【摘要】：隨著社會的發(fā)展進步，人們的生活節(jié)奏逐步加快，越來越多的環(huán)境因素、社會心理因素使惡性腫瘤的發(fā)病率逐年上升，口腔頜面部惡性腫瘤也不例外。目前頜面部惡性腫瘤術(shù)后所造成的組織缺損往往采用皮膚移植的方法加以整復(fù)，術(shù)后相繼進行化學(xué)藥物治療或放射治療，這樣不僅引起皮膚組織的嚴重損害，同時使皮瓣或皮片愈合過程延遲甚至導(dǎo)致皮瓣壞死，這就是一種典型的“放創(chuàng)復(fù)合傷”，是目前較難治愈的難愈性創(chuàng)傷之一。因此如何促進放創(chuàng)復(fù)合傷難愈性創(chuàng)面愈合是創(chuàng)傷醫(yī)學(xué)修復(fù)領(lǐng)域亟待解決的問題。 人胎盤間充質(zhì)干細胞（hunman placenta-derived mesenchymal stem cells,HPMSCs）是來源于胎盤實質(zhì)組織的一類具有自我更新能力的間充質(zhì)細胞，它作為一種新的干細胞來源近年來逐漸受到人們的關(guān)注。我實驗室朱振威等[1]已經(jīng)成功從人胎盤組織中提取出狀態(tài)較好的HPMSCs，并成功誘導(dǎo)其向成骨和成脂細胞轉(zhuǎn)化，并發(fā)現(xiàn)它能高效表達目的基因，是細胞基因治療的良好載體。劉志輝等[2]研究表明HPMSCs不僅可以作為理想的種子細胞應(yīng)用于缺血性皮瓣動物模型，而且可以作為VEGF的載體細胞發(fā)揮VEGF的長效作用。大量研究表明，HPMSCs在創(chuàng)傷修復(fù)領(lǐng)域具有重要的應(yīng)用價值，但是將其應(yīng)用于放創(chuàng)復(fù)合傷，，促進其愈合目前國內(nèi)外文獻未見相關(guān)報道。Leptin亦稱為瘦素、消脂素，是一種白色脂肪組織(White adpose tissue，WAT)特異性分泌的蛋白活性因子，它能夠促進上皮再生及新生血管的形成，在創(chuàng)傷愈合的過程中發(fā)揮了重要作用。但由于其半衰期只有（9.4±3.0）min，因此單純局部應(yīng)用Leptin很難發(fā)揮其長效作用。為了使Leptin能夠發(fā)揮其長效促愈作用，我們考慮將Leptin轉(zhuǎn)染入間充質(zhì)干細胞，使其通過間充質(zhì)干細胞獲得持續(xù)表達，從而達到其長效促愈作用。本研究的目的是在朱振威等[1]實驗基礎(chǔ)上將其成功轉(zhuǎn)染的轉(zhuǎn)Leptin HPMSCs通過局部注射移植到Wistar大鼠背部放創(chuàng)復(fù)合傷模型，觀察轉(zhuǎn)Leptin HPMSCs對放創(chuàng)復(fù)合傷難愈性創(chuàng)面的促愈作用。方法：采用雄性Wistar大鼠，在大鼠背部做3×5cm大小的隨意皮瓣，原位縫合建立動物模型，術(shù)后第1天采用電子線對大鼠進行局部照射，總劑量5Gy。對30只大鼠隨機分成三組，放射后24小時分別局部皮下注射轉(zhuǎn)染Leptin的胎盤間充質(zhì)干細胞、轉(zhuǎn)染空載體的胎盤間充質(zhì)干細胞以及生理鹽水。本實驗通過大體觀察、組織病理學(xué)觀察、免疫組織化學(xué)染色觀察等多種手段，評估Leptin及HPMSCs對放創(chuàng)復(fù)合傷的促愈作用。 結(jié)果： 1、給藥后14天內(nèi)，各組大鼠均未出現(xiàn)免疫排斥反應(yīng)。給藥后第7天，放療后皮瓣成活率：轉(zhuǎn)Leptin HPMSCs組＞轉(zhuǎn)空載體HPMSCs組＞空白對照組。 2、給藥后第14天，組織病理學(xué)檢查HE染色，發(fā)現(xiàn)Ⅰ組肉芽組織最為豐富，可見大量的新生毛細血管及成纖維細胞，血管周圍炎細胞浸潤較少，膠原排列不整齊。Ⅱ組肉芽組織較為豐富，可見較多的新生毛細血管及成纖維細胞，血管周圍炎細胞浸潤較多，膠原排列不整齊。Ⅲ組新生肉芽組織較少，新生血管較少，細胞成分少見，可見大量炎細胞浸潤，可見纖維結(jié)締組織發(fā)生玻璃樣變。 3、給藥后第14天，vWF因子多克隆抗體及Leptin多克隆抗體免疫組織化學(xué)染色。結(jié)果顯示血管內(nèi)皮細胞內(nèi)vWF因子陽性表達，胞漿染色棕黃色，鏡下觀察各組新生血管的密度：轉(zhuǎn)Leptin HPMSCs組＞轉(zhuǎn)空載體HPMSCs組＞空白對照組；Leptin陽性表達主要位于上皮角質(zhì)細胞，其次為血管內(nèi)皮細胞，胞漿染色棕黃色，鏡下觀察各組Leptin免疫組化陽性表達：轉(zhuǎn)Leptin HPMSCs組＞轉(zhuǎn)空載體HPMSCs組＞空白對照組。 結(jié)論： 1、人胎盤間充質(zhì)干細胞免疫原性極低，不同物種應(yīng)用不會引起免疫排斥反應(yīng)； 2、在局部微環(huán)境作用下HPMSCs可以分化為血管內(nèi)皮細胞，促進新生血管的形成，可以促進放創(chuàng)復(fù)合傷的愈合，提高皮瓣的成活率。 3、HPMSCs可以作為Leptin基因治療的載體細胞，它持續(xù)分泌的Leptin發(fā)揮穩(wěn)定長效促愈作用； 4、HPMSCs與Leptin協(xié)同作用促進放創(chuàng)復(fù)合傷皮瓣的愈合，HPMSCs攜帶目的基因或單獨應(yīng)用均可顯著提高放創(chuàng)復(fù)合傷皮瓣的成活率，但轉(zhuǎn)LeptinHPMSCs組作用力更強。
[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.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R782

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