The purpose of this study was to find some solutions to the problem of tendon cell proliferation control. Under the condition of in vitro culture, several materials including IGF-1 receptor antibody and mRNA antisense oligonucleotide were added to the culture medium to block the IGF-1-Receptor system. The effect of the material on the tendon cell proliferation was judged by cell count after incubation of 48 hours. The results showed that both IGF-1 Receptor antibody (IGF-1R alpha) and IGF-1 Receptor mRNA antisense oligonucleotide had negative effect on tendon cell proliferation (P lt; 0.01 and P lt; 0.05). These findings lead us to think that the above two materials could be used in the experiment of tendon adhesion preventing and living ready-made tendon producing.
Objective To study the differenation of adult marrow mesenchymal stem cells(MSCs) into vascular endothelial cells in vitro and to explore inducing conditions. Methods MSCs were isolated from adult marrow mononuclear cells by attaching growth. MSCs were divided into 4 groups to induce: the cells seeded at a density of 5×103/cm2 in 2% and 15% FCS LDMEM respectively (group1 and group 2)， at a density of 5×104/cm2 in 2% and 15% FCS LDMEM respectively (group 3 and group 4); vascular endothelial growth factor(VEGF) supplemented with Bovine pituitary extract was used to induce the cell differentiation. The differentiated cells were identified by measuring surfacemarks (CD34, VEGFR2, CD31 and vWF ) on the 14th day and 21st day and performed angiogenesis in vitroon the 21st day.The cell proliferation index（PI）of different inducing conditions were measured. Results After induced in VEGF supplemented with Bovine pituitary extract, the cells of group 3 expressed the surface marks CD34, VEGFR-2, CD31 and vWF on the 14th day, the positive rates were 8.5%, 12.0%, 40.0% and 30.0% respectively, and on the 21st day the positive ratesof CD34 and VEGFR2 increased to 15.5％ and 20.0％, while the other groups did not express these marks; the induced cells of group 3 showed low proliferating state(PI was 10.4%) and formed capillary-like structure in semisolid medium. Conclusion Adult MSCs can differentiate into vascular endothelial cellsafter induced by VEGF and Bovine pituitary extract at high cell densities and low proliferatingconditions，suggesting that adult MSCs will be ideal seed cells forthe therapeutic neovascularization and tissue engineering.
Objective To investigate the influence of the exogenouscollagen on the function of cells in construction of artificial biotendon.Methods Three materials including human hair, carbon fiber(CF) and polyglycolic acid (PGA) were combined with exogenous collagen and co-cultured with standard transferred human embryonic tenocytes at a concentration of 3×106/mm3 in vitro. The cell number and morphology were observed under inverted microscope and scanning electron microscope after 2 hours, 3 days and 5 days.Results In the artificial biotendon combined with collagen, the cells concentrated around the materials and the cells adhering to the materials turned into round after 2 hours. After 3 days, the adhering cells increased. After 5 days, the shape of the cells changed from round to spindle.ConclusionExogenous collagen will facilitate the cells to adhere onto materials and proliferate.
At present, acellular matrix is an effective replacement material for the treatment of skin damage, but there are few systematic evaluation studies on its performance. The experimental group of this study used two decellularization methods to prepare the matrix: one was the acellular matrix which sterilized with peracetic acid first (0.2% PAA/4% ethanol solution) and then treated with hypertonic saline (group A), the other was 0.05% trypsin/EDTA decellularization after γ irradiation (group B); and the control group was soaked in PBS (Group C). Then physical properties and chemical composition of the three groups were detected. Hematoxylin eosin (HE) staining showed that the acellular effect of group B was good. The porosity of group A and B were both above 84.9%. In group A, the compressive modulus of elasticity was (9.94 ± 3.81) MPa, and the compressive modulus of elasticity was (12.59 ± 5.50) MPa in group B. There was no significant difference between group A or B and group C. The total content of collagen in acellular matrix of group A and B was significantly lower than that of group C (1. 662 ± 0.229) mg/g, but there was no significant difference in the ratio of collagen Ⅰ/Ⅲ between group B and group C. Scanning electron microscopy (SEM) and atomic force microscopy (AFM) showed that there was no significant difference in microstructure. Qualitative detection of fibronectin and elastin in each group was basically consistent with that in group C. Therefore, acellular matrix of group B had better performance as scaffold material. The experimental results show that the acellular matrix prepared by γ-ray sterilization and decellularization of 0.05% Trypsin enzyme/EDTA could be used for the construction of tissue-engineered skin. It could also provide reference for the preparation and mounting of heterogeneous dermal acellular matrix. It was also could be used for electrostatic spinning or three-dimensional printed tissue engineered skin scaffold which could provide physical and chemical parameters for it.
【Abstract】 Objective To reconstruct three different kinds of tympanic membrane in vitro by tissue engineeringtechnique, and to examine their histological structures and mechanical properties. Methods The skin and dura of pig(weight 30 kg) were processed with high satuated sal ine and enzymes to make extracellular matrix. Meanwhile, fibroblasts(1×106 /mL,0.2 mL) were seeded on the surface of these two scaffolds and collagen. The composite tissues were cultured in vitro for 1 week and examined in histological structure and mechanical properties. Results Fibroblasts cultured were spindle–shaped and could grow and attach to these scaffolds with a arrangement of sarciniform and parallel. The reconstructed tissue of ECM and collagen appeared to integrate well and had better bio-compatibil ity. The mean thickness of the collagen, the skin and the dura (all covered with fibroblasts) were 9.4, 10.0 and 10.4 μm respectively. The tension of the collagen was (1.417±0.030) N/mm2, of the acellular dermal matrix was (24.500±2.040) N/mm2(being close to the tension of normal tympanic membrane, 26.700 N/ mm2),of the acellular dura was (53.300±2.600) N/mm2. Conclusion The results suggest that the tension and the thinkness of acellular dermal matrix is similar to the normal tympanic membrane of guinea pig, it is an ideal material for tympanoplasty.
目的 研究胃癌细胞SGC-7901培养上清液及转化生长因子-β1（TGF-β1）是否可促进人类腹膜间皮细胞表达βig-h3蛋白。方法 培养胃癌细胞SGC-7901，取第3天培养液上清与DMEM培养液的混合液 （1∶4）以及0、1.0、10.0和50.0ng/ml的 TGF-β1分别刺激人类腹膜间皮细胞HMrSV50、3、6、12及24h，ELISA方法检测上清液中βig-h3蛋白浓度，Western blot法检测细胞内βig-h3蛋白浓度。结果 对照组有基础量的βig-h3蛋白表达； 胃癌细胞SGC-7901培养上清液及TGF-β1均可明显增加HMrSV5细胞上清液及细胞内的βig-h3蛋白浓度（P＜0.05），且TGF-β1的刺激作用呈时间及浓度依赖性。结论 胃癌细胞SGC-7901培养上清液及TGF-β1可明显刺激HMrSV5细胞表达和分泌βig-h3蛋白。
OBJECTIVE: To study the technique and method of urethral epithelium culture in vitro, so as to lay the groundwork for reconstructing a tissue engineering urethra and to provide an experimental model of urethral mucosa in physiological, pathological, toxicological and microbiological study. METHODS: The urethral mucosa from a young male New Zealand hare that had just been out of milk, was digested into single cell liquid with Dispase II and mixed enzyme, and the fibroblast were removed. After being seeded, the cells were cultured by using L929 cells as trophoderm. The medium was changed regularly and the cells were subcultured when they grew to mix together 80% to 90%. The cultured cells were analyzed with histochemistry, immunohistochemistry dyeing and flow cytometry examination. We observed the ultrastructure of cells with scanning electron microscope and transmission electron microscope. RESULTS: The primary cultured cells fused when they had been cultured for about ten days. They were the same in size like road rocks. The cultured cells were all epithelial cells without fibroblasts and were diploid cells. The cells could be subcultured 11-13 generations, and could survive 50-60 days. CONCLUSION: The urethral epithelium of young New Zealand hare can be cultured in vitro and maintain the ability to proliferate within a certain time. The study result not only sets a role in reconstructing a tissue engineering urethral mucosa, but also provides an experimental model for the research of urethral mucosa in vitro.
【Abstract】 Objective To observe the effect of cytokines and combinations in the inducement of human umbil icalcord blood-derived CD34+ cells into hepatocyte-l ike cells. Methods The mononuclear cells (MNCs) were derived by density gradient centrifugation and the CD34+ cells were sperated from MNCs. The human umbil ical cord blood-derived CD34+ cells were cultivated through 49 different combinations of cytokines including leukemia inhibitor factor (LIF), oncostatin M, bFGF, aFGF, hepatocyte growth factor, EGF and stem cell factor for 28 days, and the concentrations of the cytokines were 10, 10, 10, 10, 20, 20 and 50 ng/mL, respectively. The mRNAs of cytokeratin 19 (CK-19), CK-18, glutamine synthetase (GS), human albumin (ALB) and α-fetoprotein (AFP) were detected every seven days. The ALB secretion abil ity, detoxification abil ity and hepatin synthesis abil ity of the induced cells were detected by immunofluorescence assay, indocyanine green (ICG) and periodic acid-schiff assay, respectively. The fresh umbil ical cord blood-derived CD34+ cells were detected at the same time as a control. Results The mRNAs of CK-19, CK-18 and GS could be transcribed in all the induced cells, but the transcription of the mRNAs of ALB and AFP which was the special mark of mature hepatocyte and l iver stem cell, respectively, was not found. All the mRNAs could not be found in freshly isolated umbil ical cord blood-derived CD34+ cells. All the cells induced in vitro could not release ALB, and not help the detoxification of ICG which was the fundamental function of mature l iver cells. These results were the same in the control group. The hepatin synthesis abil ity of all the induced cells increased by comparison to the fresh ones. Conclusion Though some mRNAs of proteins which are transcribed in hepatocytes can be found in the induced cells, umbil ical cord blood-derived CD34+ cells could not be transdifferentiated into hepatocyte-l ike cells through cytokines in vitro.
Objective To compare the myogenic differentiation abil ity in vitro of rabbit adipose-derived stem cells (ADCSs) from different sites so as to provide ideal seed cells for repair and reconstruction of urinary tract. Methods Adipose tissues were obtained from the nape of the neck, post peritoneum, and vicinity of epididymis of a 4-month-old male New Zealand rabbit and ADSCs were harvested through collagenase digestion. ADSCs were purified by differential attachment method. The protein marker CD44 of rabbit ADSCs was used to identify the stem cells by immunocytochemistry, then the5th generation of ADSCs were induced to differentiate into adipogenic, osteogenic, and myogenic cells. Multi- differentiation was confirmed by Oil red O staining, von Kossa staining, and RT-PCR. Myogenic differentiation abil ities of ADSCs from 3 different sites were compared between the control group (L-DMEM medium containing 10%FBS) and the experimental group (myogenic medium) by RT-PCR method. Results ADSCs could be easily isolated from adipose tissues of the nape of the neck, post peritoneum, and vicinity of epididymis. ADSCs displayed a typical cobblestone morphology. Brown particles could be seen in ADSCs by CD44 immunocytochemistry staining. Oil red O staining showed red fat drops in ADSCs after 14 days of adipogenic culture. Black matrix could be seen in ADSCs by von Kossa staining after 28 days of osteogenic culture. RT-PCR detection showed moderate α-actin expression in the control group and b α-actin expression in the experimental group after 42 days of myogenic culture. The growth rate of α-actin from the adipose tissue of post peritoneum (28.622% ± 4.879%) was significantly lower (P lt; 0.05) than those from the adipose tissues of the nape of the neck (35.471% ± 3.434%) and vicinity of epididymis (38.446% ± 4.852%). Conclusion The ADSCs from different sites show different myogenic differentiation abil ities in vitro. ADSCs from the adipose tissues of the nape of the neck and vicinity of epididymis can be used as ideal seed cells for tissue engineering of lower urinary tract.
In order to study the biological characteristics of tenocyte and fibroblast, the former was obtained from rabbit’s tendon, and the latter from rabbits’s skin. Both cells were cultured according Heuderson’s method. The cell morphology, strapping and expanding time, and the type of collagen fiber synthesized in culture were observed. The results showed that the strapping and expanding time of fibroblast was faster than that of tenocyte. The cellular arrangement of fibroblast was irregular, but that in tenocyte was regular. Type I and III collagen of fibers were found in cultured fibroblost while only type I collagen fibers were found in culture of tenocyte. The tenocyte and fibroblast could be identified individually by strapping and expanding time, arrangement of cells and type of collagen fiber synthesized.