於遺傳性耳聾鼠進行幹細胞移植暨移植條件之優化

Abstract

(一)研究背景與目的 近年來再生醫學蓬勃發展，在內耳的治療方面，已有相當多利用幹細胞植入治療受損之毛細胞與聽神經的研究研究報告。本研究進行幹細胞培養、分化，並將對於SLC26A4 c.919-2A>G突變的基因置換鼠(knock-in mice)進行細胞植入；此外，於細胞植入同時給與皮質類固醇(glucocorticoid)，觀察是否能減少因細胞植入所造成的聽力損失，以優化移植的條件。 (二)研究方法 第一部分：胚胎幹細胞與分化細胞植入SLC26A4基因置換鼠之耳蝸 本研究分別將胚胎幹細胞與誘導產生之分化細胞植入SLC26A4基因置換鼠(knock-in mice)之內耳中，四週後，由形態學上來看植入之細胞是否能夠嵌入內耳各個不同的構造，並對照植入幹細胞前後聽性腦幹反應檢查(auditory brainstem response)的結果。 第二部分：以皮質類固醇(glucocorticoid)減少細胞植入損傷之聽力損失 於幹細胞植入耳蝸之同時，分別加入低濃度(200 μg/ml)與高濃度(800 μg/ml)之betamethasone，由形態學上來看幹細胞在內耳之分布，並測量聽性腦幹反應檢查(auditory brainstem response)之結果。 (三)研究成果 第一部分：胚胎幹細胞與分化細胞植入SLC26A4基因置換鼠之耳蝸 本研究順利將胚胎幹細胞利用生長因子(growth factors)誘導分化形成神經先驅細胞(neural progenitor cells)與內耳分化細胞(inner ear differentiated cells)，在進行植入野生型(wild type)之小鼠之內耳後，植入之細胞多分布於鼓室階(scala tympani)，少部分之細胞會分布至中階(scala media)，聽性腦幹反應(auditory brainstem response)檢查結果發現在注射後當日、注射後7日與注射後14日之聽力閾值(thresholds)並無差異，且植入之細胞種類對於其聽力亦無影響。實驗組之SLC26A4基因置換鼠接受細胞植入後，植入之細胞多分布於鼓室階(scala tympani)，少部分之細胞會分布至中階(scala media)，聽性腦幹反應(auditory brainstem response)檢查結果發現在注射後當日、注射後7日與注射後14日之聽力閾值(thresholds)並無差異。 第二部分：以皮質類固醇(glucocorticoid)減少細胞植入損傷之聽力損失 於小鼠進行幹細胞移植後，在對照組、低濃度betamethasone組與高濃度betamethasone組皆可以觀察到其具有立即的聽力損失，三者間之聽力損失程度無統計學上之差異，可知，給與betamethasone並無法減少術後立即性之聽力損失；而於二週後測量，三組之聽性腦幹檢查之聽力閾值(thresholds)皆較為減少，而唯有高濃度betamethasone組之聽力閾值(threshold)具有統計學上之差異，進行幹細胞移植合併注射高濃度(800 μg/ml)之betamethasone，可於兩週後減輕因幹細胞植入產生內耳傷害所造成之聽力損失。

Part I: Stem Cell Transplantation in SLC26A4 c.919-2A>G Knock-in Mice To date, our laboratory had raised SLC26A4 c.919-2A>G knock-in mice. Morphologically, atrophy of the spiral ligament and stria vascularis was demonstrated. It also resulted in the changes of endocochlear potential and potassium concentration in the endolymph. Finally, it led to loss of the hair cells and hearing loss. In the present study, mice embryonic stem cells, neural progenitor cells and the inner ear differentiated cells were delivered into the cochlear of either SLC26A4 c.919-2A>G knock-in mice or the wild type B6 mice through direct injection via the round window membrane. In both groups, survival of the transplanted cells was found mainly in the scala tympani and some in the scala vestibuli two weeks later. There was no transplanted cell in the stria vascularis and the scala media. Auditory brainstem response thresholds were elevated after cell transplantation, and the thresholds right after the transplant surgery and those one week and two weeks later had no significant change. Part II: Administration of Betamethasone Attenuates Intracochlear Cell Transplantation Trauma-induced Hearing Loss After the intracochlear cell transplantation, the loss of the residual hearing develops. Application of glucocorticoid has been showed to ameliorate hearing loss resulting from the trauma of cochlear implantation in experimental animals. To investigate whether administration of the glucocorticoid can protect hearing, betamethasone of either 200 μg/ml or 800 μg/ml was injected into the cochlea of the B6 mice with the transplanted stem cells via the round window membrane simultaneously. Auditory brainstem responses (ABRs) were measured immediately, one week, and two weeks after the cell transplantation. The ABR thresholds increased right after the cell transplantation in both the control group and the betamethasone-treated group, and the hearing partially recovered in the treated group one week and two weeks after the surgery. Simultaneous injection of the betametasone of 800 μg/ml with the transplanted cell can significantly attenuate the hearing loss resulting from the trauma of intracochlear cell transplantation and is a novel strategy for preserving the residual hearing.