【作者】 赵阳；

【导师】 高建华； 鲁峰；

【作者基本信息】 南方医科大学 ， 整形外科学， 2013， 博士

【摘要】 目的世界范围内,女性乳腺癌发病率逐年增高。根据国际癌症研究中心报告,2008年调查结果显示新发乳腺癌在女性肿瘤发病的22.9%,死亡占女性肿瘤的13.7%。在中国,乳腺癌的发病趋势倾向年轻女性,新发生率和死亡率明显增高。随着乳腺癌诊疗水平的提高,早期诊断阳性率更加准确,乳腺癌的治疗方案也有所改变,保乳手术现今已成为欧美国家治疗和研究乳腺癌的热点。但仍有部分患者需要全乳切除术等治疗,术后乳腺缺如或者畸形造成患者严重的心理障碍,显著降低生活质量。乳腺癌术后对乳房整形的需求越来越多,从早期即时皮瓣乳房再造到假体置入,都有各自的缺点而让患者不能接受。材料填充技术近年在整形外科发展迅速,自体脂肪曾一度作为理想的填充材料,让整形外科医师看到希望,由于移植后脂肪其成活率低,远期效果差而几近淘汰。1995年Coleman成功完成自体颗粒脂肪移植丰乳术,让整形自体组织填充看到希望。2006年自体颗粒脂肪细胞辅助移植技术(Cell assisted lipotransfer, CAL)的应用,在整形外科领域,丰乳、面部凹陷整形和丰臀等方面的远期效果理想。移植脂肪组织的萎缩、囊肿、钙化和坏死率都有明显减少。CAL通过增加人脂肪来源干细胞(Human adipocyte-derived stem cells, hADSCs)含量,促进脂肪颗粒移植存活和脂肪细胞增殖生长,其成活率增长为30-70%[5]。hADSCs成脂分化中的调节机制研究是脂肪颗粒移植的重点,是组织塑形的关键。只有hADSCs向成熟正常细胞分化,才能保证移植的生物安全性。CAL在先天性乳房发育不全、外伤性乳腺畸形等方面取得良好的效果,可多次行CAL改善乳房外形,达到塑形效果。但对于女性乳腺癌患者治疗术后乳房整形,国内外还有争议。现今女性乳腺癌发病率有所上升,由于医疗条件的改善,早期发现乳腺癌的几率增加,乳腺外科逐渐乳腺癌保乳术作为乳腺癌手术治疗的首选术式。患者术后治疗方案个体化,使生存率得到延长,生活质量有所提高。但术后女性患者由于乳房缩小、不对称等因素影响到生活心理,对微创乳腺整形提出要求。传统二期假体置入隆乳术缺点为原有手术瘢痕明显增宽,术区因瘢痕粘连置放假体困难,术后局部反应重,乳房因瘢痕牵拉外形不满意等。此外乳房假体有可能破裂造成患者心理负担过重,影响生活,迫切需要微创有效的乳房整形。乳腺颗粒脂肪细胞辅助移植术,能解决以上不良情况,对局部隆乳可按需填充,具有最大限度的调节乳房外形、创伤小,减轻患者二次手术痛苦等优点。但对其安全性有很大争议。美国整形外科协会和法国整形外科协会对细胞辅助颗粒脂肪移植的生物安全性提出质疑,通过临床病例对照研究,仍未发现确切的证据说明其对乳腺癌复发、局部浸润以及远处转移有影响[8-10]。现今还需大量临床病例研究进行远期随访。体外实验及动物实验也没有明确说明细胞辅助的脂肪颗粒移植对乳腺癌术后局部复发、进展和远处转移,仍具有很多争议。乳腺癌细胞对hADSCs的影响是多方面的。其主要通过细胞之间旁分泌相应的细胞因子,互相刺激彼此生长、增殖、迁移。同时对hADSCs多向分化功能产生一定的影响。在CAL中,移植物主要植入皮下、乳腺脂肪及胸大肌等处。hADSCs根据植入物所处的微环境,主要促进脂肪组织生长和hADSCs成脂分化为主。在乳腺癌术后切除局部区域进行CAL,对hADSCs成脂分化产生的影响还不得而知。CAL因其增加hADSCs含量,对乳腺癌细胞的影响也是二期行CAL乳房整形手术应关注的焦点之一。移植的hADSCs是否在乳腺癌术后的微环境能成活和正常分化及其转归?对乳腺癌细胞侵袭力产生作用?主要作用机理是什么?因为考虑细胞之间的主要作用通过旁分泌机制调节细胞的生长、发育、分化和代谢,所以我们选用乳腺癌无转移的细胞株腺癌MCF-7和浸润性导管癌BT474作为研究对象,通过体外培养hADSCs、MCF-7、BT474细胞,生长旺盛时期,收集细胞无血清培养液,以及成脂条件培养液,分析细胞间的相互作用。从而为CAL的生物安全性提供理论依据。hADSCs成脂过程中,细胞转录因子影响起主导作用。现今研究得出。转录因子过氧化物酶体增殖物激活受体y(Peroxisome proliferator-activated receptor-y, PPARy)、固醇调节元件结合蛋白(sterol regulated element binding protein, SREBP1)、脂肪型脂肪酸结合蛋白(Fatty acid binding proteins,aP2)和(CCAAT/enhancer binding proteins,C/EBP)等均促进hADSCs(?)旨肪分化,各转录因子之间互相调节,促进细胞内脂肪形成,尤其以PPARy和SREBP1作用明显。各转录因子在不同细胞还存在其他功能,其中aP2在肿瘤细胞增殖有双向调节作用,影响肿瘤细胞的转移和复发。而PPARy、SREBP1能促进细胞成熟,抑制细胞异常增生。各种因子间存在互相调节,依据其细胞特性和微环境而变化,因hADSCs在成脂过程中旁分泌作用,是否对乳腺癌细胞的侵袭力有影响?主要途径是什么?都有待研究。乳腺癌细胞和hADSCs、成脂细胞(Adipocyte, AC)生长过程中,分泌大量的细胞因子和蛋白酶类,其影响细胞的生长,增殖,分化、复发和侵袭。其中与乳腺癌细胞的迁移、浸润作用明确有关的细胞因子有尿激酶型纤维蛋白酶原激活剂(Urokinase type plasminogen activator, uPA)、基质金属蛋白酶2(matrix metalloproteinase-2,MMP2)-.基质金属蛋白酶9(基质金属蛋白酶9,MMP9)和血管内皮生长因子(Vascular endothelial growth factor,VEGF)。金属蛋白酶家族主要溶解间质胶原,为肿瘤细胞侵润提供一定的空间基础,从而促进肿瘤细胞增殖和侵袭力。这些因子在正常细胞中也通过旁分泌作用产生新生血管,组织体积扩充以及促进组织生长等。同样也是肿瘤细胞生长、浸润的基础。通过对细胞相关因子的研究,可以分析细胞间旁分泌作用机制,为乳腺癌术后的脂肪颗粒移植隆乳成活机理,乳腺癌复发、浸润、转移提供分子生物学理论依据。方法1取人皮下脂肪组织,Ⅰ型胶原酶消化原代分离培养脂肪干细胞(hADSCs),通过成脂、成骨多向诱导分化和流式细胞术检测CD29、CD34. CD44、CD45、CD105鉴定hADSCs。原代hADSCs培养到第三、四代,更换成脂培养液培养,动态观察成脂情况,于第6d、12d细胞提取mRNA和总蛋白,荧光定量PCR检测PPARymRNA、SREBF1mRNA变化,Westernblot检测PPARγ、 SREBFl含量变化,分析hADSCs成脂过程中转录因子的变化。2hADSCs和AC细胞在96孔板内培养,待细胞融合为孔底面积80%以上时,按1：：2、1：8、1：16比例将MCF-7和BT474条件培养液和DMEM、成脂培养基(FBS终浓度为10%)混合进行培养,于24h、48h、72hMTT酶标仪检测,确定混合培养液最适浓度比例。3待细胞生长至瓶底80%以上时,使用成脂培养基、MCF-7和BT474条件培养基加上成脂培养基(终末FBS为10%)共同培养hADSCs和AC,待大量脂肪细胞形成后,提取提取mRNA和总蛋白,荧光定量PCR检测对转录因子SREBF-1、PPARγ; Western blot检测MCF-7、BT474条件培养基对hACSCs和AC的SREBP-1、PPARγ的影响。4hADSCs、AC细胞生长至培养瓶底80%以上时,换用无血清培养基,培养24h后收集,运用Transwell小室实验技术,预制基质胶,上室为MCF-7.BT474细胞+10%FBS,下室为hADSCs细胞培养液、AC细胞培养液(终浓度含10%FBS),培养24h后,擦去上室基底基质胶,10%多聚甲醛细胞固定后结晶紫染色,倒置显微镜下观察,采集图像,结晶紫脱色后,酶标仪测定,间接反映浸润细胞含量。5MCF-7、BT474、hADSCs、AC细胞生长至培养瓶底80%以上时,换用无酚红的无血清培养基,24h后收集培养液,ELISA方法检测培养液中血管内皮生长因子(VEGF)、金属蛋白酶2(MMP2)、金属蛋白酶9(MMP9)、尿激酶型纤溶酶原激活物(uPA)含量。6培养MCF-7、BT474、hADSCs、AC-12细胞生长至瓶底80%以上时,提取mRNA和总蛋白,荧光定量PCR检测对转录因子aP2、PPARγmRNA;Western blot检测aP2、PPARγ。结果1hADSCs第三、四代为长纤维状细胞,成簇条索状排列,细胞中无脂滴。成骨诱导12d可见成骨细胞形成,但钙盐沉积较少,细胞明显变大变圆。油红“O”染色、茜素红染色阳性,流式细胞术检测第三代人脂肪干细胞CD29、CD44、 CD105为阳性,CD34、CD45表达为阴性。成脂诱导6d开始有脂滴形成,12d脂滴明显增多,部分融合大脂滴,细胞形态为多边形。成脂诱导6d、12d细胞PPARγmRNA、SREBF1mRNA均有表达。SREBF1mRNA6d、12d表达与相应蛋白不一致,主要靠转录后调节为主。PPARγ在转录水平上调节。21：8的MCF-7、BT474条件培养基促进hADSCs和AC的增殖；MCF-7、BT474条件培养基对hADSCs分化为脂肪细胞的时间无影响。MCF-7、BT474条件培养基对细胞中hADSCs成脂前后PPARγ表达无规律,可能存在旁分泌抑制调节,MCF-7对PPARγ抑制更明显。SREBF-1仍以转录后调节为主。成脂后期均有明显增高,MCF-7中表达明显增高。与正常成脂分化和BT474条件培养液相比,具有明显统计学差异(P<0.01)。3Transwell小室实验得出hADSCs在成脂前后可增强MCF-7、BT474的侵袭性,hADSCs作用明显(P<0.05)。MCF-7结晶紫染色hADSCs条件培养液组OD为0.263±0.009；AC条件培养液组OD为0.202±0.004,作用较弱(P<0.05)；BT474hADSCs条件培养液组结晶紫染色OD为0.263±0.005；AC条件培养液组OD为0.206±0.009,作用弱(P<0.05)；4uPA、VEGF、MMP2、MMP9在hADSCs成脂分化后培养基中均有表达(分别为4.80×103±266.67pg/ml、5.10×103±91.30pg/ml、187.45±20.61pg/ml、278.97±56.89pg/ml、4.77×104±0.03pg/ml、4.93×104±0.22pg/ml、1.93×103±190.0pg/ml、0.618×103±156.0pg/ml);MCF-7、BT474条件培养基中无MMP2表达,MMP9含量无差别(为1.37×103±186.67pg/ml、0.384×103±136.0pg/ml),其MCF-7中uPA和VEGF含量呈平行趋势,含量明显高于hADSCs和成脂细胞培养液(1.7×104±566.67pg/m、320.945±28.03pg/ml),BT474中VEGF含量较高(371.955±37.512pg/ml), uPA (5.10×103±88.73pg/ml),与hADSCs和AC培养液中明显差异。在乳腺癌中血管成形能力强,侵袭力较弱：MMP2在hADSCs成脂前后表达均高(4.77×104±30pg/ml、4.93×104±220pg/ml).5荧光定量PCR、Westernblot检测MCF-7、BT474中低表达PPARymRNA及无蛋白表达；在hADSCs、AC-12中PPARy高表达,分别为0.591±0.005、0.903±0.009,有统计学差异；aP2蛋白在MCF-7、hADSCs、BT474、AC-12均有表达。其中AC-12高表达,为1.203±0.004,BT474中为0.520±0.002,呈低表达。结论CAL技术的临床应用已经趋于成熟,但适用范围较狭,美国整形外科协会提出对乳腺癌术后的脂肪移植治疗需有专家评估,根据具体评定是否适合脂肪移植治疗。本课题就乳腺癌保乳术后CAL中乳腺癌切除术后“癌床”微环境对hADSCs成脂前后的影响,以及hADSCs对乳腺癌细胞侵袭力影响进行探讨,为CAL是否适用于乳腺癌保乳术后的乳房整形提供基础细胞学理论依据。1CAL中增加hADSCs的量,在移植组织起辅助作用。在体外模拟成脂微环境发现,hADSCs成脂诱导6d,细胞内开始出现细小脂滴,12d分化为脂肪细胞。细胞内出现部分脂滴融合,形态由细长型变为多角形。转录因子PPARymRNA及其蛋白随时间逐步升高,后期达高峰,在转录水平进行调节。PPARymRNA及其蛋白参与中后期促进(?)ADSCs成脂和脂肪细胞成熟。SREBP1mRNA与SRBPF1随时间表达不一致,SRBPF1mRNA随时间逐渐增高,说明SREBP1属于转录后调节。两者在体外整个hADSCs成脂分化中参与调节,促进脂肪细胞形成及其表型表达。2乳腺癌细胞旁分泌对hADSCs成脂时间无明显影响。其旁分泌因子影响hADSCs成脂过程中细胞内成脂转录因子PPARymRNA、SREBF1mRNA及其表达产物。PPARγ无规律可循,SREBP1仍为转录后调节。充分说明细胞成脂分化仍受到微环境影响,部分呈抑制。可能存在其他通路对PPARγ抑制。但乳腺癌细胞脂肪转运功能仍受SREBP1影响,在成脂环境中,脂肪细胞为乳腺癌细胞提供“能源”。3虽然转录因子相同,但在不同细胞中作用不同。hADSCs、MCF-7、BT474和AC中,转录因子PPARymRNA在MCF-7和BT474表达较低,PPARy在乳腺癌细胞中无表达,提示PPARy在乳腺癌中影响细胞周期,间接反映细胞的恶性程度。转录因子aP2在hADSCs成脂诱导中主要影响PPRAγ的表达,具有促成脂作用；在乳腺癌肿瘤细胞中,与原癌基因结合,随癌细胞恶性程度而呈现高表达。4hADSCs成脂前后增强乳腺癌细胞侵袭性,与乳腺癌细胞、hADSCs、AC旁分泌VEGF、MMP2、MMP9、uPA等细胞因子和蛋白酶有关[19-22]。hADSCs旁分泌影响较大。hADSCs分泌MMP2、MMP9为血管和细胞生长提供空间,溶解基质后,可加速血管之间的连接,形成有效的循环,促进细胞生长。另一方面,对癌细胞来讲,也提供血运转移的基础。乳腺癌细胞自身分泌的uPA和VEGF是乳腺癌复发、浸润和远位转移的细胞因子。MCF-7中细胞旁分泌大量的VEGF和uPA,两者相互平行,是MVF-7复发和快速生长和远处转移的关键因素。而BT474细胞培养液中,uPA和VEGF不一致,VEGF分泌量多,uPA量少,与乳腺癌的病理类型有关,BT474容易在局部快速浸润生长,早期血行转移的可能性小。AC中VEGF高表达可能是脂肪颗粒移植后组织的生长过程中血管形成和组织发育的关键因素之一,保持AC含量是脂肪组织成活的关键。同时也提供了“癌床”微环境肿瘤细胞转移的血管基础。乳腺癌细胞不分泌MMP2,而hADSCs、AC分泌MMP2,由于肿瘤细胞的“窃取效应”,hADSCs成脂前后对乳腺癌细胞主要是局部影响。整个成脂过程中,脂肪库的增加,也为肿瘤细胞提供能源基础,加强乳腺癌细胞的生长、浸润以及转移需要的能源动力。5PPARy蛋白在MCF-7、BT474中无表达,其影响肿瘤细胞周期,促进细胞增殖,处于低分化状态,具有抑癌作用。hADSCs成脂分化前后,PPARy蛋白受到抑制,也许会影响hADSCs成脂分化。aP2在MCF-7、BT474均有表达,但MCF-7表达高,aP2与原癌基因结合,促进癌细胞增殖、浸润等。aP2在肿瘤细胞中无法通过PPARγ调节细胞储脂,但转运脂肪能力增加,利用周围脂肪微环境,低能量转运脂肪,在“癌床”微环境下,为乳腺癌细胞代谢、转移等提供能源。6低量的颗粒脂肪移植,hADSCs附着在脂肪颗粒支架上,可充分增加移植物和hADSCs早期与血浆的接触,促进成活。细胞活性增加后,可为血管形成提供内皮细胞分化基础,增加VEGF分泌量,新生大量血管,为脂肪颗粒成活提供有效的血供。但在“癌床”微环境下,行CAL仍存在风险性。hADSCs对休眠癌细胞的“催醒作用”以及远位转移的研究需进一步行体外实验。更多还原

【Abstract】 Objective The incidence of female breast cancer is increasing yearly all over the world. According to the International Cancer Research Center Report, survey showed new-onset breast cancer incidence in women about22.9%,female cancer deaths accounted for13.7%in2008.In China, the incidence of breast cancer is trend to young women, the new incidence and mortality was significantly higher. With the improvement of diagnosis and treatment of breast cancer, the positive rate of early diagnosis of breast cancer is more accurate and treatment options have changed. Breast-conserving surgery has now become treatment and research focus on breast cancer in Europe and the United States. But there are still some patients require d mastectomy. Breast deformities leaded to serious mental disorders and reduced quality of life significantly post-operation. After breast surgery, more and more patients demand for breast reconstruction using from immediate flap to implants. But there have some shortcomings and the patients could not be accepted them. Material for filling technology in plastic surgery has developed rapidly in recent years. As for an ideal filler material, Autologous fat once bring plastic surgeons to hope. Because of its low survival rate of transplanted fat, long-term effect is poor and almost eliminated. Coleman successfully completed mammary augumentation with transplantation of autologous fat particles in1995. And fat as filler is using in the field of plastic surgery as quickly as possible. Breast and buttock shape and facial depression is filled with fat by Cell assisted lipotransfer (CAL) and the long-term effects are satisfactory. Atrophy, cysts, calcification and necrosis rate of transplantation of adipose tissue has decreased significantly. CAL by increasing volume of human adipose-derived stem cells (Human adipocyte-derived stem cells, hADSCs) promote fat particles survival and growth of fat cells, the survival rate increased from30to70%hADSCs adipogenic differentiation of regulatory mechanisms are the research focus of fat particles transplant and the key of shaping the tissue. Only hADSCs are differentiated to mature and ensure the biological safety of fat transplantation.Breast hypoplasia, congenital, traumatic breast deformity and achieved have good results by CAL. It can shape the breasts by several times But for women with breast cancer surgery, CAL as breast plastic surgery, is controversial at home and abroad. Today the incidence of breast cancer in women is increase. Due to improvements in health care, breast conserving surgery for breast cancer is gradually as the preferred surgical treatment. Postoperative patients with individualized treatment, the survival rate is extended and the quality of life is improved. But female patients’breast reduction, asymmetry and other factors affect their psychology of life postoperation, they are desire to breast plastic surgery. Second-stage implant placement disadvantages for the original breast surgery scars became significantly wider, for the difficult to insert implants because of scar adhesion, for obvious local reaction postoperationly and for outshape of breasts because of scar stretch to unsatisfy with the shape. Occasionally Patients are afraid of implant leakage and occurring malignant, so requires effective and minimally invasive breast surgery. Breast fat granule transplantation (Cell assisted lipotransfer, CAL), can solve the above adverse circumstances. Local breast volume can be filled on its defects. There have some advantage for maximum adjustment breast shape, less traumatic, alleviating reoperation in patients with pain and so on. American Society of Plastic Surgeons and the French Society of Plastic Surgeons questioned on cell biological safety of fat grafting. Through clinical case-control study, they didnot yet find definitive evidence of their breast cancer recurrence, local invasion and distant metastasis influential. Today a large number of clinical trail need to be long-term follow-up. In vitro and In vivol there is no clear indication that the cells of CAL assisted local recurrence, progression and distant metastasis of breast cancer, still has a lots controversy.The impact of breast cancer cells hADSCs is multifaceted. Mainly through its paracrine corresponding cytokines between cells stimulate each other growth, proliferation, migration. Meanwhile hADSCs multipotent differentiation capabilities havecertain impact. In the CAL, the main graft implanted subcutaneous, mammary fat tissue and major pectorals places. Depending on the micro-environment, hADSCs mainly promote the growth of fat tissue and adipogenic differentiation. In local area of breast cancer excised, the impact adipogenic differentiation on hADSCs is still unknown by CAL.CAL for breast cancer cells should be. one of the focus of attention for the second stage breast plastic surgery Whether the hADSCs transplant are survival in microenvironment of breast cancer and normal differentiation postoperatively? Have an effect on breast cancer cell invasion force? What is the main mechanism of action? Given the major role through paracrine mechanisms to regulate cell growth, development, differentiation and metabolism between cells, So we choose without metastasis of breast cancer adenocarcinoma cell line MCF-7and invasive ductal carcinoma BT474as the research object, through in vitro culture hADSCs, MCF-7, BT474cells, in vigorous growth period, cells were collected serum-free medium, as well as mixed adipogenic culture medium and analysis of cell-cell interactions. Provide a theoretical basis for the biological safety of Grainy fat transplantation.In the hADSCs adipogenesis process, effects of transcription factors play a leading role. According to current study, PPAR y and SREBP1, aP2and C/EBP are for hADSCs fat differentiation. Promoting the formation of intracellular fat is on transcription factor regulation, especially with PPAR y and SREBP1effects obviously. Effects of transcription factors have other cell functions, which the aP2cells have double regulation, affecting tumor cell metastasis and recurrence. The major role of Metalloproteinases family can enzymolysis collagen, providing a spatial basis for tumor cell invasion, thereby facilitating tumor cell the power of proliferation and invasiveness. PPAR y and SREBP1promotes cell maturation, inhibition of cell dysplasia. whether do the paracrine materials effect on invasiveness of breast cancer cells in hADSCs adipogenesis process? What are the main ways? It is to be studied.Local effects rely mainly on paracrine regulation of cells. In the process of breast cancer cells, hADSCs, AC growth, secreting large amounts of cytokines and Proteases, the materials effect cell growth, proliferation, differentiation, and recurrent invasion. There are cytokine uPA, VEGF and MMP2and MMP9that effect the breast cancer cell migration, invasion clearly. On the other hand these factors in normal cells can develop angiogenesis, tissue volume expansion and promote organizational growth by paracrine role, as well as the base of tumor cell growth and invasion. Through the investigation of cell-associated factors, you can analyze cell paracrine mechanism to the tissue survival of CAL after augmentation mammoplasty, provide theoretical basis for the molecular biology of breast cancer recurrence, invasion, metastasis.Method1. Take subcutaneous adipose tissue, the primary adipose stem cells (hADSCs) are isolated and cultured by type I collagenase digestion. For hADSCs identification, we take a series of assays as of adipogenic, osteogenic multi-directional induction of differentiation and CD29, CD34, CD44, CD45, CD105of hADSC by flow cytometrys. The primary hADSCs were cultured to the third and fourth passage, the replacement of adipogenic culture medium, dynamic observation of adipogenic changes. At6d,12d adipocyte’s totel mRNA and protein was extracted. Quantitative PCR detects PPARymRNA, SREBF1mRNA changes; Western blot detects content change of PPARy and SREBF1protein. We analyze the role of transcription factors in the process of adipogenesis. 2. hADSCs and AC cells cultured in96-well plates until the cell confluent more than80%of the area of the bottom of each well. The condition medium were changed with the proportion of1:2,1:8,1:16of MCF-7and BT474conditions of the culture medium and DMEM or adipogenic medium (FBS final concentration of10%) mixed. At24h,48h,72h, MTT assay is detected by microplate reader to determine the mixing proportion of the optimal concentration of the culture medium.3. After the cells were confluented to more than80%of the bottom of the bottle, hADSCs and AC were cocultured in adipogenic medium, MCF-7and BT474conditioned medium and adipogenic medium (terminal FBS10%) mixed respectively. Until a lot of fat cell formation,6d andl2d, adipicytes’mRNA and total protein were harvested. Then quantitative PCR detected the transcription factor SREBF-1, PPARy mRNA; Western blot detected SREBP-1, PPARyprotein of hACSCs and AC cultured MCF-7, BT474conditions medium.4. hADSCs and AC cells were confulent more than80%of the bottom of the bottle, changed serum-free medium(DMED) and collected the medium after24h culture. By Transwell chamber experimental techniques, the bottom of upper chamber prefabricated Matrigel. MCF-7and BT474cell were cultured the upper chamber with+10%FBS DMED; lower chamber were filled with condition medium of hADSCsl and the AC containing10%FBS (final concentration). After24h, wiping the upper chamber base Matrigel, cells were fixed by10%Paraformaldehyde and stained with crystal violet. The cell were observed under the inverted microscope, captured images of crystal violet decolorizationenzyme immunoassay, which indirectly reflected the infiltrating cells content.5. MCF-7, and BT474, and hADSCs, and AC cell reached to confluence more than80%of bottom of medium dish and cultured with phenol red-free and serum-free media. After24hours the cultured medium were collected and detectd vascular endothelial growth factor (VEGF), metalloproteinase2(MMP2), metalloproteinase9(MMP9), urokinase-type plasminogen activator (uPA) content with ELISA method.6. MCF-7, BT474hADSCs, AC-12cells were cultured with the different conditions medium and conflutentedto more than80%of the bottom of the25cm2 bottle, harvested total mRNA and protein, detected aP2, PPARymRNA transcription factors by assay of fluorescence quantitative and aP2, PPAR proteins by Western blot.ResultThe third or fourth hADSCs show the long fibrous, cord-like arrangement of cell clusters without lipid droplets. After adipogenic induction6d the cell began the formation of lipid droplets, the12days lipid droplets increased significantly, and integration of large lipid droplets gradually. Cell morphology is polygon. Osteogenic induction12days, we can visible osteoblast formation, but less calcium deposition. Osteocytes become large circle. Oil Red O staining, Alizarin red staining, and CD29, CD44, CD105detection of flow cytometry to third passage adipose stem cells are positive, CD34, CD45expression was negative. Both of6d,12d adipogenic inducted cells PPARy mRNA, SREBF1mRNA were express; which SREBF1mRNA6d expression increase obviously, increase until12days. The PPARy mRNA expression increased lately. The expression of PPARy, SREBP1proteins are consistent with their mRNA.The proliferation of hADSCs and AC were promoted with the proportion of1:8between MCF-7, BT474conditions medium and10%FBS DMEM. There is no effect on the time of hADSCs adipogenesis cultured with MCF-7, BT474conditioned medium. MCF-7, BT474conditioned medium on hADSCs before and after hADSCs adipogenic, PPARy expression without the law, there may be inhibited paracrine regulation, PPARy inhibition of MCF-7groups is more obvious. SREBF-1transcriptional regulation is still based. Significantly increased adipogenic late, MCF-7expression was significantly increased. Compared Normal adipogenic differentiation with BT474conditioned medium is the significant difference (P <0.01).MCF-7, the BT474invasion are enhanced by the before and after adipogenic hADSCs according to the Transwell chambers experiment. The role of hADSCs is significantly (P<0.05). Crystal violet staining OD of MCF-7cultured hADSCs condition medium is0.263±0.009; the OD of AC condition medium group is0.202± 0.004, show less effective (P<0.05). The BT474crystal violet staining cultured hADSCs condition medium is0.263±0.005; AC conditioned medium group is of0.206±0.009, is weaken (P<0.05).There are expression of uPA, VEGF, MMP2, MMP9in the medium of hADSCs and its adipogenic differentiation (4.80×103±266.67pg/ml,5.10×103±91.30pg/ml,187.45±20.61pg/ml,278.97±56.89pg/ml,4.77×104±0.03pg/ml,4.93×104±0.22pg/ml,1.93×103±190.0pg/ml,0.618×103±156.0pg/ml); MMP2is not express in the MCF-7and BT474condition media, and MMP9content is no difference (1.37×103±186.67pg/ml,0.384×103±136.0pg/ml). uPA and VEGF levels of MCF-7condition medium showed a parallel trend, were significantly higher than the condition medium of hADSCs and adipogenic cell (1.7×104±566.67pg/ml,320.945±28.03pg/ml). And VEGF levels in BT474condition medium is also higher (371.955±37.512pg/ml), but uPA is common (5.10×103±88.73pg/ml) so that angioplasty ability is better and invasion is weak in breast cancer. MMP2expression before and after hADSCs adipogenic are always higher (4.77×30pg/ml104,4.93×104±220pg/ml), respectively.Quantitative PCR and Western blot detection of MCF-7, the BT474PPARymRNA and protein are no expression but the protein are higher in hADSCs and AC-12,(0.591±0.005,0.903±0.009). aP2protein in MCF-7hADSCs, BT474, AC-12are all expression and AC-12is high expression(1.203±0.004), but BT474showed low (0.520±0.002).ConclusionClinical application of CAL is mature, but the scope is narrow, there are some plastic surgeries in an ambiguous state. The subject is to investigate the interaction between adipocyte before and after adipogenic hADSCs and breast cancer cells in vitro.Weather or not the CAL is applicable to mammoplasty for breast cancer after breast-conserving surgery to offer theoretical basis of medicine.In CAL, hADSCs play a leading role in survival of the transplanted tissue and cell differentiation. On6d, adipogenic hADSCs began to appear small lipid droplets, then12d differentiate into mature adipocytes. Intracellular part of the lipid droplets integrate and become a polygonal shape. Transcription factors PPARymRNA, ERJBPFlmRNA in vitro modulate adipogenic hADSCs dynamically. SRBPF1mRNA adjusts adipogenic differentiation at the early stage from the transcription level. And PPARymRNA promotes hADSCs into adipocyte and then mature at the mid-late adipogenic stage.Both involved in mediating adipogenic differentiation in vitro, promote adipocyte formation and its phenotypic expression.Paracrine materials from breast cancer cells had no significant effect on the hADSCs adipogenic time. Paracrine factors influence transcription factors PPARymRNA, SREBF1mRNA their expression products during adipogenic. PPARy has no rules to follow, SREBP1remains post transcriptional regulation. hADSCs adipogenic differentiation is still subjected to micro-environmental effects and some were suppressed. There may be other pathways for PPARy suppression. But breast cancer cells fat transporter function is still influenced by SREBP1in the adipogenic environment. Fat cells provide "energy" to breast cancer.Transcription factors have different role in kinds of cells. aP2is mainly with promoting the formation of adipocyte at the procession of hADSCs adipogenic. But it has bipolar ability to regulate in tumor cells,. The each cell paracrine interactions could not effect on transcriptional factors, such as PPARymRNA, ERBPF1mRNA, aP2mRNA, of hADSCs, MCF-7, BT474and adipocyte.Before and after hADSCs adipogenic enhanced invasiveness of breast cancer cells, especially hADSCs. This phenomenon is related with some paracrine cytokines and proteases from hADSCs,, AC and breast cancer, such as VEGF, MMP2, MMP9, uPA. which have a synergistic paracrine effect between breast cancer cell and hADSCs and AC.Breast cancer cells do not secrete MMP2, but hADSCs and AC can do, so hADSCs and AC mainly effects on breast cancer cells locally. uPA and VEGF secretion by themselves is the cytokines for recurrence, invasion and distant metastasis of breast cancer. MCF-7secrete large amounts of VEGF and uPA, both are parallel, and are the key factors for MVF-7recurrence and rapid growth and distant metastasis. In the BT474culture medium, uPA is not parallel to the amount of VEGF secretion, the uPA less. It may be associated with breast cancer pathology type BT474is easily grown in local infiltration rapidly, but the possibility of hematogenous metastasis early is low. High VEGF expression in AC condition medium may be one of the key factors for the process of blood vessel formation and tissue development,after the CAL. Keeping the AC content is the key to the survival of adipose tissue. PPAR protein is no expression in MCF-7, BT474as a tumor suppressor. During the hADSCs adipogenic differentiation the PPARgamma protein is one of the self-protection regulator. aP2in MCF-7, BT474were expressed, but the expression of MCF-7is high, which is related to their higher chance of MCF-7local invasion and distant metastasis. It may promote BT474cell growth and invasion.PPARy protein in MCF-7, BT474are no expression, the effects of tumor cell cycle, promotes cell proliferation, in poorly differentiated state, show the function of tumor suppressor role. During hADSCs the cause of adipogenic differentiation, PPARy protein inhibited, may affect hADSCs adipogenic differentiation. aP2in MCF-7, BT474were expressed, but the expression of MCF-7higher. aP2conjunction to proto-oncogenes promotes cancer cell proliferation, invasion, etc. aP2can not regulate t fat reservoir through PPARy in tumor cells, but increase capacity of fat transfer. Taking advantage of the microenvironment surrounding fat. cancer cells can gain energy in the "cancer bed" for the metabolism, metastasis.Low volume of fat particles, hADSCs attache to the fat bracket and can be sufficiently increased contact with the plasma between graft and hADSCs early and promote survival. With cell activity Increased, it can provide to vascular endothelial cells and increase secretion of VEGF. Then forming a large number of new blood vessels, it can provide to an effective blood supply for the survival of the fat particles. But in the "cancer bed" micro-environment, there is still risks by CAL. hADSCs have the abilities of "wake effect" to dormant cancer cells as well as their distant metastasis needs further research in vitro. transcriptional regulation; invasion force; paracrine更多还原