BACKGROUND:Several studies have demonstrated that stem cells can differentiate into vascular endothelial cells, and then further differentiate and form blood capillary. Based on this principle, autologous bone marrow mesenchyrnal stem cell (BMSC) transplantation promotes angiogenesis to treat ischemia in lower limb.OBJECTIVE: To evaluate the angiogenesis in rabbit ischemic limbs following autologous BMSC transplantation using high-frequency two-dimensional ultrasound detection in conjunction with Doppler color-flow imaging examination. DESIGN, TIME AND SEI-FING: A randomized, controlled, animal experiment was performed in the Third People's Hospital of Wuxi between March 2007 and April 2008.MATERIALS: Twenty-four New Zealand rabbits were randomized to a control group and a cell transplantation group, with 12 rabbits in each.METHODS: Ischemia in lower limbs was induced in all rabbits. One week following ischemia induction, the cell transplantation group was injected with 0.5 mL cell suspension, comprising 2×106 BrdU-labeled autologous BMSCs cultured in vitro, through multiple sites in the region of gastrocnemius muscle. Simultaneously, the control group received the same amount of physical saline in the same region.MAIN OUTCOME MEASURES: The initial segment of rabbit femoral artery and superficial femoral artery was subjected to high-frequency two-dimensional ultrasound and Doppler color-flow imaging examinations to measure femoral artery vascular internal diameter, blood flow peak velocity, and blood flow acceleration time. Ischemic muscular tissue was taken for immunohistochemical staining to detect transplanted cell distribution and for pathological examination of angiogenesis. RESULTS: Two weeks following autologous BMSC transplantation, high-frequency ultrasound results revealed that femoral artery internal diameter and blood flow peak velocity were greater, but blood flow acceleration time was shorter, in the cell transplantation group than in the control group (P<0.01). Immunohistochemical staining results demonstrated the presence of BrdU-positive cells. Pathological sections displayed that vascular density was significantly higher in the cell transplantation group than ih the control group. CONCLUSION: Autologous BMSC transplantation is a promising, simple, and effective method of treating ischemia in lower limbs owing to its promotion of angiogenesis. Meanwhile, high-frequency ultrasound detection of femoral artery is an effective, practical method to evaluate the clinical outcomes of autologous BMSC transplantation.

BACKGROUND: Longterm therapeutic effects of routine drug treatment, intervention or vascular bypass transplantation on lower extremity arterial occlusion are not ideal. During recent years, angiogenesis of stem cells possibly becones a new method to repair or rebuild an effective collateral circulation at infarct regions.OBJECTIVE: To verify the effect of autologous bone marrow mesenchymal stem cell transplantation on promoting angiogenesis in rabbit ischemic hind limbs.DESIGN, TIME AND SETTING: A randomized controlled animal study was performed in Jiangsu Institute of Schistosomiasis Control between August 2005 and November 2006. MATERIALS: Eight rabbits were used to prepare ischemic models of hind limbs, and then they were randomly divided into experimental group (n=4) and control group (n=4).METHODS: Bone marrow mesenchymal stem cells were isolated and cultured from New Zealand rabbits in the experimental group, and they were then marked 5-bromo-2-deoxyuridine (Brdu). Suspension of bone marrow mesenchymal stem cells was injected into the ischemic hind limbs in the experimental group, while the same volume of saline was injected into the controls. MAIN OUTCOME MEASURES: After two weeks, two-dimensional and color Doppler ultrasound detection was used on rabbit femoral artery to measure inner diameter of blood vessel, peak velocity and acceleration time of blood flow before and after transplantation. Muscle tissues were obtained from ischemic regions to observe distribution of transplant cells and state of angiogenesis using immunofluorescence staining and hematoxylin-eosin (HE) staining. RESULTS: Two weeks after transplantation, the inner diameter of femoral artery and the peak velocity of blood flow in the experimental group were higher than those in the control group (P < 0.01), but the acceleration time of blood flow was shorter than that in the control group (P < 0.01). Lmmunofluorescence staining showed that anti-Brdu-staining positive cells were found out in transplant part in the experimental group; while HE staining indicated that vessel density of ischemic region in the experimental group was higher than that in the control group. CONCLUSION: Bone marrow mesenchymal stem cells can promote angiogenesis, while autologous bone marrow mesenchymai stem cell transplantation will become a simple and effective method to treat lower limb ischemia.

Objective To observe the effects of fructose,fibroblast growth supplement(FGS) and ethylamine sulfonic acid on the total number,the survival rate and the survival number of Human Retinal Pigment Epithelial(hRPE) Cell.Methods Microencapsulated hRPE cells were plated and cultured in four kinds of mediums,which contained fructose,fibroblast growth supplement(FGS),ethylamine sulfonic acid or no extra ingredient respectively.The total cell number,survival rate and viable cell number of the microencapsulated hRPE cell on day 0th,1st,3rd,7th were calculated.Results After 7days of culture,the lowest cell survival rate of microencapsulated hRPE cells in the four groups was(75.00±3.00)%,but there were no significantly differences(Ps＞0.05) among the groups.The total number of cells in the fibroblast growth factor group([8.00±0.46]×104) and ethylamine sulfonic acid group([7.20±0.36]×104) were significantly higher than the blank group(([6.10±0.56]×104),Ps＜0.05),while no statistical difference was observed in the comparison between the fructose group([6.00±0.46]×104) and blank control(P＞0.05).Conclusion The FGS and ethylamine sulfonic acid can promote the proliferation of the microencapsulated hRPE cells.

Objective To investigate the effects of mechanical stretch on the pelvic floor dysfunction and study the regulation of mechanical stretch on the morphology and extracellular matrix mRNA expression of fibroblasts derived from human uterosacral ligament.Methods 13 patients who underwent hysterectomy surgery due to uterine benign diseases in the Obstetric and Gynecological Department of the Second Hospital of Hebei Medical University from October 2009 to September 2010 and had no pelvic organ prolapse were recruited in this study.Fibroblasts of uterosacral ligament were cultured by collagenase digestion and identified by morphology and immunocytochemical methods.Fibroblasts of 3 ～ 4 generations were stretched by flexcell-4000 tension system for 0,4,15,24 hours.Total RNA was extracted from the fibroblasts and mRNA of Collagen Ⅰ,Collagen Ⅲ,matrix metalloproteinase-1 and tissue inhibitor of metalloprot einase-1were measured with real-time fluorescence quantitative PCR.Results In response to mechanical stretch,the morphology and orientation of the uterosacral ligament fibroblasts were changed as well as their synthetic function.Collagen Ⅰ mRNA for 4 or 15 hours' stretch (0.777 ± 0.251,0.730 ± 0.248)were statistically down regulated compared with 0 hour ( 1.000 ±0.284),( q =4.5786,4.6458,p ＜0.05).But for 24 hours'stretch,the mRNA expression ( 1.102 ±0.308) of collagen Ⅰ restored to the level of 0 hours.The expressions of collagen Ⅲ mRNA for 4,15,24 hours' stretch (4.771 ± 0.879,6.146 ± 1.109,5.022 ± 0.907 )were statistically up regulated compared with 0 hour (1.001 ± 0.284),(q =15.9481,21.7647,17.0099,P ＜ 0.01 ).MMP-1mRNA for 4,15,24 hours' stretch (6.420 ± 2.143,23.695 ± 2.221,20.169 ±2.794) were statistically up regulated compared with 0 hour ( 1.000 ±0.414),( q =9.3421,39.1178,33.0403,P ＜ 0.01 ).TIMP-1 mRNA for 4,15 hours' stretch (0.455 ± 0.370,0.481 ± 0.386)were statistically down regulated compared with 0 hour( 1.000 ± 0.425 ),( q =6.5265,6.3106,P ＜0.05).But for 24 hours'stretch,the mRNA expression( 0.883 ± 0.538 ) restored to the level of 0 hours.Conclusions The morphology,orientation and the synthesis function of the uterosacral ligament fibroblasts can be regulated by mechanical stress.Extracellular matrix remodeling may be adaptive changes due to the mechanical stress.

The changes in the tau protein phosphorylation and expression of bcl-2, and bax in rat parietal cortex neurons after focal cerebral ischemia-reperfusion (I/R) were explored, and the relationship between the tau protein phosphorylation and the expression of bax or apoptosis was clarified in order to elucidate the relationship between cerebral infarction and Alzheimer's disease. The rat focal cerebral I/R model was induced by occlusion of the right middle cerebral artery using the intraluminal suture method. The level of tau protein phosphorylation at Ser396, Ser404, Tyr231, Ser199/202 sites and the expression of bcl-2, bax and total tau 5 in rat parietal cortex during focal cerebral ischemia/reperfusion were detected by Western blot. The relationship between the tau protein phosphorylation and the expression of bax, or apoptosis was examined by TUNEL method and double-labeling immunofluorenscence method. The results showed that the level of tau hyperphosphorylation at Ser199 / 202, Ser396, Ser404, Tyr231 sites and the expression levels of bcl-2, and bax were significantly higher in I/R group than in the sham group, but the ratio of bcl-2/bax was decreased. Neuronal apoptosis, bax expression and the tau protein hyperphosphorylation were co-localized. It is suggested that Alzheimer's disease-like pathological changes occur after cerebral I/R. The highly abnormal phosphorylation of tau protein plays a key role in cerebral I/R-induced apoptosis. The cerebral infarction may contribute to Alzheimer's disease occurrence and development.

To study the inhibitory effect of Nogo-A shRNA on cell line PC12, the Nogo-A shRNA (short hairpin RNA, or shRNA) was designed and synthesized. The annealed shRNA template was inserted into plasmid pGenesil-1 containing enhanced green fluorescent protein (EGFP) gene by gene cloning technique to generate eukaryotic expression vector. The recombinant plasmid was transfected into PC12 cells by lipofecamine2000 and the mRNA and protein expression level of Nogo-A gene was detected by RT-PCR and Western blotting 48 h after the transfection. Gene sequencing showed that that the Nogo-A shRNA eukaryotic expression vector was successfully constructed. No significant change was found in the Nogo-A mRNA and protein expression level in empty vector-transfected group as compared with controls (P>0.05), while the expression level in shRNA-transfected group decreased significantly (P<0.05). It is concluded that the pGenesil-1/Nogo-AshRNA recombinant plasmid can effectively suppress the expression of Nogo-A gene in PC12 cells.
Cloning, Molecular ; Gene Knockdown Techniques/*methods ; Genetic Vectors ; Green Fluorescent Proteins/genetics ; Myelin Proteins/*genetics ; Myelin Proteins/metabolism ; PC12 Cells ; Plasmids ; RNA Interference ; RNA, Messenger/genetics ; RNA, Messenger/metabolism ; RNA, Small Interfering ; Transfection

This study investigated the effect of TNP-470 in combination with carmustine (BCNU) on the growth of subcutaneously implanted human glioblastoma xenografts in nude mice. Human glioblastoma U-251 cells (1×10(7)) were injected into 24 nude mice subcutaneously. The tumor-bearing mice were randomly divided into 4 groups on the seventh day following tumor implantation: TNP-470 group, in which TNP-470 was given 30 mg/kg subcutaneously every other day 7 times; BCNU group, in which 20 mg/kg BCNU were injected into peritoneal cavity per 4 days 3 times; TNP-470 plus BCNU group, in which TNP-470 and BCNU were coadministered in the same manner as in the TNP-470 group and the BCNU group; control group, in which the mice were given 0.2 mL of the mixture including 3% ethanol, 5% acacia and 0.9% saline subcutaneously every other day 7 times. The tumor size and weights were measured. The tumor microvessel density (MVD) was determined by immunostaining by using goat-anti-mouse polyclonal antibody CD105. The results showed that on the 21th day following treatment, the volume of xenografts in the TNP-470 plus BCNU group was (108.93±17.63)mm(3), markedly lower than that in the TNP-470 group [(576.10±114.29)mm(3)] and the BCNU group [(473.01±48.04)mm(3)] (both P<0.01). And the xenograft volume in these 3 treatment groups was even much lower than that in the control group [(1512.61±470.25) mm(3)] (all P<0.01). There was no significant difference in the volume of xenografts between the TNP-470 group and the BCNU group (P>0.05). The inhibition rate of the tumor growth in the TNP-470 plus BCNU group was (92.80±11.37)%, notably higher than that in the TNP-470 group [(61.91±6.29)%] and the BCNU group [(68.73±9.65)%] (both P<0.01) on the 21th day following treatment. There was no significant difference in the inhibition rate of tumor growth between the TNP-470 group and the BCNU group (P>0.05). The MVD of xenografts in the TNP-470 plus BCNU group was decreased significantly as compared with that in the TNP-470 group or the BCNU group (both P<0.05). The MVD of xenografts in the 3 treatment groups was markedly reduced as compared with that in the control group (all P<0.05). No significant changes in weights were observed before and after the treatment in each group (all P>0.05). It was concluded that the combination of TNP-470 and BCNU can significantly inhibit the growth of human glioblastoma xenografts in nude mice without evident side effects.

This study established superparamagnetic iron oxide (SPIO)-labeled nerve growth factor-beta (NGF-beta) gene-modified spinal cord-derived neural stem cells (NSCs). The E14 rat embryonic spinal cord-derived NSCs were isolated and cultured. The cells of the third passage were transfected with plasmid pcDNA3-hNGFbeta by using FuGENE HD transfection reagent. The expression of NGF-beta was measured by immunocytochemistry and Western blotting. The positive clones were selected, allowed to proliferate and then labeled with SPIO, which was mediated by FuGENE HD transfection reagent. Prussian blue staining and transmission electron microscopy (TEM) were used to identify the SPIO particles in the cells. The distinctive markers for stem cells (nestin), neuron (beta-III-tubulin), oligodendrocyte (CNPase) and astrocyte (GFAP) were employed to evaluate the differentiation ability of the labeled cells. The immunocytochemistry and western blotting showed that NGF-beta was expressed in spinal cord-derived NSCs. Prussian blue staining indicated that numerous blue-stained particles appeared in the cytoplasma of the labeled cells. TEM showed that SPIO particles were found in vacuolar structures of different sizes and the cytoplasma. The immunocytochemistry demonstrated that the labeled cells were nestin-positive. After differentiation, the cells expressed beta-III-tubulin, CNPase and GFAP. It was concluded that the SPIO-labeled NGF-beta gene-modified spinal cord-derived NSC were successfully established, which are multipotent and capable of self-renewal.
Cells, Cultured ; Dextrans/*diagnostic use ; Embryo, Mammalian ; Magnetic Resonance Imaging ; Magnetics ; Magnetite Nanoparticles/*diagnostic use ; Nerve Growth Factor/*genetics ; Nerve Growth Factor/pharmacology ; Neural Stem Cells/*cytology ; Spinal Cord/*cytology ; Transfection

Objective To investigate proteolipid protein 1 (PLP1) mutations in six pedigrees with Pelizaeus-Merzbacher disease (PMD),and to provide prenatal consulting and prenatal diagnosis.Methods Subjects were six probands with PMD admitted in Department of Pediatrics,Peking University First Hospital from July 2006 to November 2011 and their family members.Genomic DNA sarnples were extracted from peripheral bloods of probands and their family members.Multiplex ligation-dependent probe amplification (MLPA) technique was used to detect PLP1 duplication mutation.Direct DNA sequencing was used to detect point mutation.Genetic diagnosis were based on PLP1 mutation genotype from probands.Prenatal diagnosis of nine fetuses were performed from seven PLP1 mutation female carriers by fetuses' DNA extracted from amniocytes or villus cells.Results PLP1 duplications were found in probands 1-4 (P1-4) whose mothers and the aunt of proband 1 (P1) were PLP1 duplications carriers.The two cases of point mutation,c.96C＞G(p.F32L) and c.623G＞T (p.G208V),were found in proband 5 (P5) and proband 6 (P6).Hcterozygous changes of the same mutations were found in P5' and P6' mothers with normal phenotypes.Seven female PLP1 mutation carriers were pregnant again.Prenatal diagnosis of PLP1 for nine fetuses presented one PLP1 duplication,one point mutation,one PLP1 duplication carrier,and six wildtypes.A segmental crossing over of X chromosome was detected in one male fetus of PLP1 wildtype.Conclusions PLP1 mutation analysis could help to diagnose PMD pedigree and to identify female PLP1 mutation carrier in the family.The following prenatal diagnosis and proper genetic counseling are very important to prevent PMD child from being delivered.

Objective:To prepare and preliminarily identify the monoclonal antibodies(mAbs) specifically against 3D protein of Enterovirus 71(EV71),using bioinformatics to predict the epitopes of 3D,with HBc protein as a carrier.Methods: Artificial screening of 3D protein epitope sequences by bioinformatic method,inserted into the major immunodominant region(MIR) area of Hepatitis B virus core protein(HBc),to construct the recombinant protein.BALB/c mice were immunized with the recombinant virus like particles(VLPs),to prepare the mAbs against 3D protein of EV71.Affinity chromatography technology was used to purify the mAb.The indirect ELISA,ELISPOT,immunofluorescence and immunohistochemistry staining methods were used to identify the characteristic of the mAb.Results: We displayed 3D(aa34-43),3D(aa61-76) and 3D(aa151-164) epitopes by constructing fusion protein using HBc VLPs as a vector,after hybridization,one positive hybridoma cell line(3E1) was selected by ELISA.The isotype of 3E1 was IgG2a.The results of immunofluorescence and immunohistochemistry staining assay showed that the mAb 3E1 could specifically recognize EV71.Conclusion: The prepared mAb 3E1 can specifically recognizes the EV71,which laid the foundation for the detection of virus and further study on 3D protein,and verified the bioinformatics technology combined with HBc carrier displaying peptides could prepare mAb quickly and efficiently.