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 .
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.
Objective To review the mechanism of improved revascularization of free fat grafting with adipose-derived stem cells (ADSCs). Methods The literature related to the basic researches of ADSCs in free fat grafting and angiogenesis was reviewed. Results Angiogenesis is a sequence process in time and space which is regulated by various factors. ADSCs possess the capability of secreting many angiogenic growth factors and differentiating into various lineages.Conclusion ADSCs affect every process of angiogenesis with clear improved angiogenic effects, however, the mechanisms of angiogenic effects need the further researches.
【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 To observe the effect of gene expression of p53 and the polymorphism of p53 gene codon 72 on cl inical phenotype of keloids. Methods The tissue and blood samples were taken from 35 patients with keloids, 19 males and 16 females, and the course of disease was from 4 months to 8 years. Meanwhile, autologous peripheral blood was collected for genotype analysis. According to the observing scope, the tissue samples of the keloids were divided into 2 groups: the central group involving the central part of the keloids (the central area within two-thirds of the radius) and the peripheral group involving the peripheral part of the keloids (the peripheral area within one-third of the radius). According to the largest diameter of the keloids, the two groups were divided into 3 subgroups: the small size group with 5 patients (lt; 1 cm), the medium size group with 21 patients (1-3 cm) and the large size group with 9 patients (gt; 3 cm). DNA of the tissue and blood samples were extracted, and the PCR followed by DNA sequencing was used to detect the polymorphism of p53 gene codon 72. The expression change of P53 was detected by immunohistochemical staining. The fibroblast apoptosis in keloid tissues was detected by TUNEL method. Results The genetic genotype of p53 gene codon 72 in keloids included Arg/Arg in 7 cases, Pro/Arg in 21 cases, Pro/ Pro in 7 cases. The significant correlation was found between genotype and cl inical phenotype (P lt; 0.05). Immunohistochemical staining revealed that P53 was detectable in peripheral and central groups of small-medium size keloids and central groups keloids, and detectable in few cells in peripheral groups of large size keloids. The absorbency value was 3 439.359 8 ± 538.527 5 in Arg/Arg genotype, 3 273.186 2 ± 375.213 9 in Arg/Pro genotype, 1 691.372 9 ± 98.989 3 in Pro/Pro genotype. There weresignificant differences among the three genotypes (P lt; 0.05). The fibroblast apoptosis was detected by TUNEL, and the apoptotic cells were evenly distributed. The apoptosis index was 31.000 0 ± 3.266 0 in peripheral group of large size keloids, 42.300 0 ± 4.354 8 in peripheral group of medium size keloids, 44.600 0 ± 5.253 6 in peripheral group of small size keloids. There were significant differences among the three groups (P lt; 0.05). Conclusion There is close relationshi p between the cl inical phenotype of keloids and the expression of P53. The polymorphism variation of p53 gene codon 2 is beneficial for apoptosis of fibroblasts in keloids.
Objective To compare two kinds of myofascial flap encapsulating adi pose-derived stromal cells (ADSCs) in adi pogenic efficacy in vivo, and to provide experimental basis for the efficient transplantation of free adi pose tissue. Methods ADSCs were isolated from the subcutaneous adipose tissue in the neck of 10 New Zealand rabbits (aged 3-4 months old, male and female, weighing 2.0-2.5 kg), and primary culture and subculture of ADSCs were conducted. When the cells at passage 3 covered 70%-80% of the bottom of the culture flask, BrdU (10 μg/mL) was appl ied to label the cells for 48 hours before performing immunofluorescence staining. Oil red O staining observation was conducted to thosecells 2 weeks after being induced towards adi pocyte, al izarin red staining observation was performed 3 weeks after being induced towards osteoblast, and alcian blue staining was conducted 2 weeks after being induced towards chondrocyte. Besides, after being induced towards adipocyte for 2 weeks, 1 × 107 ADSCs/piece at passage 3 labeled by BrdU was seeded into Col I (10 mm × 10 mm × 5 mm/piece) to prepare cell carrier complex. The experiment was divided into two groups: group A in which vascular pedicled dextral latissimus dorsi fascial flap was adopted to encapsulate the complex; group B in which dextral gluteus maximus fascial flap with no specific vessel pedicle was appl ied to encapsulate the complex. Rabbits in each group went through autogenous ADSCs transplant and self control. The implants were dislodged 8 weeks after operation, HE staining and immunohistochemistry staining were performed to testify cambium, the wet weight and micro vessel count of the cambium in each group were tested, immunofluorescence staining was performed to determine the origin of cambium and microvascular endothel ium. Results The nucleus of ADSCs positive for BrdU label ing showed green fluorescence under fluorescence microscope, with the positive label ing ratio of ADSCs above 90%. For ADSCs at passage 3, the formation of red l ipid droplets within cells was observed 2 weeks after being induced towards adipocyte, red calcium nodules were evident 3 weeks after being induced towards osteoblast, and highly congregated cell mass positive for alcian blue staining appeared 2 weeks after being induced towards chondrocyte. Eight weeks after operation, neogenetic blood vessel grew into scaffolds and no obvious fibreencapsulation was observed in group A, while few blood vessel grew into scaffolds in group B. The wet weight of cambium in group A and B was (0.149 5 ± 0.017 3) g and (0.095 3 ± 0.012 7) g, respectively, indicating there was a significant difference between two groups (P lt; 0.01). HE staining showed the formation of neogenetic adipose tissue and the growth of micrangium in the implant, and the degradation and absorption of scaffold. The micro vessel count of group A and B was 31.2 ± 4.5 and 19.3 ± 2.6, respectively, indicating there was a significant difference between two groups (P lt; 0.01). Eight weeks after operation, the immunofluorescence staining of cambium showed that the cell nucleus of regenerated adi pocytes and partial capillary endothel ium in groups A and B presented green fluorescence. Conclusion ADSCs encapsulated by vascular pedicled latissimus dorsi fascial flap and collagen protein scaffold complex has a higher adi pogenic efficacy in vivo than the gluteus maximus fascial flap with no specific vessel pedicle.
Objective To explore the safety and complications of repeated sternotomy after mitral valve repair and prevention strategies. Methods We retrospectively analyzed the clinical data of 88 consecutive patients of non-rheumatic mitral valve disease who underwent repeated sternotomy for failure of first-time mitral valve repair in our hospital from January 2009 through June 2015. There were 53 males and 35 females with a mean age of 36.1±17.5 years in the patients who underwent repeated sternotomy. Meanwhile 88 patients who underwent the first-time sternotomy for mitral valve repair simultaneously were randomly recruited as a control group, and there were 57 males and 31 females with a mean age of 39.9±12.6 years. The clinical outcomes were analyzed retrospectively and compared between the two groups. Results No major injury was observed in the patients who underwent repeated sternotomy. Eight patients (9.1%) in the repeated sternotomy group required femoral artery cannulation. Cardiopulmonary bypass (CPB) time was longer in the femoral artery cannulation group than that in the aortic cannulation group (155.5±59.0 minvs. 119.5±39.9 min,P=0.023). While there was no statistical difference in aortic cross-clamp time (P=0.786). Eight patients (9.1%) in the repeated sternotomy group used extra-pericardium approach. There was no significant difference in CPB time (P=0.255) or aortic cross-clamp time (P=0.360) between the patients who used extra-pericardium approach and those used routine approach. There was no statistical difference in post-operative complications between the patients who used different sternotomy strategies. Although CPB time (123.0±3.0 minvs. 95.4±37.1 min,P=0.000) or aortic cross-clamp time (79.0±36.3 minvs. 67.5±29.1 min,P=0.026) was longer in the repeated sternotmy group, the major outcomes were similar between the repeated sternotmy group and the first-time sternotmy group (P＞0.05). Conclusion Repeated sternotomy after mitral valve repair is relatively safe. With appropriate strategies, repeated sternotomy is not associated with increased risk of operative morbidity.