Myosin Heavy Chain 7 Gene-derived miRNA-208b-3p Enhances the Fibrosis-related Gene Expression in Cardiac Fibroblasts / 中山大学学报(医学科学版)

ObjectiveTo investigate the effect of myosin heavy chain 7 gene-derived miRNA-208b-3p on the fibrotic phenotype of cardiac fibroblasts. MethodsmiRNA chip array was performed to detect the dysregulated miRNAs in the myocardium of diabetic db/db mice and db/m control mice. Neonatal mouse ventricular cardiomyocytes （NMVCs） and cardiac fibroblasts （CFs） were isolated from C57BL/6 mice and cultured. Real-time quantitative PCR （RT-qPCR） was conducted to determine the expression of miR-208b-3p in mouse CFs and NMVCs subjected to angiotensinⅡ（AngⅡ） and high glucose plus glucose oxidase （G/Go） treatment， respectively. Cell counting kit 8（CCk8） assay， flow cytometry and determination of fibrosis-related protein， including COL1A1， COL3A1and α-SMA， were performed in mCFs transfected with miR-208b-3p. Dual luciferase reporter assay was performed to confirm the interaction between miR-208b-3p and the 3'-UTR of metal response element binding transcription factor 2 （Mtf2） and progesterone receptor membrane component 1（Pgrmc1）， respectively. The expressions of Mtf2 and Pgrmc1 at the mRNA and protein levels in mCFs after miR-208b-3p mimic transfection were determined using RT-qPCR and Western blot assay， respectively. The small interfering RNA （siRNA） was used to inhibit Mtf2 and Pgrmc1 expression in mCFs， and the effects of Mtf2 siRNA， Pgrmc1 siRNA and miR-208b-3p on fibrosis-related protein expression in mCFs were investigated. ResultsResults of miRNA chip array and RT-qPCR assay showed that miR-208b-3p was up-regulated in the myocardium of the diabetic db/db mice. miR-208b precursor and the host gene of Myh7 were consistently increased in db/db mice. miR-208b-3p and Myh7 mRNA were expressed in mCFs and NMVCs， but the levels of miR-208b-3p and Myh7 mRNA in NMVCs were much higher than those in mCFs. miR-208b-3p was up-regulated in mCFs and NMVCs subjected to Ang Ⅱ and G/Go treatment， respectively. miR-208b-3p could significantly enhance fibrosis-related protein， including COL1A1， COL3A1 and α-SMA， in mCFs， without affecting the proliferation activity and cell cycle distribution of mCFs. Dual luciferase reporter assay revealed the interactions of miR-208b-3p with the 3'-UTR of Mtf2 and Pgrmc1. The results of RT-qPCR and Western blotting confirmed that miR-208b-3p inhibited Mtf2 and Pgrmc1 expression at the post- transcriptional level. Transfection with miR-208b-3p mimic， Mtf2 siRNA and Pgrmc1 siRNA could consistently enhance the fibrosis-related protein expression in the cardiac fibroblasts. ConclusionsmiR-208b-3p enhances fibrosis-related gene expression by targeting Mtf2 and Pgrmc1in mCFs.

PDGFRα Cell-Derived SCF Regulates Hematopoiesis of Adult Mice / 中国实验血液学杂志

OBJECTIVE@#To investigate the effect of PDGFRα stromal cells derived SCF on hematopoiesis of adult mice.@*METHODS@#Pdgfrα-CreER; R26-tdTomato mice model was constructed, and the proportion and distribution of PDGFRα cells in the liver, spleen, lung, kidney and bone marrow were analyzed by flow cytometry and confocal microscopy. Then the Pdgfrα-CreER; Scf mice model was further constructed, the Scf in PDGFRα was knocked out specifically, the effect of Scf-knocked out in PDGFRα stromal cells in the propitiation of HSPCs in the bone marrow was analyzed by flow cytometry. The effect of SCF on the proportion on number of peripheral blood cells in mice was analyzed by whole blood analyzer.@*RESULTS@#After Scf was knocked out in PDGFRα stromal cells, the propitiation and number of LKS- cell, LKS+ cell, HSC, MPP1, MKP, PreGM, PreMegE, and CFU-E in the bone marrow of mice was decreased, as well as in the number of red blood cells and hemoglobin concentration of peripheral blood. However, Scf knocked out from PDGFRα cells showed no effect on the hematopoiesis in spleen.@*CONCLUSION@#specific knocked out of Scf in PDGFRα stromal cells in adult mice can decrease the proportion of HSPCs in the bone marrow and the number of red blood cells in peripheral blood, and finally lead to anemia in mice.

Induced expression of protein kinase signaling cascade Akt after spinal cord injury in rats / 重庆医学

Objective To examine the expression of protein kinase signaling cascade protein kinase B (Akt)-which plays an im-portant role in a number of key biological functions in cellular processes after spinal cord injury (SCI) in rats ,and its effects on SCI induced motor function defects ,and to provide the molecular mechanism for repairing SCI .Methods SCI was produced by extra-dural contusion using modified Allen′s stall with damage energy .The rats were divided into three groups :sham-operated group (Sham) ,interference group (Int) ,and control group (Con) .Using immunostaining studies ,Western blot analyses and real-time qualitative RT-PCR analyses ,we detected the changes of Akt expression at the protein and mRNA level in spinal cord tissues at 1 , 7 ,and 14 d after surgery ,and evaluated the presence and extent of neurological impairment after SCI by the BBB locomotor rating scale .Results The sham operated groups exhibited low expression of the Akt signaling at the protein and mRNA level ,and the ex-pressions increased following SCI .Compared to the animals in the sham operated groups ,prominently elevated level of Akt signaling was detected in the injured spinal cords of SCI group .LY294002 ,an inhibitor of phosphatidylinositol 3-kinase (PI3K) that initiates Akt phosphorylation ,prominently inhibited the expression of Akt produced by SCI and the spontaneous recovery of motor function after SCI .Conclusion Activated protein kinase signaling cascade Akt might be the important intervention aspects of involving in neuroprotective and repair process after SCI .

EXPRESSION OF SONIC HEDGEHOG′S FUNCTIONAL RECEPTOR-PATCHED IN CULTURED NEURAL STEM CELL / 解剖学报

Objective To determine whether cultured neural stem cell expresses morphogen molecule sonic hedgehog′s functional receptor patched. Methods Cultured neural stem cell clones were subjected to RT\|PCR after passaged several times in vitro ,the amplification production was sequenced and labeled by digoxingemin and in situ hybridization technique was carried out to detect the cryosection of the neural stem cell clones. Results Most cells in the stem cell clones were positive for sonic hedgehog functional receptor patched,no significant difference was found among the positive cells and the center and peripheral of the stem cell clones.Conclusion\ The sonic hedgehog signal transduction may have important role in the proliferation and differentiation process of neural stem cell.\;[

THE GROWTH PROPERTY OF NEURAL STEM CELLS ISOLATED FROM DIFFERENT PARTS OF MICE CNS——AN IN VITRO STUDY / 解剖学报

Objective To compare the growth property of the stem cells taken from different brain regions at the same developmental stage. Methods Mice embryos at the same development stage were isolated under sterile conditions, cortex, striatum, diencephalon, mid-hind brain and spinal cord were collected and pooled separately, after single-cell suspension obtained, different regions' cell suspensions were seeded in FGF supplemented serum-free culture medium. Followed the neural stem cell clone(neurospheres) fromation, immuno-cytochemistry method was utilized to identify the cell characteristics, all these clones were passaged under same conditions, clone formation and cell migration were observed under phase-contrast microscope. Results In the FGF added serum-free medium, neural cells experienced a large scale death within 48h after being seeded, then few single cells began to proliferate and formed the floating cell clones in the medium. These clones (neurospheres) could form new clones when seeded as single cell suspension. If these clones were seeded on poly-orithine, they could differentiate into neurons and glia cells. Compare the clone formation and cell migration, we found that: cortex, striatum, diencephalon all could form floating clones with different rate, the cortex formed clones at the highest rate, striatum and diencephalon at lower rate; few neurospheres formed from cortex adhered to the culture plate substrate and few cells were found migrating out from the adhered clones, striatum and diencephalon derived neurospheres adhered the plate more easily, and there were apparent cell migration. Mid-hind brain and spinal cord formed clones at the lowest rate, floating clones were scarce, and the clones adhered to the substrate readily, there were large amount of cell migrating out from these adhered clones. Conclusion Neural stem cells could proliferate and be passaged in vitro in serum-free medium, and they could be induced to differentiate under certain conditions into major cells types of CNS, there were differences in clone forming rate and cell migration between neural stem cells derived from different CNC regions, nonetheless they were at the same development stage, this may reflect that, in some degree, these cells can keep some of their region-specified developmental intrinsic property in vitro.