Differentiation of smooth muscle progenitor cells in peripheral blood and its application in tissue engineered blood vessels / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

<p><b>BACKGROUND</b>A major shortcoming in tissue engineered blood vessels (TEBVs) is the lack of healthy and easily attainable smooth muscle cells (SMCs). Smooth muscle progenitor cells (SPCs), especially from peripheral blood, may offer an alternative cell source for tissue engineering involving a less invasive harvesting technique.</p><p><b>METHODS</b>SPCs were isolated from 5-ml fresh rat peripheral blood by density-gradient centrifugation and cultured for 3 weeks in endothelial growth medium-2-MV (EGM-2-MV) medium containing platelet-derived growth factor-BB (PDGF BB). Before seeded on the synthesized scaffold, SPC-derived smooth muscle outgrowth cell (SOC) phenotypes were assessed by immuno-fluorescent staining, Western blot analysis, and reverse transcription polymerase chain reaction (RT-PCR). The cells were seeded onto the silk fibroin-modified poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (SF-PHBHHx) scaffolds by 6x10(4) cells/cm2 and cultured under the static condition for 3 weeks. The growth and proliferation of the seeded cells on the scaffold were analyzed by 3-(4,5-dimethylthiazol-2-yl)-diphenyltetrazolium bromide (MTT) assay, scanning electron microscope (SEM), and 4,6-diamidino-2-phenylindole (DAPI) staining.</p><p><b>RESULTS</b>SOCs displayed specific "hill and valley" morphology, expressed the specific markers of the SMC lineage: smooth muscle (SM) alpha-actin, calponin and smooth muscle myosin heavy chain (SM MHC) at protein and messenger ribonucleic acid (mRNA) levels. RT-PCR results demonstrate that SOCs also expressed smooth muscle protein 22alpha (SM22alpha), a contractile protein, and extracellular matrix components elastin and matrix Gla protein (MGP), as well as vascular endothelial growth factor (VEGF). After seeded on the SF-PHBHHx scaffold, the cells showed excellent metabolic activity and proliferation.</p><p><b>CONCLUSION</b>SPCs isolated from peripheral blood can be differentiated into the SMCs in vitro and have an impressive growth potential in the biodegradable synthesized scaffold. Thus, SPCs may be a promising cell source for constructing TEBVs.</p>

Construction of tissue-engineered heart valves by using decellularized scaffolds and endothelial progenitor cells / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Tissue-engineered heart valves have the potential to overcome the limitations of present heart valve replacements. This study was designed to develop a tissue engineering heart valve by using human umbilical cord blood-derived endothelial progenitor cells (EPCs) and decellularized valve scaffolds.</p><p><b>METHODS</b>Decellularized valve scaffolds were prepared from fresh porcine heart valves. EPCs were isolated from fresh human umbilical cord blood by density gradient centrifugation, cultured for 3 weeks in EGM-2-MV medium, by which time the resultant cell population became endothelial in nature, as assessed by immunofluorescent staining. EPC-derived endothelial cells were seeded onto the decellularized scaffold at 3 x 10(6) cells/cm(2) and cultured under static conditions for 7 days. Proliferation of the seeded cells on the scaffolds was detected using the MTT assay. Tissue-engineered heart valves were analyzed by HE staining, immunofluorescent staining and scanning electron microscopy. The anti-thrombogenic function of the endothelium on the engineered heart valves was evaluated by platelet adhesion experiments and reverse transcription-polymerase chain reaction (RT-PCR) analysis for the expression of endothelial nitric oxide synthase (eNOS) and tissue-type plasminogen activator (t-PA).</p><p><b>RESULTS</b>EPC-derived endothelial cells showed a histolytic cobblestone morphology, expressed specific markers of the endothelial cell lineage including von Willebrand factor (vWF) and CD31, bound a human endothelial cell-specific lectin, Ulex Europaeus agglutinin-1 (UEA-1), and took up Dil-labeled low density lipoprotein (Dil-Ac-LDL). After seeding on the decellularized scaffold, the cells showed excellent metabolic activity and proliferation. The cells formed confluent endothelial monolayers atop the decellularized matrix, as assessed by HE staining and immunostaining for vWF and CD31. Scanning electron microscopy demonstrated the occurrence of tight junctions between cells forming the confluent monolayer. Platelets adhesion experiments suggested that the neo-endothelium was non-thrombogenic. The expression levels of eNOS and t-PA genes in the neo-endothelium were quite similar to those in human umbilical vein endothelial cells.</p><p><b>CONCLUSIONS</b>EPCs isolated from the human umbilical cord blood can differentiate into endothelial cells in vitro and form a functional endothelium atop decellularized heart valve scaffolds. Thus, EPCs may be a promising cell source for constructing tissue-engineered heart valves.</p>

Molecular cloning and expression of anti-tumor adhesion peptide (beta3) / 生物工程学报

To block tumor cell adhesion, inhibit tumor metastasis and recurrence, the anti-adhesion peptide-trimeric beta peptide (DLYYLMDLSYSMKGGDLYYLMDLSYSMKGGDLYYLMDLSYSMK, beta3) was designed. The DNA fragment of beta3 was cloned into expression vector pET-His and the fusion protein His-beta3 was expressed in E. coli. BL21(DE3)plysS. After 1.5 hours' induction with IPTG, His-beta3 peptide was expressed significantly amounting to 10% of the insoluble proteins and 4% of the total proteins. 20mg of beta3 peptide was obtained from one litter culture medium after purification by using metal-chelating sepharose 6B FF. The purity of beta3 is 92.2% according to Gel-Pro analysis. The anti-adhesion effects of beta3 peptide, beta1 peptide (DLYYLMDLSYSMK) and GRGDS on the hepatocellular carcinoma cell line SMMC-7721 and the high metastasis hepatocellular carcinoma cell line HCCLM6 were studied. The result showed the beta3 blocked the adhesion of HCCLM6 cells and SMMC-7721 cells to fibronectin (FN) specifically. The inhibition effect was dose-dependent and time-dependent and the inhibition rate of beta3 was higher than three times concentration of beta1 and GRGDS. This suggested that pET-His-beta3/BL21(DE3)plysS was a suitable expression system for beta3, and the expressed beta3 specially inhibited the adhesion of cancer cells.