通过共培养模型初步探讨脂肪干细胞促进放射性皮肤损伤修复的机制

发布时间：2018-04-20 13:30

本文选题：脂肪干细胞 + 成纤维细胞　；参考：《第二军医大学》2017年硕士论文

【摘要】：研究背景和目的:放疗、职业暴露、意外事故、战争等多种原因都可以对皮肤造成放射性损伤。大剂量的辐射可致急性放射性溃疡,小剂量辐射引起放射性皮炎,迁延不愈可造成慢性放射性溃疡,若不及时治疗,甚至可导致放射性皮肤癌。放射性皮肤损伤在临床上通常表现为潜在性、进展性、难以愈合的特点,放射性皮肤溃疡向深部发展,可以损伤肌肉、血管、神经等深部组织,严重影响患者的生活质量,甚至威胁生命。目前在临床中放射性皮肤损伤以放射性皮肤溃疡最为常见,切除病变组织,用皮瓣修复缺损是最为常见和有效的治疗方法。近年来,脂肪干细胞(ADSCs)作为再生医学和组织工程领域内的研究热点,从脂肪组织中提取,具有取材方便,供源广泛的特点。ADSCs属于间充质干细胞(MSCs),具有多项分化潜能和免疫豁免的生物学特性,并能够分泌多种细胞因子。因此,ADSCs具有广泛的临床使用前景。将ADSCs作用于放射性皮肤损伤的创面,在诸多临床实验、动物实验中都证实了ADSCs促进组织修复的能力。由于ADSCs具有向多种组织分化的潜能,因此在修复创面的过程中,可以直接分化为受损的组织细胞参与修复进程。然而随着对ADSCs研究机制的不断深入,越来越多的研究者们认为旁分泌作用在修复创面的过程中起主导作用,ADSCs可以分泌大量细胞因子包括生长因子、抗炎因子等,还可以释放具有特殊功能的细胞外囊泡(EVs),发挥抗凋亡、抗炎、促进血管生成的作用。然而,对于ADSCs逆转放射性皮肤损伤的机制还需要进一步的研究。我们通过将大鼠脂肪干细胞(r ADSCs)与辐射后的大鼠皮肤成纤维细胞(r Fbs)隔以半透膜共同培养建立r ADSCs修复放射性皮肤损伤的细胞模型,观察共培养对r Fbs放射性损伤的修复,探索ADSCs通过旁分泌作用促进放射性皮肤损伤愈合的机制,为进一步研究ADSCs修复放射性皮肤损伤奠定基础。本课题主要研究内容如下:第一部分大鼠脂肪干细胞、成纤维细胞的分离与鉴定目的分离并提取高纯度的r ADSCs、r Fbs。方法选取雄性SD大鼠,重约200-250g,腹腔注射水合氯醛麻醉后,取腹部皮下脂肪组织,将组织剪至2-4mm小块,加入配好的混合酶溶液中,按要求设置组织处理器程序,分离出r ADSCs。取大鼠腹部皮肤组织,剪去皮下粘膜,将组织剪成4mm×5mm大小的组织块,放入分散酶溶液消化过夜,分离表皮和真皮,将真皮放入胶原酶溶液反应,分离出r Fbs。将两种细胞分别在镜下观察其形态;CCK-8法描绘两种细胞的生长曲线;流式细胞术检测CD29、CD44、CD31和CD45在r ADSCs细胞表面的表达;成脂、成骨诱导培养鉴定其多向分化潜能;HE染色、波形蛋白免疫组化鉴定r Fbs。结果在倒置显微镜下,大鼠原代r ADSCs呈长梭形或多边形,与成纤维细胞相似,细胞核大而明显,位置居中,核仁明显;大鼠皮肤r Fbs在显微镜下形态清楚,呈现长梭形,核较小,呈卵圆形居中,细胞集落呈现旋涡状或者菜花状。流式细胞术显示r ADSCs中CD29、CD44呈阳性表达,表达率分别为99.42%、98.29%,CD31、CD45呈阴性表达,诱导分化鉴定发现r ADSCs具有成骨、成脂分化分能力,证明其有多向分化潜能。光镜下,HE染色切片可见r Fbs胞浆淡染,深染的细胞核呈圆形或椭圆形居于正中,免疫组化显示r Fbs的Vimentin阳性表达率几乎为100%。CCK-8表明两种细胞的都具有很强的增殖能力。结论通过酶消法提取的r ADSCs和r Fbs具有较高的纯度和活性,为后面建立细胞模型作了充分准备。第二部分体外实验检测放射对r Fbs功能的影响目的通过建立辐射对r Fbs的损伤模型,研究在不同辐射剂量、辐射后不同时间点下r Fbs的状况,为下一步共培养模型的建立确定合适的辐射剂量和时间观测点。方法用直线加速器将r Fbs分别予以2 Gy、8 Gy、16 Gy、24 Gy、32Gy的剂量进行辐照,设置未受辐照的对照组。用CCK-8法描绘各组r Fbs的生长曲线;在辐照后第2、4、6天,通过流式细胞术检测各组r Fbs的细胞周期和细胞凋亡,进行对比。结果随着辐射剂量的增加,r Fbs的增殖能力逐渐下降,细胞凋亡比率增加,细胞周期中G1期比例下降,G2期比例上升,G1/G2比值下降,在辐射后第二天变化最为明显。结论辐射会导致r Fbs增殖能力下降,细胞凋亡增加,G2期阻滞,并且具有一定的剂量—效应关系。根据实验结果,我们选择辐射剂量为8Gy建立共培养模型,在辐射后第2天进行相关指标的检测。第三部分体内、体外实验检测r ADSCs通过旁分泌作用修复r Fbs放射性损伤目的将r ADSCs与辐射后的r Fbs隔以半透膜共同培养,其培养基能够促进大鼠放射性皮肤损伤的愈合,并且r ADSCs能够通过旁分泌作用修复半透膜外r Fbs的放射性损伤,说明共培养模型的有效性,进一步探讨ADSCs的修复机制。方法:在体内实验中建立大鼠放射性皮肤损伤模型,分别将r ADSCs、r ADSCs共培养基、完全培养基在创面周围局部注射,通过创面面积测量、HE和Masson染色观察创面组织结构变化,PCNA和Tunel实验检测细胞增殖和凋亡,免疫组化方法检测创面组织CD31、TNF-α的表达,Western blot检测生长因子VEGF的水平,综合反映大鼠放射性创面的愈合状况。在体外实验中,按照分组进行细胞处理:A组—r ADSCs与辐射后r Fbs共培养组;B组—NRK与辐射后r Fbs共培养组;C组—辐射后r Fbs单独培养组;D组—r ADSCs与r Fbs共同培养组;E组—r Fbs单独培养组。通过划痕实验比较各组r Fbs迁移能力的差异,流式细胞仪检测各组r Fbs的细胞凋亡和细胞周期,Ed U检测各组r Fbs的细胞增殖能力,Western blot检测r Fbs的CCND1、P53的水平,ELISA检测培养基中TGF-β、GCSF、COLⅠ、COLⅡ等指标。结果在大鼠放射性皮肤损伤模型中,r ADSCs共培养基应用于大鼠放射性创面中,可以降低炎症反应,促进血管生成和胶原产生,增强细胞增殖能力,促进创面愈合。在体外实验中,辐射可以引起r Fbs增殖、迁移能力下降,细胞凋亡增加,G2期阻滞,CCND1蛋白水平下降,P53蛋白水平升高,分泌细胞因子TGF-β水平升高,而GCSF、CollagenⅠ、CollagenⅡ水平下降。r ADSCs共培养模型中r Fbs增殖迁移能力明显上升,细胞凋亡减少,G2期阻滞减轻,CCND1蛋白水平升高,在培养基中,TGF-β水平下降,而GCSF、CollagenⅠ、CollagenⅡ水平升高。结论r ADSCs与辐射后r Fbs的共培养基在动物模型中能够有效促进大鼠放射性皮肤损伤创面愈合。共培养模型中r ADCSs可以通过旁分泌作用增强放射后r Fbs的增殖、迁移能力,降低细胞凋亡,修复r Fbs放射性损伤。
[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.

【学位授予单位】：第二军医大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R622

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