【摘要】：實(shí)驗(yàn)?zāi)康?探索成人甲狀旁腺細(xì)胞提取和培養(yǎng)方法的可行性。為細(xì)胞移植治療甲狀旁腺功能低下提供理論依據(jù)。實(shí)驗(yàn)方法:采用膠原酶消化法消化成人甲狀旁腺組織,使細(xì)胞離散,然后將細(xì)胞進(jìn)行原代培養(yǎng)和傳代培養(yǎng),隔日觀察細(xì)胞的形態(tài)和數(shù)量變化。消化所得細(xì)胞形態(tài)完整,經(jīng)原代培養(yǎng)的細(xì)胞于2d時(shí)大部分細(xì)胞貼壁并展開(kāi),細(xì)胞形態(tài)呈梭形,細(xì)胞間形態(tài)差別不大,培養(yǎng)7d后細(xì)胞長(zhǎng)滿(mǎn)培養(yǎng)瓶瓶底。經(jīng)傳代培養(yǎng)的細(xì)胞1d細(xì)胞全部展開(kāi),細(xì)胞呈梭形,細(xì)胞體積比原代培養(yǎng)細(xì)胞大,7d后亦長(zhǎng)滿(mǎn)培養(yǎng)瓶底。消化傳代培養(yǎng)的細(xì)胞,其中一部分通過(guò)電子顯微鏡觀察細(xì)胞胞膜及胞質(zhì)形態(tài)并檢測(cè)留取培養(yǎng)液中甲狀旁腺激素(parathyroid hormone,PTH)的含量,另一部分采用聚合酶鏈反應(yīng)(PCR)的方法檢測(cè)甲狀旁腺細(xì)胞3種特異性標(biāo)志物RNA:鈣敏感性受體(calcium-sensing receptor,Ca SR)、PTH和GCM2(Glial cells missing-2)。實(shí)驗(yàn)結(jié)果:在電子顯微鏡下選取多處視野觀察傳代培養(yǎng)至第7d的細(xì)胞,鏡下可見(jiàn)細(xì)胞胞質(zhì)豐富,且胞質(zhì)內(nèi)含豐富的線粒體、內(nèi)質(zhì)網(wǎng)和高爾基體,胞質(zhì)和細(xì)胞間均可見(jiàn)分泌囊泡;細(xì)胞培養(yǎng)液上清中可檢測(cè)出PTH;甲狀旁腺細(xì)胞特異性標(biāo)志物PCR檢測(cè)結(jié)果Ca SR、PTH、GCM2 RNA均呈陽(yáng)性。實(shí)驗(yàn)結(jié)論:經(jīng)膠原酶消化法消化成人甲狀旁腺組織,提取甲狀旁腺細(xì)胞并常規(guī)培養(yǎng),所得甲狀旁腺細(xì)胞具有良好的形態(tài)并保留正常的內(nèi)分泌功能,并具有甲狀旁腺細(xì)胞的特異表型,有成為永久性甲狀旁腺功能減退癥臨床治療時(shí)同種異體細(xì)胞移植來(lái)源的潛力。實(shí)驗(yàn)?zāi)康?探索水凝膠為載體包裹甲狀旁腺細(xì)胞并應(yīng)用于臨床細(xì)胞移植治療甲狀旁腺減退癥的可行性。實(shí)驗(yàn)方法:復(fù)蘇凍存的成人甲狀旁腺細(xì)胞并常規(guī)細(xì)胞培養(yǎng),待細(xì)胞足量后,消化、離心、吹打得單細(xì)胞懸液,將殼聚糖溶液、交聯(lián)劑、細(xì)胞懸液按比例混合制得水凝膠,加培養(yǎng)液后于37℃恒溫箱中培養(yǎng),隔日留取培養(yǎng)液上清檢測(cè)甲狀旁腺激素(parathyroid hormone,PTH)含量,并對(duì)凝膠內(nèi)細(xì)胞染色觀察細(xì)胞狀態(tài)。實(shí)驗(yàn)結(jié)果:成功復(fù)蘇并培養(yǎng)成人甲狀旁腺細(xì)胞,細(xì)胞貼壁生長(zhǎng)呈梭形;水凝膠成膠時(shí)間1min;熒光掃描結(jié)果顯示24 h后膠內(nèi)活細(xì)胞比例90%,連續(xù)觀察3 d,比例沒(méi)有發(fā)生明顯改變;培養(yǎng)液中均檢測(cè)到PTH。實(shí)驗(yàn)結(jié)論:水凝膠化的成人甲狀旁腺細(xì)胞操作簡(jiǎn)單,甲狀旁腺細(xì)胞被水凝膠包裹后,不僅能夠維持細(xì)胞的正常形態(tài),且能夠穩(wěn)定釋放PTH,有成為細(xì)胞載體并應(yīng)用于移植治療甲狀旁腺功能減退癥的可能。
[Abstract]:Objective: to explore the feasibility of extraction and culture of adult parathyroid cells. To provide a theoretical basis for the treatment of hypoparathyroidism by cell transplantation. Methods: the adult parathyroid tissue was digested by collagenase digestion and the cells were separated. Then the cells were cultured in primary culture and subculture. The morphology and quantity of the cells were observed every other day. After 2 days of primary culture, most of the cells adhered to the wall and expanded. The morphology of the cells was fusiform, but there was no difference between the cells. After 7 days of culture, the cells grew up to the bottle-bottom of the culture bottle. After 1 d of culture, the cells were all expanded, the cells were fusiform, and the cell volume was larger than that of the primary culture cells for 7 days. Some of the cells digested and cultured were observed by electron microscope and the cell membrane and cytoplasm were observed and the content of parathyroid hormone (parathyroid hormone,PTH) in the culture medium was measured. In the other part, RNA: calcium sensitive receptor (calcium-sensing receptor,Ca SR) and GCM2 (Glial cells missing-2 were detected by polymerase chain reaction (PCR). The results showed that the cytoplasm of the cells was rich in cytoplasm, and the cytoplasm was rich in mitochondria, endoplasmic reticulum and Golgi body. The secretory vesicles could be seen between cytoplasm and cells. PTH; parathyroid cell specific marker PCR was detected in supernatant of cell culture medium. Ca SR,PTH,GCM2 RNA was positive. Conclusion: the human parathyroid tissue was digested by collagenase digestion and the parathyroid cells were extracted and cultured routinely. The results showed that the parathyroid cells had good morphology and maintained normal endocrine function. It also has the specific phenotype of parathyroid cells and the potential to be the source of allogeneic cell transplantation in the treatment of permanent hypothyroidism. Objective: to explore the feasibility of hydrogel encapsulating parathyroid cells and its application in the treatment of hypoparathyroidism. Methods: the frozen adult parathyroid cells were resuscitated and cultured in routine culture. After sufficient amount of cells, single cell suspension was obtained by digestion, centrifugation and blowing. The hydrogel was prepared by mixing chitosan solution, crosslinking agent and cell suspension in proportion. The supernatant of culture medium was used to detect the content of parathyroid hormone (parathyroid hormone,PTH) and the cell state was observed by staining the cells in the gel. Results: after successful resuscitation and culture of adult parathyroid cells, the adherent cells were fusiform, hydrogel gelation time was 1 min, fluorescence scanning showed that the proportion of living cells in the gel was 90% after 24 h, and the ratio was not changed obviously after 3 days of continuous observation. PTH. was detected in culture medium. Conclusion: the operation of hydrogelled adult parathyroid cells is simple. After the parathyroid cells are encapsulated by hydrogel, they can not only maintain the normal morphology of the cells. It is possible to release PTH, stably as a cell carrier and to be used in the treatment of parathyroid hypothyroidism.
【學(xué)位授予單位】：首都醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類(lèi)號(hào)】：R582

【相似文獻(xiàn)】

相關(guān)期刊論文 前10條

1 甘露;張志輝;呂美娜;王琳;;細(xì)胞培養(yǎng)技術(shù)[J];中國(guó)組織工程研究與臨床康復(fù);2008年29期

2 周麗薇;;細(xì)胞培養(yǎng)技術(shù)與防止細(xì)胞污染的方法[J];醫(yī)學(xué)信息(上旬刊);2010年11期

3 高小玲;白明;汪保英;張鐘允;;在研究生中開(kāi)設(shè)“細(xì)胞培養(yǎng)技術(shù)”課程的教學(xué)方法探索[J];世界中西醫(yī)結(jié)合雜志;2011年12期

4 向若蘭;;《組織和細(xì)胞培養(yǎng)技術(shù)》書(shū)評(píng)[J];基礎(chǔ)醫(yī)學(xué)與臨床;2011年12期

5 王晴宇;;全軍微量細(xì)胞培養(yǎng)技術(shù)學(xué)習(xí)班在我院結(jié)業(yè)[J];軍事醫(yī)學(xué)科學(xué)院院刊;1983年01期

6 趙天德;高福云;余郁;張群喜;;玻片微量細(xì)胞培養(yǎng)技術(shù)及細(xì)胞原位計(jì)量法[J];中日友好醫(yī)院學(xué)報(bào);1988年01期

7 黃珀,鄧勇,郭勇,馮志強(qiáng);腎小管細(xì)胞培養(yǎng)技術(shù)研究進(jìn)展[J];瀘州醫(yī)學(xué)院學(xué)報(bào);2001年03期

8 李焰;司徒鎮(zhèn)強(qiáng);吳軍正;劉斌;;研究生《細(xì)胞培養(yǎng)技術(shù)》教學(xué)實(shí)驗(yàn)課程實(shí)踐與思考[J];西北醫(yī)學(xué)教育;2005年01期

9 王公明;田玉科;田學(xué)愎;陳建平;安珂;楊輝;;用于包裝復(fù)制缺陷性腺病毒的低代次293細(xì)胞的培養(yǎng)[J];實(shí)用醫(yī)學(xué)雜志;2006年18期

10 楊斌;;細(xì)胞培養(yǎng)技術(shù)在耳鼻咽喉專(zhuān)業(yè)中的應(yīng)用[J];中國(guó)冶金工業(yè)醫(yī)學(xué)雜志;2008年04期

相關(guān)會(huì)議論文 前3條

1 薛建平;朱艷芳;張愛(ài)民;;藥用植物細(xì)胞培養(yǎng)影響因素的研究進(jìn)展[A];中醫(yī)藥理論與應(yīng)用研究——安徽中醫(yī)藥繼承與創(chuàng)新博士科技論壇論文集[C];2008年

2 牛新樂(lè);;細(xì)胞片層技術(shù)在心肌組織修復(fù)中的應(yīng)用[A];第八屆海峽兩岸心血管科學(xué)研討會(huì)論文集[C];2011年

3 ;序[A];植物組織培養(yǎng)與脫毒快繁技術(shù)——全國(guó)植物組培、脫毒快繁及工廠化生產(chǎn)技術(shù)學(xué)術(shù)研討會(huì)論文集[C];2001年

相關(guān)重要報(bào)紙文章 前3條

1 陳文倩;讓細(xì)胞培養(yǎng)技術(shù)走向生產(chǎn)線[N];中國(guó)醫(yī)藥報(bào);2006年

2 本報(bào)記者 吳潔;精心呵護(hù)細(xì)胞寶貝[N];科技日?qǐng)?bào);2002年

3 駐京記者　 王丹;我國(guó)首個(gè)大型哺乳類(lèi)動(dòng)物細(xì)胞培養(yǎng)技術(shù)平臺(tái)全線貫通[N];醫(yī)藥經(jīng)濟(jì)報(bào);2007年

相關(guān)博士學(xué)位論文 前3條

1 張其剛;K-ras基因?qū)氲氖蠓螑盒赞D(zhuǎn)化細(xì)胞粘附能力的改變及臨床意義[D];中國(guó)醫(yī)科大學(xué);2000年

2 王婷;扶正解毒含藥血清對(duì)鎳致癌干預(yù)作用的細(xì)胞分子機(jī)制研究[D];蘭州大學(xué);2010年

3 鄒朝春;甲狀腺激素對(duì)仔鼠大腦皮層縫隙連接蛋白表達(dá)的影響[D];浙江大學(xué);2010年

相關(guān)碩士學(xué)位論文 前9條

1 王杰;VEGF-A 165對(duì)穩(wěn)轉(zhuǎn)TDP-25質(zhì)粒的NSC34細(xì)胞的ALS細(xì)胞模型的影響及作用機(jī)制研究[D];河北醫(yī)科大學(xué);2015年

2 陳靜;Bit1靶向性小分子RNA對(duì)食管鱗癌（細(xì)胞）侵襲抑制及治療作用[D];鄭州大學(xué);2015年

3 楊心治;PRMT2對(duì)MCF-7細(xì)胞侵襲與遷移的影響及機(jī)制研究[D];南華大學(xué);2015年

4 趙越;成人甲狀旁腺細(xì)胞的培養(yǎng)鑒定及水凝膠化成人甲狀旁腺細(xì)胞的形態(tài)和功能觀察[D];首都醫(yī)科大學(xué);2016年

5 衛(wèi)元晨;基于微流控芯片對(duì)機(jī)械響應(yīng)細(xì)胞的研究[D];沈陽(yáng)工業(yè)大學(xué);2013年

6 楊貝貝;表達(dá)rhTNFR-Fc的重組CHO細(xì)胞懸浮馴化及培養(yǎng)工藝研究[D];河北科技大學(xué);2014年

7 陳冬波;CD43表達(dá)與造血干細(xì)胞發(fā)育的相關(guān)性研究[D];中國(guó)人民解放軍軍事醫(yī)學(xué)科學(xué)院;2011年

8 王北;新型維甲酸衍生物由肌球蛋白輕鏈激酶通過(guò)p38信號(hào)轉(zhuǎn)導(dǎo)途徑抑制乳腺癌MDA-MB-231細(xì)胞的遷移[D];安徽醫(yī)科大學(xué);2013年

9 張芙蓉;人垂體生長(zhǎng)激素腺瘤體外培養(yǎng)及其生物學(xué)特性的研究[D];青島大學(xué);2006年

，

本文編號(hào)：2204865