【作者】 杨会营；

【导师】 原林；

【作者基本信息】 南方医科大学 ， 人体解剖学与组织胚胎学， 2014， 博士

【摘要】 背景：筋膜学说认为,人作为一个有机整体从筋膜学角度上可划分为两个系统,即支持与储备系统和功能系统。生物通过支持储备系统来维持较长的生命周期以及维持机体稳定的内环境。功能系统在支持与储备系统支持下维持结构与功能的相对稳定,支持与储备系统则不断的为功能系统的细胞更新和功能活动提供细胞源泉和细胞活动所需的营养物质。功能系统是以功能细胞的专能特化为特点,而作为支持与储备系统的筋膜支架是以非定向干细胞为核心,通过向各种定向干细胞分化,进而分化为功能细胞。支持与储备系统不断为功能系统的更新提供细胞补充,并为功能系统的各种细胞的更新、代谢提供了一个稳定的内环境。肿瘤是机体在各种致癌因素作用下,局部组织的某些细胞在基因水平上突变,从而失去对其增殖的正常调控,导致其克隆性异常增生而形成的异常病变。主要致癌因素有：化学诱变剂、X线、放射线辐射、病毒感染以及长期物理刺激等等。这些因素皆可引起基因突变,促使细胞的生长与分裂失控。筋膜学说认为,正常功能细胞在损伤的过程中会释放细胞因子诱导定向干细胞向功能细胞分化,来补充损伤死亡的细胞,定向干细胞在分化的过程中又释放干细胞趋化因子趋化招募干细胞向定向干细胞的所在部位移动并穿过基底膜分化为定向干细胞。而在肿瘤的发生过程中,基因突变导致细胞的异常增殖分裂失控,相当于定向干细胞分化出不具备正常生理功能的功能细胞,定向干细胞的分化又产生干细胞趋化因子,诱导干细胞向定向干细胞集中和穿过基底膜补充定向干细胞的消耗,此过程的不断重复导致肿瘤的增大。同时如果大量的干细胞穿过基底膜就会导致基底膜的崩溃,干细胞与定向干细胞之间失去了基底膜的屏障,定向干细胞产生的趋化因子和定向分化因子对会在基底膜深层发生,肿瘤的侵袭转移正是这种现象在局部的体现。乳腺癌是女性常见恶性肿瘤之一,约占25%。几乎所有患者在术后要接受放射治疗,术后放射治疗已经成为临床治疗的常规手段。但是部分患者的局部控制以及生存率并没有得到很好的改善。特别是为了得到美观而进行乳房再造的患者尤其是自体脂肪移植填充再造的患者,术后存在很大复发的风险。有研究报道,保乳手术术后复发率和乳房全切术相比,保乳手术术后复发率高。脂肪间充质干细胞(adipose-derived mesenchymal stem cells, AMSCs)筋膜脂肪组织中具有干细胞特性的细胞群,是从筋膜结缔组织中获取的一类具有多向分化潜能的成体干细胞。Zuk等首次从人的脂肪组织悬液中分离出了可以在体外稳定增殖、具有多向分化能力的脂肪组织提取细胞(processed lipoaspirate cells, PLA细胞),与我们研究的AMSCs是一致的。与其它成体干细胞比较,AMSCs具有来源广泛、取材方便,创伤小且不存在免疫排斥反应等优点。大量文献报道,AMSCs具有跨胚层的多向分化潜能。AMSCs可作为干细胞种子细胞,通过分化对功能细胞进行补充,也可刺激机体分泌各种因子,促进机体的损伤修复。在诱导分化作用下,AMSCs可以分化为中胚层来源的脂肪细胞、成骨细胞、软骨细胞、心肌细胞,外胚层来源的神经细胞,也可以分化为内胚层来源的内皮细胞,及肝细胞等。由于AMSCs具备上述特性,有研究报道,用AMSCs治疗乳腺癌术后组织损伤,促进了伤口的愈合,得到了很好的临床效果。但是有研究表明,AMSCs可以促进乳腺癌术后复发。所以,AMSCs用于乳腺癌术后重建及损伤修复的安全性有待于进一步研究。目的通过观察体夕AMSCs对乳腺癌细胞电离辐射敏感性的影响以及体内移植瘤辐射抵抗的分析,探讨AMSCs对乳腺癌辐射敏感性影响以及其作用机制,为临床乳腺癌术后治疗以及术后乳房脂肪填充再造的风险性的评估提供部分实验依据,进一步为筋膜学说有关干细胞与肿瘤的假说提供部分实验佐证。1、通过对酶消化法分离培养AMSCs,观察非特异性筋膜结缔组织中是否存在AMSCs,乳腺癌患者来源与肥胖患者来源的AMSCs进行比较,观察两种患者组织来源AMSCs的不同；2、应用AMSCs上清液培养乳腺癌细胞,观察乳腺癌细胞增殖情况,AMSCs能否促进乳腺癌细胞,这是本实验的前提；3、通过Transwell法,共培养AMSCs和乳腺癌细胞,观察乳腺癌细胞能否趋化间充质干细胞,对AMSCs有趋化招募作用；4、通过克隆形成实验、DNA损伤修复实验,观察AMSCs上清液培养的乳腺癌细胞对电离辐射的抵抗情况,AMSCs上清液能否促进乳腺癌细胞对电离辐射的抵抗；5、Western Blot及Elisa酶联免疫吸附实验等技术检测AMSCs和乳腺癌细胞中IGF-1及IGF-1R的表达,初步探讨AMSCs促进辐射抵抗的机制；6、AMSCs和乳腺癌细胞悬液移植于BALB/c-nu/nu小鼠皮下,观察皮下移植瘤对电离辐射抵抗情况,进一步验证AMSCs是否促进乳腺癌对电离辐射的抵抗。方法1、抽脂来源的脂肪组织,通过酶消化法分离、培养AMSCs.通过形态学、功能学、流式细胞术鉴定所分离、培养的细胞是否具有间充质干细胞的特性,来判断所获取的脂肪组织中是否具有间充质干细胞。2、细胞培养：本实验MCF-7和BT474两株乳腺癌细胞系。AMSCs和MCF-7用高糖DMEM加10%胎牛血清(100μ/ml青霉素和100μ/ml链霉素)培养,BT474细胞系用RPMI1640加10%胎牛血清(100u/ml青霉素和100u/ml链霉素)培养。所有的细胞置于5%CO2,37℃孵育箱。3、MTT法检测乳腺癌细胞增殖情况。乳腺癌细胞种植于96孔板中。孵育24小时后,加入AMSCs上清液培养24-72小时,AMSCs上清液与普通培养基比例为1：1。按条件培养后,移除培养液加入50μl MTT2mg/ml溶液孵育2小时。终止培养,加入100μl二甲基亚砜,置摇床上低速振荡10mins,使结晶物充分溶解。在酶联免疫检测仪OD570nm处测量各孔的吸光值。实验至少重复3次。4、细胞迁移实验采用Transwell小室法。AMSCs种植于PVDF膜上层培养,乳腺癌细胞置于下层,5%CO2,37℃培养箱内培养。48小时后,用棉签拭去未穿过的AMSCs,甲醛固定,结晶紫染色,显微镜下观察,拍照。5、AMSCs及乳腺癌细胞分泌的IGF-1检测。细胞培养48小时后,上清液离心,-80℃保存待测。应用Elisa酶联免疫吸附实验试剂盒检测IGF-1的分泌情况。6、克隆形成实验、DNA损伤修复检测实验来观察乳腺癌细胞对电离辐射的抵抗情况。细胞经消化制成单细胞悬液后,按照预定数量接种于6孔板中,设0,2,4,6,8五个剂量组,每个剂量组三个复孔。照射后将细胞置于37℃、5%CO2饱和湿度培养箱内继续培养12-14天,期间根据培养液pH值变化适时更换新鲜培养液。当培养板孔中出现肉眼可见克隆时,终止培养,弃去培养液,PBS清洗细胞2遍,甲醇固定,1%结晶紫乙醇溶液染色,显微镜下计数含50个细胞以上的克隆数,计算克隆形成率和存活分数,并运用GraphPad Prism5.0软件进行多靶单击模型曲线拟合,并计算辐射增敏因子SER10。DNA损伤修复检测是通过荧光显微镜观察计数γ-H2AX点,来反映乳腺癌细胞的辐射抵抗情况。7、Western blotting法检测辐射前后AMSCs以及乳腺癌细胞IGF-1R的表达情况。处理后的细胞提取总蛋白,按照BCA法测定蛋白浓度,加入5×SDS-PAGE loading Buffer进行蛋白变性,然后经电泳、转膜、封闭后,孵育一抗和二抗,最后采用ECL法在显影仪上进行显影。8、AMSCs和乳腺癌细胞混合悬液移植于BALB/c-nu/nu、鼠皮下,分为四组：空白对照组,AMSCs组,辐射组和辐射加AMSCs组,每组5只。测量肿瘤的体积,绘制生长曲线。生长延缓分析来评价肿瘤的辐射抵抗。通过免疫组化法评价肿瘤内IGF-1R的表达情况。9、所有的结果至少重复3次。实验数据应用SPSS13.0软件分析,实验数据采用均数±标准差表示,两样本的均数比较采用两样本独立样本t检验(Independent-Sample T Test)、两组以上的均数比较采用完全随机设计资料的方差分析(one-way ANOVA)进行统计分析,析因分析采用Two-way ANOVA. P<0.05为有统计学意义。结果1、通过临床标本获得脂肪组织分离培养AMSCs,所获细胞具有长梭形、多边形的形态学特征,细胞培养可以形成细胞集落。通过体外诱导可以分化成脂肪细胞、成骨细胞。细胞具有间充质干细胞特性。两种来源所分离血管碎片细胞相比较,乳腺癌患者来源的低于肥胖患者的收获率,t=-17.725,P<0.001,具有统计学意义。在原代细胞接种于培养瓶后,24小时后贴壁细胞的贴壁收获率,乳腺癌患者来源的原代细胞贴壁收获率低于肥胖患者的原代细胞贴壁收获率,经统计学分析,两组比较均为t=15.357,P<0.001,具有统计学意义。2、AMSCs上清液促进了乳腺癌细胞的增殖。MCF-7和BT474在有AMSCs上清液培养的情况下增殖的速度要高于对照组,通过析因分析,BT474和MCF-7两组乳腺癌细胞主效应统计量分别为F=53.636, F=50.164, P值均小于0.05,说明组别与时间之间存在交互效应,随着时间的变化,上清液组与普通培养基组的增殖变化有统计学差异,AMSCs上清液促进了两组乳腺癌细胞的增殖。3、乳腺癌细胞促进了AMSCs的趋化,对其有趋化迁移作用。transwell实验检测乳腺癌细胞MCF-7对AMSCs迁移趋化能力的影响。采用One-Way ANOVA进行统计分析,F=218.491, P<0.001,说明组间有统计学差异,具有统计学意义。组间比较,结果显示,单纯培养基培养的AMSCs穿过PVDF膜的细胞较少。AMSCs与MCF-7共培养的AMSCs穿过率显著高于对照组。AMSCs与MCF-7共培养经过辐射处理后,AMSCs穿过的细胞与其他组比较更显著增高。4、AMSCs上清液促进了乳腺癌细胞的辐射抵抗。克隆形成生存分析,经过不同剂量辐射处理之后,处理组MFC-7和BT474的存活分数有所增高,放射增敏比SER10分别为0.802和0.756。免疫荧光实验,两种乳腺癌细胞经过电离辐射后,和未加AMSCs上清液培养的乳腺癌细胞相比,两组加上清液培养的乳腺癌细胞γ-H2AX损伤点明显减少,统计学分析,差异具有统计学意义(BT474：t=13.50, p<0.001; MCF-7:t=11.88, p<0.001,5、AMSCs来源的IGF-1与乳腺癌细胞的辐射抵抗相关。AMSCs可以分泌IGF-1,而且经过电离辐射后随着时间的增加,分泌IGF-1的量也在增加(F=87.069, p<0.001)。MCF-7和BT474细胞可以分泌IGF-1,但是分泌量非常少,和AMSCs相比,采用One-Way ANOVA进行统计分析,差异具有统计学意义(F=382.431, p<0.001)。Western blot结果显示,AMSCs IGF-1R的表达水平相对较低,两种乳腺癌细胞MCF-7和BT474IGF-1R的表达水平较高。克隆形成存活实验来验证,用IGF-1R阻断剂抑制AG1024阻断,观察阻断前后的变化。两组乳腺癌细胞MCF-7和BT474得出结果相类似。阻断前后比较,medium组以及medium+RT组存活分数没有统计学差异(p>0.05); IGF-1组、GF-1+RT组、AMSCs上清液组与AMSCs上清液+RT组存活分数阻断前后统计学分析,差异具有统计学意义(p<0.05)。电离辐射后,IGF-1组和AMSCs上清液组与medium组相比,具有统计学差异(p<0.05),前二者存活分数显著增高,但是这种优势可以被IGF-1R阻断剂AG1024阻断。6、体内实验AMSCs促进了乳腺癌细胞的辐射抵抗。通过对生长曲线析因分析,辐射因素与组别间存在交互作用P=0.043; FRT.FRT*天数=25.743, P<0.001; F分组*天数, P=0.004)。单独效应进行比较,对照组辐射前后随时间的变化比较,各天数点P值均小于0.05,具有统计学意义,干细胞组辐射前后随时间的变化比较结果与对照组类似。辐射前各时间点各组比较,P值均大于0.05；辐射后两组比较,0、5天没有统计学意义(F=0.092,1.278; P=0.763,0.262),10、15、20、25天有统计学意义(F=14.478,19.308,20.588,23.424；P值均小于0.001)。无辐射各组各时间点间比较,对照组与干细胞组均具有统计学意义(F=24.328,31.209；P值均小于0.001)；辐射后各组各时间点间比较,对照组与干细胞组均具有统计学意义(F=11.142,24.497；P值均小于0.001)(图3-4A,表3-5A、B)。通过以上分析,AMSCs与电离辐射之间存在拮抗作用,说明AMSCs降低了乳腺癌对辐射的敏感性,促进了乳腺癌对辐射的抵抗。射线照射后,生长延缓分析表明,干细胞组延缓时间明显短于单纯乳腺癌细胞组,统计学分析,t=3.753,P=0.006,两组之间有统计学差异。(图3-4B,表3-6)免疫组织化学法,研究了移植瘤内IGF-1R的水平。电离辐射前两组比较,AMSCs和MCF-7混合移植组IGF-1R的表达水平显著高于单纯MCF-7移植组；电离辐射后两组相比,AMSCs和MCF-7混合移植组的IGF-1R的表达水平也比MCF-7移植组高。但是,电离辐射前后比较,单纯MCF-7移植组以及AMSCs和MCF-7混合移植组IGF-1R的表达水平显著降低。结论1,本实验所获得AMSCs具有多向分化能力,经免疫表型鉴定分析,显示符合间充质干细胞的特征。2,乳腺癌患者与肥胖患者的脂肪组织相比,乳腺癌患者组织来源AMSCs收获率降低,但是获得的AMSCs在形态、多向分化性质、增殖能力和表面标志等方面没有统计学差异。3, AMSCs可以促进乳腺癌细胞的增殖,降低乳腺癌细胞对电离辐射的敏感性,增强了乳腺癌细胞的辐射抵抗。4,通过体内外实验证明,AMSCs可以通过其分泌IGF-1降低了乳腺癌细胞对电离辐射的敏感性,促进乳腺癌细胞对电离辐射的抵抗。更多还原

【Abstract】 BackgroundThe fasciaology originated from the Chinese Digital Human Research by Professor Yuanlin. This theory holds that people as an organic whole can be divided into two systems by fasciaology, which called Support and Reserve system and Function system. Creatures sustain a longer life cycle and maintain a stable internal environment of the body by Support and Reserve System. The Function system keeps a relatively stable structure and function by Support and Reserve system. And the Support and Reserve system will continue to provide a source of nutrients to cell source and cellular activities for cell renewal and activities of the Function system. Functional system is characterized by special features of functional cells. While the fascial scaffold differentiate into functional cells through various orientations of stem cells by the core of non-committed stem cells, which from Support and Reserve System. It also provides cell supplementary and a stable environment for various cells’ updating and metabolism for Function system, simultaneously.Tumor is an abnormal lesion of the result of carcinogenic factors in body that some cells lose control of its normal growth regulation at the genetic level in local tissue, which was the cause of abnormal clonal proliferation. The main factors of carcinogenicity were including: a chemical mutagen, X-ray, radiation, viral infection, long-term physical stimulation and so on. These factors could cause genetic mutations, which can promote uncontrolled growth and division of cells. From the perspective of fasciaology that the normal function will release cell differentiation factor to induce stem cells directly differentiate into functional cells during the process of damage, which will repair cell damage. And committed stem cells will release chemokines to recruited stem cells through the basement membrane to the parts of committed stem cells during their differentiation. The mutations would cause cell abnormal division during the process of the progression of tumor, as the committed stem cells differentiate into abnormal function cells. Then committed stem cells will generate stem cells chemokines, which will induce stem cells, concentrate to committed stem cells and complement the consumption of them through the basement membrane. This process will repeat more and more and lead to an increase of tumor. Moreover, if a large number of stem cells through the basement membrane will lead to the collapse of the basement membrane that the basement membrane between stem cells and committed stem cells will loss. The committed stem cells chemokines and directional barrier of stem cells will occur in the deep base film. The spread factor is partially reflected in this phenomenon.Breast cancer is considered the most commonly occurring cancers in women (approximately25%). Most patients suffering from cancer require mandatory radiotherapy after breast-conserving surgery or mastectomy, but the locoregional control and survival rate of breast cancer patients remains unsatisfactory. However, it has been reported that AMSCs might dramatically favor breast cancer recurrence. These results suggest that AMSCs are not safe for breast reconstruction or damage repair after cancer surgery in clinical applications.AMSCs are adult stem cells in the adipose tissue, which have multi-differentiation potential obtained from the fascial connective tissue. Zuk, the first one to isolate adipose tissue extracted cells (processed lipoaspirate cells, PLA cells) from human adipose tissues, which possess multipotent differentiation and can be steadily proliferating in vitro. Compared with other adult stem cells, the advantages of PLA cells are as follow: wide variety of sources, convenience of drawing, less trauma, no immunological rejection and so on. Experiments showed thatAMSCs have cross-mesoderm pluripotency.AMSCs not only can be used for functional cells complement as stem cells, but also can stimulate the body to secrete a variety of factors to promote the body’s repair. In the induction, it can differentiate into the germ layers cells, as fat cells, osteoblasts, chondrocytes, myocardial cells, neural cells derived from ectoderm, also endothelial cells and liver cells derived from endoderm.The studies have reported that AMSCs can be used for the treatment of tissue damage after breast cancer surgeries because of AMSCs have the above characteristics. But other studies have shown that AMSCs can promote breast cancer recurrence. So the safety of AMSCs for breast reconstruction and repair damage needed further study.ObjectiveThrough the observation of the effects of AMSCs in the sensitivity of ionizing radiations on breast cancer cells in vitro and the analysis of radiation resistance in vitro to explore the mechanisms and whether AMSCs can promote breast cancer development. It provides some experimental basis for risks of clinical postoperative treatment and fat reengineering fill of breast cancer. Further it also provides some experimental evidences for fascia theories, which about the hypothesis of stem cell and cancer.1To observe whether AMSCs is present in non-specific connective tissue through the acquisition of the isolation and culture of fat tissue from liposuction, comparing with the stem cell lines.2To observe the proliferation of breast cancer cells by using AMSCs supernatant to culture them. And whether AMSCs can promote breast cancer cells, which is the premise of this experiment.3To observe whether the existence of chemotaxis and recruitment of breast cancer cells to AMSCs by using the transwell method to culture AMSCs and breast cancer cells.4To observe resistance of breast cancer cells to ionizing radiation through the colony formation assay and DNA damage repair experiments, which have been cultured by AMSCs supernatant. 5The expression of IGF-1R of AMSCs and breast cancer cells were detected by Western Blot and ELISA technique to explore the mechanism of AMSCs to promote radiation resistance.6To observe the resistance of ionizing radiation by transplanting AMSCs and breast cancer cells suspension into BALB/c-nu/nu mice.There was further confirmed that AMSCs can promote the resistance of breast cancer to ionizing radiation.Methods1The fat tissues were isolated by enzyme digestion and cultured adipose derived stem cells by liposuction and using morphology, functional learning and flow cytometry to identify whether have the characteristics of AMSCs, which to determine the existence of AMSCs in non-specific fascia connective tissue.2Cell Culture: There were MCF-7and BT474two breast cancer cells in this experiment. AMSCs and MCF-7were cultured with high glucose DMEM plus10%fetal bovine serum (100u/ml penicillin and streptomycin100u/ml) and BT474cells were cultured with RPMI1640plus10%fetal bovine serum (100u/ml penicillin and100u/ml streptomycin). All cells were placed in5%CO2,37℃incubator.3Cell proliferation was measured using the3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium (MTT) dye reduction method. In brief, breast cancer cells (2×103/100μL) were plated into each well of96-well plates. After24h incubation, AMSCs culture supernatants were added into wells for24-72h and incubated an additional2h with50μL MTT solution (2mg/mL). The medium was subsequently removed, and the dark blue crystals were dissolved with100μL DMSO. The absorbance was measured using a microplate reader with reference wavelengths of570nm. The percent growth was determined relative to untreated controls. Each experiment was performed with triplicate samples and at least three times independently.4Cell migration assays were performed using the modified Boyden chamber method with an8-μm pore filter separating the top and bottom Transwell chambers (Corning, America). The AMSCs (104cells/200μL DMEM-HG) were added to the top chamber, and MCF-7cells (5×104cells/500μL DMEM-HG+10%FBS) were added to the bottom chambers under a humidified atmosphere of5% CO2at37℃. After48h incubation, the cells that failed to migrate from the top surface of the filters were removed with cotton swabs. The migrating cells on the surface of the filters were fixed with methanol and stained with crystal violet. The migration was quantitated by counting cells in six selected fields randomly on each filter under a microscope at a x200magnification, and the graphs were depicted as the mean of three independent experiments.5The testing of IGF-1, which were secreted from AMSCs and breast cancer cells. AMSCs, MCF-7or BT-474cells (5×106) were incubated for48h in5-mL culture medium. The supernatants were centrifuged and stored at-80℃IGF-1was quantitated by ELISA in accordance with the manufacturer’s procedure (Human IGF-I Quantikine ELISA Kit; R&D). The detection limit of IGF-1was0.1ng/mL. All samples were tested in triplicate independently.6Cells were trypsinized, harvested and counted. Cells were seeded in triplicate at varying concentrations in6-well plates according to the dose irradiated with or without AMSC supernatants or IGF-1. After24-h incubation, the cells were exposed at2,4,6or8Gy of6MV X-rays generated by linear accelerator (Varian2300EX, Varian, Palo Alto, CA) at a dose rate of5Gy/min. The cells were then incubated for10-14days at37℃to form colonies. After that, the colonies were fixed with100% methanol, and then stained with crystal violet. Colonies containing≥50cells were counted by microscopic inspection. The surviving fractions were calculated with following equation:(mean number of colonies)/(number of inoculated cellsxplating efficiency). Plating efficiency was defined as follows:(mean number of colonies)/(the number of inoculated control cells, which were not exposed to radiation). Survival curves were fitted using the classic multi-target single-hit model (SF=1-(1-e-D/D0)N) using GraphPad prism soft. Treatment with IGF-1R antagonist AG1024(Calbiochem, San Diego, CA, USA) was2h before radiation.The repair of DNA damage was detected by fluorescent microscope counting gamma-H2AX points, to reflect the resistance of radiation of breast cancer cells. 7After incubated, cells were lysed in cell lysis buffer with a phosphatase inhibitor cocktail and proteinase inhibitor cocktail (Sigma), and the protein concentrations were quantified with a bicinchoninic acid protein assay kit (Pierce Biotechnology). Cell lysates were harvested, and western blot analysis was performed. Proteins were resolved by SDS-polyacrylamide gel (Bio-Rad) electrophoresis and transferred onto polyvinylidene difluoride membranes (Bio-Rad). Afterward, the membranes were blocked with nonfat milk for1hour at room temperature and followed by incubation with primary antibodies: rabbit anti-IGF-1R (Abcam) or mouse anti-β-actin (ProteinTech) overnight at4℃. After washing thrice, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies for1h at room temperature. Immunoreactive bands were visualized with ECL western blotting substrate (Pierce, Rockford, IL, USA). Each experiment was performed at least three times independently.8Suspensions of5x106MCF-7cells with or without AMSCs in a2:1ratio were injected into the subcutaneous tissue of the right hindlimbs of4-to6-week-old female BALB/c-nu/nu nude mice. After tumor cells implantation,33micrograms of17β-estradiol (Sigma) was administered by intramuscular injection weekly. When tumor volumes reached about150mm3, mice with MCF-7cells and mice with MCF-7cells and AMSCs were randomly assigned to control and treated groups (5mice per group), respectively. Mice in the treated groups were irradiated at a single dose of8Gy with a linear accelerator (Varian2300EX, USA) with a dose rate of500cGy/min with copper shielding. The source-to-target distance was100cm. Tumor volumes were calculated every5days using the formula (lengthxwidth2)/2. The length and width of the tumors were measured with calipers. When tumor volumes reached about2000mm3, the mice were killed, and tumor tissues were collected for immunohistochemistry (IHC).9Results represent the mean of at least3independent experiments. Student’s t-test was used for comparison of the difference between two groups. One-way ANOVA was used for comparison of the difference between more than two groups. Two-way ANOVA were carried out for factorial analysis. All p values lower than0.05 were considered statistically significant.Results1AMSCs exhibited long spindle and polygonal morphology and cell colonies. The two passage cells were adherent and had a spindle-shaped morphology; the detection of cell surface markers showed that the cells with ability of multiplication of mesenchym in vitro with a mesenchymal stem cell properties. The comparative results of the yield of stromal vascular fraction cells between obese-derived fat tissue and breast cancer-derived fat tissue was significantly, t=l7.725, P<0.001. The comparative results of the yield of adherent cells between obese-derived fat tissue and breast cancer-derived fat tissue was significantly, t=15.357, P<0.001.2AMSC supernatants promoted proliferation of human breast cancer cells. To investigate whether the susceptibility of breast cancer cells was affected by AMSCs, we cultured MCF-7and BT474cells with AMSC supernatants. We observed that the proliferation of MCF-7and BT474cells was increased in the presence of supernatants of AMSCs compared with the control group F=53.636, F=50.164, all P<0.05. These results suggested that there seem to be some cytokines secreted by AMSCs promote proliferation of breast cancer cells. This study provided an experimental premise for our subsequent experiments.3Human breast cancer cells induce recruitment of AMSCs. AMSCs have been shown to possess the ability to be recruited. These cells originate from adipose tissue throughout the whole body, which can produce secretions to promote the migration of these cells to the target damaged organs. We speculated that breast cancer cells might affect the behavior of AMSCs, particularly their recruitment. We therefore assessed the effect of breast cancer cells on AMSCs migration. In the presence of medium alone, only a few AMSCs migrated through the filters. In contrast, MCF-7cells dramatically induced AMSCs migration, especially after radiation, F=218.491, P<0.001. These data suggested that breast cancer cells might recruit AMSCs to increase their radiation resistance.4AMSC supernatants induced radioresistance in breast cancer cells. To confirm the effect of the supernatants of AMSCs on the radioresistance of breast cancer cells, we performed a clonogenic survival assay with MCF-7and BT474cells. Survival fraction curves indicated that pretreatment with supernatants promoted the clonogenic survival of both MCF-7and BT474cells after varying doses of radiation (SER10=0.802,0.756). To further evaluate the effect of supernatants of AMSCs on the radioresistance of MCF-7and BT474cells, we investigate the DNA damage response by measuring the number of y-H2AX foci after irradiation, which is a well-known marker of DNA double-strand breakage and repair. We observed that pretreatment with supernatants in combination with IR (6Gy) led to a dramatic reduction in the number of y-H2AX foci24h post-IR administration compared with exposure to IR alone(BT474: t=13.50, p<0.001; MCF-7:t=11.88, p<0.001). These results indicated that AMSCs supernatants induced radiation resistance in breast cancer cells.5IGF-1derived from AMSCs was correlated with ion radiation (IR) resistance in human breast cancer cells. The breast cancer cell lines MCF-7and BT474secrete lower levels of IGF-1into their culture supernatants as compared with AMSCs. However, the expression of IGF-1R in MCF-7and BT474cells were higher than that in AMSCs (F=382.431, p<0.001). Twenty-four hours after various dose of radiation, IGF-1R expression in MCF-7and BT474cells were gradually increased (F=87.069, p<0.001). While the secreted level of IGF-1in the culture supernatants of AMSCs after radiation was gradually increased in a time-dependent manner.To further confirm the effect of IGF-1derived from AMSCs on radioresistance in human breast cancer cells, we performed a clonogenic survival assay with MCF-7and BT474cells with or without IGF-1, anti-IGF-1R, or culture supernatants of AMSCs. After8Gy radiation, cultured with the supernatants of AMSCs or IGF-1, the survival fraction of both MCF-7and BT474cells were significantly increased (p<0.05). Under these experimental conditions, both MCF-7and BT474cells became highly resistant to radiation in the presence of culture supernatants of AMSCs (p<0.05), but this effect was inhibited by treatment with anti-IGF-1R neutralizing antibody (p<0.05). Similar results were obtained in the presence of IGF-1(p<0.05).6AMSCs induced radiation resistance of breast cancer cells in vivo.To investigate whether the radiation resistance of breast cancer cells could be affected by AMSCs in vivo, we co-injected MCF-7cells with or without AMSCs into BALB/c-nu/nu nude mice subcutaneously. Without ion radiation (IR), the growth curves indicated that the tumors in mice injected with MCF-7plus AMSCs grew significantly faster than the tumors in mice injected with MCF-7cells alone. Similarly, in mice subjected to ion radiation (IR), the tumors with MCF-7plus AMSCs grew significantly faster than the tumors with MCF-7cells alone. Two-way ANOVA were carried out for factorial analysis that indicated the crosstalk between AMSCs and the radioresistance of breast cancer, and AMSCs enhance the radiation resistance of breast cancers. Subsequently, tumor growth delay of the tumors with MCF-7plus AMSCs were significantly shorter than that of tumors with MCF-7alone (t=3.753, P=0.006). We further investigated the levels of IGF-1R of MCF-7tumor xenografts by immunohistochemical Staining. With or without IR, the tumors in mice injected with MCF-7cells alone expressed low detectable levels of IGF-1R, whereas the tumors in mice injected with MCF-7cells and AMSCs produced higher levels of IGF-1R. In irradiated mice, although the levels of IGF-1R of MCF-7tumors with or without AMSCs were significantly lower than in without IR group, the tumors injected with MCF-7and AMSCs cells produced higher levels of IGF-1R than the tumors injected with MCF-7cells alone. These results indicated that AMSCs in the tumor microenvironment might enhance radiation resistance in breast cancer cells in vivo.Conclusions1In our study, the isolated and cultured AMSCs showed similar cell morphology that was homogeneous, polygonal or fusiform shape as reported. The induction and differentiation have showed the ability to differentiate into adipogenic and osteogenic. Phenotypic identification was as reported. There were undifferentiated mesenchymal stem cells in human adipose tissue.2The comparative results of the yield of stromal vascular fraction cells and adherent cellsbetween obese-derived fat tissue and breast cancer-derived fat tissue was significantly, but the cell morphology, induction and differentiation, phenotypic identification were not significantly.3The AMSCs can promote the resistance of breast cancer cells to ionizing radiation, in vitro.4, The IGF-1could promote the resistance of breast cancer cells to ionizing radiation, which were derived from AMSCs, in vivo.更多还原