本文選題：生物發(fā)光成像 + 人脂肪干細(xì)胞��； 參考：《蘇州大學(xué)》2014年碩士論文

【摘要】：【背景】盆底重建手術(shù)植入人工合成材料后，可出現(xiàn)侵蝕（erosion）、暴露(exposure)、收縮(constraction)、性交痛(dyspareunia)、瘺管（fistula）等術(shù)后并發(fā)癥。研究者們一直在尋找一種既能保證療效，降低術(shù)后復(fù)發(fā)率及并發(fā)癥，又能改善其潛在病理生理損傷的治療措施，以更好的治療女性盆底功能障礙性疾病。細(xì)胞治療和組織工程技術(shù)在女性盆底重建外科學(xué)的研究中逐漸受到關(guān)注，是女性盆底重建外科學(xué)的一個新的發(fā)展領(lǐng)域。然而組織工程生物活性材料修復(fù)時，其移植的干細(xì)胞在體內(nèi)的生存時間、長期存活量、分布，以及支架材料在體內(nèi)的降解情況仍未闡明。本研究先體外雙重標(biāo)記綠色熒光蛋白和熒光素酶于脂肪干細(xì)胞，復(fù)合ABP移植裸鼠體內(nèi)，然后利用先進(jìn)的小動物活體成像系統(tǒng)活體內(nèi)實時動態(tài)追蹤移植細(xì)胞的存活、分布，利用組織學(xué)及免疫組化評估生物活性材料的轉(zhuǎn)歸，探索其修復(fù)的可行性，為其在女性盆底重建中的應(yīng)用提供依據(jù)。 【方法】 1、生物活性補片的體外制備 1.1人ASCs的分離培養(yǎng)、鑒定、標(biāo)記及刷選 （1）從整形外科女性患者（患者知情同意并簽知情同意書）抽脂手術(shù)中取出脂肪組織20ml，采用酶消化法、差速貼壁法體外分離培養(yǎng)人脂肪干細(xì)胞，在普通倒置顯微鏡下觀察記錄其形態(tài)特點、增殖特征，流式細(xì)胞儀鑒定脂肪干細(xì)胞的表面標(biāo)記物，證明多向分化能力。 （2）雙報告基因——增強(qiáng)型綠色熒光蛋白（enhance green fluorescent protein,eGFP)和熒光素酶蛋白(Luciferase, Luc)以攜帶嘌呤霉素抗性基因的慢病毒為載體感染第三代人脂肪干細(xì)胞，轉(zhuǎn)染后倒置熒光顯微鏡觀察eGFP的表達(dá)情況。 （3）利用嘌呤霉素對eGFP·Luc-hASCs進(jìn)行刷選，提高慢病毒轉(zhuǎn)染率，利用流式細(xì)胞技術(shù)檢測eGFP·Luc-hASCs的轉(zhuǎn)染率。 1.2建立體內(nèi)、外生物發(fā)光顯像量效標(biāo)準(zhǔn)曲線 （1）體外將eGFP·Luc-hASCs按照細(xì)胞個數(shù)(10,100,500,1000,5000,10000)個/100μL鋪于96孔板，加入熒光素在活體成像儀中生物發(fā)光成像，繪制細(xì)胞量和發(fā)光量的線性曲線。 （2）體內(nèi)將eGFP·Luc-hASCs按照細(xì)胞個數(shù)（1×106/100μL,1×105/100μL,1×104/100μL,1×103/100μL,100/100μL）注射裸鼠背部，腹腔注射熒光素活體成像，繪制體內(nèi)細(xì)胞量和生物發(fā)光量的標(biāo)準(zhǔn)曲線為后續(xù)實驗定量分析體內(nèi)移植細(xì)胞的數(shù)量做準(zhǔn)備。 1.3體外ASCs和ABP共培養(yǎng) （1）采第八代eGFP·Luc-hASCs在體外與脫細(xì)胞牛心包膜（ABP）共培養(yǎng)（上海欣吉特生物科技有限公司提供）。 （2） CCK-8法檢測eGFP·Luc-hASCs在脫細(xì)胞牛心包膜上的增殖。將第8代eGFP·Luc-hASCs接種于ABP后，連續(xù)7天加入CCK-8測吸光度值，制作細(xì)胞的生長曲線。 2、生物活性補片的體內(nèi)轉(zhuǎn)歸 2.1生物活性補片的體內(nèi)移植：30只裸鼠隨機(jī)分為三組，CO-CULTURE組（n=10）：體外共培養(yǎng)eGFP·Luc-hASCs和ABP再植入裸鼠背部； LOCAL-INJE組（n=10）：ABP植裸鼠背部后，eGFP·Luc-hASCs直接注射移植部位，無體外共培養(yǎng)；BLANK組（n=10）：單純植入ABP裸鼠背部。 2.2生物活性補片的體內(nèi)評估： （1）分別于移植后1天，1周，，2周，3周，4周，6周，8周，12周在裸鼠腹腔注射熒光素活體生物發(fā)光成像實時動態(tài)觀察eGFP·Luc-hASCs在體內(nèi)的存活、分布； （2）移植12周后裸鼠體內(nèi)取出移植物、心、肝、肺等組織快速冰凍DAPI染色觀察eGFP的表達(dá)；病理HE染色，Masson染色、免疫組織化學(xué)染色觀察評價生物活性補片體內(nèi)的轉(zhuǎn)歸。 【結(jié)果】 1、傳代培養(yǎng)的hASCs形態(tài)呈旋渦狀、輻射狀排列，體外增殖穩(wěn)定。慢病毒介導(dǎo)雙報告基因eGFP和Luc轉(zhuǎn)染hASCs，體外熒光顯微鏡可觀察到eGFP高表達(dá)。流式細(xì)胞儀檢測示eGFP·Luc-hASCs的轉(zhuǎn)染率達(dá)91.12%。 2、熒光顯微鏡觀察示eGFP·Luc-hASCs在ABP上黏附生長良好，CCK-8顯示活性生物補片中細(xì)胞增殖能力與未接種于脫細(xì)胞牛心包膜的hASCs無明顯區(qū)別。 3、體內(nèi)外細(xì)胞梯度生物發(fā)光成像顯示，細(xì)胞量和活體成像的發(fā)光量成線性關(guān)系，繪制量效關(guān)系曲線后可量化體內(nèi)移植細(xì)胞的數(shù)量。 4、活性生物補片移植后12周活體連續(xù)成像顯示，移植的脂肪干細(xì)胞可長期存活，移植前3周細(xì)胞存活數(shù)量急劇下降，3周后達(dá)到平臺期，12周仍可監(jiān)測到存活的細(xì)胞；移植細(xì)胞主要分布移植部位，未發(fā)現(xiàn)向其他重要臟器明顯遷移。 5、快速冰凍DAPI染色后可在細(xì)胞移植部位發(fā)現(xiàn)eGFP的表達(dá)，證實移植細(xì)胞的存在，主要存在皮下組織，心、肺、肝未見明顯eGFP的表達(dá)；HE染色發(fā)現(xiàn)LOCAL-INJE組移植材料內(nèi)細(xì)胞數(shù)量、血管密度最豐富的， CO-CULTURE組其次，對照組最少。Masson染色LOCAL-INJE組含量較CO-CULTURE組高，對照組含量最低。免疫組化染色中，LOCAL-INJE組可觀察到Desmin、α-SMA陽性表達(dá)。 【結(jié)論】生物活性補片植入裸鼠體內(nèi)后人脂肪干細(xì)胞能長期在移植部位存活。脂肪干細(xì)胞與脫細(xì)胞牛心包膜不經(jīng)體外共培養(yǎng)同時移植體內(nèi)，更有利于細(xì)胞長入ABP，減緩ABP的降解，促進(jìn)血管、平滑肌的合成，有利于組織的修復(fù)，是未來女性盆底重建材料的理想選擇。
[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.
【學(xué)位授予單位】：蘇州大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R711.5

【共引文獻(xiàn)】

相關(guān)期刊論文 前10條

1 曹杰;趙純?nèi)?;全盆底重建術(shù)相關(guān)并發(fā)癥的研究進(jìn)展[J];重慶醫(yī)學(xué);2012年35期

2 張曦;張誠;陳幸華;;重視造血干細(xì)胞移植中的造血微環(huán)境問題[J];第三軍醫(yī)大學(xué)學(xué)報;2012年24期

3 楊光;程慶礫;;干細(xì)胞與糖尿病腎病[J];北京大學(xué)學(xué)報(醫(yī)學(xué)版);2013年03期

4 崔岳毅;宋純理;譚杰;付鑫;陳仲強(qiáng);劉忠軍;黨耕町;;局部植入辛伐他汀誘導(dǎo)自體骨髓間充質(zhì)干細(xì)胞歸巢修復(fù)大鼠顱骨缺損[J];國際骨科學(xué)雜志;2013年06期

5 楊潔瑩;陳南梁;張佩華;;吊帶在手術(shù)治療女性壓力性尿失禁上的應(yīng)用[J];產(chǎn)業(yè)用紡織品;2014年02期

6 趙江民;劉佳;林雁冰;宗根林;吳利忠;錢海珊;游建雄;;SPIO標(biāo)記濃度對BMSC_s生物學(xué)特性的影響及體外MRI表現(xiàn)[J];磁共振成像;2014年02期

7 于祥茹;韓曉謙;袁浩天;董志恒;程梁;吳哲;;PLGA/CPC支架材料復(fù)合骨髓基質(zhì)干細(xì)胞構(gòu)建組織工程骨的體外效果評價[J];吉林大學(xué)學(xué)報(醫(yī)學(xué)版);2014年02期

8 李江璇;肖麗玲;;脂肪干細(xì)胞分泌功能的研究進(jìn)展及應(yīng)用前景[J];廣東醫(yī)學(xué);2014年02期

9 RIBEIRO Viviana Pinto;RIBEIRO Ana Soares;SILVA Carla Joana;DUR飴ES Nelson Feio;BONIF嚶CIO Gra

本文編號：2108390