Objective To investigate the possibility of differentiation of theisolated and cultured adipose-derived adult stem cells into chondrocytes, which is induced by the recombinant human bone morphogenetic protein 2 (rhBMP-2). Methods The rabbit adipose tissue was minced and digested by collagenase Type Ⅰ. The adposederived adult stem cells were obtained and then they were cultured inthe micropellet condition respectively in the rhBMP-2 group, the rhTGF-β1 group, the combination group, and the control group for 14 days. The differentiation of the adiposederived stem cells into chondrocytes was identifiedby the histological methods including HE, Alcian blue, Von kossa, and immunohistochemical stainings. Results After the continuous induction by rhBMP-2 and continuous culture for 14 days, the HE staining revealed a formation of the cartilage lacuna; Alcian blue indicated that proteoglycan existed in the extracellular matrix; the immunohistochemical staining indicated that collagen Ⅱ was in the cellular matrix; and Von kossa indicated that the adipose-derived stem cells couldnot differentiate into the osteoblasts by an induction of rhBMP-2. Conclusion In the micropellet condition, the adipose-derived adult stemcells can differentiate into the chondrocytes, which is initially induced by rhBMP-2. This differentiation can provide a foundation for the repair of the cartilage injury.
Objective To construct inducible lentiviral vector containing human bone morphogenetic protein 2 (hBMP-2) gene and to study its expression in human umbil ical cord blood mesenchymal stem cells (HUMSCs). Methods hBMP-2 gene was ampl ified by PCR from a plasmid and was cloned into pDown by BP reaction. pLV/EXPN2-Neo-TRE-hBMP-2 and pLV/EXPN2-Puro-EF1A-reverse transactivator (rtTA) were obtained with GATEWAY technology, and then were sequenced and analyzed by PCR. The recombinant vectors were transfected into 293FT cells respectively through l ipofectamine, and the lentiviral viruses were harvested from 293FT cells, then the titer was determined. Viruses were used to infect HUMSCs in tandem. In order to research the influence of induction time and concentration, one group of HUMSCs was induced by different doxycl ine concentrations (0, 10, 100 ng/mL, and 1, 10, 100 μg/mL) in the same induction time (48 hours), and the other by the same concentration (10 μg/mL) in different time points (12, 24, 48, and 72 hours). The expression of target gene hBMP-2 was indentified by ELISA method. After 2-week osteogenic induction of transfected HUMSCs, the mineral ization nodes were detected with Al izarin bordeaux staining method. Results Therecombinant inducible lentiviral vectors (pLV/EXPN2-Neo-TRE-hBMP-2 and pLV/EXPN2-Puro-EF1A-rtTA) were successfully constructed. The lentiviruses were also obtained and mediated by 293FT cells, and the virus titers were 3.5 × 108 TU/mL and 9.5 × 107 TU/mL respectively. HUMSCs could expression hBMP-2 by induction of doxycycl ine. The expression of hBMP-2 reached the peak at 10 μg/mL doxycl ine at 48 hours of induction. After 2-week osteogenic induction, a lot of mineral ization nodes were observed. Conclusion The recombinant inducible lentiviral vectors containing hBMP-2 gene can be successfully constructed, which provide an effective and simple method for the further study of stem cells and animal experiment in vivo.
ObjectiveTo investigate the relationship between the nucleotide binding oligomerization domain like receptor protein 3 (NLRP3) inflammasome and inflammatory reaction of venous ulcer of lower extremity.MethodsTwenty-four patients with active venous ulcer of lower extremity (active ulcer group), 24 patients with non exudative venous ulcer of lower extremity as positive control (non-active ulcer group), and 24 patients with traumatic wound as negative control (traumatic-wound group) were enrolled. The clinical data of the three groups were compared, the tissue samples around the wound were harvested, and the expressions of NLRP3 protein were detected by immunohistochemistry among the three groups. Enzyme linked immunosorbent assay (ELISA) was used to detect the IL-1β and IL-18 protein levels, RT-PCR was used to detect the mRNA expressions of apoptosis associated speck like protein containing CARD (ASC), caspase-1, c-Jun N-terminal kinase (JNK), p38, nuclear factor (NF)-κB p65 and NF-κB inhibitor alpha (NF-κB IkBα), and Western blotting was performed to evaluate the level of NLRP3 inflammasome in wound tissues.ResultsThe inflammatory response in the non-active ulcer group and trauma-wound group were milder than that in the active ulcer group. The levels of IL-1β and IL-18 proteins in the active ulcer group were higher than those in the non-active ulcer group and the traumatic-wound group [IL-1β: (146.621±11.597) ng/L vs. (80.967±14.213) ng/L vs. (84.962±19.484) ng/L, F=136.200, P<0.001; IL-18: (119.814±12.788) ng/L vs. (72.899±17.220) ng/L vs. (48.131±10.407) ng/L, F=167.910, P<0.001]. The results of RT-PCR showed that the mRNA expressions of ASC [(0.030±0.012) ng/L vs. (0.021±0.005) ng/L vs. (0.016±0.004) ng/L, F=18.106, P<0.001], caspase-1 [(0.054±0.012) ng/L vs. (0.013±0.009) ng/L vs. (0.018±0.006) ng/L, F=130.372, P<0.001], NF-κB p65 [(0.093±0.015) ng/L vs. (0.038±0.013) ng/L vs. (0.043±0.014) ng/L, F=110.950, P<0.001], NF-κB IkB-α [(0.085±0.015) ng/L vs. (0.078±0.015) ng/L vs. (0.041±0.016) ng/L, F=53.070, P<0.001], and JNK [(0.075±0.018) ng/L vs. (0.042±0.013) ng/L vs. (0.039±0.014) ng/L, F=41.271, P<0.001] in the wound tissues of the active ulcer group were higher than those in the non-active ulcer group and the traumatic-wound group. And the mRNA expression of p38 in the wound tissues of the active ulcer group was lower than that in the non-active ulcer group [(0.050±0.008) ng/L vs. (0.064±0.014) ng/L, P<0.05]. The result of Western blotting showed that the relative expression level of NLRP3 protein in the wound tissues of the active ulcer group was higher than that in the trauma-wound group and non-active ulcer group (0.767±0.272 vs. 0.605±0.212 vs. 0.556±0.183, F=4.804, P=0.012).ConclusionNLRP3 inflammasome is closely related to the wound in venous ulcer of lower extremity and provides a new target to the therapy of venous ulcer of lower extremity.
Objective To determine whether fibroblasts can be used to promote endochondral bone formation in vivo by transfer of human bone morphogenetic protein-2(hBMP-2) into fibroblasts. Methods pcDNA3-hBMP-2 was constructed by use of gene clone and recombined technique.NIH3T3 fibroblasts were transfected with pcDNA3hBMP-2. The positive cell clones were selected with G418. In NIH3T3 fibroblaststransferred with pcDNA3-hBMP-2, the expression of hBMP-2 was determined by in situ hybridization and immunohistochemical analysis; alkaline phosphatase activity was measured. hBMP-2producing fibroblasts were implanted into nude mouse muscle to observe endochondral bone formation in vivo. Results pcDNA3-hBMP-2 was successfully constructed. In NIH3T3 fibroblasts transfected with -pcDNA3-hBMP-2,the BMP-2 expression was stable; alkaline phophatase activity was much higher than that in nontransfectedNIH3T3 cells. Endochondral bone formation invivo was observed at the site of implantation 4 weeks later.Conclusion Fibroblasts transfected by hBMP-2 gene can be used to promote endochondral bone formation in vivo.
Thirteen patients with intractable nonunions of fractures of long bones were sucessfully treated by a combination of internal fixation and implantation of bBMP. There was an average of 1.5 operative procedures per patient in an attempting to establish reunion prior to bBMP implantation. Union was obtained in 12 of the 13 patients exapt in one who gained success from establish the second attempt. The average time requited to union was 4.7 months. No complication was seen.
ObjectiveTo investigate the molecular mechanism by which metastasis-associated protein 3 (MTA3) participates in glioma resistance through reactive oxygen species. Methods Protein expression in glioma stem cells (GSCs) and non-GSCs was detected using Western blotting. GSCs included U87 and SHG44 cells, while non-GSCs included U87s and SU-2 cells. After overexpressing MTA3, U87 and SHG44 cells were divided into Lv-scr and Lv-MTA3 groups. The self-renewal capacity of glioma cells was assessed through a neurosphere formation assay. Cell survival fractions were examined following exposure to 0, 2, 4, 6, 8, and 10 Gy X-ray irradiation under normoxic or hypoxic conditions. Apoptosis and reactive oxygen species expression were analyzed using flow cytometry. Immunofluorescence staining was performed to detect the stem cell markers CD133 and nestin, as well as the differentiation markers glial fibrillary acidic protein (GFAP, for astrocytes) and neuronal class Ⅲ β-tubulin. Results In GSCs, MTA3 expression was lower in the U87s and SU-2 groups. After MTA3 overexpression, Lv-MTA3 expression was higher in U87s and SU-2 compared to the Lv-scr group. Under normoxic or hypoxic conditions, U87 and SU-2 showed greater radioresistance compared to glioma cell lines U87 and SHG44. Compared to non-GSCs, basal reactive oxygen species formation was reduced in GSCs, while reactive oxygen species generation was increased in non-GSCs. Following exposure to different doses of X-rays under normoxic or hypoxic conditions, GSCs with MTA3 overexpression exhibited greater radiosensitivity than those with stable integration. Additionally, MTA3 overexpression slightly increased the oxygen enhancement ratio (OER) in GSCs. MTA3 overexpression reduced the immunoreactivity of CD133 and nestin in both stem cell lines, and increased immunofluorescence staining of GFAP and neuronal class Ⅲ β-tubulin, with statistically significant differences (P<0.05). Conclusions MTA3 is downregulated in GSCs. Overexpression of MTA3 reduces the radioresistance and stemness of GSCs both in vitro and in vivo. MTA3 plays a crucial role in regulating the radiosensitivity and stemness of GSCs through reactive oxygen species.
Objective To clarify the relationship between inhibition of proliferation and cxpression of Ki-67 in cultured human retinal pigment epithelial(RPE) cells. Methods The cultured human RPE cells were treated with daunoblastina at a dose of 180 mu;g/L for 12h.Twenty-four hours later,DNA inhibiting rate was studied by using tritium-labelled thymidine deoxyribose(3H-TdR)incorporation assay.The expression of Ki-67 was evaluated by immunocytochemical staining technique and image analysis system.Flow cytometry was used to analyse cell cycle. Results DNA inhibiting rate was directly proportional to the dosage of daunoblastina.The proportion of the cells positive staining to Ki-67 in the control and the daunoblastina-treated group were 89.3% and 45.6%(Plt;0. 01),and the integral optical density values for expression of Ki-67 in the two groups were 68.1plusmn;6.2 and 27.3plusmn;5.5(Plt;0.01),respectively.The percen tage of cells in G2 phase of cell cycle increased from 8.9% to 29.5%. Conclusion G2 block was induced and poliferation was inhibited by daunoblastina in cultured human RPE cells.There is a relatively good correlation between Ki-67 immunostaining and inhibition of RPE cell proliferation. (Chin J Ocul Fundus Dis，2000，16：1-70)
Objective In order to study the mechanism of cholesterol gallstone formation through rabbit model which was induced by high cholesterol diet (HCD)Methods the activities of the high density lipoprotein receptor (HDLR) and low density lipoprotein receptor (LDLR) of hepatocytes were investigated. Results The results were as follows: The HDLR activity increased significantly after taking HCD for one week, at the same time, the LDLR activity only increased slightly. Thereafter, the activities of HDLR and LDLR all decreased markedly. As the time of animals taking HCD went on, serum total cholesterol, LDL cholesterol and hepatic cholesterol increased, but bile acids of biliary tract decreased gradually. Conclusion The results suggest that the changes of HDLR and LDLR activities of hepatocytes had no significant effect on bile cholesterol and the decreased HDLR and LDLR activities may cause the reduction some of substrate for bile acids synthesise and play an important role in the formation of gallstone.
Objective To observe the expression of matrix metalloproteinase-9 (MMP-9), its tissue inhibitor of matrix metalloproteinase (TIMP-1), inducible nitric oxide synthase (iNOS) and contents of nitric oxide (NO) in the ocular tissues of Sprague-Dawley (SD) rats with endotoxin induced uveitis（EIU）. Methods Ninety SD rats were randomly divided into experimental (81 rats) and control group (9 rats). The model of EIU was induced in rats in experimental group by injecting with lipoplysaccharide (LPS) 200 μl into the hind feet pads, while the rats in the control group were not injected. Nine rats were executed 0, 6, 12, 18, 24, 48, 72, 96 hours and 7 days, respectively, after injecting with LPS; the NO content and concentration of protein in the aqueous humor in blood plasma, aqueous humor, and uveal tissues were detected. The expressions of MMP-9, TIMP-1 and iNOS in the ocular tissues were detected by immunohistochemistry, and the average absorbance (A) value was evaluated by computer medical image analysis system. Results iNOS, MMP-9 and TIMP-1 expressed in the epithelial cells of iris and ciliary body and exudated inflammatory cells of rats. The concentration of protein in the aqueous humor, the contents of NO in blood plasma, aqueous humor, and uveal tissues, and A value of MMP-9 had obvious relativity with the inflammatory extent, while no positive correlation was found between the inflammatory extent and the A value of iNOS and TIMP-1. Expression of iNOS was found 6 hours after injection, reached the peak after 12 hours, and then dropped gradually. The expression of TIMP-1 could be seen 24 hours after injection, and reached its peak after 72 hours. Conclusion The content of NO and expressions of iNOS, MMP-9 and TIMP-1 changes from the beginning and during the development of EIU, which suggests that NO, iNOS, MMP-9 and TIMP-1 are involved in the pathologic process of EIU. (Chin J Ocul Fundus Dis, 2005, 21: 371-374)
Objective To explore the expression of CD105 protein in esophageal squamous cell carcinoma and it＇s relationship with P53 protein. Methods Using streptavidin biotinperoxidase （SP） method, the expression of CD105 protein and P53 protein in esophageal squamous cell carcinoma were examined in normal esophageal tissues （n=10） and esophageal squamous cell carcinoma tissues（n=86）. Results The expression positive rate of CD105 protein was 74. 4%（64/86） in esophageal squamous cell carcinoma , 0% in normal esophageal epithelium. Expression positive rate of CD105 protein was 66. 1%（37/56） in early stage （stage Ⅰ-Ⅱ ）, 90.0% （27/30） in later stages （stage Ⅲ-Ⅳ ）. The expression of CD105 protein were bly associated with P53 protein（P〈0. 05）. Conclusion CD105 protein may participate in the onset and progression of esophageal squamous cell carcinoma. CD105 protein could he a new diagnostic /therapeutic target in esophageal squamous cell carcinoma.