本文選題：脂肪干細胞 + 成纖維細胞�。� 參考：《第二軍醫(yī)大學(xué)》2017年碩士論文

【摘要】：研究背景和目的:放療、職業(yè)暴露、意外事故、戰(zhàn)爭等多種原因都可以對皮膚造成放射性損傷。大劑量的輻射可致急性放射性潰瘍,小劑量輻射引起放射性皮炎,遷延不愈可造成慢性放射性潰瘍,若不及時治療,甚至可導(dǎo)致放射性皮膚癌。放射性皮膚損傷在臨床上通常表現(xiàn)為潛在性、進展性、難以愈合的特點,放射性皮膚潰瘍向深部發(fā)展,可以損傷肌肉、血管、神經(jīng)等深部組織,嚴重影響患者的生活質(zhì)量,甚至威脅生命。目前在臨床中放射性皮膚損傷以放射性皮膚潰瘍最為常見,切除病變組織,用皮瓣修復(fù)缺損是最為常見和有效的治療方法。近年來,脂肪干細胞(ADSCs)作為再生醫(yī)學(xué)和組織工程領(lǐng)域內(nèi)的研究熱點,從脂肪組織中提取,具有取材方便,供源廣泛的特點。ADSCs屬于間充質(zhì)干細胞(MSCs),具有多項分化潛能和免疫豁免的生物學(xué)特性,并能夠分泌多種細胞因子。因此,ADSCs具有廣泛的臨床使用前景。將ADSCs作用于放射性皮膚損傷的創(chuàng)面,在諸多臨床實驗、動物實驗中都證實了ADSCs促進組織修復(fù)的能力。由于ADSCs具有向多種組織分化的潛能,因此在修復(fù)創(chuàng)面的過程中,可以直接分化為受損的組織細胞參與修復(fù)進程。然而隨著對ADSCs研究機制的不斷深入,越來越多的研究者們認為旁分泌作用在修復(fù)創(chuàng)面的過程中起主導(dǎo)作用,ADSCs可以分泌大量細胞因子包括生長因子、抗炎因子等,還可以釋放具有特殊功能的細胞外囊泡(EVs),發(fā)揮抗凋亡、抗炎、促進血管生成的作用。然而,對于ADSCs逆轉(zhuǎn)放射性皮膚損傷的機制還需要進一步的研究。我們通過將大鼠脂肪干細胞(r ADSCs)與輻射后的大鼠皮膚成纖維細胞(r Fbs)隔以半透膜共同培養(yǎng)建立r ADSCs修復(fù)放射性皮膚損傷的細胞模型,觀察共培養(yǎng)對r Fbs放射性損傷的修復(fù),探索ADSCs通過旁分泌作用促進放射性皮膚損傷愈合的機制,為進一步研究ADSCs修復(fù)放射性皮膚損傷奠定基礎(chǔ)。本課題主要研究內(nèi)容如下:第一部分大鼠脂肪干細胞、成纖維細胞的分離與鑒定目的分離并提取高純度的r ADSCs、r Fbs。方法選取雄性SD大鼠,重約200-250g,腹腔注射水合氯醛麻醉后,取腹部皮下脂肪組織,將組織剪至2-4mm小塊,加入配好的混合酶溶液中,按要求設(shè)置組織處理器程序,分離出r ADSCs。取大鼠腹部皮膚組織,剪去皮下粘膜,將組織剪成4mm×5mm大小的組織塊,放入分散酶溶液消化過夜,分離表皮和真皮,將真皮放入膠原酶溶液反應(yīng),分離出r Fbs。將兩種細胞分別在鏡下觀察其形態(tài);CCK-8法描繪兩種細胞的生長曲線;流式細胞術(shù)檢測CD29、CD44、CD31和CD45在r ADSCs細胞表面的表達;成脂、成骨誘導(dǎo)培養(yǎng)鑒定其多向分化潛能;HE染色、波形蛋白免疫組化鑒定r Fbs。結(jié)果在倒置顯微鏡下,大鼠原代r ADSCs呈長梭形或多邊形,與成纖維細胞相似,細胞核大而明顯,位置居中,核仁明顯;大鼠皮膚r Fbs在顯微鏡下形態(tài)清楚,呈現(xiàn)長梭形,核較小,呈卵圓形居中,細胞集落呈現(xiàn)旋渦狀或者菜花狀。流式細胞術(shù)顯示r ADSCs中CD29、CD44呈陽性表達,表達率分別為99.42%、98.29%,CD31、CD45呈陰性表達,誘導(dǎo)分化鑒定發(fā)現(xiàn)r ADSCs具有成骨、成脂分化分能力,證明其有多向分化潛能。光鏡下,HE染色切片可見r Fbs胞漿淡染,深染的細胞核呈圓形或橢圓形居于正中,免疫組化顯示r Fbs的Vimentin陽性表達率幾乎為100%。CCK-8表明兩種細胞的都具有很強的增殖能力。結(jié)論通過酶消法提取的r ADSCs和r Fbs具有較高的純度和活性,為后面建立細胞模型作了充分準備。第二部分體外實驗檢測放射對r Fbs功能的影響目的通過建立輻射對r Fbs的損傷模型,研究在不同輻射劑量、輻射后不同時間點下r Fbs的狀況,為下一步共培養(yǎng)模型的建立確定合適的輻射劑量和時間觀測點。方法用直線加速器將r Fbs分別予以2 Gy、8 Gy、16 Gy、24 Gy、32Gy的劑量進行輻照,設(shè)置未受輻照的對照組。用CCK-8法描繪各組r Fbs的生長曲線;在輻照后第2、4、6天,通過流式細胞術(shù)檢測各組r Fbs的細胞周期和細胞凋亡,進行對比。結(jié)果隨著輻射劑量的增加,r Fbs的增殖能力逐漸下降,細胞凋亡比率增加,細胞周期中G1期比例下降,G2期比例上升,G1/G2比值下降,在輻射后第二天變化最為明顯。結(jié)論輻射會導(dǎo)致r Fbs增殖能力下降,細胞凋亡增加,G2期阻滯,并且具有一定的劑量—效應(yīng)關(guān)系。根據(jù)實驗結(jié)果,我們選擇輻射劑量為8Gy建立共培養(yǎng)模型,在輻射后第2天進行相關(guān)指標(biāo)的檢測。第三部分體內(nèi)、體外實驗檢測r ADSCs通過旁分泌作用修復(fù)r Fbs放射性損傷目的將r ADSCs與輻射后的r Fbs隔以半透膜共同培養(yǎng),其培養(yǎng)基能夠促進大鼠放射性皮膚損傷的愈合,并且r ADSCs能夠通過旁分泌作用修復(fù)半透膜外r Fbs的放射性損傷,說明共培養(yǎng)模型的有效性,進一步探討ADSCs的修復(fù)機制。方法:在體內(nèi)實驗中建立大鼠放射性皮膚損傷模型,分別將r ADSCs、r ADSCs共培養(yǎng)基、完全培養(yǎng)基在創(chuàng)面周圍局部注射,通過創(chuàng)面面積測量、HE和Masson染色觀察創(chuàng)面組織結(jié)構(gòu)變化,PCNA和Tunel實驗檢測細胞增殖和凋亡,免疫組化方法檢測創(chuàng)面組織CD31、TNF-α的表達,Western blot檢測生長因子VEGF的水平,綜合反映大鼠放射性創(chuàng)面的愈合狀況。在體外實驗中,按照分組進行細胞處理:A組—r ADSCs與輻射后r Fbs共培養(yǎng)組;B組—NRK與輻射后r Fbs共培養(yǎng)組;C組—輻射后r Fbs單獨培養(yǎng)組;D組—r ADSCs與r Fbs共同培養(yǎng)組;E組—r Fbs單獨培養(yǎng)組。通過劃痕實驗比較各組r Fbs遷移能力的差異,流式細胞儀檢測各組r Fbs的細胞凋亡和細胞周期,Ed U檢測各組r Fbs的細胞增殖能力,Western blot檢測r Fbs的CCND1、P53的水平,ELISA檢測培養(yǎng)基中TGF-β、GCSF、COLⅠ、COLⅡ等指標(biāo)。結(jié)果在大鼠放射性皮膚損傷模型中,r ADSCs共培養(yǎng)基應(yīng)用于大鼠放射性創(chuàng)面中,可以降低炎癥反應(yīng),促進血管生成和膠原產(chǎn)生,增強細胞增殖能力,促進創(chuàng)面愈合。在體外實驗中,輻射可以引起r Fbs增殖、遷移能力下降,細胞凋亡增加,G2期阻滯,CCND1蛋白水平下降,P53蛋白水平升高,分泌細胞因子TGF-β水平升高,而GCSF、CollagenⅠ、CollagenⅡ水平下降。r ADSCs共培養(yǎng)模型中r Fbs增殖遷移能力明顯上升,細胞凋亡減少,G2期阻滯減輕,CCND1蛋白水平升高,在培養(yǎng)基中,TGF-β水平下降,而GCSF、CollagenⅠ、CollagenⅡ水平升高。結(jié)論r ADSCs與輻射后r Fbs的共培養(yǎng)基在動物模型中能夠有效促進大鼠放射性皮膚損傷創(chuàng)面愈合。共培養(yǎng)模型中r ADCSs可以通過旁分泌作用增強放射后r Fbs的增殖、遷移能力,降低細胞凋亡,修復(fù)r Fbs放射性損傷。
[Abstract]:Background and objective: radiotherapy, occupation exposure, accidents, war and other reasons can cause radiation damage to the skin. High doses of radiation can cause acute radiation ulcer, small doses of radiation caused by radioactive dermatitis, delayed healing can cause chronic radiation ulcer, if not timely treatment, and even can lead to skin cancer. Put radioactive Radioactive skin injury in clinic usually manifests as potential, progress, characteristics of difficult to heal, radiation-induced skin ulcers to the deep development, can damage the muscle, blood vessels, nerves and other deep tissue, seriously affecting the quality of life of patients, and even life-threatening. At present in the near bed of radioactive skin damage to radiation-induced skin ulcers most often See, the removal of diseased tissue, with skin flaps is the most common and effective treatment methods. In recent years, adipose derived stem cells (ADSCs) as a research hotspot in the field of regenerative medicine and tissue engineering, extracted from adipose tissue, it is a convenient,.ADSCs for source widely belongs to mesenchymal stem cells (MSCs), with multiple differentiation potential The biological characteristics and immune immunity, and can secrete a variety of cytokines. Therefore, ADSCs has the prospect of widespread clinical use. The wound ADSCs effects on radiation-induced skin injury, in many clinical experiments, animal experiments have confirmed that ADSCs can promote tissue repair. Because ADSCs has a variety of tissue to differentiate from In the process of repairing the wound, it can directly differentiate into damaged tissue cells to participate in the repair process. However, more and more researchers believe that the paracrine effect plays a leading role in repairing the wound, and ADSCs can secrete a large number of cytokines including growth factors and anti-inflammatory factors as the ADSCs research mechanism continues to deepen. We can also release special functional extracellular vesicles (EVs) to play the role of anti apoptosis, anti-inflammatory, and angiogenesis. However, further research is needed for the mechanism of ADSCs reversal of radiation skin damage. We are separated by half of the rat adipose stem cells (R ADSCs) with the irradiated rat skin fibroblasts (R Fbs). To establish a cell model of R ADSCs to repair radioactive skin injury, observe the repair of radioactivity damage of R Fbs by co culture, explore the mechanism of ADSCs to promote the healing of radioactive skin injury through paracrine effect of ADSCs, and lay the foundation for further study of ADSCs repair of radioactive skin injury. The main contents of this topic are as follows: Part of the rat adipose stem cells, the isolation and identification of fibroblasts, separation and identification of high purity R ADSCs, R Fbs. method was used to select male SD rats and weighed about 200-250g. After intraperitoneal injection of chloral hydrate, the abdominal subcutaneous adipose tissue was taken and the tissue was cut to 2-4mm small pieces and added to the mixed enzyme solution to set the tissue according to the requirement. The processor program separates the R ADSCs. from the abdominal skin tissue of the rat, cuts the skin of the skin of the rat, cuts the subcutaneous mucous membrane, cuts the tissue into the tissue block of 4mm x 5mm, and Digestis for the night, separates the epidermis and the dermis, and puts the dermis into the collagenase solution, and separates the R Fbs. to observe the morphology of the two cells under the microscope, and the CCK-8 method depicts two fines. Cell growth curve; flow cytometry was used to detect the expression of CD29, CD44, CD31 and CD45 on the surface of R ADSCs cells; lipid, osteogenic induction to identify its multidirectional differentiation potential; HE staining, and vimentin immunohistochemical identification of R Fbs. results under inverted microscope, the primary R ADSCs was spindle shaped or polygonal, similar to fibroblasts, and nuclei. It was large and obvious, the position was in the middle and the nucleolus was obvious. The skin R Fbs of the rat skin was clear under the microscope, showed a long shuttle shape, the nucleus was small, the egg was oval in the middle, the cell colony appeared whirlpool or cauliflower. The flow cytometry showed that CD29 and CD44 were positive expression in R ADSCs, and the expression rate was 99.42%, 98.29%, CD31, CD45 showed negative expression, induced differentiation, respectively. It was found that R ADSCs had the ability of osteogenesis and lipid differentiation, which proved to have multiple differentiation potential. Under light microscope, HE staining section showed R Fbs cytoplasm light dyeing, deep dyed nuclei were round or oval in the middle, and immunohistochemistry showed that the positive rate of Vimentin positive expression of R Fbs was almost 100%.CCK-8 indicating that all two kinds of cells had strong proliferation. Conclusion R ADSCs and R Fbs, extracted by enzyme digestion, have high purity and activity, and make full preparation for the subsequent establishment of cell models. Second in vitro, the effect of radiation on the function of R Fbs was detected by establishing the radiation damage model to R Fbs, and the study of R Fbs at different radiation doses and at different time points after radiation. In order to establish the appropriate radiation dose and time observation point for the establishment of the next co culture model, the R Fbs was irradiated with the dose of 2 Gy, 8 Gy, 16 Gy, 24 Gy and 32Gy by linear accelerator. The growth curve of R Fbs in each group was depicted with CCK-8 method, and the flow cell was passed by the flow cell on the 2,4,6 day after irradiation. The cell cycle and apoptosis of R Fbs in each group were measured and compared. Results with the increase of radiation dose, the proliferation ability of R Fbs decreased gradually, the ratio of apoptosis increased, the proportion of G1 in the cell cycle decreased, the proportion of G2 phase increased, the ratio of G1/G2 decreased, and the most obvious change was second days after radiation. Conclusion radiation will lead to Fbs proliferation of R. Decreased capacity, increased apoptosis, G2 phase block, and a dose effect relationship. According to the results of the experiment, we chose a radiation dose of 8Gy to establish a co culture model and test the related indexes at second days after radiation. In the third part, in vitro, the R ADSCs was detected by paracrine effect to repair the R Fbs radioactivity damage order. The co culture of R ADSCs and R Fbs after radiation is co cultured with a semi permeable membrane. The medium can promote the healing of radiation skin injury in rats, and R ADSCs can repair the radioactivity damage of the semi permeable R Fbs through paracrine effect, indicating the effectiveness of the co culture model and further exploring the repair mechanism of ADSCs. Method: in the experiment in vivo The rat model of radiation skin injury was established. The R ADSCs, R ADSCs co culture medium, complete medium were injected around the wound surface, the wound area was measured, the tissue structure changes were observed by HE and Masson staining. The proliferation and apoptosis of the cells were detected by PCNA and Tunel, and the immunohistochemical method was used to detect the expression of CD31, TNF- a, Wes, and Wes. Tern blot measured the level of growth factor VEGF and comprehensively reflected the healing status of radiation wound in rats. In vitro experiments, cell processing was carried out in groups: group A - R ADSCs and post radiation R Fbs co culture group; B group - NRK and post - radiation R Fbs co culture group; Group E - R Fbs alone culture group. The difference in migration ability of R Fbs was compared by scratch test. Flow cytometry was used to detect the cell apoptosis and cell cycle of R Fbs in each group. Ed U detected the cell proliferation ability of R Fbs. In the rat model of radiation skin injury, the R ADSCs co culture medium is applied to the radiation wound of rats, which can reduce the inflammatory response, promote angiogenesis and collagen production, enhance the proliferation of cells and promote the healing of the wound. In vitro, radiation can cause the proliferation of R Fbs, the decrease of migration ability, the increase of cell apoptosis, G2 phase block, C The level of CND1 protein decreased, the level of P53 protein increased and the level of secreted cytokine TGF- beta was elevated, while the level of GCSF, Collagen I and Collagen II decreased in the.R ADSCs co culture model, and the proliferation and migration of R Fbs increased significantly, the apoptosis decreased, the G2 phase retardation decreased, the CCND1 protein level increased. I concluded that the level of Collagen II was elevated. Conclusion the co culture of R ADSCs and R Fbs after radiation can effectively promote the healing of radioactive skin injury in rats. In co culture model, R ADCSs can enhance the proliferation of R Fbs, transfer ability, decrease apoptosis and repair R Fbs radioactive damage through paracrine effect.

【學(xué)位授予單位】：第二軍醫(yī)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R622

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