidata 1322A-pClamp 9.0 data acquisition system. Ketamine decreased I(to) of human atrial myocytes in a dose-dependent manner. The current-voltage curve was significantly lowered, 30, 100, 300, and 1000 micromol x L(-1) ketamine decreased respectively I(to) current density about (13.62 +/- 0.04)%, (38.92 +/- 0.05)%, (72.24 +/- 0.10)% and (83.84 +/- 0.05)% at the potential of 50 mV, with an IC50 of 121 micromol x L(-1). The I(to) activation curve, inactivation curve and the recovery curve were not altered by ketamine. So, ketamine concentration-dependently decreased I(to) of human atrial myocytes.

Role of nitro oxide in immunosuppressive effect of human mesenchymal stem cells on allogenic proliferative response of lymphocytes / 中国实验血液学杂志

The purpose of this study was to investigate the influence of nitro oxide (NO) from mesenchymal stem cells (MSC) on the proliferative responses of allogeneic lymphocytes and its mechanism. MSCs were isolated and cultured from human bone marrow. Selected surface antigens of MSCs were detected by flow cytometry and their morphologic characteristics were determined by microscopy. Mitomycin C-treated MSCs were plated in dishes and then mixed lymphocyte cultures (MLC) were set up. After 4 days, lymphocyte proliferation was determined by CCK-8 assays; NO secretion in coculture supernatant was determined by Griess reagent kit; the level of FOXP3 mRNA expression was detected by real-time quantitative PCR. The results indicated that in MSC/MLC coculture experiment, the lymphocyte proliferation decreased significantly with of IOD value 0.49+/-0.03, NO production increased obviously (21.05+/-1.14 micromol/L) and FOXP3 mRNA expression was increased [(1.56+/-0.34)%] as compared with MLC coculture without MSC. There were significant difference between these two groups. It is concluded that NO production in human MSC culture up-regulates FOXP3 mRNA expression and thus inhibits lymphocyte proliferation response.

Identification of the interactions between the truncated fragments of macrophage migration inhibitory factor and CD74 using a yeast two-hybrid system / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the interaction domains between macrophage migration inhibitory factors (MIF) and the extracellular segment of type-II trans-membrane protein CD74 using a yeast two-hybrid system.</p><p><b>METHODS</b>By using molecular cloning techniques, the DNA fragments encoding MIF, MIF(50-65) and MIF(1-50/65-115) were introduced into the pGBKT7 vector to construct the corresponding recombinant bait plasmids, and the DNA fragments encoding CD74(73-232), CD74(73-109), CD74(1109-149) and CD74(149-232) into the pGADT7 vector to construct the recombinant activation domain (AD) plasmids. PEG/LiAC method was employed to transform the above 3 recombinant bait plasmids paired with each of the 4 recombinant AD plasmids into the chemical competent yeast AH109 cells. The transformed yeast AH109 cells were screened consecutively on SD/-Trp-Leu and SD/-Trp-Leu-Ade-His/X-alpha-gal nutritional media.</p><p><b>RESULTS</b>The results of restriction endonuclease digestion and DNA sequencing verified the correct construction of all the recombinant plasmids. The yeast AH109 cells transformed with each of the 3 recombinant bait plasmids could grow on SD/-trp nutritional media without autonomous activation effect on the reporter gene MEL1. The cells transformed with each of the 4 recombinant AD plasmids could also grow on SD/-leu nutritional media without activation of the reporter gene MEL1. Only the yeast AH109 cells co-transformed with MIF, MIF(50-65), or MIF(1-50/65-115) plasmid and CD74(73-232) plasmid could grow on SD/-Trp-Leu-Ade-His nutritional media with transcription activation of the reporter gene MEL1.</p><p><b>CONCLUSION</b>MIF interacts with the intact extracellular segment of CD74 (CD74(73-232)) independent of the functional domain of MIF(50-65).</p>

An efficient method for screening effective siRNAs using dual-luciferase reporter assay system / 南方医科大学学报

<p><b>OBJECTIVE</b>To establish an efficient method for screening effective small interference RNA (siRNA) using dual-luciferase reporter assay system.</p><p><b>METHODS</b>Based on the siRNA expression vector pSilencer-4.1, 3 candidate green fluorescence protein (GFP) gene siRNA expression plasmids, namely pSi-GFPsiRNA1, pSi-GFPsiRNA2, and pSi-GFPsiRNA3, along with the negative control pSi-Negative, were constructed. Using the pGL3-promoter vector, the GFP-luciferase (GFP-LUC) expression plasmid pGL3-GFPf was constructed with the same Kozak consensus translation initiation site and start codon ATG for GFP-LUC coding sequence. The GFP fragment containing the target sequences of 3 GFP siRNAs was introduced into the 3' untranslate region of LUC in the modified pGL3-promoter vector to construct the plasmid pGL3-GFPp. The GFP siRNAs expression plasmids and Renilla luciferase reporter vector pRL-TK were co-transfected with pGL3-GFPf or pGL3-GFPp into the HEK293 cells, respectively. The luciferase activities were determined by dual-luciferase reporter assay, and the GFP mRNA expressions were detected by real-time quantitative PCR.</p><p><b>RESULTS</b>In the groups cotransfected with GFP siRNAs expression plasmids and pGL3-GFPf, the luciferase activities were reduced obviously, and the reduction was more significant in cells transfected with GFPsiRNA1 compared with the control cells (P<0.01).GFP mRNA levels were also markedly lowered in cells transfected with GFPsiRNA1 as shown by real-time PCR (P<0.01). In addition, the results of dual-luciferase reporter assay and real-time PCR showed that among the groups cotransfected with GFP siRNAs expression plasmids and pGL3-GFPp, the GFP expression was inhibited most obviously by GFPsiRNA1 (P<0.01).</p><p><b>CONCLUSION</b>The dual-luciferase reporter assay system provides a useful method for screening effective siRNAs targeting specific genes.</p>

Establishment of a rat model of bone marrow mesenchymal stem cell transplantation for repairing full-thickness skin defect / 南方医科大学学报

<p><b>OBJECTIVE</b>To establish a rat model of full-thickness skin defect to receive bone marrow mesenchymal stem cell transplantation for wound repair.</p><p><b>METHODS</b>A full-thickness skin defect measuring 4 cmx4 cm in 36 F344 rats, which were divided into 3 groups with the wound covered with alloskin graft, acellular dermal matrix, or petrolatum gauze. In vitro cultured BMSCs in the 5th passage were transplanted into the skin defect, and the time of wound dressing dissociation and number of transplanted Brdu-positive cells in the wound were observed 14 days later.</p><p><b>RESULTS</b>The alloskin graft resulted in significantly longer time before dressing dissociation, with greater number of Brdu-positive cells in the wound than the other two wound dressings (P<0.001). The acellular dermal matrix showed better effect than petrolatum gauze in terms of the dressing dissociation time and the viable transplanted cell number in the wound.</p><p><b>CONCLUSION</b>Alloskin graft can be ideal for covering the wound surface to protect the transplanted BMSCs in rats.</p>

Efficacy comparison between transplanting microenvironmental induced and non-induced bone marrow mesenchymal stem cells in ischemic rat hearts / 中华心血管病杂志

<p><b>OBJECTIVE</b>To compare the efficacy of transplanting bone marrow mesenchymal stem cell (BMSC) or microenvironmental induced BMSC (iBMSC) into the ischemic myocardium of rats with myocardial infarction.</p><p><b>METHODS</b>iBMSC was defined as BMSC co-cultured with myocardial cells for 2 weeks. The stem cells or equal volume PBS were injected into ischemic border zone 1 wk after experimental infarction. Cardiac performance was evaluated at 1, 2, and 4 wk after cell transplantation by echocardiography and analyzed histologically at 4 wk after cell transplantations.</p><p><b>RESULTS</b>Compared with PBS group, both BMSC and iBMSC transplantations reduced infarct size. iBMSC enhanced the beneficial effects of BMSC on improving cardiac function (FS: 28.5% +/- 4.3% in PBS, 29.0% +/- 2.0% in BMSC and 45.1% +/- 3.1% in iBMSC group at 4 weeks post transplantation, iBMSC group vs. PBS group P < 0.05, iBMSC group vs. BMSC group P < 0.05). Immunofluorescence microscopy results revealed co-localization of SPIO-labeled transplanted cells with cardiac markers for cardiomyocytes, indicating regeneration of damaged myocardium.</p><p><b>CONCLUSION</b>Our data suggest that iBMSC implantation is more effective on improving cardiac function than BMSC implantation in this model. iBMSC might serve as a new promising therapeutic cell source for regenerating ischemic myocardium in patients with post-infarction heart failure.</p>

Effect of vascular endothelial growth factor small interfering RNA (siRNA) on retinal microvascular endothelial cells under hypoxia condition in vitro / 中华儿科杂志

<p><b>OBJECTIVE</b>To explore VEGF siRNA's effect on the immature fetal retinal microvascular endothelial cells in vitro.</p><p><b>METHOD</b>The fresh retinal micrangium was primarily cultured to obtain microvascular endothelial cells. CoCl2 was used to simulate oxygen-deficient conditions. siRNA directed against human VEGF was designed and chemically synthesized. There were 3 groups in our experiment: VEGF siRNA group, hypoxia control group, and negative siRNA control group. The fetal retinal micrangium vascular endothelial cells were transfected by using liposome. The expression levels of VEGF mRNA and protein were evaluated by RT-PCR and Western blotting 24, 48, 72 h after transfection, cell proliferation was evaluated by MTT method.</p><p><b>RESULT</b>The expression levels of VEGF mRNA decreased by 21.05%, 79.67%, and 90.48% 24 h, 48 h, and 72 h after transfection as compared to those in hypoxia control group, the expression level of VEGF protein had decreased by 14.58%, 66.97%, and 81.61% as compared to those in hypoxia control group. The siRNA could decrease cell proliferation under hypoxia too, the multiplication rate after 12, 24, 48, and 72 h decreased by 15.0%, 42.9%, 78.3% and 65.9%.</p><p><b>CONCLUSION</b>VEGF siRNA could down-regulate the expression of VEGF in immature fetal retinal microvascular endothelial cells and suppressed cell proliferation. Application of siRNA to inhibit expression of VEGF may be a hopeful way to prevent and cure ROP.</p>

Plasmid-mediated miRNA-1-2 specifically inhibits Hand2 protein expression in H9C2 cells / 南方医科大学学报

<p><b>OBJECTIVE</b>To construct an eukaryotic expression vector for miRNA-1-2 that can be expressed in rat H9C2 cardiomyocytes.</p><p><b>METHODS</b>The precursor miRNA (pre-miRNA) DNA template for miRNA-1-2 was designed and generated by PCR amplification. The DNA template was inserted into the hairpin RNA expression vector pSilence-4.1-neo and identified by DNA sequencing analysis. The recombinant plasmid DNA was then transfected into H9C2 cells via Lipofectamine, and the green fluorescence protein expression vector pEGFP-N3 served as the transfection marker. Twenty-four hours after transfection, the total cellular RNA was extracted using TRIzol reagent, and thermoscript reverse transcriptase (RT)-PCR was performed to determine miRNA-1-2 precursor expression.</p><p><b>RESULTS AND CONCLUSION</b>DNA sequencing indicated that the miR-1-2 expression plasmid was correctly constructed. The precursor miRNA-1-2 was successfully expressed in the H9C2 cells, and the expression of Hand2 protein could be efficiently inhibited by miRNA-1.</p>

Effect of adenoviral vectors on the differentiation potential of human bone marrow mesenchymal stem cells / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the effect of adenoviral vector infection on the differentiation potential of human bone marrow mesenchymal stem cells (hMSCs).</p><p><b>METHODS</b>The third-passage hMSCs were infected with the recombinant adenovirus expressing green fluorescence protein (GFP) for 2, 4, 8 and 16 days. RT-PCR was used to detect the mRNA expression of endodermal marker CYP 51, mesodermal marker SM22alpha, ectodermal marker nestin, pluripotent marker oct-4 and the alternative splicing factor nPTB. Seven days after adenovirus infection, the hMSCs were cultured in the presence of adipogenic agents for 14 days, and the adipose cells differentiated from hMSCs were detected with oil red O staining.</p><p><b>RESULTS</b>Compared with normal hMSCs, the cell infected with the adenovirus for 2, 4, 8 and 16 days showed no obvious down-regulation of CYP51, SM22alpha, nestin, OCT4 or nPTB. The hMSCs 7 days after adenovirus infection were induced to differentiate into adipose cells, with a similar differentiation rate to that of normal hMSCs. CONCLUSION The differentiation potential of hMSCs is not affected by adenovirus infection, suggesting that adenovirus can be used as the gene delivery vector in MSC differentiation studies.</p>

Human bone marrow mesenchymal stem cells cocultured with semi-permeable membrane separated neonatal rat ventricular myocytes differentiated into cardiomyocyte phenotype / 中华心血管病杂志

<p><b>OBJECTIVE</b>To investigate the ability of human bone marrow mesenchymal stem cells (hBMSCs), cocultured with semi-permeable membrane separated neonatal rat ventricular myocytes, to differentiate into cardiomyocytes.</p><p><b>METHODS</b>hBMSCs were isolated and purified by density gradient centrifugation and adherence screening method. Cells were expanded as undifferentiated cells in culture for more than 3 passages and their phenotypes were identified with flow cytometer. hBMSCs were cocultured with neonatal rat ventricular myocytes in a rate of 1:10 separated by semi-permeable membrane. GATA4 mRNA was detected by RT-PCR; Immunocytochemistry, and Immunostaining were used to detect sarcomeric alpha-actinin, desmin, cTnT, and cTnI protein level.</p><p><b>RESULTS</b>CD29 (98.64% +/- 0.80%) and CD44 (96.70% +/- 1.50%) were the major surface markers of hBMSCs. After coculturing with semi-permeable membrane separated neonatal rat ventricular myocytes, the first contraction of single cells was noted at day 7 and GATA4 expression was detected on these cells by RT-PCR after 1 to 3 weeks coculture. Desmin, sarcomeric alpha-actinin, cTnI and cTnT could be detected by immunocytochemistry and immunostaining on some of these cells.</p><p><b>CONCLUSION</b>hBMSCs possess the potential to differentiate into myocardial cell phenotype in the cardiac microenvironment. Direct contact with cardiomyocytes was not necessary required for hBMSCs differentiation.</p>

MicroRNAs can be expressed in cardiomyocyte-like cells differentiated from human mesenchymal stem cells / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the expression of representative heart-specific primary microRNAs (pri-miRNAs) in the cardiomyocyte-like cells differentiated from human mesenchymal stem cells (hMSCs).</p><p><b>METHODS</b>The phenotype of hMSCs isolated was identified by flow cytometry using monoclonal antibodies against FITC-conjugated CD29, CD34, and CD11b. The third-passage hMSCs were induced to differentiate into cardiomyocyte-like cells by 5-azacytidine and indirect coculture with neonatal rat myocytes, respectively. Immunocytochemical analysis was performed to detect the expression of the cardiac-specific proteins, namely cardiac troponin I (cTnI) and sarcomeric alpha-actinin, in the cardiomyocyte-like cells differentiated from hMSCs. RT-PCR and DNA sequencing were used to identify the expression of the 5 representative heart-specific pri-miRNAs.</p><p><b>RESULTS</b>High hMSC marker CD29 expression rate (98.87%) and low hematopoietic cell markers CD34 (5%) and CD11b (0.4%) expression rates were identified in the hMSCs isolated. cTnI and sarcomeric alpha-actinin expression occurred in the hMSCs following induction with the 2 differentiation-inducing methods. miRNA-143 and -181 expressions were induced in the hMSCs by 5-azacytidine and miRNA-143, -181, -206, and -208 expressions were induced by indirect coculture with neonatal rat myocytes, but pri-miRNA-1-2 expression failed to be induced by these two induction methods.</p><p><b>CONCLUSION</b>Expressions of the representative heart-specific pri-miRNAs in different patterns can be induced in cardiomyocyte-like cells differentiated from hMSCs by 5-azacytidine and indirect coculture with neonatal rat myocytes.</p>

Significance of quantitative detection of bcr-abl mRNA in chronic myeloid leukemia patients after allogeneic hematopoietic stem cell transplant / 中国实验血液学杂志

The objective of this study was to analyze the level of bcr-abl mRNA in peripheral blood (PB) after allogeneic stem cell transplantation (allo-SCT) in chronic myeloid leukemia patients, providing a experimental basis for diagnosing early relapse. bcr-abl mRNA levels in 78 PB and bone marrow (BM) samples from 15 CML patients after allo-SCT were detected by using real-time quantitative PCR. The results indicated that levels of bcr-abl mRNA before transplantation were high (median 29.303%) and decreased greatly (median 0) at the first month after allo-SCT. During the first year after allo-SCT, the patterns of serial bcr-abl transcripts varied in number, but the overall bcr-abl transcript levels significantly decreased at 6 months after allo-SCT. Majority of patients with undetectable or very low levels of bcr-abl mRNA were monitored after 1 year following transplantation. The hematological features of BM and PB in all detected patients remained normal. PB and BM bcr-abl values were not different significantly and had the similar trend of changes. It is concluded that the bcr-abl mRNA levels in CML patients change greatly early after allograft. Serial monitoring measurements for bcr-abl mRNA contribute to understanding the trend of change and effect of transplantation, also can be a guidance for starting therapy. But detectable levels of bcr-abl mRNA during the first 6 months do not indicate relapse. Measurements of bcr-abl mRNA of PB may be more suitable for routine monitoring long-term disease status in CML after allo-HSCT.

An method for small hairpin RNA expression vector reconstruction for easy single restriction endonuclease identification / 南方医科大学学报

<p><b>OBJECTIVE</b>To develop an effective method for screening recombinant hairpin RNA expression plasmids using single restriction endonuclease analysis.</p><p><b>METHODS</b>The double-strand DNA fragment containing a ClaI site (the flanking sequences of which were not complementary) was annealed and ligated into small hairpin RNA (shRNA) expression vector pSilencer-4.1 that did not contain ClaI site to construct the circular pSilencer-4.1-ClaI vector. With BamHI and HindIII, the pSilencer-4.1-ClaIwas digested and ligated with the DNA template of green fluorescence protein (GFP) shRNA that did not include a ClaI site. The plasmid DNA of the positive clones was extracted and digested with ClaI, and the inserted DNA sequence of the non-linearized plasmid was identified by sequence analysis.</p><p><b>RESULT AND CONCLUSION</b>DNA sequencing showed that pSilencer-4.1-ClaI was correctly constructed and the plasmids resistant to ClaI digestion were all recombinant vectors encoding GFP shRNA. The constructed pSilencer-4.1-ClaI can be used as a universal vector to construct the shRNA expression plasmid, and the incorporated ClaI sites may allow efficient screening of recombinant shRNA expression vectors.</p>

Association of the polymorphism of macrophage migration inhibitory factor gene with coronary heart disease in Chinese population / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>Inflammation is involved in the process of coronary heart disease (CHD). Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine which can inhibit the random migration of macrophages and concentrate macrophages at the inflammatory site, and is thought to play an important role in cell mediated immunity. The present study is to investigate the association of the -173 G/C polymorphism of MIF gene with the outcome of the CHD.</p><p><b>METHODS</b>One hundred and thirty-eight patients with coronary angiography (CAG) proved CHD were studied, and 163 healthy matched controls in Guangdong were studied. Patients and controls were genotyped for a single nucleotide polymorphism in the 5'-flanking region at position -173 of the MIF gene, using PCR-RFLP analysis, followed by DNA sequencing identification.</p><p><b>RESULTS</b>Only MIF -173G/G and MIF -173G/C genotypes were detected in CHD patients and controls. The MIF -173 G allele was detected in 0.966 of normal controls and 0.917 of patients, while MIF -173 C allele was detected in 0.034 of normal controls and 0.083 of patients. Individuals possessing a MIF-173*C genotype have an increased risk of CHD (16.7% versus 6.8%) (OR: 2.764, 95% CI: 1.295-5.899; P= 0.007).</p><p><b>CONCLUSION</b>These results suggest that MIF -173G /C polymorphism was associated with CHD in Chinese population, the MIF -173C allele might be a risk factor for CHD in Chinese Han nationality.</p>