BACKGROUND:The use of mesenchymal stem cels in the field of tissue engineering for osteoarticular injury repair is a very promising tool since these cels are readily expandable and able to differentiate into chondrocytes. Abundant evidence suggests that microRNAs play critical roles in chondrogenic differentiation of mesenchymal stem cels.
OBJECTIVE:To observe the chondrogenic effect of human bone marrow mesenchymal stem cels transfected with lentiviral vectors bearing miR-221-3p/222-3p inhibition, thereby provding new strategies for cartilage injury.
METHODS: miRNA microarray technology was applied to detect microRNAs expression profiles at three different stages of chondrogenic differentiation induction after transforming growth factor-β3 treatment and verified by real-time fluorescence quantitative PCR (RT-qPCR). Human bone marrow mesenchymal stem cels were infected with lentivirus bearing miR-221-3p/222-3p inhibition. After co-suppressing the expression of miR-221/222-3p, cel counting kit-8 was used to determine the cel proliferation, the differentiation of bone marrow mesenchymal stem cels towards chondrocytes was verified by type II colagen protein expression through immunohistochemistry and glycosaminoglycan accumulation was also elevated by sarranine O staining. RT-PCR was used to detect type II colagen and aggrecan mRNA expression at 21 days of chondrogenic induction.
RESULTS AND CONCLUSION: The expression of miR-221-3p/222-3p was inhibited after Lv-miR221-3p/222-3p inhibition co-transfected into bone marrow mesenchymal stem cels. microRNA microarray and RT-qPCR results showed that the expression of miR-221-3p/222-3p was declined significantly at the anaphase of chondrogenic differentiation. The expression levels of chondrogenic markers, Aggrecan and type II colagen were significantly increased in the miR-221-3p/222-3p inhibition group and cel proliferation was also inhibited significantly compared with non-transduced cels or transduced with the empty lentiviral vector group. miR-221-3p/222-3p knockdown in bone marrow mesenchymal stem cels could inhibit proliferation but promote chondrogenic differentiation of bone marrow mesenchymal stem cels.

BACKGROUND:Many studies have showed that enough blood supply is an essential condition of bone repair and regeneration. OBJECTIVE:To construct the endothelial progenitor cells/bone marrow mesenchymal stem cells (EPCs/BMSCs) composite sheet. METHODS:After isolation and culture, EPCs and BMSCs were co-cultured directly to form EPCs/BMSCs sheet by cellsheet-inducing medium. After 10 days of induction, the sheet was investigated by gross observation, inverted microscope and hematoxylin-eosin staining. The distribution and communication of EPCs and BMSCs during the process of cellsheet induction were observed after the fluorescence labeling separately. Alkaline phosphatase assay and alizarin red staining were applied to examine the ability of osteogenic differentiation of EPCs/BMSCs sheet.
RESULTS AND CONCLUSION:EPCs/BMSCs sheet was harvested after 10-day induction. Cel-cellcontact between EPCs and BMSCs could be observed during the process of the cellsheet preparation. The harvested sheet was composed of multiple layers of cells and cel-produced extracellular matrix. Alkaline phosphatase assay and alizarin red staining both demonstrated that EPCs/BMSCs sheet had good osteogenic differentiation ability. These results suggested that EPCs/BMSCs sheet can be constructed successful y, and the sheet has strong osteogenic differentiation capability in vitro, providing the foundation for the repair of bone defects.

Objective To study the changes of plasma cystatin C level (PcyC),and evaluate the effects of the joint use of atorvastatin and probucol on PcyC and severity of coronary lesion in patients with borderline lesion of coronary artery.Methods One hundred and thirty consecutive patients with borderline coronary lesion assessed by quantitative coronary angiography were enrolled into borderline coronary lesion group (BCL),and another 136 subjects without coronary lesion were enrolled as controls (CTR).And in the meantime,the subjects in BCL group were randomized (closed envelope method) into routine treatment subgroup ( RTT,n =60),and combined treatment subgroup in which patients were treated with atorvastatin 20 mg plus probucol 1.0 g daily in addition to routine medication ( CBT,n =70) for 6 months.There were no statistical differences in basic clinical features between two subgroups.PcyC,high-sensitive C-reactive protein (hs-CRP),total cholesterol (TC),low-density lipoprotein cholesterol (LDL-C),high-density lipoprotein cholesterol ( HDL-C ) and triglycerides (TG) were determined.Of them,104 patients in BCL group rechecked by coronary angiography.Comparison of biomarkers carried out between two groups by using a number of independent-sample t-test and analysis of variance.For enumeration data,chi-square test was used to compare mean values of biomarkers between groups. P ＜ 0.05 was considered statistically significant.Results PcyC levels were significantly higher in BCL group than those in CTR group ( P ＜ 0.05 ).Compared with RTT subgroup,levels of PcyC,TC,LDL-C,TG and hs-CRP were more significantly decreased in CBT subgroup (P ＜ 0.05,P ＜ 0.01 ).Moreover,there was a trend of slight decrease in the mean percent of stenosis (MPS) of coronary artery with borderline lesion in RTT subgroup treated for 6 months,whereas more marked decrease in the MPS of coronary artery with borderline coronary lesion in CBT subgroup treated for 6 months ( P ＞ 0.05 ; P ＜ 0.05 ).Conclusions Cystatin C plays an important role in the pathogenesis of coronary artery,and PcyC is associated with severity of coronary lesion,the combination of atorvastatin and probucol decreases the PcyC level,and it may be the treatment of choice for borderline lesion of coronary artery.

Objective To evaluate the relationship between plasma cystatin C concentration (PcyC) and coronary artery diseases (CAD). Method A total of 126 subjects with CAD evidenced by coronary angiography admitted from April 2007 to March 2009 were divided into three groups: stable angina pectoris (SAPs, n = 34),unstable angina pectoris (UAPs, n = 56) and acute myocardial infarction (AMIs, n = 36), according to the diag-nostic criteria of CAD set by WHO. Another 34 subjects without CAD were taken as controls. There were no statis-tical differences in demographics among four groups. Serum lipids profile, uric acid (UA), PcyC and high-sensi-tive C-reactive protein (hs-CRP) were determined. And in the meantime, all patients were followed up for six months and adverse cardiovascular events were recorded. Comparisons were made between groups with a number of independent-sample t -tests. Data were processed with analysis of variance to test the differences in means among four groups, and the means were compared with chi-square test. Statistical significance was established at a P val-ue of less than 0.05. Results Cystatin C levels were significantly higher in UAPs than that in SAPs and in controls (P < 0.05), but were much lower than that in AMIs (P < 0.05). And much higher concentration of hs-CRP was found in UAPs (P < 0.05) and in AMIs (P < 0.01). Cystatin C was positively and significantly corre-lated with age, hs-CRP, WBC, creatinine and UA (r > 0, P < 0.05), whereas a significantly negative correla-tion with high-density lipoprotein cholesterol was found (r = - 0.227, P < 0.05). These coefficients were obvi-ously high for creatinine (r = + 0. 612), and WBC (r = + 0.459). During the period of six-month follow-up, 26 patients with adverse cardiovascular events were found, and had significantly higher cystatin C levels than 22 con-trols at admission (P < 0.01). Conclusions Cystatin C plays a pivotal role in the course of CAD, and the PcyC is a strong predictor for the risk of cardiovascular events.

BACKGROUND: Group pre-test has confirmed that amnion endothelial cell conditioned medium can induce human umbilical cord blood mesenchymal stem cells into dopaminergic neuron-like cells. In this process, neurotrophic factors and their receptors may play an important role. OBJECTIVE: To study the function of neurotrophic factors secreted by amniotic epithelial cells in the differentiation of human umbilical cord blood mesenchymal stem cells into neurons.METHODS: P1 human umbilical cord blood mesenchymal stem cells at 2×10~8 /L were incubated and assigned to 3 group. Control group was added with HG-DMEM medium. Induction group received human amniotic epithelial cell medium. Blocking agent group underwent blocking agent K252a fluid, and the incubated was conducted at 36 ℃ for 40 minutes, and then amniotic epithelial cell medium was added. Immunofluorescence chemistry was used to determine neuron specific enolase and dopamine transporter expression in human umbilical cord blood mesenchymal stem cells. Real-time quantitative PCR was employed to detect neuron specific enolase, dopamine transporter and tyrosine hydroxylase expression in human umbilical cord blood mesenchymal stem cells. RESULTS AND CONCLUSION: Nerve growth factor and brain-derived neurotrophic factor were observed in human amniotic supernatant. P1 human umbilical cord blood mesenchymal stem cells expressed Trka and Trkb. Forty-eight hours following induction, compared with the control group, positive expression of neuron specific enolase and dopamine transporter was significantly increased in the induction and blocking agent groups (P < 0.05), especially in the induction group (P < 0.05). Neuron specific enolase, dopamine transporter and tyrosine hydroxylase mRNA levels were significantly greater in the induction and blocking agent groups compared with the control group (P < 0.01), and each gene mRNA levels were significantly greater in the induction group than in the blocking agent group (P < 0.01). Results verified that neurotrophic factor in the human amniotic epithelial cells plays important effects on differentiation of human umbilical cord blood mesenchymal stem cells into neurons. The promotion effects are mediated by activating Trk receptor.

Objective To compare the sensitivities of the stimulation effect of human cord blood mesenchymal stem cells(CB-MSCs) and CB-MSCs of neuronal differentiation to lymphocytes(LCs) detected with carboxyfluorescein didcetate(CFSE) and MTT. Methods To prepare LCs from SD rat and divided into four group stimulating cells: 1. CB-MSCs;2. Dif-CB-HSCs;3. SH-SY5Y(positive control);4. Auto-LC(negative control).Stimulating cells were respectively Co-cultured with LCs. The proliferation of LCs was detected with MTT and CFSE ( n =3). Results CB-MSCs and Dif-CB-HSCs stimulated LCs to proliferate more weakly than positive control detected with MTT and CFSE. The quantity of proliferation of lymphocytes Co-cultured with CB-MSCs and Dif-CB-HSCs were higher than that of Auto-LC detected with CFSE. But MTT OD value of CB-MSCs and Dif-CB-HSCs was a little lower than that of Auto-LC. Statistical analysis results showed no significant difference.Conclusion CFSE can reflect proliferation status of lymphocytes better than MTT. CFSE shows more advantages in practical use.

Background and purpose: Osteopontin (OPN) is a glycophosphoprotein that is expressed by numerous human cancer cells. The function of OPN in skeletal modeling and remodeling, bone resorption, angiogenesis and tumor cell metastasis and progression through binding with integrin and CD44 receptors were studied. Our purpose of the study was to detect the level of osteopontin(OPN) and CD44 variant isoforms(CD44v6) in multiple myeloma (MM) patients, and to explore the relationship between OPN and CD44v6 with the progress of MM. Methods: 32 MM patients were admitted to our hospital from Sep. 2007 to Dec. 2008. The patients were divided into two groups, group A (untreated and relapsed MM patients) and B (stable MM patients), and the control group including 15 subjects were the benign anemia patients or healthy people who suffered bone fracture. Bone marrow mononuclear cells (MNCs) and bone marrow stromal cells (BMSCs) from MM patients and subjects were investigated as potential OPN and CD44v6 producers. The level of OPN and CD44v6 of the conditioned media from MM patients and subjects were analyzed by ELISA. Results: The OPN level in group A (19 cases) was significantly higher than group B (13 cases) and control group (P<0.05). The CD44v6 level of 14 patients in group A was significantly higher than that of 10 cases in group B and control group (P<0.05); The OPN level of MM patients was correlated with the level of CD44v6 (r=0.52, P=0.000), the percentage of plasma cells in the bone marrow (r=0.74, P=0.000), M protein (r=0.53, P=0.014), and β2-microglubin (r=0.62, P=0.002). Conclusion: The increase of OPN and CD44v6 is associated with progress and pathogenesis of MM,and may be involved with tumor burden, stage and tumor invasion.

Objective To explore the application values of effect on the expression of growth associated protein-43 (GAP-43) and myelin basic protein(MBP) by electrical stimulation in different sites of spinal cord injured rats after the human umbilical cord blood stem cells transplantation. Methods In this study the subjects adopted are clean grade rats, which were performed with Allen's method by NYU blow device, resulting in SCI models. 192 rats successfully modeled were divided into groups according to the random table. Group A received electric stimulation in the scalp surface projection area of motor area: Group A1 received electric stimulation and transplantation of umbilical cord blood stem cell, while Group A2 only received electric stimulation; Group B received local electric stimulation at damaged site: Group B1 received electric stimulation and transplantation of umbilical cord blood stem cell, while Group B2 only received electric stimulation; Group C received electric stimulation in the scalp surface projection area of motor area and local electric stimulation at damaged site: Group C1 received electric stimulation and transplantation of umbilical cord blood stem cell, while Group C2 only received electric stimulation; Group D are just the SCI models: Group D1 received transplantation of umbilical cord blood stem cell without electric stimulation, while Group D2 neither received cell transplantation nor electric stimulation. The sacrifice time of rats was the 1st week, 2nd week, 3rd week, 4th week, 8th week, and 12th week after operation respectively. The distribution of the remained or regenerative neurons at damaged areas by corticospinal tract anterograde tracer with Biotinylated dextran amine (BDA), and we also detected the content changes of GAP-43, MBP and the MAB1281 specific expression of the human nucleus of human umbilical cord blood stem cells. Results 1). NYU is a blow device for Allen's rat model of SCI, which could quantify the crash potential energy, and gain a stable and repeated model with high successful rate. 2). At each time site in this study, the factors that benefit for the neurofunction restoration after electric stimulation in the microenvironment into which the stem cells have been transplanted are GAP-43 and MBP, and they are all positive; the effects of the combination of head acupuncture with body acupuncture are much better than single head acupuncture or body acupuncture. 3). At each time site in this study, the factors that benefit for the neurofunction restoration after injury are GAP-43 and MBP, and the effects of them are all positive, which do not depend on whether the stem cell transplantation has been done. Conclusion 1). After the stem cell transplantation have been done, the changes of the microenvironment develops in the direction of benefiting for restoring neurofunction, which means that stem cell transplantation can promote the restoration of neurofunction and the stem cell transplantation is successful in this study. 2). Electric stimulation is significantly positive for the factors (GAP-43, MBP) which benefit for restoring neurofunction in the microenvironment after stem cell transplantation, the effects of the combination of head acupuncture with body acupuncture is better than single head acupuncture or body acupuncture. The significantly positive effects of the electric stimulation on microenvironment after injuries are independent, which do not depend on whether the stem cell transplantation has been done. Electric stimulation and stem cell transplantation are significantly synergistic at the microenvironment.

Objective To explore the effects of electrical stimulation in different acupoints on the expression of growth associated protein-43 (GAP-43), basic fibroblast growth factor (bFGF), insulin-like growth factor (IGF) in spinal cord injured rats after the human umbilical cord blood stem cells transplantation.Methods 192 SD rats were injured at T10-11 level with NYU, and divided randomly into groups: Group A received electric stimulation in the scalp of motor area (A1 group received electric stimulation and transplantation of umbilical cord blood stem cell, the rats of A2 group only received electric stimulation); group B received local electric stimulation at damaged site (B1 with electric stimulation and transplantation, B2 only with electric stimulation); group C received electric stimulation in the scalp of motor area and at damaged site (C1 with electric stimulation and transplantation, C2 only with electric stimulation); D1 received transplantation without electric stimulation, D2 neither with transplantation nor electric stimulation. The expression of GAP-43, bFGF and IGF were detected 1, 2, 3, 4, 8 and 12 weeks after operation. Results Electric stimulation increased the expression of GAP-43, bFGF, IGF, stimulating scalp and body were more than stimulated scalp or body alone, combined with the stem cells transplantation were more than no transplantation. Conclusion Both electric stimulation and stem cells transplantation can improve the microenvironment for neural recovery synergistically. Stimulating scalp and body is more effective than alone.

Objective To explore the effect of monosodium glutamate (MSG) on the expression of 5\|HT in gastroenteric mucosa of rat. Methods Immunohistochemical technique was used to detect the expression of 5\|HT. The immuno\|activity and the density of positive cells were measured by image analysis. Results The immuno\|activity and the density of 5\|HT positive cells(EC cell) in the experimental group are higher than that in control group. The effect by 120d is the most significant, followed by that of 52d. Statistical analysis showed a significant difference ( P