VEGF-expressing Bone Marrow Mesenchymal Stem Cells Transplantation Improved Heart Function of Myocardial Infarct Rabbits

Objectives To treat myocardial infarction with MSCs transplantation combined with VEGF gene therapy in rabbits and to study its mechanisms. Methods Forty-eight rabbits were randomly divided into MI group （n=12）, MSCs group （n=12）, VEGF group （n=12）, MSCs＋VEGF group （M＋V group, n=12）. Rabbit myocardial infarction models were founded by the ligation of left anterior descending artery. 107 MSCs were injected into the infarct-zone in four sites 2 weeks later in MSCs and M＋ V group, phVEGF gene were injected in infarct-zone in VEGF group and MSCs transfected with phVEGF gene were injected in M＋V group. Heart function including LVEDP, LVSP, LVDP, -dp/dtmax, ＋dp/dtmax, were measured in vivo. The hearts were harvested at 4 weeks after transplantation and sectioned for HE stain, immunohistochemical stain of BrdU and VIII factor antigen. Results The left ventricular hemodynamics parameters showed that heart function were improved more in M＋V group than MSCs group, MI group and VEGF group. The numbers of BrdU positive cells in M＋ V group（61±8）were more than in MSCs group （44±8, P 〈 0.01）. The numbers of vessels in infarcted zone were more in M＋V group （49±8） than in MSCs group （33±6, P 〈 0.01）,VEGF group（30±8, P 〈 0.01）and Mlgroup （18±4, P〈0.01）. Conclusions VEGF-expressing MSCs transplantation could improve heart function after myocardial infarction, and they were more effective than sole MSCs transplantation. Keeping more MSCs survival and ameliorating the blood supply of infarct-zone might be involved in the mechanisms.

CO-TRANSFECTION OF RAT BONE MARROW MESENCHYMAL STEM CELLS WITH HUMAN BMP2 AND VEGF165 GENES

Objective To explore the feasibility and efficacy of lentivirus-mediated co-transfection of rat bone marrow mesenchymal stem cells （MSCs） with human vascular endothelial growth factor 165 （hVEGFI65） gene and human bone morphogenetic protein 2 （hBMP2） gene. Methods The hVEGF165 and hBMP2 cDNAs were obtained from human osteosarcoma cell line MG63 and cloned into lentiviral expression vectors designed to co-express the copepod green fluorescent protein （copGFP）. The expression lentivector and packaging Plasmid Mix were co-transferred to 293TN cells, which produced the lentivirus carrying hVEGF165 （Lv-VEGF） or hBMP2 （ Lv-BMP） , respectively. MSCs of Wistar rats were co-transfected with Lv-BMP and Lv-VEGF （BMP ＋ VEGF group）, or each alone （BMP group and VEGF group）, or with no virus （ Control group）. The mRNA and protein expressions of hVEGF165 and hBMP2 genes in each group were detected by real-time PCR and enzyme linked immunosorbent assay （ELISA）. Results Lentiviral expression vectors carrying hVEGF165 or hBMP2 were correctly constructed and confirmed by restriction endonucleses analysis and DNA sequencing analysis. A transfer efficiency up to 90% was archieved in all the transfected groups detected by the fraction of fluorescent cells using fluorescent microscopy. From the results generated by real-time PCR and ELISA, VEGF165 and BMP2 genes were co-expressed in BMP ＋ VEGF group. No significant difference of BMP2 expression was detected between BMP ＋ VEGF and BMP groups （ P 〉 0. 05）. Similarly, there was no significant difference of VEGF165 expression between BMP ＋ VEGF and VEGF groups （ P 〉 0. 05）. Conclusion VEGF165 and BMP2 genes were successfully co-expressed in MSCs by lentivirus-mediated co-transfection, which provided a further foundation for the combined gene therapy of bone regeneration.

A novel and feasible way to cultivate and purify endothelial progenitor cells from bone marrow of children with congenital heart diseases

Background Endothelial progenitor cells （EPCs） are used in vascular tissue engineering and clinic therapy. Some investigators get EPCs from the peripheral blood for clinic treatment, but the number of EPCs is seldom enough. We have developed the cultivation and purification of EPCs from the bone marrow of children with congenital heart disease, to provide enough seed cells for a small calibre vascular tissue engineering study. Methods The 0.5-ml of bone marrow was separated from the sternum bone, and 5-ml of peripheral blood was collected from children with congenital heart diseases who had undergone open thoracic surgery. CD34＋ and CD34＋NEGFR＋ cells in the bone marrow and peripheral blood were quantified by flow cytometry. CD34＋/VEGFR＋ cells were defined as EPCs. Mononuclear cells in the bone marrow were isolated by Ficoll density gradient centrifugation and cultured by the EndoCult Liquid Medium KitTM. Colony forming endothelial cells was detected. Immunohistochemistry staining for Dil-ac-LDL and FITC-UEA-1 confirmed the endothelial lineage of these cells. Results CD34＋ and CD34＋NEGFR＋ cells in peripheral blood were （0.07±0.05）% and （0.05±0.02）%, respectively. The number of CD34＋ and CD34＋/VEGFR＋ cells in bone marrow were significantly higher than in blood, （4.41±1.47）% and （0.98±0.65）%, respectively （P 〈0.0001）. Many colony forming units formed in the culture. These cells also expressed high levels of Dil-ac-LDL and FITC-UEA-I. Conclusion This is a novel and feasible approach that can cultivate and purify EPCs from the bone marrow of children with congenital heart disease, and provide seed cells for small calibre vascular tissue engineering.

Clinical significance of HBME-1,Galectin-3,and CK19 expression and the status of BRAF mutation in papillary thyroid carcinoma

Objective The aim of this study was to explore the clinical significance of the expression of proteins human bone marrow endothelial cell markers(HBME-1), Galectin-3, and cytokeratin19(CK19), as well as the status of v-raf murine sarcoma viral oncogene homolog B1(BRAF) mutation in papillary thyroid carcinoma(PTC). Methods Immunohistochemical staining was performed in 82 specimens each of PTC and papillary benign lesions to detect the expression of HBME-1, Galectin-3, and CK19. Polymerase chain reaction(PCR) and gene sequencing were performed on 60 specimens each of PTC and papillary benign lesions to detect the status of BRAF mutation. Results The positive expression ratios of HBME-1, Galectin-3, and CK19 in PTC were 98.8%, 97.6% and 100% respectively, which were significantly higher than the expressions in papillary benign lesions(P < 0.05). No significant relationship was observed between the expression of these makers and the clinicopathological features of PTC. The sensitivity of co-expression of HBME-1 and CK19 or HBME-1 and Galectin-3 as diagnostic criteria of PTC was 99.9%, with a specificity of 95.4%. BRAF mutation was detected in 40 of 60 PTC(66.7%) specimens. There was a statistical difference in BRAF mutations between PTC and papillary benign lesions(P < 0.05); there were no associations between BRAF mutation and the clinicopathological features of PTC. Conclusion Combined immunohistochemical staining of HBME-1, Galectin-3, and CK19 can further improve the sensitivity and specificity of differential diagnosis of PTC. BRAF mutation is a significant genetic event, which may have diagnostic value for PTC.

Inducing effects of macrophage stimulating protein on the expansion of early hematopoietic progenitor cells in liquid culture

Background Macrophage stimulating protein （MSP） is produced by human bone marrow endothelial cells. In this study we sought to observe its effects on inducing the expansion of early hematopoietic progenitor cells which were cultured in a liquid culture system in the presence of the combination of stem cell factor （SCF）, interleukin 3 （IL-3）, interleukin 6 （IL-6）, granulocyte macrophage-colony stimulating factor （GM-CSF）, erythropoietin （EPO） （Cys） and MSP or of Cys and bone marrow endothelial cell conditioned medium （EC-CM）. Methods Human bone marrow CD34^＋ cells were separated and cultured in a liquid culture system for 6 days. Granulocyte-macrophage colony forming unit （CFU-GM） and colony forming unit-granulocyte, erythrocyte, macrophage, megakaryocyte （CFU-GEMM） were employed to assay the effects of different treatment on the proliferation of hematopoeitic stem/progenitor cells. The nitroblue tetrazolium （NBT） reductive test and hoechest 33258 staining were employed to reflect the differentiation and apoptosis of the cells respectively. Results MSP inhibited the proliferation of CFU-GM and CFU-GEMM in semi-solid culture and the inhibitory effect on CFU-GEMM was stronger than on CFU-GM. MSP inhibited the differentiation of early hematopoietic progenitor cells induced by hematopoietic stimulators. Bone marrow （BM） CFU-GEMM was 2.3-fold or 1.7-fold increase or significantly decreased in either Cys＋EC-CM, Cys＋MSP or Cys compared with 0 hour control in liquid culture system after 6 days. Conclusion MSP, a hematopoietic inhibitor, inhibits the differentiation of early hematopoietic progenitor cells induced by hematopoietic stimulators and makes the early hematopoietic progenitor cells expand in a liquid culture system.

The protective effect of icariin on glucocorticoid-damaged BMECs explored by microfluidic organ chip

Osteonecrosis of the femoral head(ONFH)is a devastating musculoskeletal disease characterized by the impaired circulation of bone.The purpose of this study was to explore the underlying mechanisms of the protective effect of icariin on the glucocorticoid-induced injury of bone microvascular endothelial cells(BMECs).Normal BMECs were extracted from the femoral heads by enzymatic isolation and magneticactivated cell sorting methods.Dexamethasone and icariin were used to intervene BMECs in microfluidic organ chips,and phalloidin staining was conducted to observe the cell morphology and viability.Then next-generation transcriptome sequencing and real-time quantitative reverse transcription-polymerase chain reaction(q RT-PCR)were performed to identify the differentially expressed genes(DEGs)in different groups.Through the microfluidic organ chip,it can be observed that after dexamethasone intervention,the filamentous structure in cell fibers disappeared and the cell morphology changed from spindle to round until death.Icariin could relieve these changes and showed a protective effect on glucocorticoiddamaged BMECs.In addition,201 DEGs were detected between the icariin protection group and the dexamethasone group,which were significantly enriched in 17 signaling pathways.8 of the top ten selected hub genes(IL6,PTGS2,VEGFA,etc.)were confirmed by q RT-PCR.Transcription factors(TFs)-gene network showed 63 connections between 18 TFs and 12 DEGs.For instance,GATA2 could regulate 5 DEGs.The associations between 92 mi RNA and 12 DEGs were visualized in a mi RNA-gene network.The hub mi RNA,has-mir-335–5 p was predicted to interact with 8 DEGs(PTGS2,VEGFA,etc.).Microfluidic organ chips could provide excellent morphological results for cell experiments,by which it could be observed that icariin showed a protective effect on the glucocorticoid-induced injury of BMECs.Beside,these DEGs,possible regulatory TF(GATA2,FOXC1,etc.)and mi RNA(has-mir-335–5 p)might be dysregulated in the initiation of ONFH and have prospective importance in ONFH diagnosis and therapy.