Yanjun Zeng

Comparing the chondrogenic potential in vivo of autogeneic mesenchymal stem cells derived from different tissues.

Abstract Objective: To compare the chondrogenic ability of mesenchymal stem cells (MSCs) derived from different tissues in rabbits’ full-thickness articular cartilage defects. Methods: Sixty New Zealand white rabbits of ordinary grade with a body weight of 2.5∼3.5kg were selected for this study. Six were sacrificed for preparation of deminerized bone matrix (DBM) as scaffold. Fifty-four were used for cartilage defects model. Full-thickness cartilage defect of knee joint was created on trochlear groove at two sides of the femur with a diameter of 4 mm and thickness of 3 mm. All 54 rabbits were randomly divided into 6 groups and treated by autogeneic MSCs isolated from bone marrow, periosteum, synovium, adipose tissue and muscle, respectively. The 6th group was a control group with nothing plugged into the defects. Every three rabbits were killed at three time points, which were 4, 8, and 12 weeks after the operation in each group. The reparative tissue samples were evaluated grossly, histologically, immunohistochemically, and graded according to gross and histological scales 12 weeks postoperatively. We input the scores into SPSS 11.5 software and the analysis of variance (one-way-ANOVA) and student-newman-keuls (SNK-q) test were used to process statistical analysis and find out if the differences between each group had statistical significance. Results: Fifty-four rabbits are included in the final analysis. The defects are all repaired by hyaline-like tissue except the control group. The bone-marrow-MSCs produced much more cartilage matrix than that of other groups. Gross and histological grading scale indicates that the defects repaired by MSCs isolated from bone marrow are superior to that repaired by MSCs isolated from periosteum, synovium, adipose tissue, and muscle (p < 0.05). In adipose-MSCs and muscle-MSCs group, some defects are even repaired by fibrous tissue. Conclusion: Bone-marrow-MSCs have greater in vivo chondrogenic potential than periosteum-, synovium-, adipose- and muscle-MSCs.

Circulating MicroRNAs as Novel Potential Biomarkers for Early Diagnosis of Acute Stroke in Humans

BACKGROUND Many diseases include microRNAs (miRNAs) as reported biomarkers. The significance of circulating miRNAs for early diagnosis of acute stroke in humans is unknown. We aim to determine whether circulating miRNAs potentially serve as novel biomarkers for acute stroke. METHODS We prospectively recruited patients with acute stroke and those with nonstroke disease. Patients with acute stroke were identified using magnetic resonance imaging (MRI) for early diagnosis. If the patient suffered from acute stroke that was detected with diffusion-weighted imaging, the patient was defined as an MRI(+) patient. Otherwise, it was defined as an MRI(-) patient. Circulating miRNAs were measured by miRNA microarray and real-time polymerase chain reaction (PCR) analysis. RESULTS A total of 136 patients were included in the study. Testing by miRNA microarray and real-time PCR analyses showed that hsa-miR-106b-5P and hsa-miR-4306 were present with markedly high abundance in patients of acute stroke, whereas hsa-miR-320e and hsa-miR-320d were present with quite low abundance in patients compared with healthy individuals. Compared with healthy individuals, the miRNAs were increased as in patients with acute stroke as follows: hsa-miR-106b-5P, 3.63-fold in MRI(-) patients and 23.90-fold in MRI(+) patients; hsa-miR-4306, 3.19-fold in MRI(-) patients and 5.30-fold in MRI(+) patients; hsa-miR-320e, .33-fold in MRI(-) patients and .13-fold in MRI(+) patients; and hsa-miR-320d, .23-fold in MRI(-) patients and .07-fold in MRI(+) patients. CONCLUSIONS Elevated hsa-miR-106b-5P and hsa-miR-4306 and decreased hsa-miR-320e and hsa-miR-320d in plasma may be novel biomarkers for the early detection of acute stroke in humans.

miR-125b Inhibitor May Enhance the Invasion-Prevention Activity of Temozolomide in Glioblastoma Stem Cells by Targeting PIAS3

BackgroundTemozolomide, an alkylating agent, is a promising chemotherapeutic agent for treating glioblastoma. Although chemotherapy with temozolomide may restrain tumor growth for some months, invariable tumor recurrence suggests that cancer stem cells maintaining these tumors persist. Previous research has shown that temozolomide can inhibit the proliferation of human glioblastoma stem cells (GSCs); however, no research has focused on the invasion of GSCs, which is an important factor for glioblastoma recurrence. Accumulating evidence indicates that microRNA (miR)-125b over-expression in GSCs may increase their invasiveness.ObjectiveOur objective was to identify the effects and mechanism of action of an miR-125b inhibitor combined with temozolomide in the invasive pathogenesis of GSCs.MethodsWe modified the levels of miR-125b expression in primary GSCs in order to observe the effect on sensitivity to temozolomide on invasion, and we further analyzed the differences in mechanism between miR-125b treatment alone and treatment with miR-125b plus temozolomide using the Cancer PathwayFinder PCR Array.ResultsOur results demonstrated that either an miR-125b inhibitor or temozolomide could modestly inhibit the invasiveness of GSCs. Furthermore, GSCs that were pre-transfected with an miR-125b inhibitor, then treated with temozolomide, showed significantly decreased invasiveness when compared with GSCs treated with an miR-125b inhibitor or temozolomide alone. Further research into the underlying mechanism demonstrated that the miR-125b inhibitor enhanced the invasion-prevention activity of temozolomide in GSCs through targeting PIAS3 (protein inhibitor of activated STAT [signal transducer and activator of transcription]), which contributed to reduced STAT3 transcriptional activity and subsequent decreased expression of matrix metalloproteinase (MMP)-2 and -9.ConclusionsmiR-125b could play a role in the development of temozolomide resistance in GSCs. Inhibition of miR-125b expression may enhance sensitivity of GSCs to temozolomide by targeting PIAS3 on cell invasion.

Expression of cytokines in rat brain with focal cerebral ischemia after grafting with bone marrow stromal cells and endothelial progenitor cells

BACKGROUND AIMS This study aimed to observe nine factors expressed in rat ischemic brain after transplantation of bone marrow stromal cells (BMSC) and/or endothelial progenitor cells (EPC). These factors were vascular endothelial growth factor (VEGF), stromal cell-derived factor-1 (SDF-1), basic fibroblast growth factor (bFGF), insulin-like growth factor (IGF-l), transforming growth factor-β (TGF-β), platelet-derived growth factor-BB (PDGF-BB), brain-derived neurotrophic factor (BDNF), glial cell line-derived neurotrophic factor (GDNF) and nerve growth factor (NGF). METHODS Adult Wistar rats were divided randomly into four groups: a vehicle group, BMSC group, EPC group and BMSC combined with EPC group. The rats were subjected to middle cerebral artery occlusion (MCAO) then implanted intravenously with 3 × 10(6) BMSC, EPC, BMSC/EPC or phosphate-buffered saline (PBS) 24 h after MCAO. Neurologic functional deficits were measured on days 1, 7, 14, 28 after transplantation. On day 7 after transplantation, quantitative reverse transcription (qRT)-polymerase chain reaction (PCR) and Western blot were employed to detect the expression of VEGF, SDF-1, bFGF, IGF-l, TGF-β, PDGF-BB, BDNF, GDNF and NGF. RESULTS The neurologic evaluation found that the neurologic severity scores were no different between the four groups on day 1, and the scores of rats in the BMSC/EPC group were significantly lower than those of rats in the other groups on days 7, 14 and 28 after transplantation. The expressions of bFGF, VEGF and BNDF were significantly higher in the BMSC/EPC group compared with the other groups. CONCLUSIONS The intravenous transplantation of BMSC combined with EPC could promote the functional rehabilitation of rats with focal cerebral ischemia, and the mechanism may be related to the enhanced expression of factors.

Multidrug resistance-associated biomarkers PGP, GST-π, Topo-II and LRP as prognostic factors in primary ovarian carcinoma

Abstract This study aims to investigate the expression of P-glycoprotein (PGP), glutathione S-transferase π (GST-π), DNA topoisomerase II (Topo-II) and lung resistance-related protein (LRP) in ovarian carcinoma, thus providing better chemotherapy choice and post-operative prognosis for ovarian carcinoma patients. A total of 80 primary ovarian carcinoma, 16 benign ovarian epithelial neoplasm, and 12 normal ovarian tissue samples were collected. Immunohistochemistry was used to detect the expression of PGP, GST-π, Topo-II and LRP, and the results were analysed by correlation with clinicopathological parameters. Positive expression rates of PGP, GST-π, Topo-II and LRP in patients with ovarian carcinoma (57.5%, 58.8%, 76.3% and 73.8%, respectively) were all higher than those found in normal and benign tissue (P<0.05). In clinical stages I/II vs. III/IV, the expression rates of PGP, GST-π, Topo-II and LRP were 40.7% vs. 66% (P<0.05), 40.7% vs. 67.9% (P<0.05), 66.7% vs. 81.1% (P>0.05) and 55.6% vs. 83.0% (P<0.05), respectively. Carcinoma differentiation ranged from well to poor, and expression levels of each marker were as follows: PGP, 57.9%, 62.1% and 53.1% (P>0.05); GST-π, 36.8%, 55.2% and 75.0% (P<0.05); Topo-II, 52.6%, 79.3% and 87.5% (P<0.05); and LRP, 84.2%, 69.0% and 71.9% (P>0.05). Ovarian carcinoma patients with PGP-, GST-π-, Topo-II- and LRP-positive expression had a shorter median survival time than those who were negative for these markers (PGP: 36 months vs. 48 months [P=0.0017]; GST-π: 36 months vs. 41 months [P=0.0103]; Topo-II: 37 months vs. 39 months [P=0.3811]; LRP: 37 months vs. 55 months [P=0.002]). COX regression analysis demonstrated that the clinical stage of the tumour, and the expression of PGP, GST-π or LRP, may influence patient survival time after surgery. The relative death risk for patients with clinical stage III/IV tumours increased 9.46-fold compared to those with stage I/II tumours. The relative death risk in the PGP-, GST-π- and LRP-positive groups increased by 2.049-, 2.452- or 2.609-fold, respectively, compared with the corresponding negative groups. PGP, GST-π, Topo-II and LRP are all expressed in primary ovarian carcinoma, indicating the presence of multidrug resistance in this disease. Combined evaluation of PGP, GST-π, Topo-II and LRP expression may enable better chemotherapeutic choice and provide an accurate prognosis for ovarian carcinoma patients.

Biomechanical properties of ascending aorta and pulmonary trunk in pigs and humans.

Abstract:  Background:  This study aims to obtain the biomechanical properties of ascending aorta and pulmonary trunk between healthy humans and pigs of different months, so as to provide necessary biomechanical experimental basis for anastomosing blood vessel in pig‐to‐human heart xenotransplantation.

Inhibition of tumor cell migration and invasion through knockdown of Rac1 expression in medulloblastoma cells.

Medulloblastoma is one of the common malignant brain tumors in children or young adults and its overall disease-free 5-year survival rate is approximately 50% due to tumor progression, invasion, and metastasis. This study aimed to determine whether one of Rho GTPases, Rac1 can affect the morphology, motility, and invasion of medulloblastoma cells through knockdown of Rac1 expression. Medulloblastoma Daoy cells were used to manipulate Rac1 expression using Rac1 shRNA, Rac1N17, and Rac1L61 constructs. Reverse transcriptase-PCR and western blot were used to detect expression of Rac1 mRNA and protein, respectively. Invasion and migration assays were performed to assess invasion and migration capacity of Daoy cells, respectively. The data showed that Rac1 mRNA and protein were overexpressed in Daoy cells. Deletion of Rac1 decreased the cross-linked actin network and pseudopodia and also inhibited the number of migration cells migrated or invaded to the other side of the filter compared to control cells. These data indicated that the invasion and migration in Daoy cells were inhibited by deletion of Rac1, and suggest that targeting Rac1 by Rac1 shRNA may further be evaluated and used as a potential anticancer strategy to treat medulloblastoma.

Experimental Study of Nano-Hydroxyapatite/Recombinant Human Bone Morphogenetic Protein-2 Composite Artificial Bone

Abstract Objective: To investigate the repairing ability of nano-hydroxyapatite/recombinant human bone morphogenetic protein-2 (Nano-HA/rhBMP-2) composite artificial bone for bone defect and to provide evidence for its application in clinical repair of bone defect. Methods: The animal model of bone defect was made on unilateral radius of 90 New Zealand white rabbits, which were randomly divided into experimental group (group A, bone defect was repaired with Nano-HA/rhBMP-2 composite artificial bone), control group (group B, bone defect was repaired with Nano-HA artificial bone), and blank group (group C, bone defect was unrepaired). The repairing ability for bone defect was evaluated by gross observation, X-ray examination, scanning electron microscope, radionuclide bone imaging and biomechanical analysis at 4th, 8th, and 12th week postoperatively. Results: Both Nano-HA/rhBMP-2 composite artificial bone and Nano-HA artificial bone could stimulate new bone formation, but the former could stimulate more new bone formation and had better repairing ability for bone defect than that of the latter, with statistically significant difference (P <0.05). Conclusion: Nano-HA/rhBMP-2 composite artificial bone has good repairing ability for bone defect and it is hopeful to become an ideal repairing material for bone defect.

The Study on Biocompatibility of Porous nHA/PLGA Composite Scaffolds for Tissue Engineering with Rabbit Chondrocytes In Vitro

Objective. To examine the biocompatibility of a novel nanohydroxyapatite/poly[lactic-co-glycolic acid] (nHA/PLGA) composite and evaluate its feasibility as a scaffold for cartilage tissue engineering. Methods. Chondrocytes of fetal rabbit were cultured with nHA/PLGA scaffold in vitro and the cell viability was assessed by MTT assay first. Cells adhering to nHA/PLGA scaffold were then observed by inverted microscope and scanning electron microscope (SEM). The cell cycle profile was analyzed by flow cytometry. Results. The viability of the chondrocytes on the scaffold was not affected by nHA/PLGA comparing with the control group as it was shown by MTT assay. Cells on the surface and in the pores of the scaffold increased in a time-dependent manner. Results obtained from flow cytometry showed that there was no significant difference in cell cycle profiles between the coculture group and control (P > 0.05). Conclusion. The porous nHA/PLGA composite scaffold is a biocompatible and good kind of scaffold for cartilage tissue engineering.

Establishing an experimental model of photodynamically induced anterior ischemic optic neuropathy.

Numerous methods and drugs have been used to treat anterior ischemic optic neuropathy (AION); however, further investigations to determine the value of treatments for AION have been impeded by the lack of appropriate animal models of AION, significantly impacting on in-depth study of the disease. A rat model of AION was established, and corresponding functional changes of the fundus were observed using fundus fluorescein angiography (FFA), optical coherence tomography (OCT), and flash visual-evoked potential (F-VEP) in order to confirm the reliability of the AION model histopathologically. One day after model establishment, histopathology demonstrated that portions of the optic disc were highly edematous, with edema of nerve fibers and loose tissue, accompanied by displacement of the surrounding retina. At 23 days, the optic disc and surrounding nerve fiber layers had become thinner. None of the above-mentioned changes was observed in the laser, hematoporphyrin derivative (HPD), or naive groups. The results of fundus, FFA, F-VEP, and OCT-within 90 days after model establishment-confirmed that krypton red laser irradiation (647 nm), applied 2 h after HPD injection, can establish an ideal animal model of AION.