成人甲状旁腺细胞的培养鉴定及水凝胶化成人甲状旁腺细胞的形态和功能观察

发布时间：2018-08-26 12:25

【摘要】：实验目的:探索成人甲状旁腺细胞提取和培养方法的可行性。为细胞移植治疗甲状旁腺功能低下提供理论依据。实验方法:采用胶原酶消化法消化成人甲状旁腺组织,使细胞离散,然后将细胞进行原代培养和传代培养,隔日观察细胞的形态和数量变化。消化所得细胞形态完整,经原代培养的细胞于2d时大部分细胞贴壁并展开,细胞形态呈梭形,细胞间形态差别不大,培养7d后细胞长满培养瓶瓶底。经传代培养的细胞1d细胞全部展开,细胞呈梭形,细胞体积比原代培养细胞大,7d后亦长满培养瓶底。消化传代培养的细胞,其中一部分通过电子显微镜观察细胞胞膜及胞质形态并检测留取培养液中甲状旁腺激素(parathyroid hormone,PTH)的含量,另一部分采用聚合酶链反应(PCR)的方法检测甲状旁腺细胞3种特异性标志物RNA:钙敏感性受体(calcium-sensing receptor,Ca SR)、PTH和GCM2(Glial cells missing-2)。实验结果:在电子显微镜下选取多处视野观察传代培养至第7d的细胞,镜下可见细胞胞质丰富,且胞质内含丰富的线粒体、内质网和高尔基体,胞质和细胞间均可见分泌囊泡;细胞培养液上清中可检测出PTH;甲状旁腺细胞特异性标志物PCR检测结果Ca SR、PTH、GCM2 RNA均呈阳性。实验结论:经胶原酶消化法消化成人甲状旁腺组织,提取甲状旁腺细胞并常规培养,所得甲状旁腺细胞具有良好的形态并保留正常的内分泌功能,并具有甲状旁腺细胞的特异表型,有成为永久性甲状旁腺功能减退症临床治疗时同种异体细胞移植来源的潜力。实验目的:探索水凝胶为载体包裹甲状旁腺细胞并应用于临床细胞移植治疗甲状旁腺减退症的可行性。实验方法:复苏冻存的成人甲状旁腺细胞并常规细胞培养,待细胞足量后,消化、离心、吹打得单细胞悬液,将壳聚糖溶液、交联剂、细胞悬液按比例混合制得水凝胶,加培养液后于37℃恒温箱中培养,隔日留取培养液上清检测甲状旁腺激素(parathyroid hormone,PTH)含量,并对凝胶内细胞染色观察细胞状态。实验结果:成功复苏并培养成人甲状旁腺细胞,细胞贴壁生长呈梭形;水凝胶成胶时间1min;荧光扫描结果显示24 h后胶内活细胞比例90%,连续观察3 d,比例没有发生明显改变;培养液中均检测到PTH。实验结论:水凝胶化的成人甲状旁腺细胞操作简单,甲状旁腺细胞被水凝胶包裹后,不仅能够维持细胞的正常形态,且能够稳定释放PTH,有成为细胞载体并应用于移植治疗甲状旁腺功能减退症的可能。
[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.
【学位授予单位】：首都医科大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R582

【相似文献】