【摘要】：實驗目的:探索成人甲狀旁腺細胞提取和培養(yǎng)方法的可行性。為細胞移植治療甲狀旁腺功能低下提供理論依據。實驗方法:采用膠原酶消化法消化成人甲狀旁腺組織,使細胞離散,然后將細胞進行原代培養(yǎng)和傳代培養(yǎng),隔日觀察細胞的形態(tài)和數量變化。消化所得細胞形態(tài)完整,經原代培養(yǎng)的細胞于2d時大部分細胞貼壁并展開,細胞形態(tài)呈梭形,細胞間形態(tài)差別不大,培養(yǎng)7d后細胞長滿培養(yǎng)瓶瓶底。經傳代培養(yǎng)的細胞1d細胞全部展開,細胞呈梭形,細胞體積比原代培養(yǎng)細胞大,7d后亦長滿培養(yǎng)瓶底。消化傳代培養(yǎng)的細胞,其中一部分通過電子顯微鏡觀察細胞胞膜及胞質形態(tài)并檢測留取培養(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(PCR)的方法檢測甲狀旁腺細胞3種特異性標志物RNA:鈣敏感性受體(calcium-sensing receptor,Ca SR)、PTH和GCM2(Glial cells missing-2)。實驗結果:在電子顯微鏡下選取多處視野觀察傳代培養(yǎng)至第7d的細胞,鏡下可見細胞胞質豐富,且胞質內含豐富的線粒體、內質網和高爾基體,胞質和細胞間均可見分泌囊泡;細胞培養(yǎng)液上清中可檢測出PTH;甲狀旁腺細胞特異性標志物PCR檢測結果Ca SR、PTH、GCM2 RNA均呈陽性。實驗結論:經膠原酶消化法消化成人甲狀旁腺組織,提取甲狀旁腺細胞并常規(guī)培養(yǎng),所得甲狀旁腺細胞具有良好的形態(tài)并保留正常的內分泌功能,并具有甲狀旁腺細胞的特異表型,有成為永久性甲狀旁腺功能減退癥臨床治療時同種異體細胞移植來源的潛力。實驗目的:探索水凝膠為載體包裹甲狀旁腺細胞并應用于臨床細胞移植治療甲狀旁腺減退癥的可行性。實驗方法:復蘇凍存的成人甲狀旁腺細胞并常規(guī)細胞培養(yǎng),待細胞足量后,消化、離心、吹打得單細胞懸液,將殼聚糖溶液、交聯劑、細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)液上清檢測甲狀旁腺激素(parathyroid hormone,PTH)含量,并對凝膠內細胞染色觀察細胞狀態(tài)。實驗結果:成功復蘇并培養(yǎng)成人甲狀旁腺細胞,細胞貼壁生長呈梭形;水凝膠成膠時間1min;熒光掃描結果顯示24 h后膠內活細胞比例90%,連續(xù)觀察3 d,比例沒有發(fā)生明顯改變;培養(yǎng)液中均檢測到PTH。實驗結論:水凝膠化的成人甲狀旁腺細胞操作簡單,甲狀旁腺細胞被水凝膠包裹后,不僅能夠維持細胞的正常形態(tài),且能夠穩(wěn)定釋放PTH,有成為細胞載體并應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.
【學位授予單位】：首都醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2016
【分類號】：R582

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