Objective To investigate the possibility of creation of tissue engineered heart valve leaflets in vitro . Methods Aorta were obtained from 9 hybrid young pigs. The endothelial cell, fibroblast and smooth muscle cells were isolated and cultured to get enough cell. The expanded fibroblast, smooth muscle cell,and endothelial cells were seeded on the polymers sequentially. The cell polymer constructs were sent for scanning electron microscopy(SEM) examination after cultured for 7, 14, and 28 days. Histological examination were performed after the cell polymer constructs cultured for 28 days. Results SEM showed that the number of cells on the polymers increased as the culture time prolonged, with the formation of matrix. After 28 days, there were a great number of cells and large amount of matrix on the scaffolds. The confluent cell had covered a large area of the polymers. Hematoxylin and eosin(HE) stain showed large amount of cells attached to the polymers. Conclusion With the viability of the cultured cellular scaffolds,it is possible to create tissue engineered heart valve leaflets in vitro.
【摘要】 目的 观察不同种培养基中重组人色素上皮衍生因子(rPEDF)融合蛋白的表达。 方法 将前期研究已构建的pET28aPEDF原核表达重组体转化E.coli BL21大肠杆菌表达宿主菌,酶切鉴定阳性菌落后,分别在M9和LB培养基中用异丙基βD硫代半乳糖(IPTG,IsopropylbetaDthiogalactoside)诱导表达,SDSPAGE电泳检测表达的PEDF蛋白, 美国ImagePro Plus 分析系统进行蛋白定量分析。结果 LB和M9培养基中均获得相对分子质量约54×103的rPEDF融合蛋白。但LB培养基获得的是rPEDF融合蛋白的包涵体,目的蛋白占总蛋白含量为21046%,M9培养基获得的是可溶性的rPEDF的融合蛋白,目的蛋白占总蛋白含量的1231%。结论 不同种培养基中均有rPEDF 融合蛋白的表达。【Abstract】 Objective To observe the express of recombinant pigment epithelial derivative facto (rPEDF) in the different medium. Methods The pET28aPEDF was transformed into E.coli BL21. After the colonies were positive identification which were induced by IsopropylbetaDthiogalactoside in medium M9 and LB. The PEDF protein were detected by SDSPAGE and analyzed by American ImagePro Plus system. Results LB and M9 medium obtained the relative molecular mass about 54×103 rPEDF fusion protein. But LB medium obtained the inclusion bodys of rPEDF fusion protein，the purpose protein account for 21.046%；LB medium obtained the soluble rPEDF fusion protein，the purpose protein account for 12.31%. Conclusion The rPEDF protein was expressed in the different medium.
Abstract An experiment was carried out to investigate the possibility of the establishment of an osteoblasts bank which could supply osteoblasts in repairing bone defect. Osteoblasts were isolated from thetibial periosteum of eight New-Zealand rabbits and cultured in votro. A bone defect, 1.5cm in length was made in both radii of each of the 8 rabbits. The cultivated osteoblasts, gelfoam as a carrier were randomly implanted into the defects of the radii of rabbits. Accordingly, the contralateral radial defects wereimplanted with gelfoam absorbed with the Hanks solution as control. The healing of bone defects was evaluated by roentgenographic examination at 2, 4, 8 and 12 weeks after operation, respectively. It was shown that the implanted cells had osteogenetic capability and could be possible to promote healing of the bone defects. It was suggested that further study needed to be carried out in this field.
OBJECTIVE: To investigate the biological characteristics of human muscle satellite cell cultured in vitro. METHODS: Human muscle satellite cells were obtained from skeletal muscle biopsies of six patients during corrective orthopedic surgery, cultivated in growth medium for ten days, then in differentiation medium for additional five days. Human satellite cells were identified with monoclonal antibody against desmin. Cells were observed under phase contrast microscopy. RESULTS: Human muscle satellite cells proliferated in growth medium, and fused to form myotubes in differentiation medium. After 24 hours in differentiation medium, the confluent satellite cells began to fuse actively and achieved the top level at 72 hours. CONCLUSION: Human muscle satellite cell can proliferate and differentiate in appropriate culture condition. Immunocytochemical detection of desmin is the effective early method to determine satellite cell.
Objective To determine whether the transforminggrowth factor β1 (TGF-β1) is a key regulatory molecule required for an increase or a balance of extracellular matrix (ECM) and DNA synthesis in the goat passaged nucleus pulposus (NP) cells. Methods The NP cells isolated from the goat intervertebral discs were cultured in vitro for a serial of passages and transfected with the replicationincompetent adenoviral vectors carrying the human TGF-β1 (hTGF-β1) or lacZ genes. Then, they were cultured in monolayer or alginate bead 3dimensional (3-D) systems for 10 days.The changes in the production and the molecular components of ECM that occurredin the NP cells transfected with Ad/hTGF-β1 or the controls were evaluated by Westernblot and absorbance of glycosaminoglycan (GAG)-Alcian Blue complexes. Differences of DNA synthesis in the variant cells and culture systems were assessed by fluorometric analysis of the DNA content. ResultsA quantitation in the variant culture systems indicated that in monolayers the NP cells at Passage 3 transfected with Ad/hTGF-β1 had a much higher cell viability and more DNA synthesis(P＜0.05); however, in the alginate 3-D culture system, the NP cells transfected with Ad/hTGF-β1 did not have any significant difference from the controls(P＞0.05). The Western blotting analysis ofthe protein sample isolated from the variant cells for TGF-β1, type Ⅱ collagen, and Aggrecan expression indicated that in the monolayers and alginate 3-D culture systems the NP cells at Passage 3 transfected with Ad/hTGF-β1 revealed much higher protein levels than the controls(P＜0.05)； whereas the type Ⅰcollagen content was much lower than the controls (P＜0.05), but a significatly increased ratio of type Ⅱ/type Ⅰ collagen was found in both of the cell culture systems(P＜0.05). The GAG quantification also showed a positive result in both the cell culture systems and the NP cells at Passage 3 transfected with Ad/hTGF-β1 had a much higher GAG content than the controls(P＜0.05). Conclusion To a greaterextent, hTGF-β1 can play a key role in maintaining the phenotype of the NP cells and can still have an effect of the phenotypic modulation after a serial of the cell passages. The NP cells that are genetically manipulated to express hTGF-β1 have a promising effect on the restoration of the intervertebral disc defects. The NP cells transfected with Ad/hTGF-β1 cultured in the 3-D alginate bead systems can show a nearly native phenotype.
OBJECTIVE: To observe the effects of silks on attachment, shape and function of chondrocytes cultured in vitro. METHODS: The silks from silk worm cocoons were digested by trypsin and coated with polylactic acid to from three dimensional scaffolds for rabbit rib chondrocyte culture. The growth and shape of chondrocytes were observed with phase contrast microscopy, scanning electron microscopy. RESULTS: The chondrocytes were adhered to silks slowly after chondrocytes were seeded into silk scaffolds and cells fixed on silks well 1 or 2 days later. Cells began to proliferate after 3 days and multiplicative growth was observed on the 6th day. Microholes of silk scaffolds were filled with chondrocytes 2 weeks later. Scanning electron microscopy showed that there was a lot of extracellular matrix surrounding cells. CONCLUSION: Silks are ideal for attachment, growth and function maintenance of chondrocytes, and silks can be used as scaffolds for chondrocytes in three dimensional culture.
Objective To investigate the method of cultivation and the feature of differentiation of spinal cordderived stem cells in vitro.Methods The neural stemcells from spinal cord of 15 days fetal rats were harvested and cultivated in aserumfree limited medium. The stem cells were induced to differentiate and theresults were identified by cellular immunohistochemistry. Results Lots of stem cells were obtained from the spinal cord of fetal rats and the sphere of stemcells was formed about 10 days. Neural stem cells can give rise to mature neurons and astrocytes.Conclusion Epidermal growth factor/basic fibroblast growth factor serum-free limited medium can promote the proliferation activity ofthe stem cells. Spinal cord-derived stem cells can differentiate into glial cells and neurons.
Abstract A new kind of soft tissue expander with exteriorized injection valve was reported. Two types of expanders were designed: one with single cyst, single tube and single chamber, and the other with single cyst, single tube but double chambers, i.e, an additional drainage chamber. The drainage orifice could be controlled by a valve. An animal experiment had been conducted, and the outcome suggested that the new expander could provide rapid expansion of the soft tissue, sufficient drainage of the expanded area. The bacterial culture showed that among the 42 samples obtained from the expanded cyst, only 3 were positive. (2 of bacteria and 1 of fungus), suggesting comparatively low cyst infection.
Objective To observe the effects of culture medium of amniotic cells on NO and NOS in retinal tissues of rabbits in vitro in order to provide a protective method for antioxidation in retina transplantation. Methods Thirty adult healthy rabbits (30 right eyes) were divided into 3 groups. Group I: fresh retinal tissue; group II: routine culture medium; group III: culture medium of amniotic cells. The retinal tissues in group II and III were cultured in the corresponding culture medium for 1 week. The content of NO and NOS in retinal tissues in the 3 groups were determined. Results Compared with group I, the content of NO and NOS of group II increased obviously (t=3.821, 3.854; P＜0.001). There was no statistical difference of content of NO and NOS between group I and III (t=1.657, 1.745; P＞0.05). Conclusion Culture medium of amniotic cells may remove free radicals and enhance the ability of antioxidation. (Chin J Ocul Fundus Dis，2004，20：366-368)
OBJECTIVE: To investigate the characteristic and phenotype of ectomesenchymal stem cells of human fetal facial processes and the procedure of spontaneous differentiation to smooth muscle cells. METHODS: The primary ectomesenchymal cells of E 50 human fetal facial processes were isolated by 2.5 g/L trypsin and cultured with DMEM/F 12 with 10(-6) U/L leukemia inhibitor factor(LIF). The morphology and growth rate were observed by inverted microscop. After being withdrawn LIF, the characteristic of cells were identified by immunohistochemistry and RT-PCR. Ultrastructure was observed by transmission electron microscope. RESULTS: The cultured cells displayed monolayer growth and were fibroblast-like with 2-4 processes. The cells were stainely positived for anti-human natural killer cell marker-1, Vimentin, S-100, neuron specific enolase, myoglobin and VIII factor, but negatively for glial fibrillary acidic protein, neural fiblament, alpha-SMA and cytokeratin in immunohistochemistry. Two days after being withdrawn the LIF, cells expressed alpha-SMA in protein and mRNA levels. The cells were rich in muscular filament-like structure and dense bodies under transmission electron microscope. CONCLUSION: Cultured cells are undifferentiated ectomesenchymal stem cells. The cells have the potential for differentiating spontaneously to smooth muscle cell.