转Leptin的胎盘间充质干细胞对放创复合伤促愈作用的实验研究
发布时间：2018-09-19 14:23
【摘要】：随着社会的发展进步，人们的生活节奏逐步加快，越来越多的环境因素、社会心理因素使恶性肿瘤的发病率逐年上升，口腔颌面部恶性肿瘤也不例外。目前颌面部恶性肿瘤术后所造成的组织缺损往往采用皮肤移植的方法加以整复，术后相继进行化学药物治疗或放射治疗，这样不仅引起皮肤组织的严重损害，同时使皮瓣或皮片愈合过程延迟甚至导致皮瓣坏死，这就是一种典型的“放创复合伤”，是目前较难治愈的难愈性创伤之一。因此如何促进放创复合伤难愈性创面愈合是创伤医学修复领域亟待解决的问题。 人胎盘间充质干细胞（hunman placenta-derived mesenchymal stem cells,HPMSCs）是来源于胎盘实质组织的一类具有自我更新能力的间充质细胞，它作为一种新的干细胞来源近年来逐渐受到人们的关注。我实验室朱振威等[1]已经成功从人胎盘组织中提取出状态较好的HPMSCs，并成功诱导其向成骨和成脂细胞转化，并发现它能高效表达目的基因，是细胞基因治疗的良好载体。刘志辉等[2]研究表明HPMSCs不仅可以作为理想的种子细胞应用于缺血性皮瓣动物模型，而且可以作为VEGF的载体细胞发挥VEGF的长效作用。大量研究表明，HPMSCs在创伤修复领域具有重要的应用价值，但是将其应用于放创复合伤，，促进其愈合目前国内外文献未见相关报道。Leptin亦称为瘦素、消脂素，是一种白色脂肪组织(White adpose tissue，WAT)特异性分泌的蛋白活性因子，它能够促进上皮再生及新生血管的形成，在创伤愈合的过程中发挥了重要作用。但由于其半衰期只有（9.4±3.0）min，因此单纯局部应用Leptin很难发挥其长效作用。为了使Leptin能够发挥其长效促愈作用，我们考虑将Leptin转染入间充质干细胞，使其通过间充质干细胞获得持续表达，从而达到其长效促愈作用。本研究的目的是在朱振威等[1]实验基础上将其成功转染的转Leptin HPMSCs通过局部注射移植到Wistar大鼠背部放创复合伤模型，观察转Leptin HPMSCs对放创复合伤难愈性创面的促愈作用。方法：采用雄性Wistar大鼠，在大鼠背部做3×5cm大小的随意皮瓣，原位缝合建立动物模型，术后第1天采用电子线对大鼠进行局部照射，总剂量5Gy。对30只大鼠随机分成三组，放射后24小时分别局部皮下注射转染Leptin的胎盘间充质干细胞、转染空载体的胎盘间充质干细胞以及生理盐水。本实验通过大体观察、组织病理学观察、免疫组织化学染色观察等多种手段，评估Leptin及HPMSCs对放创复合伤的促愈作用。 结果： 1、给药后14天内，各组大鼠均未出现免疫排斥反应。给药后第7天，放疗后皮瓣成活率：转Leptin HPMSCs组＞转空载体HPMSCs组＞空白对照组。 2、给药后第14天，组织病理学检查HE染色，发现Ⅰ组肉芽组织最为丰富，可见大量的新生毛细血管及成纤维细胞，血管周围炎细胞浸润较少，胶原排列不整齐。Ⅱ组肉芽组织较为丰富，可见较多的新生毛细血管及成纤维细胞，血管周围炎细胞浸润较多，胶原排列不整齐。Ⅲ组新生肉芽组织较少，新生血管较少，细胞成分少见，可见大量炎细胞浸润，可见纤维结缔组织发生玻璃样变。 3、给药后第14天，vWF因子多克隆抗体及Leptin多克隆抗体免疫组织化学染色。结果显示血管内皮细胞内vWF因子阳性表达，胞浆染色棕黄色，镜下观察各组新生血管的密度：转Leptin HPMSCs组＞转空载体HPMSCs组＞空白对照组；Leptin阳性表达主要位于上皮角质细胞，其次为血管内皮细胞，胞浆染色棕黄色，镜下观察各组Leptin免疫组化阳性表达：转Leptin HPMSCs组＞转空载体HPMSCs组＞空白对照组。 结论： 1、人胎盘间充质干细胞免疫原性极低，不同物种应用不会引起免疫排斥反应； 2、在局部微环境作用下HPMSCs可以分化为血管内皮细胞，促进新生血管的形成，可以促进放创复合伤的愈合，提高皮瓣的成活率。 3、HPMSCs可以作为Leptin基因治疗的载体细胞，它持续分泌的Leptin发挥稳定长效促愈作用； 4、HPMSCs与Leptin协同作用促进放创复合伤皮瓣的愈合，HPMSCs携带目的基因或单独应用均可显著提高放创复合伤皮瓣的成活率，但转LeptinHPMSCs组作用力更强。
[Abstract]:With the development of society, the rhythm of people's life is quickening gradually, more and more environmental factors, social and psychological factors make the incidence of malignant tumors rising year by year, oral and maxillofacial malignant tumors are no exception. Chemotherapy or radiotherapy in succession not only causes serious damage to skin tissue, but also delays the healing process of skin flap or skin flap and even leads to flap necrosis. This is a typical "combined radiation wound" which is one of the more difficult wounds to heal at present. Healing is an urgent problem in the field of wound healing.
Human placenta-derived mesenchymal stem cells (HPMSCs) are a kind of self-renewing mesenchymal cells derived from placental parenchyma. As a new source of stem cells, HPMSCs have attracted more and more attention in recent years. HPMSCs were successfully induced to transform into osteoblasts and adipocytes. It was found that HPMSCs could efficiently express the target gene and were a good carrier for cell gene therapy. Many studies have shown that HPMSCs play an important role in the field of wound healing. However, it has not been reported that HPMSCs can be used in the field of wound healing after combined radiation injury. Protein-active factors, which can promote epithelial regeneration and neovascularization, play an important role in wound healing. However, because the half-life of Leptin is only (9.4 (+) 3.0) minutes, it is difficult to exert its long-term effect only by topical application of Leptin. The aim of this study was to observe the effect of Leptin HPMSCs transfected successfully into Wistar rats by local injection and to observe the effect of Leptin HPMSCs transfected on the wound healing. Methods: 30 Wistar rats were randomly divided into three groups at a total dose of 5 Gy. Le was transfected subcutaneously 24 hours after irradiation with electron beam on the first day after operation. Placental mesenchymal stem cells transfected with ptin, placental mesenchymal stem cells transfected with empty vectors and normal saline were used to evaluate the healing effect of PTIN and HPMSCs on combined radiation injury by gross observation, histopathological observation and immunohistochemical staining.
Result:
1. Within 14 days after administration, there was no immunological rejection in all groups. On the 7th day after administration, the survival rate of flaps was as follows: Leptin HPMSCs group > empty carrier HPMSCs group > blank control group.
2. On the 14th day after administration, HE staining showed that the granulation tissue in group I was the most abundant, with a large number of new capillaries and fibroblasts, less infiltration of perivascular inflammatory cells and irregular collagen arrangement. In group III, there were less granulation tissue, less neovascularization, less cell components, and a large number of inflammatory cells infiltration. Fibrous connective tissue had hyaline degeneration.
3. Immunohistochemical staining of polyclonal antibodies against vWF factor and Leptin polyclonal antibodies was performed on the 14th day after administration. The results showed that the expression of vWF factor was positive in vascular endothelial cells and the cytoplasm was brown yellow. The density of neovascularization in each group was observed under microscope: trans-Leptin HPMSCs group > trans-empty carrier HPMSCs group > blank control group. Leptin immunohistochemical positive expression was observed under microscope: Leptin HPMSCs group > empty vector HPMSCs group > blank control group.
Conclusion:
1, the immunogenicity of human placenta derived mesenchymal stem cells is very low, and the application of different species will not cause immune rejection.
2. HPMSCs can differentiate into vascular endothelial cells under local microenvironment, promote the formation of new blood vessels, promote the healing of combined radiation injury and improve the survival rate of flap.
3. HPMSCs can be used as carrier cells for Leptin gene therapy, and the sustained secretion of Leptin plays a stable and long-term role in promoting healing.
4. The synergistic effect of HPMSCs and Leptin can promote the healing of composite wound flaps. The survival rate of composite wound flaps can be significantly improved by HPMSCs carrying the target gene or by using HPMSCs alone, but the effect of Leptin HPMSCs is stronger.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R782
本文编号：2250388
[Abstract]:With the development of society, the rhythm of people's life is quickening gradually, more and more environmental factors, social and psychological factors make the incidence of malignant tumors rising year by year, oral and maxillofacial malignant tumors are no exception. Chemotherapy or radiotherapy in succession not only causes serious damage to skin tissue, but also delays the healing process of skin flap or skin flap and even leads to flap necrosis. This is a typical "combined radiation wound" which is one of the more difficult wounds to heal at present. Healing is an urgent problem in the field of wound healing.
Human placenta-derived mesenchymal stem cells (HPMSCs) are a kind of self-renewing mesenchymal cells derived from placental parenchyma. As a new source of stem cells, HPMSCs have attracted more and more attention in recent years. HPMSCs were successfully induced to transform into osteoblasts and adipocytes. It was found that HPMSCs could efficiently express the target gene and were a good carrier for cell gene therapy. Many studies have shown that HPMSCs play an important role in the field of wound healing. However, it has not been reported that HPMSCs can be used in the field of wound healing after combined radiation injury. Protein-active factors, which can promote epithelial regeneration and neovascularization, play an important role in wound healing. However, because the half-life of Leptin is only (9.4 (+) 3.0) minutes, it is difficult to exert its long-term effect only by topical application of Leptin. The aim of this study was to observe the effect of Leptin HPMSCs transfected successfully into Wistar rats by local injection and to observe the effect of Leptin HPMSCs transfected on the wound healing. Methods: 30 Wistar rats were randomly divided into three groups at a total dose of 5 Gy. Le was transfected subcutaneously 24 hours after irradiation with electron beam on the first day after operation. Placental mesenchymal stem cells transfected with ptin, placental mesenchymal stem cells transfected with empty vectors and normal saline were used to evaluate the healing effect of PTIN and HPMSCs on combined radiation injury by gross observation, histopathological observation and immunohistochemical staining.
Result:
1. Within 14 days after administration, there was no immunological rejection in all groups. On the 7th day after administration, the survival rate of flaps was as follows: Leptin HPMSCs group > empty carrier HPMSCs group > blank control group.
2. On the 14th day after administration, HE staining showed that the granulation tissue in group I was the most abundant, with a large number of new capillaries and fibroblasts, less infiltration of perivascular inflammatory cells and irregular collagen arrangement. In group III, there were less granulation tissue, less neovascularization, less cell components, and a large number of inflammatory cells infiltration. Fibrous connective tissue had hyaline degeneration.
3. Immunohistochemical staining of polyclonal antibodies against vWF factor and Leptin polyclonal antibodies was performed on the 14th day after administration. The results showed that the expression of vWF factor was positive in vascular endothelial cells and the cytoplasm was brown yellow. The density of neovascularization in each group was observed under microscope: trans-Leptin HPMSCs group > trans-empty carrier HPMSCs group > blank control group. Leptin immunohistochemical positive expression was observed under microscope: Leptin HPMSCs group > empty vector HPMSCs group > blank control group.
Conclusion:
1, the immunogenicity of human placenta derived mesenchymal stem cells is very low, and the application of different species will not cause immune rejection.
2. HPMSCs can differentiate into vascular endothelial cells under local microenvironment, promote the formation of new blood vessels, promote the healing of combined radiation injury and improve the survival rate of flap.
3. HPMSCs can be used as carrier cells for Leptin gene therapy, and the sustained secretion of Leptin plays a stable and long-term role in promoting healing.
4. The synergistic effect of HPMSCs and Leptin can promote the healing of composite wound flaps. The survival rate of composite wound flaps can be significantly improved by HPMSCs carrying the target gene or by using HPMSCs alone, but the effect of Leptin HPMSCs is stronger.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R782
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