Activity of intervertebral disc stem cells and nucleus pulposus mesenchymal stem cells in intervertebral disc niche condition / 中国组织工程研究

BACKGROUND: Disc microenvironment plays an important role in the biological behavior of stem cells. It is proposed that the microenvironment can be used to achieve the repair of disc tissue independent of seed cells by niche regulation. OBJECTIVE: To explore the difference in the activity of intervertebral disc stem cells and nucleus pulposus mesenchymal stem cells under the intervertebral disc niche. METHODS: Ten healthy male Sprague-Dawley rats were used. The intervertebral disc bone tissue was isolated according to the anatomical area. Intervertebral disc stem cells were digested with type II collagenase and cultured in vitro. Nucleus pulposus was isolated, and nucleus pulposus mesenchymal stem cells were cultured in vitro using enzyme digestion. Intervertebral disc stem cells and nucleus pulposus mesenchymal stem cells were cultured and amplified in normal condition and intervertebral disc niche. The proliferation curves of cultured cells were measured by MTT method at 1-6 days after culture. The expression level of CD29 was detected by flow cytometry at 1, 3 and 6 days. RESULTS AND CONCLUSION: Under different culture conditions, the proliferation of intervertebral disc stem cells and nucleus pulposus mesenchymal stem cells showed an opposite trend. Under normal culture conditions, intervertebral disc stem cells and nucleus pulposus mesenchymal stem cells were proliferated logarithmically from day 4 to day 6, with no statistical significance (P > 0.05). In the intervertebral disc niche condition, the survival rate of nucleus pulposus mesenchymal stem cells was significantly lower than that of intervertebral disc stem cells (P < 0.05). In intervertebral disc niche condition, at 3 and 6 days, the surface positive antigen CD29 of intervertebral disc stem cells was significantly higher than that of nucleus pulposus mesenchymal stem cells (P < 0.05). These results suggest that in the intervertebral disc niche condition, the activity of intervertebral disc stem cells and nucleus pulposus mesenchymal stem cells is inhibited to some extent, but intervertebral disc stem cells are more active than nucleus pulposus mesenchymal stem cells.

Garlicin Post-Conditioning Suppresses Adhesion Molecules in a Porcine Model of Myocardial Ischemia-Reperfusion Injury / 中国结合医学杂志

OBJECTIVES@#To evaluate whether garlicin post-conditioning can attenuate myocardial ischemiareperfusion injury in a catheter-based porcine model of acute myocardial infarction (AMI) by affecting adhesion molecules integrin β1/CD29 and platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31).@*METHODS@#Twenty-two swine were devided into 3 groups: 6 in a sham-operation group, and 8 each in the model and garlicin groups. AMI porcine model was established in the model and garlicin groups. The distal parts of the left anterior descending coronary artery in the animals of the model and garlicin groups were occluded by dilated balloon for 2 h, followed by reperfusion for 3 h. Garlicin (1.88 mg/kg) was injected over a period of 1 h, beginning just before reperfusion, in the garlicin group. Real-time polymerase chain reaction, immunohistochemistry and Western blot were carried out to detect mRNA and protein expressions of CD29 and CD31 3 h after reperfusion.@*RESULTS@#Hematoxylin-eosin staining showed a better myocardial structure in the garlicin group after reperfusion. Compared to the model group, garlicin inhibited both the mRNA and protein expression of CD29 and CD31 in reperfusion area and no-reflflow area (P<0.05 respectively).@*CONCLUSIONS@#Garlicin post-conditioning induced cardio-protection against myocardial ischemia-reperfusion injury in this catheter-based porcine model of AMI. The cardio-protective effect of garlicin is possibly owing to suppression of production of CD29 and CD31, by inhibition of the mRNA expression of CD29 and CD31.

Correlation between CD4+CD29+T cells and metastasis and radiotherapy for patients with pulmonary ade- nocarcinoma / 实用医学杂志

Objective To observe the correlation between CD4+ CD29+ T cells and metastasis and radiotherapy for patients with pulmonary adenocarcinoma. Method Seventy-one patients with lung adenocarcinoma, 93 patients with lung adenocarcinoma ,76 cases of chronic obstructive pulmonary disease (COPD),63 cases of healthy volunteers were enrolled. Frequencies of blood CD4+ CD29+ T cells and their intracellular necrosis factor alpha(TNF-α)and interleukin 1(IL-1)were compared. Compare TNF-α,IL-1,integrin beta 1 and vascular endothelial growth factor(VEGF)levels in the patients with transferred pulmonary adenocarcinoma or with non-transferred pulmonary adenocarcinoma and their changes with the treatment of radiotherapy. Results the patients with lung adenocarcinoma and non lung adenocarcinoma were significantly higher than that of COPD and health group,and patients with lung adenocarcinoma is significantly higher than patients with non lung adenocarcinoma (P<0.05);Integrin beta 1,VEGF and CD4+CD29+T cells,TNF-αand IL-1 level in patients with lung adeno-carcinoma metastasis were significantly higher than non-transferred group(P < 0.05);After radiotherapy,CD4+CD29+T cells,TNF-αand IL-1 in patients with lung adenocarcinoma were significantly lower than before(P<0.05);CD4+ CD29+ T cells,TNF alpha and IL-1 with integrin beta 1 and VEGF had significantly positive correlations. Conclusion CD4+CD29+T cells and cytokines increase significantly in the blood of patients with lung adenocarci-noma,and are related to the prognosis of metastasis and radiation therapy,which has important clinical significance.

Molecular association of CD98, CD29, and CD147 critically mediates monocytic U937 cell adhesion

Adhesion events of monocytes represent an important step in inflammatory responses induced by chemokines. The β1-integrin CD29 is a major adhesion molecule regulating leukocyte migration and extravasation. Although several adhesion molecules have been known as regulators of CD29, the molecular interactions between CD29 and its regulatory adhesion molecules (such as CD98 and CD147) have not been fully elucidated. Therefore, in this study, we examined whether these molecules are functionally, biochemically, and cell-biologically associated using monocytic U937 cells treated with aggregation-stimulating and blocking antibodies, as well as enzyme inhibitors. The surface levels of CD29, CD98, and CD147 (but not CD43, CD44, and CD82) were increased. The activation of CD29, CD98, and CD147 by ligation of them with aggregation-activating antibodies triggered the induction of cell-cell adhesion, and sensitivity to various enzyme inhibitors and aggregation-blocking antibodies was similar for CD29-, CD98-, and CD147-induced U937 cell aggregation. Molecular association between these molecules and the actin cytoskeleton was confirmed by confocal microscopy and immunoprecipitation. These results strongly suggest that CD29 might be modulated by its biochemical and cellular regulators, including CD98 and CD147, via the actin cytoskeleton.

Correlation between CD4+CD29+T cells and the pathological typing and staging for patients with primary pulmonary carcinoma / 实用医学杂志

Objective To explore the correlation between CD4+CD29+T cells and the pathological typing and staging for patients with primary pulmonary carcinoma. Methods Flow cytometry was used to detect peripheral blood CD4+CD29+T cells, gamma interferon (IFN-γ) and tumor necrosis factor alpha (TNF-α) of 60 patients with lung cancer. The relationships between them and the pathological typing and staging were studied. Results (1) The CD4+CD29+T cellsand TNF-α percentages in thecancer patients were significantly higher than those of the control group (P 0.65, P < 0.05). Conclusion Theincreased CD4+CD29+T cells and TNF-α, together with decreased IFN-γ arehighly indicative of immunological characteristics of lung cancer , and closely related to the pathological typing and staging.

Study of Reversing Invasion of Human Gastric Cancer Cell Line BGC-823 by Targeting Angiopoietin-1 Using siRNA / 天津医药

Objective To knock down angiopoietin-1 expression in human gastric cancer cell line BGC-823 and to observe its effect of reversing tumor invasion. Methods siRNA sequence fragments was designed to target angiopoietin-1 and transferred into human gastric cancer cell line BGC-823. RT-PCR was used to assess the transcription level of angio-poietin-1 mRNA, then western blot and immunofluorescence were used to examine the expression level of three invasion-as-sociated proteins include integrinβ1, CD44V6 and Ang-1. Cell adhesion ability was evaluated by cell adhesion assay and cell invation was determined by matrigel and transwell plastic dual-chamber culture system. Results Ang-1 mRNA was knocked down by siRNA showed by RT-PCR. The expression of integrinβ1, CD44V6 and Ang-1 were significantly lower than control group(P<0.05), so did the cellular adhesion and invasion abilities(P<0.05). Conclusion Knocking down angiopoietin-1 by siRNA can reverse invasion of human gastric cancer cell line BGC-823 and may provide new ideas and reference for gene therapy of gastric cancer in the future.

Pluripotent lineage of CD133 stem cells isolated from human skin samples.

Skin stem cells are very important in cosmetics, pharmacological and regenerative medicine and burn cases. Foreskin samples surgically removed after circumcision from boys below 7 years were collected and primary epidermal cells were prepared by enzymatic and mechanical tituration method. Selecting CD133 (prominin-1) multipotent stem cell marker, enriched stem cells were analyzed by MACS using CD133 antibodies conjugated with magnetic beads. CD133 positive and negative cells with specific skin stem cells markers like - CD34 (Universal stem cells marker), CD29 (integrin beta-1) and CD49f (integrin alpha-6) immunophenotypes were screened and sorted in flowcytometer. Further the expression of four embryonic genes or transcription factors of pluripotent stem cells were analyzed for pluripotent character of sorted cells. It was found that skin stem cell markers associated with CD133 cells, differentially expressed CD34, CD29 and CD49f immunophenotyes on both positive and negative CD133 cells in FACS analysis. The embryonic stem cell markers (induced pluripotent stem cell markers) like Oct4, SOX2, Notch-2 and K19 genes were expressed in CD133 positive epidermal cells. It is therefore evident that foreskin derived epidermal stem cells showed pluripotent or multipotent nature. This finding opens up avenues for new uses of these stem cells for direct cell seeding in wound healing, surgical suturing and drug screening.

The study of resveratrol protective effects and mechanism on tissue-engineered cartilage / 中华风湿病学杂志

Objective To investigate the mechanism of protective effects of resveratrol on tissueengineered cartilage.Methods The chondrogenesis of alginate-encapsulated bone marrow mesenchymal stem cells (BMSCs) were evaluated by toluidine blue staining and immunostain.The morphology of BMSCs-derived chondrocytes cultured on chitosan-gelatin scaffolds (CGS) was evaluated by scanning electron microscope and laser confocal microscope.When these cells on CGS were pre-stimulated with interleukin-1β (IL-1β) or cotreated with IL-1β and resveratrol in the absence and presence of specific β1-integrin blocking antibody,collagen type Ⅱ,aggrecan,matrix metalloproteinase-13 (MMP-13) expression,and the translocation of nuclear factor kappaB (NF-κB) were analyzed by Western blotting.ANOVA was used for statistical analysis.Results Alginate bead culture plus conditional medium together could induce the cartilage-specific collagen type Ⅱ,aggrecan expression and extracellular matrix accumulation in differentiated chondrocytes.CGS supported differentiated cell attachment,proliferation,and migration.When those cells cultured on CGS were stimulated with IL-1β alone,collagen type Ⅱ and aggrecan expression was inhibited.However,MMP-13 expression increased.By Western blotting semi-quantitative analysis,the expression level of cartilage-specific collagen type Ⅱ of the control group was 0.484±0.006; the expression level of resveratrol intervention group was 0.474±0.014.The difference between these two groups was not statistically significant (P＞0.05).The expression level of the IL-1β intervention group reduced to 0.155±0.009,which was statistically significant different from the above two groups(P＜0.05).Resveratrol could antagonist the negative effect of IL-1β,and increase collagen type Ⅱ to 0.468±0.014,the difference between these two was statistically significant (P＜0.05),and no significant difference when compared to the control group (P＞0.05).Specific β1-integrin blocking antibody could abrogate these effects of resveratrol,decrease collagen Ⅱ expression to 0.169±0.011,the difference was significant (P＜0.05),but there was no difference when compared to the IL-1β group (P＞0.05).Aggrecan semi-quantitative expression has the same trend in the expression of type Ⅱ collagen while the expression of MMP-13,NF-κB had the reversal trend.These indicated that the resveratrol reversed the catabolic effects by reducing the nuclear translocation of NF-κB.Specific β1-integrin blocking antibody abrogated these effects of resveratrol.Conclusion Resveratrol,by regulating β1-integrin,acts as a NF-κB nuclear trans-location inhibitor to protect tissue-engineered cartilage.

Repair of ulcer with rhEGF sustained-release microspheres in diabetic rats / 中华创伤杂志

Objective To prepare recombinant human epidermal growth factor (rhEGF) sustained-release microspheres and evaluate their morphology, rhEGF releasing activities and cell proliferation activity in vitro and compare difference of rhEGF sustained-release microspheres and rhEGF in facilitaring ulcer healing in diabetic rats. Methods (1) rhEGF sustained-release microspheres were prepared by the modified double emulsion method. Morphology of the microspheres was detected by transmission electron microscope and size distribution measured by laser granularity meter/Zeta electric potential meter. ELISA assays were applied to determine rhEGF releasing. (2)Proliferation of mouse fibroblasts was analyzed by MTr method. (3) Diabetic rat models were prepared and divided into four groups, ie, rhEGF sustained-release mierospheres group (Group A), rhEGF stock solution group (Group B), blank sustainedrelease mierospheres group (Group C) and PBS meustruum control group (Group D), which were given drug once a day. The wound healing rate was calculated by taking photographs at days 3,7,14 and 21. Skin specimens from the wound edge were harvested partially for observation of hydroxyproline (HYP) contents. Immunohistochemistry was employed to detect integrin 131 and keratin-19 and measure their positive staining area ratio. Results (1) The particle diameter of rhEGF sustained-release microspheres was 193.5 nm, with relative uniform particle diameter distribution. There showed no conglutination among rhEGF susrained-release microspheres, with good dispersibility. Releasing drug lasted for 24 hours and accorded with Higuchi release kinetic model. (2) Different concentrations of rhEGF sustained-release microspheres could promote the proliferation of mouse fibroblast, especially the concentration of 10 μg/L (P <0.05, compared with the control). (3) From the 7th day after treatment, Group A had the fastest wound healing rate, with statistical difference compared with other three groups (P < 0.05). Group A had higher HYP contents and positive area ratio of integrin β1 and keratin-19 than Group B. Conclusions rhEGF sustained-release microspheres prepared by the modified double emulsion method have uniform particle size and can last release for 24 hours. Compared with rhEGF stock solution, rhEGF sustained-release microspheres have faster and better ulcer healing and higher healing quality in diabetic rats.

The expressions of hypoxia-inducible factor-1α and cell adhesion molecules in pancreatic adenocarcinoma and their clinical significance / 肿瘤

Objective: To investigate the relationship between the expressions of hypoxia inducible factor-1α (HIF-1α) and cellular adhesion molecules in human pancreatic adenocarcinoma, and discuss the mechanism for the promoting effects of HIF-1α on cancer invasiveness and metastasis and the clinical significance. Methods: Expressions of HIF-1α, E-cadherin and integrin β1 were detected by immunohistochemistry in 34 cases of radically resected pancreatic cancer tissues and 10 normal pancreatic tissues. The association of the expression of HIF-1α with the expression of E-cadherin and integrin β1 and the relationship between expression of HIF-1α and clinical features were analyzed. Results: There were overexpressions of HIF-1α, abnormal expression of E-cadherin, and high expression of integrin in pancreatic cancer tissues. Expression of HIF-1α negatively correlated with E-cadherin but had no significant relationship with expression of integrin. Expression of HIF-1α correlated with pathological staging and lymph node metastasis. Abnormal expression of E-cadherin was associated with tumor differentiation, pathological staging, and lymph node metastasis. Expression of integrin had no correlation with clinical features. Conclusion: HIF-1α promotes cancer invasiveness and metastasis by regulating abnormal expression of E-cadherin. Immunhistochemistry results indicated that detection of HIF-1α and E-cadherin may be valuable for judging the potential malignancy of pancreatic cancer.

Expression of Cell Surface Marker on Human Bone Marrow Derived Stromal Cells during Chondrogenic Differentiation / 대한류마티스학회지

OBJECTIVE: Multipotent bone marrow stromal cells have the ability to differentiate toward a variety of connective tissue lineages including cartilage. The future use of adult mesenchymal stem cells (MSCs) for human therapies depends on the establishment of preclinical studies. Therefore, in this preclinical study we demonstrated the expression of MSC surface markers CD29, CD105, and CD44 on human bone marrow derived stromal cells during chondrogenic differentiation. METHODS: Adult human bone marrow was collected from the iliac crest of 7 donors following informed consent. Mononuclear cells were isolated, incubated in monolayers, and embedded in alginate beads for three-dimensional cultures. Cellualr viability was assessed by MTT assay. Flow cytometry of alginate bead cultures was performed on days 0, 7, 14, 21, and 28 using monoclonal antibody against surface molecules, CD105, CD29, CD44, CD34 and CD45. Total contents of collagen and glycosaminoglycan (GAG) of the alginate beads was measured. SPSS 11.0 was used for data analysis. RESULTS: After 7 days of culture, 89% of the cells expressed the human integrin beta 1 antibody, CD29. The CD29-positive cells remained elevated at 83% on days 28. However, while only 18% expressed the type II TGF-beta receptor endoglin, CD105 on day 7, the CD105-positive cells increased abruptly 65% on day 14 remaining elevated up to day 28. The expression of CD44 was maximal in the first passage cell (63%). High concentration of TGF-beta 3 (10 ng/mL) was more favorable for sustaining cell viability than a low concentration (0.5 ng/mL)(n=4, p= 0.002, day 21). The total contents of collagen and GAG in the MSC-alginate beads increased during the three-dimensional culture (n=4, p=0.02, p=0.006) suggesting its differentiation into a chondrogenic lineage. CONCLUSION: CD29 was expressed earlier than CD105 during chondrogenic differentiation of human bone marrow MSC. CD44 expression was highest in the first passage cells and gradually decreased afterwards.

Studies on the expression of fibronectin and integrins in epiretinal membranes / 中华眼底病杂志

Objective To study the expression of the fibronectin (FN) and ? 1 integrin (? 1) in epiretinal membranes(ERM) of eyes with proliferative vitreoretinopathy(PVR). Methods Twenty epiretinal membranes were obtained from eyes undergone vitrectomy for retinal detachment complicated with PVR and observed by immunohistochemical methods. Results Overexpression of FN and ? 1 were observed in 18 and 16 membranes respectively. Conclusion The synergism of FN and ? 1 in their action mignt be one of the important roles in the development of PVR.

Relationship between expression of integrin ?1,?1 and apoptosis in placenta of pregnancy induced hypertension syndrome / 中华围产医学杂志

Objective To investigate the expression of integrin ?1 and ?1 and to study their role on cell apoptosis of placenta with pregnancy induced hypertension syndrome. Methods Immuno histochemistry was used for 40 placental samples with PIH and 43 normal placental samples to detect the expression of integrin ?1、?1; TDT mediated dUTP nick end labeling(Tune1) was used for 12 placental samples with PIH and 24 normal placental samples to examine apoptosis index in cytotrophoblast, syncytiotrophoblast and decidual cell. Results The apoptosis index in placental cytotrophoblast, syncytiotrophoblast and decidual cells in PIH group were (11.04 ? 3.46)%, (12.2 ? 3.67)%, (13.03 ? 4.38)% respectively and was significantly higher than of control group [(3.91 ? 1.65)%,(5.39 ? 1.76)%, (4.08 ? 1.97)%] ( P