Objective To observe the eotaxin expression of rat airway smooth muscle cells ( ASMCs) induced by serum from asthmatic rats, and explore the possible mechanism. Methods ASMCs isolated fromrat tracheas were cultured in vivo. Then they were treated with serum from asthmatic rats, or treated with serum and dexamethasone simultaneously. The level of eotaxin protein in supernatant and eotaxin mRNA in ASMCs were measured by ELISA and reverse transcription-polymerase chain reaction. The expression of cAMP in ASMCs was examined by radioimmunoassay. Results After the treatment with sensitized serum, the eotaxin level in supernatant and mRNA expression in ASMCs were significantly higher [ ( 107. 09 ±7. 12) ng/L vs. ( 0. 63 ±0. 56) ng/L, P lt; 0. 05; 1. 39 ±0. 04 vs. 0. 05 ±0. 01, P lt;0. 05] , and the level of cAMP in ASMCs was significantly lower compared with the control group [ ( 17. 58 ±3. 62) ng/L vs. ( 32. 39 ±3. 36) ng/L, P lt; 0. 05] . After intervened by the sensitized serum and dexamethasone simultaneously, the protein and mRNA expressions of eotaxin were lower compared with those intervened by sensitized serumalone [ ( 64. 18 ±4. 04) ng/L and 0. 77 ±0. 19] . The level of eotaxin in supernatant was negatively correlated with cAMP level in ASMCs ( r = - 0. 788, P lt; 0. 01) . Conclusions There is anautocrine function in ASMCs as inflammatory cells after stimulation with sensitized serum. Eotaxin may play an important roll in the pathogenesis of asthma via a cAMP-dependent pathway.
Objective To explore the feasibility of allogeneic marrow stromal stem cells(MSCs) as seed cells to construct tissue engineered bone bydetecting the expressions of interleukin 2(IL-2) and IL-2 receptor in rhesus monkeys after implanting these tissue engineered bones.Methods Engineered bones were constructed with osteoblasts which derived from allogeneic MSCs and bio-derived materials in vitro, and then were implanted to bridge 2.5 cm segmental bone defects of left radius in 15 rhesus monkeys as experimental group, bioderived materials only were implanted to bridge same size defects of right radius as control group. Every 3 monkeys were sacrificed in the 1st, the 2nd, the 3rd, the 6th andthe 12th weeks postoperatively and the expressions of IL-2 and IL-2 receptor in blood and graft samples were detected quantitatively by enzymelinked immuneosorbent assay (ELISA).Results There was no significant difference in the contents of IL-2 and its receptor between 2 groups（P＞0.05）. The contents ofIL-2 and its receptor increased from the 2nd week and maintained high level from the 2nd to the 6th week, but decreased after 6 weeks.ConclusionTissue engineered bones constructed with allogeneic MSCs and bio-derived materials show low immunogenicity. Allogeneic MSCs may be used as seed cells to construct tissue engineered bone.
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 explore the effect of the platelet-rich plasma (PRP) on proliferation and osteogenic differentiation of the bone marrow mesenchymal stem cells (MSCs) in China goat in vitro. Methods MSCs from the bone marrow of China goat were cultured. The third passage of MSCs were treated with PRP in the PRP group (the experimental group), but the cells were cultured with only the fetal calf serum (FCS) in the FCS group (the control group). The morphology and proliferation of the cells were observed by an inverted phase contrast microscope. The effect of PRP on proliferation of MSCs was examined by the MTT assay at 2,4,6 and 8 days. Furthermore, MSCs were cultured withdexamethasone（DEX）or PRP; alkaline phosphatase (ALP) and the calcium stainingwere used to evaluate the effect of DEX or PRP on osteogenic differatiation of MSCs at 18 days. The results from the PRP group were compared with those from the FCS group. Results The time for the MSCs confluence in the PRP group was earlier than that in the FCS group when observed under the inverted phase contrast microscope. The MTT assay showed that at 2, 4, 6 and 8 days the mean absorbance values were 0.252±0.026, 0.747±0.042, 1.173±0.067, and 1.242±0.056 in the PRP group, but 0.137±0.019, 0.436±0.052, 0.939±0.036, and 1.105±0.070 in the FCS group. The mean absorbance value was significantly higher in the PRP group than in the FCS group at each observation time (P＜0.01). Compared with the FCS group, the positive-ALP cells and the calcium deposition were decreased in the PRP group; however, DEX could increase boththe number of the positiveALP cells and the calcium deposition. Conclusion The PRP can promote proliferation of the MSCs of China goats in vitro but inhibit osteogenic differentiation.
Schwanns cells were obtained from the distal end of the sciatic nerve following Wallerian degeneration of SD rats. These cells were cultured with the anteriorhorn neuron of spinal cord of 14dayold SD rat fetus. The two kinds of cells were separated by a slice. Through the microscope, the dendrites and the morphology changes at the 24th, 48th, 72th, and 96 th hour after culture were observed. It was demonstrated that the Schwanns cells played the role of maintaining the survival of neuron and promoting the growth of dendrites. It was said that the Schwanns cells could secrete neurotrophic factor which made the body enlarged and caused the dendrites enlonged to several times of the body.
Schwanns cell (SC) was isolated from sciatic nerve of adult rat with Wallerine degeneration. After culture, SC-serum free culture media (SCSFCM) was obtained. By ultrafiltration with PM-10 Amicon Membrane, electrophoresis with DiscPAGE,and electrical wash-out with Biotrap apparatus, D-band protein was isolated from the SC-SFCM. The D-band protein in the concentration of 25ng/ml could affect the survival of the spinal anterior horn neuron in vitro, prominently and itsactivity was not changed after being frozen. The molecular weight of the protein ranged from 43 to 67 Kd. The D-band protein might be a neurotrophic substancedifferent from the known SCderived neurotrophic factors (NTF). Its concentration with biological activity was high enough to be detected. The advantages of MTT in assessment of NTF activity were also discussed.
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.
Objective To investigate the effect of surface propertyof different polyether-ester block copolymers［poly(ethylene glycol-terephthalate)/poly(butylene terephthalate), PEGT/PBT］ on the growth of smooth muscle cells (SMCs) and endothelial cells(ECs). Methods Three kinds of copolymers were synthesized, which were 1000-T20 (group A), 1000PEGT70/PBT30 (group B) and 600PEGT70/PBT30 (group C). The water-uptake and contact angle of three polyether-ester membranes were determined. The canine aorta smooth muscle cells and external jugular vein endothelial cells were primarily harvested, subcultured, and then identified. The proliferation of SMCs and ECs on the different polyether-ester membranes were investigated. Results The water-uptake of three copolymers arranged as the sequence of group C＜group A＜group B, and contact angle as the sequence of group C＞group A＞group B, indicating group B being more hydrophilic. However, smooth musclecells andendothelial cells grew poorly on the membrane of group B after low density seeding, but proliferated well on the membranes of group A and group C. Conclusion In contrast with more hydrophilic 1000PEGT70/PBT30, moderately hydrophilic 1000-T20 and 600PEGT70/PBT30 has better compatibility with vascular cells. The above results indicate that the vascular cells can grow well on moderately hydrophilic PEGT/PBT and that PEGT/PBT can be used in vascular tissue engineering.
Objective To investigate the effect of the drug-resistance characteristic of neoplasm cell on the expression of Fas during the chemical medi-cure.Methods The adriamycin-resistance hepatic carcinoma cells (HepG2 cell lines) were estabilished by cell biology. Changes of expression of the HepG2 cell lines was determined by immunohistochemistry. Results When the HepG2 cell lines were not induced by adriamycin, the expression of Fas of them was weak and Fas mainly existed in cell membrane. When induced by adriamycin, the expression was enhanced and Fas mainly existed in cytochylema. Simultaneously, the death rate of the cell lines changed. The death rate of the drug-resistance cell lines in 0.1 μg/ml ADM was almost as same as that of non-drug-resistance cell lines without ADM (Ｐ＞0.05) and was significantly different from that of non-drug-resistance cell lines in 0.1 μg/ml ADM (Ｐ＜0.05). Conclusion Changes of the expression of Fas may be one of the drug-resistance mechanisms of carcinoma cell.
OBJECTIVE: To observe the effects of hyaluronic acid (HA) and basic fibroblast growth factor (bFGF) on the proliferation of the cells from medial collateral ligament (MCL) and anterior cruciate ligament (ACL) cells. METHODS: The MCL cells and ACL cells of mature New Zealand white rabbit were cultured, while HA, bFGF or HA and bFGF were added to the cell culture media, the cellular proliferation was assayed by MTT method. RESULTS: HA only had no effect on the preoliferation of ACL cells, but had a small stimulatory effect on the proliferation of MCL cells. The addition of 1 ng/ml bFGF enhanced the proliferation of both MCL and ACL cells significantly, and this enhancement was maximal in the concentration of 50 ng/ml. However, the enhancement of proliferation of MCL and ACL cells could be achieved when the combination of HA in concentration of 100 micrograms/ml and bFGF in concentration of 100 ng/ml. CONCLUSION: It is evident that bFGF can enhance the proliferation of the ligament cells. HA can maintain the normal growth of ACL cells with no effect on the proliferation of the cells, while HA has a small stimulatory effect on the proliferation of MCL cells. However, when bFGF is coordinated with HA, more improvement of cellular proliferation can be achieved. HA can be used as a potential carrier for bFGF to enhance the healing of ligamentous tissue injuries.