發(fā)布時間：2018-05-27 09:32

  本文選題：脂肪間充質(zhì)干細胞 + 臍帶間充質(zhì)干細胞��； 參考：《南開大學(xué)》2014年博士論文

【摘要】：第一部分： 目的：尋找獲得用于移植的間充質(zhì)干細胞的適宜方法；培養(yǎng)豬脂肪間充質(zhì)干細胞(ADMSC).豬骨髓間充質(zhì)干細胞(BMSC).人臍帶間充質(zhì)干細胞(UMSC).人脂肪間充質(zhì)干細胞；對培養(yǎng)的間充質(zhì)干細胞進行鑒定；篩選適合對榮昌豬進行移植的間充質(zhì)干細胞類型。 方法：膠原酶消化法原代分離培養(yǎng)豬脂肪間充質(zhì)干細胞,用WST-1法比較豬ADMSC在37度和38度條件下的生長曲線,向成骨、成脂和成軟骨方向誘導(dǎo)分化ADMSC,鑒定其多能性。密度梯度離心法原代分離培養(yǎng)豬骨髓間充質(zhì)干細胞,用細胞計數(shù)法描繪其生長曲線,向成骨和成脂方向誘導(dǎo)分化鑒定其多能性。組織塊貼壁法原代培養(yǎng)人臍帶間充質(zhì)干細胞,用流式法鑒定干細胞表面抗原。擴增培養(yǎng)成人脂肪間充質(zhì)干細胞,用WST-1法比較在5%和15%血清濃度下培養(yǎng)人ADMSC的生長曲線。 結(jié)果：用膠原酶消化法可以從豬頸部脂肪組織中分離培養(yǎng)出脂肪間充質(zhì)干細胞,貼壁生長,P2代以后形態(tài)均一,穩(wěn)定增殖,約5天可傳代,在一定條件誘導(dǎo)下可以向成骨、成脂或成軟骨方向分化,適宜在5%CO2,38℃,飽和濕度條件下培養(yǎng)。用密度梯度離心法可以從豬股骨骨髓中提取分離出骨髓間充質(zhì)干細胞,貼壁生長,P3代以后形態(tài)均一,P4代以內(nèi)增殖穩(wěn)定,約10天可傳代,在一定條件誘導(dǎo)下可以向成骨及成脂方向分化。用組織塊貼壁法可以從人新鮮臍帶組織中分離出臍帶間充質(zhì)干細胞,組織貼壁約20天后可見UMSC爬出,細胞形態(tài)均一,呈漩渦樣排列,P1-P10代生長迅速,約4天即可傳代,經(jīng)流式細胞儀檢測其表型為CD73(+), CD105(+),CD31(-),CD34(-),符合間充質(zhì)干細胞特點。 結(jié)論：原代培養(yǎng)的人臍帶間充質(zhì)干細胞,分離方便、增殖速度快,狀態(tài)穩(wěn)定,適合用于移植治療。 第二部分： 目的：熟悉小型豬脊柱的解剖結(jié)構(gòu),模擬腦脊液循環(huán)途徑,探討經(jīng)鞘內(nèi)注射細胞移植途徑的可行性。 方法：對小型豬腰、骶椎脊柱區(qū)進行局部解剖,了解其結(jié)構(gòu)特點,測量腰椎棘突大小及椎間隙的寬度。行腰椎穿刺向蛛網(wǎng)膜下腔注射美蘭染液,2小時后處死動物觀察染料分布情況。腰椎穿刺向蛛網(wǎng)膜下腔注射DAPI染液,24小時后處死動物,取聽神經(jīng)及耳蝸制作組織冰凍切片,激光顯微鏡下觀察。 結(jié)果：小型豬的腰椎棘突向頭側(cè)傾斜,高度1cm,厚度0.2cm,寬度1.2-1.5cm,腰5-骶1椎間隙最寬,約0.5cm。經(jīng)蛛網(wǎng)膜下腔注射的美蘭染料可以將聽神經(jīng)內(nèi)聽道段染為藍色。經(jīng)蛛網(wǎng)膜下腔注射DAPI染料,24小時后在激光顯微鏡下觀察可見聽神經(jīng)內(nèi)聽道段有藍色熒光,而耳蝸脫鈣處理1個月后行冰凍組織切片后再觀察未見熒光。 結(jié)論：小型豬脊柱解剖結(jié)構(gòu)與人類相似,在麻醉恰當(dāng)?shù)那闆r下,腰椎穿刺手術(shù)順利,可以完成腦脊液的取樣和蛛網(wǎng)膜下腔藥物注射,經(jīng)此途徑注射的染料可以對腦脊液進行短期示蹤,可對聽神經(jīng)內(nèi)聽道段進行活體染色標(biāo)記。 第三部分： 目的：檢測經(jīng)腰椎穿刺蛛網(wǎng)膜下腔注射移植的間充質(zhì)干細胞在動物內(nèi)耳及全身的分布,以及這種移植方法對聽性腦干反應(yīng)電位的影響。 方法：原代分離培養(yǎng)人臍帶間充質(zhì)干細胞。選擇Mitf-M突變的白化耳聾榮昌豬作為模型,正常榮昌豬為對照。將P5-P6代的人臍帶間充質(zhì)干細胞經(jīng)蛛網(wǎng)膜下腔注射的途徑移植到正常及耳聾的榮昌豬體內(nèi),每只豬移植細胞量為10～6-10～7個。分別在移植前、移植后2小時、3天、1周、2周、3周、4周直至動物處死前檢測其聽性腦干反應(yīng)電位。在移植后2小時、3天、1周、2周、4周、8周處死動物取其耳蝸,制作冰凍組織切片行免疫組織熒光染色,用人特異性抗體檢測供體細胞。在移植后3天、1周和3周處死動物取其全身器官標(biāo)本制作冰凍組織切片、提取DNA,用免疫組織熒光以及聚合酶鏈反應(yīng)的方法,用人特異性抗體及引物檢測供體細胞在動物體內(nèi)分布。 結(jié)果：經(jīng)蛛網(wǎng)膜下腔注射的人臍帶間充質(zhì)干細胞在移植后3天、1周、2周、3周、4周可以在動物內(nèi)耳的血管紋、螺旋神經(jīng)節(jié)及基底膜等處檢出；在移植后3天和1周可以在大腦、小腦、延髓、心、肝和肺中檢出。通過PCR方法在3天、1周后可以在神經(jīng)系統(tǒng)、心、肝、肺組織中檢出供體細胞的DNA。在移植后3天和1周,白化榮昌豬ABR出現(xiàn)了與移植前明顯不同的波形。 結(jié)論：經(jīng)蛛網(wǎng)膜下腔注射移植人臍帶間充質(zhì)干細胞后,8周內(nèi)不會導(dǎo)致宿主動物死亡,移植細胞可以進入耳蝸及中樞神經(jīng)系統(tǒng)以及部分內(nèi)臟,可以對耳聾榮昌豬的聽性腦干反應(yīng)電位造成短期影響。 第四部分： 目的：總結(jié)顳骨巨細胞肉芽腫的臨床表現(xiàn)、病理特點、診斷與治療方法以提高其療效。 方法：回顧性分析我科2001年至2010年十年間發(fā)生在顳骨的巨細胞肉芽腫8例,總結(jié)其臨床和影像學(xué)表現(xiàn)、手術(shù)進路方法、病理及隨訪結(jié)果。 結(jié)果：8例中男性4例、女性4例,年齡21-50歲,中位數(shù)年齡37歲,左側(cè)4例、右側(cè)4例,病程從5-60個月,中位數(shù)病程21個月,其中1例為復(fù)發(fā)病例,其余均為首次就診。主訴聽力下降5例、耳鳴4例、局部包塊2例。影像學(xué)檢查可見局部腫物、顳骨及顱底骨壁破壞缺損。術(shù)前均未明確診斷,全部采用手術(shù)治療,其中顱中窩進路5例,顱-耳頸聯(lián)合進路3例,術(shù)后3例出現(xiàn)不全面癱,1例腦水腫,經(jīng)治療后均恢復(fù)。術(shù)后病理診斷均為巨細胞肉芽腫。隨訪7例,12至58個月無一例復(fù)發(fā),另有1例失訪。 結(jié)論：顳骨巨細胞無特異性臨床和影像學(xué)表現(xiàn),常以聽力下降為首發(fā)癥狀,也可表現(xiàn)為耳鳴、眩暈、耳痛以及局部包塊等,術(shù)前診斷困難,治療以手術(shù)徹底清除病變?yōu)闉槭走x,術(shù)后復(fù)發(fā)率較低。
[Abstract]:Part one:
Objective: to find a suitable method for obtaining mesenchymal stem cells for transplantation; to cultivate porcine adipose mesenchymal stem cells (ADMSC), pig bone marrow mesenchymal stem cells (BMSC), human umbilical cord mesenchymal stem cells (UMSC), human adipose mesenchymal stem cells (human adipose mesenchymal stem cells), identification of cultured mesenchymal stem cells (MSCs), and to screen for the transplants suitable for Rongchang pigs. The type of mesenchymal stem cells.
Methods: pig fat mesenchymal stem cells were isolated and cultured by collagenase digestion. WST-1 method was used to compare the growth curve of pig ADMSC at 37 and 38 degrees. It was induced to differentiate into bone, lipid and cartilaginous direction to differentiate ADMSC and identify its pluripotent activity. Density gradient centrifugation was used to separate bone marrow mesenchymal stem cells from pig to be cultured, and the cell count method was used. The growth curve was drawn to identify the pluripotent activity of the osteogenic and fat induced differentiation. The tissue block adherence method was used to culture human umbilical cord mesenchymal stem cells. The stem cell surface antigen was identified by flow method. Adult adipose mesenchymal stem cells were amplified and cultured. The growth curve of human ADMSC was compared with the serum concentration of 5% and 15% by WST-1 method.
Results: the adipose mesenchymal stem cells can be isolated and cultured from porcine neck adipose tissue by collagenase digestion. After P2 generation, the cells can grow evenly and proliferate steadily. It can be passaged for about 5 days, and can be differentiated into osteogenesis, fat or cartilage under certain conditions. It is suitable to be cultivated at 5%CO2,38 C and saturated humidity. The gradient centrifugation method can extract and separate bone marrow mesenchymal stem cells from the bone marrow of pig's femur. After P3 generation, the proliferation of mesenchymal stem cells is uniform, and the proliferation is stable within P4 generation. It can be passaged for about 10 days, and can differentiate into osteogenesis and fat formation under certain conditions. The umbilical cord mesenchymal cells can be separated from human umbilical cord tissue by tissue block wall method. The stem cells were found to climb out of UMSC after 20 days of tissue adherence. The cell morphology was uniform, and the cells were arranged in a whirlpool like arrangement. The growth of the P1-P10 generation was rapid, about 4 days. The phenotype of the cells was CD73 (+), CD105 (+), CD31 (-), CD34 (-), conforming to the characteristics of mesenchymal stem cells.
Conclusion: primary cultured human umbilical cord mesenchymal stem cells are easy to isolate, proliferate fast and stable, and are suitable for transplantation therapy.
The second part:
Objective: to understand the anatomy of the spinal column of the miniature pig and to simulate the circulation of cerebrospinal fluid, and to explore the feasibility of intrathecal injection of cell transplantation.
Methods: the local anatomy of the small pig's waist and the sacral spine area was carried out to understand the structural features of the lumbar spine, and to measure the size of the spine and the width of the intervertebral space. The lumbar puncture was injected into the subarachnoid cavity and the dye was injected into the subarachnoid cavity. After 2 hours, the animals were sacrificed to observe the distribution of the dyestuffs. The lumbar puncture was injected into the arachnoid membrane and the animals were killed after 24 hours. The frozen tissues of the auditory nerve and cochlea were collected and observed under laser microscope.
Results: the lumbar spine of the small pig was inclined to the head side, the height of 1cm, the thickness of 0.2cm, the width 1.2-1.5cm, the width of the lumbosacral 1 vertebra in the lumbar 5-, and the methylene blue dye which was injected by the subarachnoid cavity could dye the auditory canal in the auditory nerve blue. The DAPI dye was injected into the subarachnoid cavity, and the auditory nerve was observed under the laser microscope after the laser microscope. There was blue fluorescence in the passage, but no fluorescence was observed in the frozen tissue after 1 months of cochlear decalcification.
Conclusion: the anatomical structure of the spinal column is similar to that of human. In the case of appropriate anesthesia, the lumbar puncture operation is smooth and the cerebrospinal fluid can be sampled and the subarachnoid drug injection can be completed. The dye injected through this way can trace the cerebrospinal fluid in short term, and can be used to stain the auditory segment of the auditory nerve.
The third part:
Objective: to detect the distribution of mesenchymal stem cells (MSCs) in the inner ear and the whole body of the subarachnoid injection through the lumbar puncture, and the effect of this method on the auditory brainstem response potential.
Methods: human umbilical cord mesenchymal stem cells were isolated and cultured. The Mitf-M mutated albino deafness Rongchang pig was selected as the model and the normal Rongchang pig as the control. The P5-P6 generation human umbilical cord mesenchymal stem cells were transplanted into the normal and deafness Rongchang pigs by subarachnoid injection into 10 to 6-10 to 7 pigs. Before transplantation, 2 hours, 3 days, 1 weeks, 2 weeks, 3 weeks, 4 weeks until the animals were executed, the auditory brainstem response potential was detected before the animals were executed. The cochlea was taken for 2 hours, 3 days, 1 weeks, 2 weeks, 4 weeks and 8 weeks. The frozen tissue sections were stained with immunofluorescence, and the donor cells were detected by human specific antibodies. Frozen tissue sections were taken from the dead animals of Zhou Dynasty to produce frozen tissue sections to extract DNA. The immunofluorescence and polymerase chain reaction (PCR) were used to detect the distribution of donor cells in animals by human specific antibodies and primers.
Results: human umbilical cord mesenchymal stem cells were injected into the subarachnoid space for 3 days, 1 weeks, 2 weeks, 3 weeks, and 4 weeks to detect the vascular lines, spiral ganglion and basal membrane in the inner ear, and can be detected in the brain, cerebellum, medulla, heart, liver and lungs at 3 days and 1 weeks after transplantation. The PCR method can be used in the nerve system for 3 days and 1 weeks after the transplantation. The DNA. of donor cells was detected in the heart, liver and lung tissues. 3 days and 1 weeks after transplantation, the ABR of albino Rongchang pig appeared distinct waveforms before transplantation.
Conclusion: the transplantation of human umbilical cord mesenchymal stem cells by subarachnoid injection can not lead to the death of the host animal within 8 weeks. The transplanted cells can enter the cochlea and the central nervous system as well as some viscera, which can have a short-term effect on the auditory brainstem response potential of the deaf Rongchang pigs.
The fourth part:
Objective: To summarize the clinical manifestations, pathological features, diagnosis and treatment of giant cell granuloma of temporal bone in order to improve its curative effect.
Methods: a retrospective analysis of 8 cases of giant cell granuloma occurring in the temporal bone in our department from 2001 to 2010 was reviewed. The clinical and imaging findings, surgical approach, pathology and follow-up results were summarized.
Results: 4 of the 8 men, 4 women, 21-50 years old, the median age 37, the left 4, the right 4, the course from 5-60 months, the median course 21 months, 1 cases for the first time, the rest were all for the first time. The main complaints were 5, tinnitus 4 cases, and partial lump 2 cases. Imaging examination showed local mass, temporal bone and skull base wall No definite diagnosis was performed before the operation, all of them were treated with surgical treatment, including 5 cases of cranial fossa approach, 3 cases with craniofacial neck combined approach, 3 cases of incomplete facial paralysis after operation, 1 cases of brain edema and recovery after treatment. All the postoperative pathological diagnosis was giant cell granuloma. 7 cases were followed up, 12 to 58 months had no recurrence, and 1 cases were lost.
Conclusion: there is no specific clinical and imaging manifestations of giant cells in the temporal bone, often with hearing loss as the first symptom. It can also show tinnitus, vertigo, ear pain and local mass. It is difficult to diagnose before operation. The first choice is to remove the disease thoroughly by operation, and the recurrence rate is low after operation.
【學(xué)位授予單位】：南開大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2014
【分類號】：R764.431

【參考文獻】

相關(guān)期刊論文 前10條

1 呼云之，沈銘昌;腮腺區(qū)嗜伊紅細胞增多性淋巴肉芽腫（附37例分析）[J];北京口腔醫(yī)學(xué);1996年02期

2 秦賀;楊仕明;翟所強;;骨髓間充質(zhì)干細胞治療感音神經(jīng)性聾的基礎(chǔ)研究進展[J];中國耳鼻咽喉頭頸外科;2009年01期

3 王先寶;蔡德鴻;張樺;楊建明;孫嘉;張振;馬昭杰;陳宏;;磁標(biāo)記豬骨髓間充質(zhì)干細胞肝臟移植示蹤研究[J];廣東醫(yī)學(xué);2009年03期

4 方亮;施曉雷;褚薛慧;方勝;丁義濤;;磁標(biāo)記豬骨髓間充質(zhì)干細胞自體肝內(nèi)移植后MR活體示蹤研究[J];肝膽外科雜志;2008年03期

5 劉軍;頭頸部巨細胞修復(fù)性肉芽腫[J];國外醫(yī)學(xué).耳鼻咽喉科學(xué)分冊;2000年01期

6 史宗道;焦錫葳;周志瑜;;頜骨及頜骨巨細胞修復(fù)性肉芽腫[J];口腔醫(yī)學(xué);1984年01期

7 雷愛軍,崔香艷;顳骨及鼻骨巨細胞修復(fù)性肉芽腫2例[J];臨床耳鼻咽喉科雜志;2004年11期

8 葉茂奎,高思佳,馬忠禮,徐克;多發(fā)性骨巨細胞修復(fù)性肉芽腫(2例報告并文獻復(fù)習(xí))[J];臨床放射學(xué)雜志;2001年10期

9 吳沛橋;巴曉革;胡海;趙靜;;綠色熒光蛋白GFP的研究進展及應(yīng)用[J];生物醫(yī)學(xué)工程研究;2009年01期

10 袁先道;閆曦;楊華;高志強;李康華;韓欽;盧姍;陳曉巍;亓放;姜鴻;趙春華;;人脂肪來源間充質(zhì)干細胞植入受損耳蝸后定向歸巢并分化為毛細胞樣細胞[J];中國生物工程雜志;2010年08期

相關(guān)博士學(xué)位論文 前2條

1 秦賀;骨髓間充質(zhì)干細胞治療藥物性聾的基礎(chǔ)研究[D];中國人民解放軍軍醫(yī)進修學(xué)院;2010年

2 任麗麗;白化榮昌豬耳聾的分子病理機制研究[D];中國人民解放軍軍醫(yī)進修學(xué)院;2013年

，

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