誘導(dǎo)人乳腺脂肪來源干細(xì)胞向乳腺上皮樣細(xì)胞分化的實(shí)驗(yàn)研究
發(fā)布時(shí)間：2018-10-12 12:55
【摘要】：第一部分 人肥大乳房乳腺組織中脂肪來源干細(xì)胞的分離、培養(yǎng)及鑒定 目的建立人乳腺脂肪來源干細(xì)胞(hbASCs)原代培養(yǎng)的方法,并對(duì)其進(jìn)行體外培養(yǎng)及鑒定,觀察其形態(tài)特點(diǎn)、生物學(xué)特性及間充質(zhì)來源干細(xì)胞表面相關(guān)標(biāo)志的表達(dá)。 方法本實(shí)驗(yàn)于2011.03-2012.04在華中科技大學(xué)附屬協(xié)和醫(yī)院外科實(shí)驗(yàn)中心整形外科實(shí)驗(yàn)室完成。①對(duì)象：實(shí)驗(yàn)期間在我科行乳房縮小術(shù)患者7例,年齡23-53歲,BMI指數(shù)20.01-23.26kg/m2,術(shù)后病理檢查無惡性腫瘤發(fā)生,患者知情同意。②方法：于無菌條件下獲取人乳腺脂肪組織塊約10-15毫升,采用Ⅰ型膠原酶消化+貼壁法分離原代脂肪來源干細(xì)胞,生長(zhǎng)至85%融合時(shí)傳代,倒置相差顯微鏡觀察形態(tài)特點(diǎn)、生物學(xué)特性,采用CCK-8法繪制細(xì)胞生長(zhǎng)曲線,計(jì)算細(xì)胞倍增時(shí)間；選擇生長(zhǎng)狀態(tài)良好的第3代hbASCs分別行熒光免疫法和流式細(xì)胞儀檢測(cè)間充質(zhì)來源干細(xì)胞特異性表面標(biāo)志物的表達(dá),并向成脂、成骨定向誘導(dǎo)。 結(jié)果hbASCs呈長(zhǎng)梭形貼壁生長(zhǎng),可陽性表達(dá)間充質(zhì)干細(xì)胞生長(zhǎng)特性相對(duì)特異性標(biāo)志物,體外可成脂、成骨定向分化。經(jīng)凍存復(fù)蘇后仍可維持細(xì)胞活性、生長(zhǎng)和增值能力。 結(jié)論可從人乳腺脂肪組織中成功分離獲取干細(xì)胞,為組織工程化乳房提供了新的取材方便、不增加供區(qū)的種子細(xì)胞來源。 第二部分 共培養(yǎng)誘導(dǎo)hbASCs向乳腺上皮樣細(xì)胞定向分化的實(shí)驗(yàn)研究 目的探討通過Transwell建立hbASCs和HBL-100細(xì)胞系共培養(yǎng)體系,誘導(dǎo)hbASCs向乳腺上皮樣細(xì)胞轉(zhuǎn)化的可能性。 方法分對(duì)照組和實(shí)驗(yàn)組,每組3復(fù)孔。實(shí)驗(yàn)組：將第3代hbASCs和HBL-100細(xì)胞系分別接種于Transwell共培養(yǎng)系統(tǒng)的下室和上室中,調(diào)整細(xì)胞比例為1：1,使用DMEM/F12培養(yǎng)基；對(duì)照組：分別在Transwell上、下室接種第3代hbASCs,使用DMEM/F12培養(yǎng)基；以上兩組均隔日換液。共培養(yǎng)15天后,觀察實(shí)驗(yàn)組和對(duì)照組Transwell下室中hbASCs的光鏡特征、電鏡結(jié)構(gòu)和熒光免疫細(xì)胞化學(xué)染色,對(duì)下室被誘導(dǎo)細(xì)胞進(jìn)行鑒定。 結(jié)果Transwell體系中各細(xì)胞均可良好貼壁生長(zhǎng)。共培養(yǎng)15天后,實(shí)驗(yàn)組下室hbASCs細(xì)胞數(shù)量較對(duì)照組明顯增多,細(xì)胞外基質(zhì)合成豐富,部分細(xì)胞形態(tài)上表現(xiàn)出乳腺上皮樣細(xì)胞特征；電鏡下可見微絨毛、橋粒和張力絲等典型上皮細(xì)胞結(jié)構(gòu)特征；免疫熒光結(jié)果顯示,實(shí)驗(yàn)組共培養(yǎng)環(huán)境下分化細(xì)胞可陽性表達(dá)乳腺上皮細(xì)胞特異性標(biāo)記物CK-18CK-19和間充質(zhì)細(xì)胞標(biāo)記物Vimentin;對(duì)照組下室hbASCs上述檢測(cè)結(jié)果均為陰性； 結(jié)論本實(shí)驗(yàn)在體外成功建立了hbASCs和HBL-100細(xì)胞系的Transwell共培養(yǎng)體系,并證實(shí)在該體系中可誘導(dǎo)hbASCs向乳腺上皮樣細(xì)胞定向分化。 第三部分 EGF促進(jìn)hbASCs向乳腺上皮樣細(xì)胞定向分化的實(shí)驗(yàn)研究 目的探討在體外間接共培養(yǎng)體系中添加表皮生長(zhǎng)因子(EGF)對(duì)促進(jìn)hbASCs向乳腺上皮樣細(xì)胞定向分化的影響。 方法研究分兩組。實(shí)驗(yàn)組：取生長(zhǎng)良好第3代hbASCs,加入條件培養(yǎng)基(HBL-100細(xì)胞系上清液)+10ug/L EGF+1％雙抗；對(duì)照組：取生長(zhǎng)良好第3代hbASCs,僅加入條件培養(yǎng)基(HBL-100細(xì)胞系上清液)+1%雙抗；兩組細(xì)胞均隔日換液。共培養(yǎng)15天后,觀察各組中hbASCs的光鏡特征、熒光免疫細(xì)胞化學(xué)染色、流式細(xì)胞儀檢測(cè)CK-18陽性細(xì)胞比例以及實(shí)時(shí)定量PCR (real-time quantitative reverse-transcription polymerase chain reaction, QRT-PCR)檢測(cè)CK-18mRNA水平,對(duì)結(jié)果進(jìn)行配對(duì)t檢驗(yàn)。結(jié)果共培養(yǎng)體系中各細(xì)胞均可良好貼壁生長(zhǎng)。共培養(yǎng)15天后,兩組中均可發(fā)現(xiàn)部分hbASCS細(xì)胞形態(tài)、排列方式發(fā)生改變,實(shí)驗(yàn)組中細(xì)胞各項(xiàng)改變較對(duì)照組明顯；實(shí)驗(yàn)組誘導(dǎo)細(xì)胞熒光免疫細(xì)胞化學(xué)染色乳腺上皮細(xì)胞特異性角蛋白-18、角蛋白-19陽性細(xì)胞數(shù)和QRT-PCR基因表達(dá)量(△△CT法)分別為683.000±58.014個(gè)/視野和3.5350+0.3737倍,明顯高于對(duì)照組的298.167±27.272個(gè)/視野和1倍,有統(tǒng)計(jì)學(xué)意義(P＜0.05)。結(jié)論表皮生長(zhǎng)因子(EGF)可促進(jìn)體外間接共培養(yǎng)體系中hbASCs向乳腺上皮樣細(xì)胞的定向分化。
[Abstract]:The first part Isolation and culture of adipose-derived stem cells from breast tissue of human hypertrophic breast Objective To establish primary culture method of human breast fat-derived stem cells (hbASCs) and to culture and identify them in vitro. Its morphological characteristics, biological characteristics and the surface-related surface of mesenchymal stem cells Expression of markers. Methods This experiment was conducted in 2011. 03-2012. 04 at Central Huazhong University of Science and Technology Co., Ltd. and Hospital Surgical Experiment Center. Results: 7 patients with breast reduction, 23-53 years of age and 20. 01-23.26kg/ m2 of BMI were performed during the experiment. There was no malignant tumor in postoperative pathological examination. Method: Obtain human mammary adipose tissue mass of about 10-15 ml under aseptic conditions, isolate primary adipose-derived stem cells with type I collagenase digestion + apposition method, grow to 85% fusion, and observe the morphology by inverted phase contrast microscope. Characteristics, biological characteristics, using CCK-8 method to plot the cell growth curve, calculating the cell doubling time, selecting the third generation hbASCs with good growth state, respectively carrying out fluorescence immunoassay and flow cytometry to detect the expression of the specific surface marker of the mesenchymal stem cells, The results showed that hbASCs had a long shuttle-shaped adherent growth, and the growth characteristics of positive expression mesenchymal stem cells were relatively specific markers. It can be made into fat or bone-oriented differentiation. After frozen storage, it can still be maintained fine Conclusion The stem cells can be isolated from human breast adipose tissue successfully, and new materials are provided for tissue engineered breast.-No, no, no, no, no. The source of seed cells in the donor region. The second portion was co-cultured to induce hb. An experimental study of ASCs directed differentiation to mammary epithelial-like cells was conducted to investigate the co-culture system of hbASCs and HBL-100 cell lines by Transwell inducing hbASCs to epithelial-like cells in breast The possibility of transformation was divided into two groups: control group and experimental group, 3 complex pores in each group. The third generation hbASCs and HBL-100 cell lines were respectively inoculated into the lower and upper chambers of the Transwell co-culture system, the cell ratio was 1: 1, DMEM/ F12 medium was used, and the control group: the 3rd generation hbAS was inoculated on Transwell and the lower chamber respectively. Cs, DMEM/ F12 medium were used; two groups of the above two groups were used to exchange liquid. After 15 days of co-culture, the light microscope characteristics of hbASCs in the experimental group and control group were observed, and the electron microscope Structure and fluorescence immunocytochemistry staining to identify cells to be induced in the lower chamber Results After 15 days of co-culture, the number of hbASCs in the experimental group was higher than that in the control group, the extracellular matrix was rich, and the morphology of some cells showed the mammary epithelium-like cells. Characteristics: The structural characteristics of typical epithelial cells, such as microvilli, bridges and tension wires, were observed under electron microscope. The results showed that the differentiated cells in the experimental group could express the specific markers CK-18CK-19 and the marker V of the mesenchymal epithelial cells in the co-culture environment of the experimental group. The results of hbASCs and hbASCs were negative in the control group. Conclusion Transwell co-culture of hbASCs and HBL-100 cell lines was successfully established in vitro. the system, and It was confirmed that hbASCs were induced to differentiate into breast epithelial-like cells in the system. The third part of EGF promotes the directional differentiation of hbASCs to mammary epithelial-like cells, and discusses the indirect co-culture in vitro. Add epidermis in the system The effects of growth factor (EGF) on the orientation and differentiation of hbASCs to breast epithelial-like cells were studied. The experimental groups were divided into two groups: the third generation hbASCs, the addition condition medium (supernatant of HBL-100 cell line) + 10ug/ L EGF + 1% double resistance, the control group: the third generation of growth was good. BASCs were added only with conditioned medium (supernatant of HBL-100 cell line) + 1% double resistance; two groups of cells were incubated on day-to-day exchange. After 15 days of co-culture, the light microscope characteristics of hbASCs in each group were observed, the chemical staining of fluorescence immunocytochemistry, the ratio of CK-18 positive cells detected by flow cytometry and real-time quantitative PCR (real-time quantitative reverse-trans-transcription policy) were observed. ain reaction, QRT-PCR) CK-18mRNA level was detected and paired t-test was performed on the results. Results All cells in co-culture system could grow well. After 15 days of co-culture, the morphology and arrangement of partial hbASCS cells could be found in both groups. In the experimental group, the specific cytokeratin-18, keratin-19 positive cells and QRT-PCR gene expression were 683,000 and 584.014/ visual field and 3,5350 + 0.3737 times, respectively. It was significantly higher than that of the control group (298. 167, 27. 272)/ field of view (P <0.05).
【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2012
【分類號(hào)】：R329
本文編號(hào)：2266191
[Abstract]:The first part Isolation and culture of adipose-derived stem cells from breast tissue of human hypertrophic breast Objective To establish primary culture method of human breast fat-derived stem cells (hbASCs) and to culture and identify them in vitro. Its morphological characteristics, biological characteristics and the surface-related surface of mesenchymal stem cells Expression of markers. Methods This experiment was conducted in 2011. 03-2012. 04 at Central Huazhong University of Science and Technology Co., Ltd. and Hospital Surgical Experiment Center. Results: 7 patients with breast reduction, 23-53 years of age and 20. 01-23.26kg/ m2 of BMI were performed during the experiment. There was no malignant tumor in postoperative pathological examination. Method: Obtain human mammary adipose tissue mass of about 10-15 ml under aseptic conditions, isolate primary adipose-derived stem cells with type I collagenase digestion + apposition method, grow to 85% fusion, and observe the morphology by inverted phase contrast microscope. Characteristics, biological characteristics, using CCK-8 method to plot the cell growth curve, calculating the cell doubling time, selecting the third generation hbASCs with good growth state, respectively carrying out fluorescence immunoassay and flow cytometry to detect the expression of the specific surface marker of the mesenchymal stem cells, The results showed that hbASCs had a long shuttle-shaped adherent growth, and the growth characteristics of positive expression mesenchymal stem cells were relatively specific markers. It can be made into fat or bone-oriented differentiation. After frozen storage, it can still be maintained fine Conclusion The stem cells can be isolated from human breast adipose tissue successfully, and new materials are provided for tissue engineered breast.-No, no, no, no, no. The source of seed cells in the donor region. The second portion was co-cultured to induce hb. An experimental study of ASCs directed differentiation to mammary epithelial-like cells was conducted to investigate the co-culture system of hbASCs and HBL-100 cell lines by Transwell inducing hbASCs to epithelial-like cells in breast The possibility of transformation was divided into two groups: control group and experimental group, 3 complex pores in each group. The third generation hbASCs and HBL-100 cell lines were respectively inoculated into the lower and upper chambers of the Transwell co-culture system, the cell ratio was 1: 1, DMEM/ F12 medium was used, and the control group: the 3rd generation hbAS was inoculated on Transwell and the lower chamber respectively. Cs, DMEM/ F12 medium were used; two groups of the above two groups were used to exchange liquid. After 15 days of co-culture, the light microscope characteristics of hbASCs in the experimental group and control group were observed, and the electron microscope Structure and fluorescence immunocytochemistry staining to identify cells to be induced in the lower chamber Results After 15 days of co-culture, the number of hbASCs in the experimental group was higher than that in the control group, the extracellular matrix was rich, and the morphology of some cells showed the mammary epithelium-like cells. Characteristics: The structural characteristics of typical epithelial cells, such as microvilli, bridges and tension wires, were observed under electron microscope. The results showed that the differentiated cells in the experimental group could express the specific markers CK-18CK-19 and the marker V of the mesenchymal epithelial cells in the co-culture environment of the experimental group. The results of hbASCs and hbASCs were negative in the control group. Conclusion Transwell co-culture of hbASCs and HBL-100 cell lines was successfully established in vitro. the system, and It was confirmed that hbASCs were induced to differentiate into breast epithelial-like cells in the system. The third part of EGF promotes the directional differentiation of hbASCs to mammary epithelial-like cells, and discusses the indirect co-culture in vitro. Add epidermis in the system The effects of growth factor (EGF) on the orientation and differentiation of hbASCs to breast epithelial-like cells were studied. The experimental groups were divided into two groups: the third generation hbASCs, the addition condition medium (supernatant of HBL-100 cell line) + 10ug/ L EGF + 1% double resistance, the control group: the third generation of growth was good. BASCs were added only with conditioned medium (supernatant of HBL-100 cell line) + 1% double resistance; two groups of cells were incubated on day-to-day exchange. After 15 days of co-culture, the light microscope characteristics of hbASCs in each group were observed, the chemical staining of fluorescence immunocytochemistry, the ratio of CK-18 positive cells detected by flow cytometry and real-time quantitative PCR (real-time quantitative reverse-trans-transcription policy) were observed. ain reaction, QRT-PCR) CK-18mRNA level was detected and paired t-test was performed on the results. Results All cells in co-culture system could grow well. After 15 days of co-culture, the morphology and arrangement of partial hbASCS cells could be found in both groups. In the experimental group, the specific cytokeratin-18, keratin-19 positive cells and QRT-PCR gene expression were 683,000 and 584.014/ visual field and 3,5350 + 0.3737 times, respectively. It was significantly higher than that of the control group (298. 167, 27. 272)/ field of view (P <0.05).
【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2012
【分類號(hào)】：R329
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