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IMMUNOMODULATORY EFFECTS OF HUMAN PLACENTAL-DERIVED MESENCHYMAL STEM CELLS ON IMMUNE REJECTION IN MOUSE ALLOGENEIC SKIN TRANSPLANTATION

Objective To investigate the effect of human placental-derived mesenchymal stem cells (PMSCs) on immunological rejection in mouse allogeneic skin transplantation. Methods The placenta fetal tissues from voluntary donors were used to isolate and culture the PMSCs, and the 3rd passage PMSCs were used in the experiment. Thirty Vr ∶ CD1 (ICR) mice at age of 1-2 days were used as skin donors for allogeneic skin transplantation. Thirty C57BL/6 mice at age of 6-8 weeks as recipients were made back skin defect of 12 mm in diameter and were randomly divided into 3 groups (n=10): group A, autograft; group B, allogeneic graft + PBS tail vein injection; and group C, allogeneic graft + human PMSCs (1 × 105 cells/mouse) tail vein injection. The flap survival was observed. At 7 days after skin transplantation, blood leukocyte counting, abdominal fluid macrophage activation, and the expression levels of interleukin 4 (IL-4), interleukin 17 (IL-17), and interferon γ (INF-γ) in blood and spleen were detected by ELISA and RT-PCR, respectively. Results The flap survival time was significantly longer in group A [(58.33 ± 4.04) days] than in groups B and C [(3.80 ± 0.92) days and (6.80 ± 0.82) days] (P lt; 0.05), and in group C than in group B (P lt; 0.05). At 7 days after transplantation, the blood leukocyte number was (6.32 ± 0.45) × 109/L in group A, (7.45 ± 0.52) × 109/L in group B, and (6.35 ± 0.39)× 109/ L in group C, and it was significantly more in group B than in groups A and C (P lt; 0.05). The macrophage activation rate of the abdominal fluid was 6.87% ± 2.40% in group A, 7.84% ± 0.44% in group B, and 15.98% ± 2.87% in group C; group C was significantly higher than groups A and B (P lt; 0.01). ELISA results showed that there was no significant difference in the concentrations of IL-4 among 3 groups (P gt; 0.05). Compared with group B, the concentrations of IL-17 and IFN-γ were significantly reduced in group C (P lt; 0.05), while the concentration of IFN-γ was significantly increased in group B when compared with group A (P lt; 0.05). RT-PCR results showed that there were significant differences in the expressions of IL-4, IL-17, and IFN-γ mRNA between groups B, C and group A (P lt; 0.05); the expressions of IL-4 and IFN-γ mRNA were significantly lower in group C than in group B (P lt; 0.05). Conclusion Human PMSCs transplantation can suppress the acute immunological rejection in allogeneic skin transplantation. The possible mechanism may be partially related to the inhibitory effect on the secretion of IL-17 and IFN-γ.

EXPERIMENTAL STUDY ON INDUCTION OF SKIN ALLOGRAFT TOLERANCE IN MICE BY HEAT SHOCK PROTEIN 60

Objective To investigate the role and mechanism of heat shock protein 60 (HSP60) in induction of murine skin allograft tolerance. Methods At the age of 8-12 weeks, inbred female BALB/C (H-2d) mice (n=45) and CBA/N (H-2k)mice (n=15) were used as transplantation donors and C57BL/6 (H-2b) mice (n=60) as recipients. Recipients C57BL/6 (H-2b) mice were randomized into 4 groups (n=15). In group A, 1 cm × 1 cm Wolfe-Krause skin graft was excised from the back of BALB/C (H-2d) mice and hypoderma was scraped off aseptically, and then transplanted to the back of C57BL/6 (H-2b)mice. The method of skin transplantation in the other 3 groups was the same as to group A. In group B, C57BL/6 (H-2b) mice were treated with imcompleted Freund’s adjuvant (IFA) administration into the back 2 weeks before transplantation of BALB/C (H-2d) mice skin. In group C, C57BL/6 (H-2b) mice were administered HSP60 emulsified in IFA into the back 2 weeks before transplantation of BALB/C (H-2d) mice skin. In group D, C57BL/6 (H-2b) mice were treated with HSP60 emulsified in IFA into the back and followed by skin transplantation of CBA/N (H-2k) mice 2 weeks later. The delayed type hypersensitivity was determined 7 days after transplantation. One-way mixed lymphocyte reaction, the concentration of cytokines in the mixed lymphocyte reaction culture supernatant was determined 7 days and 25 days after transplantation. The survival time of skin allograft was observed. Results The survival time of skin allograft in groups A, B, C and D was 12.4 ± 0.5, 11.6 ± 0.8, 29.3 ± 2.6 and 27.6 ± 2.1 days, respectively. There was significant difference between groups A, B and groups C, D (P﹤0.05), while there was no significant difference between group A and group B as well as between group C and group D (P gt; 0.05). The counts of per minute impulse (cpm) of mixed lymphocyte reaction 7 days after transplantation in groups A, B, C and D was 12 836 ± 1 357, 11 876 ±1 265, 6 581 ± 573 and 6 843 ± 612, respectively. There was significant difference between groups A, B and group C and group D (P lt; 0.05), while there was no significant difference between group A and group B as well as between group C and group D (P gt; 0.05). The cpm of mixed lymphocyte reaction at 25 days after transplantation in group A, B, C and D was 13 286 ±1 498, 12 960 ± 1 376, 11 936 ± 1 265 and 12 374 ± 1269, respectively. There was no significant difference among 4 groups (P gt;0.05).The concentration of IL-10 in the mixed lymphocyte reaction culture supernatant in groups C, D were higher than that in groups A, B, and IL-2 and IFN-γ were lower than that in groups A, B 7 days after transplantation (P lt; 0.05), while there was no significant difference between group A and group B as well as between group C and group D (P gt; 0.05). There was no significant difference in cytokines among the 4 groups 25 days after transplantation (P gt; 0.05). The delayed type hypersensitivity in groups A, B, C and D 7 days after transplantation was 0.84 ± 0.09, 0.81 ± 0.07, 0.43 ± 0.05 and 0.46 ± 0.03 mm, respectively. There was significant differences between groups A, B and groups C, D (P lt; 0.05). While there was no significant difference between group A and group B as well as between group C and group D (P gt; 0.05). Conclusion HSP60 may play a role in induction and maintenance of murine skin allograft tolerance.

AN EXPERIMENTAL STUDY ON CHANGES OF EPIDERMAL STEM CELLS DURING SURVIVAL PROCESS AFTER FULLTHICKNESS SKIN AUTOGRAFT

Objective To provide theoretical evidence for clinical application of the epidermal stem cells after an investigation on changes of the epidermal stem cells during the survival process after the fullthickness skin autograft. Methods On the backs of 42 Wistar rats, orthotopic transplantation models (1.5 cm×1.5 cm) of the fullthickness skin autograft were made. According to the time of the specimen taking, at 1, 3, 5, 7, 14, 21 and 30 days after operation, the rats were randomly divided in 7 groups (Groups 1-7). Specimens taken in each group before operation were used as controls. At each time point, the gross observation was made on the transplanted skin flaps, from which the skin tissues were harvested at each time point before and after operation. The routine pathological and the immunohistochemical examinations were performed on the specimens, which were stained by HE and were observed for immunohistochemical changes and the changes in the cells positive for integrinβ-1 and p63. Results All the fullthickness skin autografts survived 3 days after operation except the skin autograft in 1 rat in both Group 5 and Group 6, which was infected around the transplanted skin flap. In Groups 1-4, cell edema, inflammatory cell infiltration, and increased fibrocytes were observed. In Groups 5-7, the maturity degree of the epithelial cells became higher and higher, and the fibrocyte proportion was lowered. In each group the cell positivity rate for integrin β1 was lower than the cell positivity rate for p63. The positive cells were arranged in disorder, distributed into the layers of the epidermis and gradually concentrated in the basal layer of the epidermis and the bulge of the folliculus pili. The positive cells were also found in the other layers of the epidermis.The positive cells were gradually decreased in number, and reached the lowest level in Group 2. There was a significant difference in the above variables in Groups 1,2,3,5,6 and 7 between before and after operations (P＜0.05). Conclusion During the survival process of the fullthickness skin autograft, the proportion of theepidermal stem cells is gradually decreased at first; Then, the proportion isgradually increased, even beyond the normal level; finally, the proportion is decreased again. The distribution of the epidermal stem cells appear in disorder, almost distributed in the layers of the epidermis; finally, the almost normal distribution can be found. 

COMPARISON OF THE CLINICAL EFFECT OF COMPLEX TRANSPLANTATION WITH HETERGENEOUS ACELLULAR DERMAL MATRIX (ADM) AND HETEROCATAL ADM

Objective To explore the shortterm clinical effects of complex transplantation among the acellular dermal matrix(ADM) of heterogenic or heterocatal and autogenic split on the burnt wound as to find out a permanent substitution for the treatment on full skin thickness defect without scar. Methods Two kinds of ADM were used on the 18 patients with full thicknessburn wound through complex transplantation with autogenic splits. The patients with medialthickness autograft was used as control group. Survival rate was obtained 2 weeks after operation; contraction rate and the scores of Vancouver burn scale were obtained 8 weeks after operation. Results No significant difference was observed in survival rate among the three groups 2 weeks after operation(P＞0.05); no significant difference was observed in contraction rate of autografts and scores of Vancouver burn scale among the three groups 8 weeks after operation(P＞0.05). Conclusion ADM of heterogenic and ADM of heterocatal have similar effect on the reconstruction of skin, so the piglet ADM made in this way could be used as a substitution.

复合皮移植修复功能部位肉芽创面的临床研究

EFFECT OF AUTOSKIN GRAFTING IN FAULT HYPODERMIS WOUND OF GRANULATION EXCISION ON FULL-THICKNESS BURN HEALING

Objective To explore the mechanism of full-thickness burn wound healing with autoskin grafting in fault hypodermis wound of granulation excision and to evaluate its effect.Methods By the techniques of clinical observation, histopathology, immunohistochemistry,TEM and FCM,we observed changes of the activity andstructure of grafted skin and the granulation tissue,collagnous fiber,microvessels,the ultramicrostructure of fibroblasts and the expression of basic fibroblast growth factor(bFGF) in the base of autoskin grafting in fault hypodermis wound in burned adult minipigs(Group A), and compared with traditional method of autoskingrafting on the basilar fibrous tissue wound of scraped partly granulation being(Group B) and control group (Group C, without treatment except de-fur).Results The grafted skin survived after 3 days of operation, and it had less injury and higher proliferative index(PI) in group A than in group B. The hyperplasiaof granulation tissue and vascular endothelial and the expression of bFGF were more evident in group A. After 5 days, the proliferation of endothelial cells and granulation and the protein synthesis of fibroblasts were more active in groupA, and at this moment, fresh collagen appeared and proliferated more actively in group B. After 7-14 days, epidermic structure and dermic microvascular density became normal gradually, the granulation on grafting base matured and transformed into fibrous connective tissue in group A. The same change deferred about 2 days in group B. After 21 days, the above pathologic change in group A was less than that in group B. After 3060 days of operation, Group A achieved much less contraction and transfiguration than Group B, and the grafted skin was tender and movable. Conclusion Autoskin grafting in fault hypodermis wound of granulation excision has a better effect than traditional operation.

The Effect of Kupffer Cells Expressing Indoleamine 2,3Dioxygenase on Mouse Allografted Skin

摘要:目的:探讨表达吲哚胺2,3二氧化酶(IDO)的KC对同种异体小鼠移植皮片存活时间的影响及其机制。方法:构建BABL/c →C57BL/6的皮肤移植模型,分别于移植术后第2、7、14天输注KC,于移植术后第7天每组各取2只皮瓣行HE染色和TUNEL以检测淋巴细胞浸润和凋亡情况。KaplanMeier对数秩检验对各组进行生存分析。结果:输入表达IDO和FasL的KC能明显延长BABL/c →C57BL/6皮肤移植模型中皮肤移植物的存活时间,1－甲基色氨酸能阻断此效应。IFNγ组皮瓣浸润淋巴细胞的凋亡率较高(Plt;0.05)。结论:表达IDO和FasL的KC在体内能明显延长同种异体小鼠皮片的存活时间,IDO在KC维持外周免疫耐受中发挥重要作用。Abstract: Objective: To investigate kupffer cells(KC) expressing indoleamine 2,3dioxygenase(IDO) on the survival of grafted skin in mouse and its underlying mechanism. Methods: BABL/c skin was transplanted to C57BL/6. Donor KC were injected i.v. at days 2,7, 14 before transplantation. HE and TUNELAP were used to identify infiltrating cells and apoptotic cells in section of skin allografts from 7 days posttransplantation respectively. The survival rate of recipients among groups were analyzed by Logrank test. Results: Injection of KC expressing IDO and FasL from BABL/c mice into C57BL/6 could prolong a skin graft survival from the donor, but 1methyltryptophan could block the effect in vivo. The apoptosis rate of lymphocyte among skin graft in IFNγ group is more than other group(Plt;0.05). Conclusion: IDO and FasLexpressing KC from the donor of mouse can significantly prolong the skin graft survival. IDO may play an important role in KC to induce immune tolerance.

AN EXPERIMENTAL STUDY ON REPAIR OF FULL THICKNESS SKIN WOUND WITH GRAFT OF AUTOLOGOUS KERATINOCYTE SUSPENSION IN RATS

ObjectiveTo investigate the application of autologous keratinocyte suspension transplantation in skin reconstruction. MethodsForty adult Sprague Dawley rats (weighing, 210-230 g) were randomly divided into 4 groups (n=10): high density keratinocyte suspension transplantation (1×106 cells/cm2) group (group A), middle density (1×105 cells/cm2) group (group B), low density (1×104 cells/cm2) group (group C), and control group (group D). Skin samples were harvested from the rats in groups A, B, C for the isolation of keratinocytes. The model of anti-contracture of full thickness skin wound was made in rats and autologous keratinocyte suspension was transplanted into the wound. The wound was covered by the allogeneic skin (allogeneic skin derived from Wistar rats). The survival of rats was observed after operation. The survival of allogeneic skin was observed at 7, 14, and 21 days after operation, and wound healing rate was calculated after allogeneic skin dropped off. Histological staining and immunohistochemical staining were carried out at 21 days after operation. ResultsAll the rats survived to the end of the experiment. The allograft skins survived in all groups, dried and dropped off. The epithelium sheet could be seen in groups A and B at 21 days, a few very thin epithelium in group C, and no epithelization in group D. The wound healing rate of groups A (62.9%±9.6%) and B (64.2%±9.1%) were significantly higher than that of groups C (38.5%±5.7%) and D (22.7%±5.5%) (P<0.05), and significant difference was found between groups C and D (P<0.05), but there was no significant difference between groups A and B (P>0.05). The results of histological observation showed that squamous epithelial cells were observed in groups A, B, and C, but not in group D; obvious layers of epidermis were observed in groups A and B, thin epidermis and inflammatory cell infiltration in group C, and granulation tissue in group D. The immunohistochemistry staining showed that the expressions of collagen type IV and collagen type VII in groups A, B, and C; the percentage of collagen type IV and collagen type VII positive cells in groups A and B were significantly higher than that of group C (P<0.05), but there was no significant difference between groups A and B (P>0.05). The expressions of collagen type IV and collagen type VII in group D were negative. ConclusionThe repair of full thickness skin wound with graft of autologous keratinocyte suspension can achieve reconstruction of the skin. The appropriate density of keratinocyte suspension for wound healing is 1×105 cells/cm2.

Short-term effectiveness of hypospadias repair with free inner prepuce tube

ObjectiveTo explore the short-term effectiveness of hypospadias repairs with free inner prepuce tube. MethodsForty-two males with hypospadias were repaired with free inner prepuce tube between May and October 2015. The age ranged from 1 year and 1 month to 28 years and 2 months with a median of 2 years and 9 months. Initial operation for hypospadias was performed in 41 cases and re-operation in 1 case of chordee after 16 months of hypospadias repair. There were 36 cases of hypospadias (6 cases of proximal penile type, 1 case of penile scrotal type, 26 cases of scrotal type, and 3 cases of perineal type) and 6 cases of typeⅣchordee according to Donnahoo classification criteria. ResultsThe length of reconstructed urethra was 2.0-5.5 cm (mean, 3.6 cm). The patients were followed up 6-10 months (mean, 8 months). Urinary fistulae was noted in 10 patients, which healed spontaneously in 5 cases within 3 months after surgery, and was cured after repair in 5 cases. Urethral stenosis occurred in 4 cases (2 external orifice stricture and 2 distal urethral stricture) and urethral incision was performed. Wound infection was noted in 3 cases, 2 of them needed further surgery for a permanent fistula. Skin ischemic necrosis was found in 3 cases, 1 of them had infection and fistula, and needed further surgical repair. Diverticulum of scrotum developed in 1 case. Ten patients need re-operation, and the success rate was 76.2% (32/42). At 3 months after surgery, the HOSE score ranged from 11 to 16 (mean, 14.7). The maximum urinary flow rate was 1.9-28.4 mL/s (mean, 10.5 mL/s) in 22 cases of outpatient at 3 months after surgery. ConclusionFree inner prepuce tube can survive and play a role of normal neourethra. The short-term effectiveness has shown a relatively high complication rate and suggests this procedure should be carefully applied.