Please use this identifier to cite or link to this item:
http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/59898
Title: | 近端腎小管細胞之組織工程研究 Tissue Engineering of Renal Proximal Tubule Cell |
Authors: | CHIANG I-NI 姜宜妮 |
Advisor: | 楊台鴻 |
Keyword: | 近端腎小管細胞、組織工程,遠紅外線,幾丁聚醣,角膜內皮細胞, renal proximal tubule cell,tissue engineering,far infrared radiation,chitosan,corneal endothelial cell, |
Publication Year : | 2017 |
Degree: | 博士 |
Abstract: | 近端腎小管細胞負責接受腎絲球過濾液,具有再吸收的功能,藉以維持生物體內水分和電解質的平衡,是腎臟中相當重要的細胞組成。然而近端腎小管細胞相當脆弱,感染發炎、氧氣不足、環境內的毒物、肥胖和糖尿併發症都會造成近端腎小管細胞的損傷。因此近端腎小管的再生與組織工程亦是一個具有發展性的研究課題。對於近端腎小管的再生與組織工程來說,足量的細胞來源和正常的細胞功能是最重要的。許多學者投入心力研究培養近端腎小管細胞的培養液、培養液補充成分、分離取下細胞的消化酶和用以培養細胞的細胞外間質,希望能更有效率地培養出具有正常功能的近端腎小管細胞。
遠紅外線是自然界光譜中的一個分支,目前已被證實有助於組織修復、抗發炎、促進細胞生長、調控睡眠品質、加速末梢血液循環和緩解疼痛。本研究嘗試將遠紅外線應用於近端腎小管細胞的培養,觀察遠紅外線定期照射下,細胞的生長分化、功能特性和免疫螢光表現。本研究顯示,培養兔近端腎小管細胞時,每日接受三十分鐘遠紅外線照射的組別,其細胞活性顯著高於沒有照射遠紅外線的組別。且經細胞染色和西方點墨法證實,接受遠紅外線照射的近端腎小管細胞會表現出顯著較強的細胞特性,包括:鈉鉀幫浦和葡萄糖運輸子。本研究進一步培養人類近端腎小管細胞株作藥毒性實驗。在近端腎小管細胞的培養液中加入具有腎毒性的化療藥物順鉑會造成細胞活性降低和細胞凋亡。若給予細胞株每日三十分鐘的遠紅外線照射,加入順鉑後雖然細胞活性也會下降,但其活性下降幅度和細胞凋亡的比例都會低於沒有照射的組別。根據本研究的結果,遠紅外線照射有助於近端腎小管細胞培養,可增加細胞活性,並且可藉由降低細胞凋亡,來減少腎毒性藥物順鉑對近端腎小管細胞造成的腎毒性。 本研究同時也在尋找適合近端腎小管細胞生長分化的生醫材料。過去的文獻中,通常使用膠原蛋白作為培養近端腎小管細胞的基底。然而膠原蛋白的機械強度不高,且將近端腎小管細胞培養於膠原蛋白上,需另外加入血清以促進細胞的生長分化,考量到異體血清可能帶來排斥和感染的問題,本研究嘗試將近端腎小管細胞培養在幾丁聚醣薄膜上,並使用無血清培養液,人類近端腎小管細胞可生長於幾丁聚醣薄膜上,維持長達一百五十天的穩定活性。幾丁聚醣是培養上皮細胞和軟組織的新興生醫材料,具有以下優點:生物相容性、可分解、抗菌和促進傷口癒合。本研究比較近端腎小管細胞分別培養在膠原蛋白和幾丁聚醣薄膜上的生長分化,發現培養於幾丁聚醣上的細胞表現出較高的鈉鉀幫浦、較低的纖維母細胞相關因子和較低的電阻,此結果顯示近端腎小管細胞於無血清的環境中生長於幾丁聚醣薄膜上具有較好的功能性。除了將近端腎小管細胞養於平面薄膜之外,本研究亦將幾丁聚醣製成細管,將近端腎小管細胞培養於幾丁聚醣管腔中,細胞於幾丁聚醣管內生長良好。 除了以幾丁聚醣為基底的近端腎小管培養之外,本研究也常是將近端腎小管細胞用於自體細胞治療。角膜內皮細胞和近端腎小管細胞一樣具有調控水分的功能並且都具有鈉鉀幫浦和水通道。本研究在兔模組上將自體近端腎小管細胞培養於幾丁聚醣薄膜上植入修補受損的角膜內皮細胞。植入一周後,自體近端腎小管細胞仍在眼球中存活。 然而,兔眼球在植入近端腎小管細胞/幾丁聚醣複合體後,眼球呈現不透明和腫脹的情況。因此進一步比較了有機矽膜和幾丁聚醣膜植入眼球內的組織變化。有機矽膜植入後直觀上角膜的透明度較佳,因此進一步將人類近端腎小管細胞培養餘有機矽膜上植入兔眼球,植入十天後進行解剖,發現人類近端腎小管細胞仍能存活於兔眼球中。後續仍將繼續此研究,希望能改善細胞/有機矽膜與眼球的貼附度,並期望能進行更多功能性的試驗。 本研究以近端腎小管細胞為主軸,藉由照光和改變培養細胞之生醫材料來改善細胞培養,並嘗試將培養之近端腎小管細胞應用於藥毒性觀察、培養於薄膜和管狀鷹架上,並嘗試用於自體細胞治療,以近端腎小管細胞修復受損之角膜內皮細胞。 Renal proximal tubule cells (RPTCs) are responsible for glomerular filtration and maintenance of water/electrolyte balance. RPTCs are vulnerable to infections, hypoxia, environmental toxins, obstetric, and diabetic complications. To regenerate a proximal tubule, sufficient cell numbers and normal cell function are requisite. Efforts have been made to improve RPTC cultivation with medium adjustment, medium supplements, digesting methods, and pre-coated extracellular matrix (ECM) Far infrared radiation (FIR), a subdivision of the electromagnetic spectrum, has been proved to be beneficial for long-term effects of tissue healing, anti-inflammation, promoting growth, modulating sleep, acceleration of microcirculation, and pain relief. We attempt to study whether FIR would be beneficial RPTC cultivation and renal tissue engineering. We observed the beneficial effects of FIR on RPTCs, including cell viability, functional characteristics, immunofluorescence presentations, and subcellular findings. And the FIR protective effects were further examined with HK-2 cell (Human proximal tubule cell line) against cisplatin, a nephrotoxic agent. Our study showed that daily exposure to FIR for 30 minutes could significantly increase rabbit RPTC viability in vitro. FIR is not only beneficial in RPTC cell viability. RPTCs with FIR exposure presented higher expression of Na-K ATPase and GLUT1 (Glucose Transporter 1). The finding was documented with western blot analysis with statistical significance. With Q-PCR, CDK5R1, GNAS, NPPB, and TEK expressions were significantly enhanced. With HK-2 cell, the proximal tubule cell line, FIR had protective effects against cisplatin nephrotoxicity through reducing apoptosis. FIR is a potential photomodulation therapy to facilitate RPTC cultivation and would possibly be applied in further nephrotoxicity protection and other cisplatin-sensitive cell protection. As for choosing a suitable biomaterial as the substrate, we compared human RPTCs (HRPTCs) cultivated on collagen and chitosan. Collagen has been routinely used as a substrate for culturing HRPTCs. However, HRPTCs cultivation on collagen requires serum addition, which brings the concerns of infection and immune responses. Chitosan is a promising biomaterial in tissue engineering approaches for primary culture of epithelial cells and soft tissue due to its biocompatibility, biodegradability, antibacterial, and wound healing activity. In this study, primary HRPTCs retrieved were cultivated on chitosan as a substrate in serum-free condition for up to 150 days. HRPTCs could maintain a typical epithelial morphology and the specific differentiation feature of transporting epithelia with dome formation after such long-term culture. As compared to HRPTCs cultivated on collagen, those cultivated on chitosan showed more dome formation, higher Na-K ATPase expression, lower vimentin expression, and lower transepithelial electrical resistance (TEER), indicating that HRPTCs cultivated on chitosan presented better differentiation status and would be more functional with better active transportation. Thus, the study indicates that chitosan is suitable for HRPTC cultivation in serum-free condition. We further cultivated HRPTCs on a 3D chitosan tubular scaffold, a chitosan conduit. HRPTCs also grew to confluence and differentiated with dome formation in the 3D chitosan tubular scaffold to form a chitosan-based tissue-engineered renal proximal tubule conduit. After completing stationary RPTC cultivation, we further came up with the idea of autologous cell therapy. RPTCs and CECs are both capable of water pumping, which is a very unique ability in the same way. Since CECs and RPTCs share similar physiological property in water balance, presentations of Na-K ATPase and aquaporin-1 (AQP-1). We tried to use the autologous expanded RPTCs to replace dysfunctional CECs with a biodegradable carrier in a rabbit model. Autologously derived RPTCs cultivated on chitosan membrane were used to repair damaged cornea endothelium. The RPTC/chitosan construct was still viable with functional characteristics after one week transplantation However, in the rabbit model with RPTC/chitosan construct, the eye transparency was not satisfying. We further compared biocompatibility of chitosan membrane and poly-dimethylsiloxane (PDMS) membrane. The transparency of the cornea was better with PDMS implantation. Thus, we cultivated HRPTCs on PDMS membrane to fabricate a HRPTC/PDMS construct for implantation. With the HRPTC/PDMS implantation, HRPTCs are viable in the rabbit eye after 10-day implantation. We would progress the study in the future to improve the attachment between cornea and the HRPTC/PDMS construct and to observe the functions of the implanted construct. The study was mainly about RPTCs. We improve RPTC cultivation with FIR and different biomaterials. Further applications in nephrotoxicity assay, RPTC/biomaterial constructs, and autologous cell therapy would be investigated. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/59898 |
DOI: | 10.6342/NTU201700273 |
Fulltext Rights: | 有償授權 |
Appears in Collections: | 醫學工程學研究所 |
Files in This Item:
File | Size | Format | |
---|---|---|---|
ntu-106-1.pdf Restricted Access | 3.48 MB | Adobe PDF |
Items in DSpace are protected by copyright, with all rights reserved, unless otherwise indicated.