负载脂肪干细胞的生物活性补片在体内转归的研究

发布时间：2018-07-08 18:41

本文选题：生物发光成像 + 人脂肪干细胞　；参考：《苏州大学》2014年硕士论文

【摘要】：【背景】盆底重建手术植入人工合成材料后，可出现侵蚀（erosion）、暴露(exposure)、收缩(constraction)、性交痛(dyspareunia)、瘘管（fistula）等术后并发症。研究者们一直在寻找一种既能保证疗效，降低术后复发率及并发症，又能改善其潜在病理生理损伤的治疗措施，以更好的治疗女性盆底功能障碍性疾病。细胞治疗和组织工程技术在女性盆底重建外科学的研究中逐渐受到关注，是女性盆底重建外科学的一个新的发展领域。然而组织工程生物活性材料修复时，其移植的干细胞在体内的生存时间、长期存活量、分布，以及支架材料在体内的降解情况仍未阐明。本研究先体外双重标记绿色荧光蛋白和荧光素酶于脂肪干细胞，复合ABP移植裸鼠体内，然后利用先进的小动物活体成像系统活体内实时动态追踪移植细胞的存活、分布，利用组织学及免疫组化评估生物活性材料的转归，探索其修复的可行性，为其在女性盆底重建中的应用提供依据。【方法】 1、生物活性补片的体外制备 1.1人ASCs的分离培养、鉴定、标记及刷选（1）从整形外科女性患者（患者知情同意并签知情同意书）抽脂手术中取出脂肪组织20ml，采用酶消化法、差速贴壁法体外分离培养人脂肪干细胞，在普通倒置显微镜下观察记录其形态特点、增殖特征，流式细胞仪鉴定脂肪干细胞的表面标记物，证明多向分化能力。（2）双报告基因——增强型绿色荧光蛋白（enhance green fluorescent protein,eGFP)和荧光素酶蛋白(Luciferase, Luc)以携带嘌呤霉素抗性基因的慢病毒为载体感染第三代人脂肪干细胞，转染后倒置荧光显微镜观察eGFP的表达情况。（3）利用嘌呤霉素对eGFP·Luc-hASCs进行刷选，提高慢病毒转染率，利用流式细胞技术检测eGFP·Luc-hASCs的转染率。 1.2建立体内、外生物发光显像量效标准曲线（1）体外将eGFP·Luc-hASCs按照细胞个数(10,100,500,1000,5000,10000)个/100μL铺于96孔板，加入荧光素在活体成像仪中生物发光成像，绘制细胞量和发光量的线性曲线。（2）体内将eGFP·Luc-hASCs按照细胞个数（1×106/100μL,1×105/100μL,1×104/100μL,1×103/100μL,100/100μL）注射裸鼠背部，腹腔注射荧光素活体成像，绘制体内细胞量和生物发光量的标准曲线为后续实验定量分析体内移植细胞的数量做准备。 1.3体外ASCs和ABP共培养（1）采第八代eGFP·Luc-hASCs在体外与脱细胞牛心包膜（ABP）共培养（上海欣吉特生物科技有限公司提供）。（2） CCK-8法检测eGFP·Luc-hASCs在脱细胞牛心包膜上的增殖。将第8代eGFP·Luc-hASCs接种于ABP后，连续7天加入CCK-8测吸光度值，制作细胞的生长曲线。 2、生物活性补片的体内转归 2.1生物活性补片的体内移植：30只裸鼠随机分为三组，CO-CULTURE组（n=10）：体外共培养eGFP·Luc-hASCs和ABP再植入裸鼠背部； LOCAL-INJE组（n=10）：ABP植裸鼠背部后，eGFP·Luc-hASCs直接注射移植部位，无体外共培养；BLANK组（n=10）：单纯植入ABP裸鼠背部。 2.2生物活性补片的体内评估：（1）分别于移植后1天，1周，，2周，3周，4周，6周，8周，12周在裸鼠腹腔注射荧光素活体生物发光成像实时动态观察eGFP·Luc-hASCs在体内的存活、分布；（2）移植12周后裸鼠体内取出移植物、心、肝、肺等组织快速冰冻DAPI染色观察eGFP的表达；病理HE染色，Masson染色、免疫组织化学染色观察评价生物活性补片体内的转归。【结果】 1、传代培养的hASCs形态呈旋涡状、辐射状排列，体外增殖稳定。慢病毒介导双报告基因eGFP和Luc转染hASCs，体外荧光显微镜可观察到eGFP高表达。流式细胞仪检测示eGFP·Luc-hASCs的转染率达91.12%。 2、荧光显微镜观察示eGFP·Luc-hASCs在ABP上黏附生长良好，CCK-8显示活性生物补片中细胞增殖能力与未接种于脱细胞牛心包膜的hASCs无明显区别。 3、体内外细胞梯度生物发光成像显示，细胞量和活体成像的发光量成线性关系，绘制量效关系曲线后可量化体内移植细胞的数量。 4、活性生物补片移植后12周活体连续成像显示，移植的脂肪干细胞可长期存活，移植前3周细胞存活数量急剧下降，3周后达到平台期，12周仍可监测到存活的细胞；移植细胞主要分布移植部位，未发现向其他重要脏器明显迁移。 5、快速冰冻DAPI染色后可在细胞移植部位发现eGFP的表达，证实移植细胞的存在，主要存在皮下组织，心、肺、肝未见明显eGFP的表达；HE染色发现LOCAL-INJE组移植材料内细胞数量、血管密度最丰富的， CO-CULTURE组其次，对照组最少。Masson染色LOCAL-INJE组含量较CO-CULTURE组高，对照组含量最低。免疫组化染色中，LOCAL-INJE组可观察到Desmin、α-SMA阳性表达。【结论】生物活性补片植入裸鼠体内后人脂肪干细胞能长期在移植部位存活。脂肪干细胞与脱细胞牛心包膜不经体外共培养同时移植体内，更有利于细胞长入ABP，减缓ABP的降解，促进血管、平滑肌的合成，有利于组织的修复，是未来女性盆底重建材料的理想选择。
[Abstract]:[background] after the pelvic floor reconstruction is implanted into synthetic materials, the postoperative complications such as erosion (erosion), exposure (exposure), contraction (constraction), sexual pain (dyspareunia), and fistula (fistula) have been found. The researchers have been searching for one kind that can ensure the curative effect, reduce the postoperative recurrence rate and complications, and improve the potential pathophysiology. The treatment of injury to better treatment of female pelvic floor dysfunction. Cell therapy and tissue engineering are becoming more and more concerned in the research of female pelvic floor reconstruction surgery. It is a new development field of female pelvic floor reconstructive surgery. However, when tissue engineering bioactive materials are repaired, the transplanted stem cells are in body The survival time, long-term survival, distribution, and the degradation of scaffolds in the body have not been elucidated. In this study, we first double labeled green fluorescent protein and luciferase in adipose stem cells, transplanting ABP in nude mice, and then using the advanced small animal living body imaging system to track the transplanted cells in real time. The survival and distribution of bioactive materials were evaluated by histology and immunohistochemistry, and the feasibility of its repair was explored to provide a basis for its application in the reconstruction of female pelvic floor.
[method]
1, in vitro preparation of bioactive patch
Isolation, identification, marking and cleaning of 1.1 people ASCs
(1) from the plastic surgery female patients (patients informed consent and informed consent book) extraction of fat tissue 20ml in liposuction surgery, using enzyme digestion method, differential adhesion method in vitro isolation and culture of human fat stem cells in vitro, observe the morphological characteristics, colonization characteristics under the common inverted microscope, the flow cytometry to identify the surface markers of fat stem cells It proves the ability of multiple differentiation.
(2) the double reporting gene, enhanced green fluorescent protein (enhance green fluorescent protein, eGFP) and luciferase protein (Luciferase, Luc), infected third generation fat stem cells with the lentivirus carrying the purinamycin resistance gene, and the expression of eGFP was observed by inverted fluorescence microscope after transfection.
(3) using puromycin to screen eGFP? Luc-hASCs and increase the transfection rate of lentivirus. The transfection rate of eGFP Luc-hASCs was detected by flow cytometry.
1.2 establish the in vivo and external bioluminescence imaging dose effect standard curve.
(1) eGFP Luc-hASCs was spread on the 96 hole plate according to the number of cells (101005001000500010000) /100 mu L in vitro, and fluorescein was added to the bioluminescence imaging in the living imager, and the linear curve of cell volume and luminescence was drawn.
(2) eGFP Luc-hASCs was injected into the back of nude mice in vivo according to the number of cells (1 x 106/100 mu L, 1 x 105/100 mu L, 1 x 104/100 mu L, 1 x 103/100 mu L, 100/100 micron). The standard curve of cell volume and bioluminescence in vivo was injected into the body to prepare the quantitative analysis of the number of transplanted cells in the body.
1.3 co culture of ASCs and ABP in vitro
(1) the eighth generation eGFP? Luc-hASCs was co cultured with decellularized bovine pericardium (ABP) in vitro (Shanghai hingji Biotechnology Co., Ltd.).
(2) CCK-8 method was used to detect the proliferation of eGFP Luc-hASCs on the capsule of acellular bovine heart. After eighth generation of eGFP / Luc-hASCs were inoculated to ABP, the absorbance value of CCK-8 was added to CCK-8 for 7 days, and the growth curve of the cell was made.
2, in vivo transformation of bioactive patch
2.1 in vivo transplantation of bioactive patch: 30 nude mice were randomly divided into three groups, group CO-CULTURE (n=10): co culture eGFP / Luc-hASCs and ABP in the back of nude mice; LOCAL-INJE group (n=10):ABP implanted in the back of the nude mice, eGFP Luc-hASCs direct injection site, no external co culture; BLANK group (n=10): simple implantation of bare nude mice back Department.
2.2 in vivo evaluation of bioactive patch:
(1) the survival and distribution of eGFP Luc-hASCs in the body was observed at 1 days, 1 weeks, 2 weeks, 3 weeks, 4 weeks, 6 weeks, 8 weeks, and 12 weeks in nude mice by intraperitoneal injection of fluorescein bioluminescence imaging.
(2) after 12 weeks of transplantation, the grafts were taken out of the nude mice. The expression of eGFP was observed by rapid freezing DAPI staining in the heart, liver and lung tissues. Pathological HE staining, Masson staining and immunohistochemical staining were used to evaluate the outcome of bioactive patch in vivo.
[results]
1, the form of hASCs in the subculture was vortexed, radiated and stable in vitro. The lentivirus mediated double reporter gene eGFP and Luc transfected hASCs, and the high expression of eGFP was observed by the fluorescence microscope in vitro. The flow cytometry showed that the transfection rate of eGFP Luc-hASCs reached 91.12%..
2, the fluorescence microscope showed that the adhesion growth of eGFP Luc-hASCs on ABP was good, and CCK-8 showed that the cell proliferation ability of the active biopatch was not significantly different from that of the uninoculated bovine pericardium capsule.
3, cell gradient bioluminescence imaging in vitro and in vivo showed that the volume of cells was linear with the luminescence of the living body imaging, and the quantity of the transplanted cells in the body could be quantified after the volume effect relationship curve was drawn.
4, continuous imaging of living body after 12 weeks of bioactive patch showed that the transplanted fat stem cells could survive for a long time, the number of cells survived 3 weeks before the transplantation and reached the platform stage after 3 weeks, and the surviving cells could still be monitored in 12 weeks, and the transplanted cells were mainly distributed in the transplant site, and no obvious migration to other important organs was found.
5, after fast freezing DAPI staining, the expression of eGFP could be found at the site of cell transplantation, which confirmed the existence of the transplanted cells. There was no obvious eGFP expression in the subcutaneous tissue, heart, lung and liver. HE staining found the number of cells in the LOCAL-INJE group, the most abundant blood vessel density, the next of the CO-CULTURE group, and the least.Masson dyed LOCAL-I in the control group. The content of NJE group was higher than that of CO-CULTURE group and the control group was the lowest. Immunohistochemical staining showed that Desmin and -SMA expression could be observed in LOCAL-INJE group.
[Conclusion] the human adipose stem cells can survive in the transplant site for a long time after implantation of biological active patch in nude mice. Adipose stem cells and acellular bovine pericardium membrane are not co cultured in vitro and transplanted in vitro, which is more beneficial to cells to grow into ABP, slow down the degradation of ABP, promote the synthesis of blood vessels, smooth muscle, and be beneficial to the repair of tissue. It is a future female. Ideal choice for the reconstructive material of the pelvic floor.
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R711.5

【共引文献】