| 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... |
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