Objective To replace dysfunctional Fas gene and reconstruct the blocked Fas signal by using two kinds of prepared recombinantAdenovirus which have human Fas gene. Methods After the keloids derived from fibroblasts were infected by the Adenovicus, the expressions of Fas protein before the exposure and after the exposure was compared. Then the function of the newly produced Fas protein was detected. Results The highly improve expression of Fas protein in the infected keloid derived fibroblasts was detected. Obvious apoptosis was also detected in the infected keloid derived from fibroblasts under the condition of exposing to FasMcab. Conclusion ①The recombinant Adenovirus with Fas gene can transfect the Fas gene into keloidderived fibroblasts and highly improved the expression of Fas protein. The newly expressed Fas gene can reconstruct the blocked Fas signal. ②Ad-Fas(B) has better therapeutic effect in vitro gene therapy. ③ The correlation between keloid and Fas gene was further proved and it may pave the way for further gene therapy in keloid .
ObjectiveTo summarize recent progress in surgical management of progressive hemifacial atrophy (PHA), to analyse the key features of various methods of treatment, and to define subjects worthy of further researches. MethodsThe publications concerning the etiology and surgical management of PHA were reviewed, analyzed, and summarized. ResultsSurgical management serves as the primary treatment, including flap transposition, tissue flap reconstruction, free tissue grafting, prosthetic implants, and other surgical treatments. Each method has its own advantages and limitations. At present, comprehensive treatment are considered to be the most commonly method for PHA. ConclusionThe combined use of various surgical methods is the trend of surgical management of PHA; effective treatments specific to the etiology and minimally invasive surgical methods are still to be developed.
Objective To compare gene express difference ofkeloid and normal skin tissues by using the suppression subtractive hybridization (SSH) so asto find the differential express gene in keloid. Methods mRNA extracted fromkeloid and normal skin tissues was used as the template to synthesis cDNA of keoid and normal skin. The cDNA of keloid served as a tester, the cDNA of normal skin as a driver. cDNA was digested with RsaⅠ. Adaptor-ligated tester cDNA was prepared. Then first hybridization, second hybridization and PCR amplificationwere done. Differentially expressed cDNA was selectively amplified during thesereactions. After SSH, the PCR mixture was ligated with T-vector. The positive clones were selected and the insert gene fragments were analyzed. Southern hybridization identified the keloid differential express genes. The positive clones ofSouthern hybridization were selected, and these sequences were analyzed. The results were compared with that of GeneBank. Results Thirteen differential genes were found in keloid, of which 11 gene clones have been known their function, and 2 clones have not known their function. 〖WTHZ〗Conclusion Keloid differentially expressed gene was screened successfully by SSH.
【Abstract】 Objective To explore the optimal dosage, timing and cytotoxicity of bromodeoxyuridine (BrdU) labelling for rabbit adipose-derived stromal stem cells (ADSCs) in vitro so as to confirm its feasibil ity for stem cells labell ing and tracer means. Methods Six rabbits were used in this experiment, aged 8-12 weeks, weighing 1.5-2.0 kg and neglecting their gender. 1-2 mL fat was removed, the ADSCs were isolated and cultured using the adherence method in vitro . The 3rd passage of ADSCs was incubated with BrdU at 5, 10, 15 and 20 μg/mL (groups A, B, C and D）for 12, 24, 48 and 72 hours to identify the optimal BrdU concentration and incubating time for cell labell ing. Immunohistochemistry and trypanblau strain were performed respectively to calculate the labell ing index (positive rate) and the cells’ activity for different time after BrdU labell ing. The ADSCs without BrdU labell ing were used as control (Group E）. Results The main appearance of primary ADSCs was short fusiform shape, and of the 3rd passage ADSCs long fusiform shape. The 3rd passage of ADSCs could differentiate into osteoblastsand adipocytes under corresponding inductive medium. The ADSCs’ nucleus show green fluor under fluorescence microscope after labeled by the BrdU. The labell ing ratio increased in groups A, B, C and D after incubating 12 hours, the mean labell ing ratio were 30.6％ ±2.3％，32.4％ ±1.9％，45.8％ ±1.8％，50.8％ ±3.1％ , respectively, and the labell ing ratio of Group E was 0. There were significant differences between groups C, D and Group A (P lt; 0.01). The labell ing ratio of groups A, B, C and D were 45.9％ ±2.0％，87.9％ ±3.3％，90.6％ ±2.9％，91.7％ ±3.2％，respectively after 24 hours and the labell ing ratio of Group E was 0. There were significant differences between groups B, C, D and Group A (P lt; 0.01). The results of all groups after incubating48 hours and 72 h ours were similar to that after incubating 24 hours. The cell counting of groups A, B, C and D were better than that of Group E, but showing no siginificant differences（P gt; 0.05）. Conclusion The most appropriate time for BrdU labell ing ADSCs is 48 hours, the most appropriate concentration is 10 μg/mL. The labell ing rate is high and cytotoxicity is l ittle.
Objective Seed cells are the hotspot of tissue engineering research. To study the seed cells with high potential of adipogenic differentiation for applying the adipose tissue engineering and increasing the constructing efficiency of adipose tissue engineering. Methods Mature adipocytes (MA) and adipose-derived stromal cells (ADSCs) were harvestedfrom human fat aspirates via l iposuction by collagenase digestion. MA were cultured and induced to dedifferentiated adipocytes (DA) by ceil ing adherent culture method. DA and ADSCs were induced to adipogenic differentiation. The adipogenic abil ities of DA and ADSCs were compared by inverted phase contrast microscope observation, absorption spectrometry assay of oil red O staining, and cell counting of oil red O staining. Results MA could dedifferentiate into fibroblast-shaped DA. After adi pogenic differentiation, the inverted phase contrast microscope observation showed that there were much more l i pid droplet in DA than in ADSCs. Absorption spectrometry assay of oil red O staining showed there were significant l ipid droplet aggregation in DA 4 days of adipogenic induction. However, the same phenomenon could be observed in ADSCs at 10 days after differentiation. After 12 days, the absorption value of DA was higher than that of ADSCs, showing significant difference (P lt; 0.05). The cell counting of oil red O staining demonstrated that the adipogenic rates of DA and ADSCs were 65% ± 6% and 35% ± 5%, respectively, showing significant difference (P lt; 0.05). Conclusion The potential of adipogenic differentiation of DA is ber than that of ADSCs. DA is a promising seed cell of adipose tissue engineering.
Objective To investigate the mechanism of vascular stromal fraction (SVF) at the early stage after aspirated fat transplantation. Methods Fat was harvested from 5 cases of women undergoing abdominal liposuction operation, and SVF was isolated. Aspirated fat with (group B) or without (group A) SVF was injected subcutaneously into the back of nude mice, and the grafts were harvested at 1, 3, 5, and 7 days. Graft wet weight was measured; and immunohistochemical method (CD31) was performed and the secretion of vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF) were qnantified by Western blot assay. Results The wet weight of transplanted adipose tissue showed an increasing tendency in groups A and B with time, and no significant difference was found between groups A and B (P gt; 0.05). At 1 and 3 days after transplantation, no CD31 positive cells was seen in 2 groups; the CD31 positive cells of group B were significantly more than those of group A at 5 and 7 days (P lt; 0.05), and the CD31 positive cells at 7 days were significantly more than those at 5 days in 2 groups (P lt; 0.05). Western blot test showed that VEGF expression reached peak at 3 days , then decreased gradually; the expression of VEGF protein in group B was significantly higher than that in group A at 1, 3, and 5 days (P lt; 0.05). The expression of HGF protein in groups A and B remained at a high level within 5 days, but it tended to decrease at 7 days, which was significantly higher in group B than that in group A (P lt; 0.05). Conclusion SVF can enhance angiogenesis by secretion of growth factors at the early stage after aspirated fat transplantation.