Evaluation of Epitheliotrophic Capacity of Four Different Blood Derivatives on Bovine Corneal Epithelial cells
Date Issued
2007
Date
2007
Author(s)
Cheng, Shao-Hui
DOI
zh-TW
Abstract
實驗目的：
犬隻、馬以及人類的自體血清已經成功且廣泛地使用在嚴重的角膜疾病以及促進角膜傷口癒合。人類的臍帶血清在臨床上被認為可以有效地治療乾眼症與持續性的角膜上皮缺損。相對於人類血清與臍帶血清而言，冷凍血漿是一個取得方便，又有良好品質管理的血液製劑，但目前尚未有使用在臨床上的報告。本實驗目的為比較臍帶血清、冷凍血漿與人類血清促進角膜上皮傷口癒合、細胞增生和細胞移行的效果，評估在臨床上這些血液製劑是否可以取代自體血清，或擁有比自體血清更佳的效果；胎牛血清常規地使用在各實驗中，協助試管內 (in vitro) 培養的細胞生長，但近年來因為狂牛病的肆虐，讓此血液製劑在使用上有安全上的疑慮，因此尋找替代的血液製劑變得格外重要，所以本實驗比較了使用這四種血液製劑培養的牛角膜上皮細胞之細胞形態與繁殖效果，進而評估這些血液製劑用來培養細胞的可能性。
材料與方法：
使用的細胞為試管內培養的第二代牛角膜上皮細胞。細胞培養液是用DMEM/F-12稀釋成10%的胎牛血清、人類血清、臍帶血清與冷凍血漿。實驗主要分為傷口癒合試驗、細胞增生試驗、細胞移行試驗、細胞形態的觀察以及血液製劑中生長因子與細胞外基質含量的測定。傷口癒合試驗是在單層的牛角膜上皮細胞上，製造機械性的傷口，比較這四種不同的血液製劑，造成角膜上皮傷口癒合的速度；細胞增生試驗是利用可評估活細胞數的MTS assay，來測試以不同血液製劑培養的牛角膜上皮細胞，經過1、2、3天的培養後，細胞數增加的情形。細胞移行試驗則是利用Boyden chamber來評估這四種血液製劑促進細胞穿過固定直徑孔洞的能力。細胞形態學的觀察是使用這四種血液製劑，培養牛角膜細胞四天後，利用倒立顯微鏡觀察且紀錄比較。最後利用ELISA套組，測定各種人類來源的血液製劑中上皮生長因子 (EGF)、轉化生長因子β1 (TGF-β1) 和fibronectin的濃度。
結果：
在傷口癒合試驗中，胎牛血清使傷口癒合最為快速，其次為三種人類來源的血液製劑；在細胞增生試驗中，胎牛血清造成的細胞增生效果最為顯著、人類血清與臍帶血清次之，冷凍血漿效果最差；在細胞移行試驗中，胎牛血清促進細胞移行的效果最佳、人類血清次之、臍帶血清和冷凍血漿較差；細胞學形態的觀察方面，四種血液製劑培養的牛角膜上皮細胞，在倒立顯微鏡下形態相似。至於三種人類來源血液製劑生長因子的濃度，以臍帶血清最多，人類血清次之，冷凍血漿最少；而細胞外基質fibronectin的含量則是冷凍血漿最多、人類血清次之、臍帶血清最少。
結論：
臍帶血清在傷口癒合試驗、細胞增生試驗、細胞移行試驗以及細胞形態上的表現，都可與人類血清相比，且臨床上已經使用在人類乾眼症和持續性角膜上皮缺損的病患身上，未來可嘗試使用在犬隻或馬身上。冷凍血漿在促進細胞增生和移行的效果稍差，但因取得方便，未來可嘗試使用本實驗的製備方式，輔助治療角膜上皮缺損的病患。至於在培養細胞上，這三種人類來源的血液製劑，在牛角膜上皮細胞上皮化能力的表現，雖沒有像胎牛血清那樣突出，但仍舊可以促進細胞的增生和移行，且維持一定的形態，若要培養未來使用在人類眼睛上的細胞，或許也可考慮這三種人類來源的血液製劑。
PURPOSE： Human serum eye drops have been successfully used in treatment of severe ocular surface disorders and enhancement of corneal wound healing. Umbilical cord serum is effective in treatment of dry eye and persistent corneal epithelial defects. Fresh frozen plasma has not yet been tested for use as eye drops in patients, although it is easily available as quality-controlled products from blood banks. Fresh frozen plasma or umbilical cord serum could be used for substituting human serum when the source of human serum lacks. The use of fetal bovine serum (FBS) in the culture medium has been a major concern worldwide because of bovine spongiform encephalitis (BSE). The use of other human source blood-derived products to replace FBS in the culture process would help. We compared these four blood products by investigating the epitheliotrophic capacity in an in vitro model of bovine epithelial cell monolayer.
MATERIALS AND METHODS：Primary cultured bovine corneal epithelial cells were used to investigate wound healing, cell proliferation and migration by means of scratch corneal wound evaluation, MTS assay and Boyden chamber migration assay in response to human serum, umbilical cord serum, fresh frozen plasma and FBS. The concentrations of EGF, TGF-β1, and fibronectin in human serum, umbilical cord serum and fresh frozen plasma were evaluated by ELISA kits.
RESULTS： The effect of promoting corneal epithelial wound healing of FBS was better than three other human sourced blood products. Cell proliferation and migration were best enhanced by FBS, followed by umbilical cord serum and human serum, and were worst in fresh frozen plasma. Growth factor (EGF and TGF-β1) concentrations were significantly higher in umbilical cord serum than in human serum and were lowest in frozen plasma. The concentration of fibronectin in frozen plasma was significantly higher than in human serum and was lowest in umbilical cord serum.
CONCLUSIONS： Umbilical cord serum and fresh frozen plasma may possess potential for substitution of human serum to treatment ocular surface disease and enhance corneal wound healing. Although the enhancement of epitheliotrophic capacity in human-sourced blood products was not as excellent as FBS, these three blood products could also promote proliferation and migration of corneal epithelial cells. These products could be useful materials in cultivating cells for clinical use.
Subjects
上皮化能力
人類血清
胎牛血清
冷凍血漿
臍帶血清
Epitheliotrophic capacity
Human serum
Fetal bovine serum (FBS)
Frozen plasma
Umbilical cord serum
SDGs
Type
thesis