促进微粒上皮修复创面减轻挛缩的动物实验研究
发布时间：2019-07-05 17:01
【摘要】：第一部分猪自体皮肤微粒修复体表创面与皮肤微粒量的关系 目的： 观察阶梯比例自体微粒皮移植后对创面的修复效果,明确不同比例微粒皮移植于创面后的愈合速度及修复质量,寻找微粒皮游离移植修复创面的最佳比例。 材料与方法： 在实验用猪的体表制备若干2x2cm2全层皮肤缺损创面,将自体微粒皮扩张面积比例(即移植微粒面积与创面面积比例)分别设定为10%、20%、30%、40%、50%、60%、70%、80%、90%、100%,共10组,每组6个创面。分别将各组微粒皮移植于创面,术后2、3、4周观察各组创面愈合情况,术后4、8、12周观察各组挛缩情况,并行HE染色观察上皮及皮下胶原的分布。 结果： 1.10组中,10%、20%和30%三组均愈合较慢,术后3周时愈合率仅约为45%,组间无明显差异(P0.05)；而40%-100%七组的愈合速度相对较快,术后3周时愈合率约为75%,组间无明显差异(P0.05)；后七组的愈合率均高于前三组,具有统计学差异(P0.05)。 2.移植术后4周各组创面基本完全上皮化,至12周时创面的大小趋于稳定。此时10%、20%和30%三组的挛缩率约为70%,组间无明显差异(P0.05)；40%-100%七组的挛缩相对较轻,挛缩率约为55%,组间无明显差异(P0.05)；后七组的挛缩率均小于前三组,具有统计学差异(P0.05)。 3.HE染色显示12周时,40%-100%七组的上皮细胞层数较多,极性良好,真皮乳头层丰富,皮下可见大量成熟胶原纤维排列有序,其间散在分布纤维细胞；而10%-30%三组基本为瘢痕愈合,可见上皮细胞层数较少,无上皮角,皮下仍可见大量纤维细胞,胶原尚未完全成熟。 结论： 自体微粒皮扩张面积比例(即移植微粒面积与创面面积比例)为40%时,达到修复创面的最佳比例。即利用相对较少的皮肤可以达到相对较快的愈合速度和较轻的后期挛缩。 第二部分猪自体微粒皮+微粒黏膜+人工真皮复合移植修复体表创面的远期疗效观察 目的： 观察自体微粒皮+微粒黏膜+人工真皮复合移植修复创面的远期效果,评价此方法是否可改善单纯微粒皮+微粒黏膜修复创面远期挛缩较重的问题。 材料与方法： 在实验用猪的体表制备若干5x6cm2全层皮肤缺损创面,体表创面根据修复组织的不同随机分为口腔黏膜微粒+皮肤微粒组(A组)、口腔黏膜微粒+皮肤微粒+人工真皮组(B组)、人工真皮组(C组)、厚中厚植皮组(D组),共4组,每组6个创面。将微粒扩张面积比例(即移植微粒面积与创面面积比例)设定为40%。术后2周、4周、8周、12周比较各组愈合情况和创面挛缩情况,并行HE染色观察上皮化情况和皮下胶原的分布,第12周行扫描电镜观察表皮真皮结合的情况以及修复质量。第12周将创面标本行生物力学检测比较各组皮肤的粘弹性和抗拉强度的差异。 结果： 1.移植术后10天、2周、3周,A、B、C三组逐渐上皮化,C组人工真皮大部分脱落,创面挛缩明显,D组完全愈合。愈合率方面D组优于A、B、C三组(P0.05),A、B两组优于C组(P0.05),A、B组间无明显差异(P0.05)。 2.移植术后4、8、12周分别比较各组创面挛缩率。术后4周A、B、C三组间无明显差异(P0.05)。D组挛缩较轻,与前三组差异明显(P0.05)。术后8周、12周各组的挛缩率比较：DBAC,组间比较均有差异(P0.05)。 3.组织学观察：术后2、3、4周,可见各组创面修复动态过程,上皮层数逐渐增多,极性排列变好,炎症细胞逐渐减少,皮下胶原逐渐增多,排列逐渐有序。C组愈合较慢,炎症持续时间长,人工真皮大部分脱落。术后12周,B组和D组与正常皮肤相近,皮下胶原排列规则,疏松；A组和C组真皮乳头层较少,皮下胶原致密、紊乱。 4.生物力学检测：D组的皮肤在粘弹性与抗拉能力方面与正常皮肤相近,A、B、C三组与D组存在很大差距,B组在弹性方面有所改善,但抗拉能力显著下降。 结论： 自体微粒皮+微粒黏膜+人工真皮修复创面方法简单,可以改善创面愈合的远期质量,但与厚中厚植皮法仍存在一定差距。 第三部分脂肪间充质干细胞联合皮肤微粒移植修复裸鼠创面的实验研究 目的： 观察脂肪间充质干细胞联合自体皮肤微粒游离移植修复创面的情况,明确脂肪间充质干细胞是否可促进自体皮肤微粒游离移植修复创面的上皮化过程及愈合速度,探讨脂肪干细胞移植创面后的存活和转归。 材料与方法： 10只6周龄SPF级BALB/c裸鼠随机分为两组,在背部两侧制作直径为1cm的圆形全层皮肤缺损,自体皮肤微粒移植面积比例为40%,分别将各组皮肤微粒移植于创面。实验组创面下注射PBS缓冲液悬浮的GFP+ADSCs,对照组注射相同体积的PBS缓冲液。术后7、9、12天观察并分析创面愈合情况,HE染色观察上皮化情况、皮下胶原分布及皮下新生小血管密度。免疫荧光观察移植细胞的存活和转归情况。Real-time PCR检测创面组织中促炎因子白细胞介素-1(IL-1)、白细胞介素-6(1L-6)以及抗炎因子白细胞介素-10(IL-10)的表达；同时检测创面愈合相关细胞因子如血管内皮细胞生长因子(VEGF)、转化生长因子β(TGF-β)、肝细胞生长因子(HGF)、成纤维细胞生长因子2(FGF2). I型胶原蛋白(Collagen I)的表达。 结果： 1.术后7、9天实验组的愈合率均高于对照组(P0.05)。术后9、12天实验组挛缩率均低于对照组(P0.05)。 2.组织学观察发现术后7天各组创面尚未愈合,实验组较对照组肉芽形成丰富,皮下可见成纤维细胞增生明显,胶原层较厚,创面内新生血管更为明显。术后12天时实验组创面己完全修复,成纤维细胞开始减少,胶原开始增多,对照组仍可见大量的成纤维细胞和纤维细胞,胶原分泌较少。术后7、12天实验组新生小血管的密度均高于对照组,具有统计学差异(p0.05)。 3.免疫荧光检查发现术后7、12天能观察到同时表达GFP和CD31的双阳性细胞,即ADSCs分化成血管内皮细胞,未观察到ADSCs分化成表皮细胞。同时,ADSCs存活率较低,向内皮细胞分化的效率也较低。 4. Real-time PCR测试：与对照组相比,实验组创面组织中IL-1、IL-6显著下降,IL-10、VEGF、FGF2、TGF-β、Collagen I显著升高(P0.05)。 结论： 通过同种异体局部注射脂肪间充质干细胞可以显著促进皮肤创面的愈合。ADSCs可少量存活并向血管内皮细胞分化,参与创面新生组织血管形成过程,主要作用机制可能与ADSCs调节局部的炎症反应和细胞因子分泌相关。
[Abstract]:The relationship between the surface wound and the amount of skin particles in the first part of porcine autologous skin order Objective: To observe the effect of step-scale autograft on the repair of the wound, and to determine the healing speed and repair of the different proportion of the microskin graft in the wound. The most important method for the repair of the wound by finding the free graft of the particle skin by the complex mass A good proportion. The method comprises the following steps of: preparing a plurality of 2x2cm2 whole-layer skin defect wounds on the surface of a pig in an experiment, and setting the proportion of the expansion area of the self-particle skin (i.e., the proportion of the area of the transplanted particles and the area of the wound surface) to be 10%,20%,30%,40%,50%,60% and 70%, respectively, 80%,90%,100%,10 groups The wound healing of each group was observed at 2,3 and 4 weeks after operation. The contracture of each group was observed at 4,8 and 12 weeks after operation. Subcutaneous glue Results: In the 10%,20% and 30% group, the healing rate was only about 45% at 3 weeks after the operation, and there was no significant difference between the groups (P0.05), while the healing rate of 40% to 100% group was relatively fast, and the healing rate was about 75% at 3 weeks after operation. There was no difference between the groups. The healing rate of the latter group was higher than that of the first three groups (P0.05), and the healing rate of the latter group was higher than that of the first three groups. The results showed that the wound surface of each group was completely epithelialized after 4 weeks after the transplantation. At 12 weeks, the size of the wound was stable. At this time, the contracture rate of 10%,20% and 30% of the three groups was about 70%. There was no significant difference between the groups (P0.05). The contracture of 40% to 100% group was relatively light and the contracture rate was about 55%. There was no significant difference between the groups (P0.05). The contracture rate of the latter group was less than that of the first three groups. There was a statistical difference (P0.05).3. HE staining showed that the number of epithelial cells in 40% to 100% of the seven groups was more, the polarity was good, the dermal papilla layer was rich, and a large number of mature collagen fibers in the subcutaneous layer were arranged in order, interspersed among the fibroblasts, while 10% to 30% of the three groups were basically Tracheal wound healing, the number of the visible epithelial cells is small, no epithelial angle, and the subcutaneous is still visible. large amount of fiber It was concluded that the ratio of the area of the expanded area of the autograft (i.e., the area of the implanted particles to the area of the wound) in the case of 40%, the optimal proportion of the repair wound is reached. i. e., with relatively small skin, A relatively fast rate of healing and a relatively light post-contracture. + Labor Long-term effect of dermal composite graft in the repair of the surface of the surface of the surface of the body: the observation of the self-micro- The long-term effect of the granulation + particle mucosa and the artificial dermis composite graft in the repair of the wound surface, and whether the method can be used for evaluating the long-term effect of the method improve pure particle The problem of long-term contracture of skin and particulate mucosa was studied. Materials and methods: a number of 5 x 6 cm 2 whole-layer skin defect wound was prepared on the surface of the pig, and the surface wound was randomly divided into the oral mucosa particles and the skin according to the different repair tissue. Particle group (group A), oral mucosa microparticles + skin particles + artificial dermis group (group B), artificial Dermal group (group C), thick medium-thickness skin graft (group D),4 groups,6 wounds in each group. The proportion of the area of particle expansion (i.e., the area of the implanted particles and the area of the wound area) was set to 40%. The healing and the contracture of the wound were compared in 2 weeks,4 weeks,8 weeks and 12 weeks after the operation. The epithelialization and the subcutaneous collagen were observed in parallel HE staining. The distribution, the scanning electron microscope at the 12th week to observe the combination of the epidermis and the skin, and the quality of the repair. The 12th week will wound scale The results were as follows:1.10 days after the transplantation,2 weeks,3 weeks, A, B and C The results showed that the group D was better than A, B and C (P 0.05), A, B and C (P0.05). Both groups were better than group C (P0.05). There was no significant difference between group A and group B. The contracture rate of the wound was compared in 4,8 and 12 weeks after the transplantation. There was no significant difference (P0.05). The contracture of D group was light, and the difference between the three groups was significant (P0.05). At 8 and 12 weeks, the contracture rate of each group was higher than that of the DBAC group (P0.05).3. Histological observation:2,3 and 4 weeks after the operation, the dynamic process of the wound repair and the epithelial layer in each group were observed. The numbers are gradually increased, the polarity arrangement is good, the inflammatory cells are gradually reduced, and the subcutaneous glue In group C, the healing of C group was slow, the duration of inflammation was long, and the artificial dermis mostly fell off. The group B and D and the normal skin at 12 weeks after the operation. The results showed that the skin of the D group was similar to the normal skin in the aspects of viscoelasticity and tensile strength. ,A, The group B and C had a large gap with the D group, and the group B was improved in elasticity, but the tensile strength decreased significantly. The method is simple and can improve the long-term quality of wound healing. middle-and-thick planting There is still a certain gap in the skin method. The third part of the adipose-derived mesenchymal stem cells combined with the skin particles The purpose of the experimental study on the repair of the wound in nude mice was to observe the free transfer of the adipose-derived mesenchymal stem cells and the free-graft repair of the wound, and to clarify the adipose-derived mesenchymal stem cells. Is it possible to promote self-adaptation Methods: Ten 6-week-old SPF BALB/ c nude mice were randomly divided into two groups. A circular full-layer skin defect with a diameter of 1 cm was made on both sides of the back, and the proportion of the graft area of the autologous skin particles was 40%, respectively. The skin particles were transplanted into the wound. In the experimental group, the PBS buffer was injected with PBS buffer, and the same volume of PBS was injected into the control group. Buffer. Observe and analyze the wound at 7,9 and 12 days. Surface healing, HE staining, epithelialization, subcutaneous collagen distribution and small vessel density under the skin were observed. The survival and outcome of the transplanted cells were observed by immunofluorescence. Real-time PCR was used to detect the pro-inflammatory factors, interleukin-1 (IL-1), interleukin-6 (1 L-6), and anti-inflammatory factor interleukin-10 (IL-10) in wound tissue. 10) expression of wound healing-related cytokines such as vascular endothelial cells Factor (VEGF), transformed growth factor (TGF- for example), hepatocyte growth factor (HGF), fibroblast growth factor 2 (FGF2) . Expression of type I collagen (Colagen I). Results: 1. The healing rate of the experimental group was higher than that in the control group (P0.05). The contracture rate of the experimental group was lower than that of the control group (P0.05). 7 days after operation, the wound of the group had not yet healed, and the experimental group was rich in granulation of the control group, and the proliferation of the subcutaneous visible fibroblasts was obvious, the collagen layer was thicker, and the new blood vessels in the wound were more obvious. After 12 days, the wound surface of the experimental group was completely repaired, the fibroblasts began to decrease, the collagen began to increase, and the control group was still visible. In the experimental group, the density of the new small blood vessels in the experimental group was higher than that of the control group, and there was a statistical difference (p0.05). Two positive cells expressing GFP and CD31 were observed at 7 and 12 days In other words, ADSCs differentiated into vascular endothelial cells, and ADSCs were not observed to differentiate into epidermal cells. At the same time, the survival of ADSCs was low and the efficiency of differentiation to endothelial cells was also low. group creation IL-1, IL-6 significantly decreased in surface tissue, IL-10, VEGF, and FGF Conclusion: The local injection of the adipose-derived mesenchymal stem cells can promote the healing of the wound of the skin.
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R-332;R641
本文编号：2510678
[Abstract]:The relationship between the surface wound and the amount of skin particles in the first part of porcine autologous skin order Objective: To observe the effect of step-scale autograft on the repair of the wound, and to determine the healing speed and repair of the different proportion of the microskin graft in the wound. The most important method for the repair of the wound by finding the free graft of the particle skin by the complex mass A good proportion. The method comprises the following steps of: preparing a plurality of 2x2cm2 whole-layer skin defect wounds on the surface of a pig in an experiment, and setting the proportion of the expansion area of the self-particle skin (i.e., the proportion of the area of the transplanted particles and the area of the wound surface) to be 10%,20%,30%,40%,50%,60% and 70%, respectively, 80%,90%,100%,10 groups The wound healing of each group was observed at 2,3 and 4 weeks after operation. The contracture of each group was observed at 4,8 and 12 weeks after operation. Subcutaneous glue Results: In the 10%,20% and 30% group, the healing rate was only about 45% at 3 weeks after the operation, and there was no significant difference between the groups (P0.05), while the healing rate of 40% to 100% group was relatively fast, and the healing rate was about 75% at 3 weeks after operation. There was no difference between the groups. The healing rate of the latter group was higher than that of the first three groups (P0.05), and the healing rate of the latter group was higher than that of the first three groups. The results showed that the wound surface of each group was completely epithelialized after 4 weeks after the transplantation. At 12 weeks, the size of the wound was stable. At this time, the contracture rate of 10%,20% and 30% of the three groups was about 70%. There was no significant difference between the groups (P0.05). The contracture of 40% to 100% group was relatively light and the contracture rate was about 55%. There was no significant difference between the groups (P0.05). The contracture rate of the latter group was less than that of the first three groups. There was a statistical difference (P0.05).3. HE staining showed that the number of epithelial cells in 40% to 100% of the seven groups was more, the polarity was good, the dermal papilla layer was rich, and a large number of mature collagen fibers in the subcutaneous layer were arranged in order, interspersed among the fibroblasts, while 10% to 30% of the three groups were basically Tracheal wound healing, the number of the visible epithelial cells is small, no epithelial angle, and the subcutaneous is still visible. large amount of fiber It was concluded that the ratio of the area of the expanded area of the autograft (i.e., the area of the implanted particles to the area of the wound) in the case of 40%, the optimal proportion of the repair wound is reached. i. e., with relatively small skin, A relatively fast rate of healing and a relatively light post-contracture. + Labor Long-term effect of dermal composite graft in the repair of the surface of the surface of the surface of the body: the observation of the self-micro- The long-term effect of the granulation + particle mucosa and the artificial dermis composite graft in the repair of the wound surface, and whether the method can be used for evaluating the long-term effect of the method improve pure particle The problem of long-term contracture of skin and particulate mucosa was studied. Materials and methods: a number of 5 x 6 cm 2 whole-layer skin defect wound was prepared on the surface of the pig, and the surface wound was randomly divided into the oral mucosa particles and the skin according to the different repair tissue. Particle group (group A), oral mucosa microparticles + skin particles + artificial dermis group (group B), artificial Dermal group (group C), thick medium-thickness skin graft (group D),4 groups,6 wounds in each group. The proportion of the area of particle expansion (i.e., the area of the implanted particles and the area of the wound area) was set to 40%. The healing and the contracture of the wound were compared in 2 weeks,4 weeks,8 weeks and 12 weeks after the operation. The epithelialization and the subcutaneous collagen were observed in parallel HE staining. The distribution, the scanning electron microscope at the 12th week to observe the combination of the epidermis and the skin, and the quality of the repair. The 12th week will wound scale The results were as follows:1.10 days after the transplantation,2 weeks,3 weeks, A, B and C The results showed that the group D was better than A, B and C (P 0.05), A, B and C (P0.05). Both groups were better than group C (P0.05). There was no significant difference between group A and group B. The contracture rate of the wound was compared in 4,8 and 12 weeks after the transplantation. There was no significant difference (P0.05). The contracture of D group was light, and the difference between the three groups was significant (P0.05). At 8 and 12 weeks, the contracture rate of each group was higher than that of the DBAC group (P0.05).3. Histological observation:2,3 and 4 weeks after the operation, the dynamic process of the wound repair and the epithelial layer in each group were observed. The numbers are gradually increased, the polarity arrangement is good, the inflammatory cells are gradually reduced, and the subcutaneous glue In group C, the healing of C group was slow, the duration of inflammation was long, and the artificial dermis mostly fell off. The group B and D and the normal skin at 12 weeks after the operation. The results showed that the skin of the D group was similar to the normal skin in the aspects of viscoelasticity and tensile strength. ,A, The group B and C had a large gap with the D group, and the group B was improved in elasticity, but the tensile strength decreased significantly. The method is simple and can improve the long-term quality of wound healing. middle-and-thick planting There is still a certain gap in the skin method. The third part of the adipose-derived mesenchymal stem cells combined with the skin particles The purpose of the experimental study on the repair of the wound in nude mice was to observe the free transfer of the adipose-derived mesenchymal stem cells and the free-graft repair of the wound, and to clarify the adipose-derived mesenchymal stem cells. Is it possible to promote self-adaptation Methods: Ten 6-week-old SPF BALB/ c nude mice were randomly divided into two groups. A circular full-layer skin defect with a diameter of 1 cm was made on both sides of the back, and the proportion of the graft area of the autologous skin particles was 40%, respectively. The skin particles were transplanted into the wound. In the experimental group, the PBS buffer was injected with PBS buffer, and the same volume of PBS was injected into the control group. Buffer. Observe and analyze the wound at 7,9 and 12 days. Surface healing, HE staining, epithelialization, subcutaneous collagen distribution and small vessel density under the skin were observed. The survival and outcome of the transplanted cells were observed by immunofluorescence. Real-time PCR was used to detect the pro-inflammatory factors, interleukin-1 (IL-1), interleukin-6 (1 L-6), and anti-inflammatory factor interleukin-10 (IL-10) in wound tissue. 10) expression of wound healing-related cytokines such as vascular endothelial cells Factor (VEGF), transformed growth factor (TGF- for example), hepatocyte growth factor (HGF), fibroblast growth factor 2 (FGF2) . Expression of type I collagen (Colagen I). Results: 1. The healing rate of the experimental group was higher than that in the control group (P0.05). The contracture rate of the experimental group was lower than that of the control group (P0.05). 7 days after operation, the wound of the group had not yet healed, and the experimental group was rich in granulation of the control group, and the proliferation of the subcutaneous visible fibroblasts was obvious, the collagen layer was thicker, and the new blood vessels in the wound were more obvious. After 12 days, the wound surface of the experimental group was completely repaired, the fibroblasts began to decrease, the collagen began to increase, and the control group was still visible. In the experimental group, the density of the new small blood vessels in the experimental group was higher than that of the control group, and there was a statistical difference (p0.05). Two positive cells expressing GFP and CD31 were observed at 7 and 12 days In other words, ADSCs differentiated into vascular endothelial cells, and ADSCs were not observed to differentiate into epidermal cells. At the same time, the survival of ADSCs was low and the efficiency of differentiation to endothelial cells was also low. group creation IL-1, IL-6 significantly decreased in surface tissue, IL-10, VEGF, and FGF Conclusion: The local injection of the adipose-derived mesenchymal stem cells can promote the healing of the wound of the skin.
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R-332;R641
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