臺灣博碩士論文加值系統

哺乳類的腎臟發育主要區分為三個階段，第三個階段稱為後腎期用於形成後腎系統，它開始於人類孕期的第五週，相當於實驗小鼠孕期的第10.5至11天，並且於懷孕的第13.0天，完全形成後腎。
後腎是將來成熟腎臟的前趨組織，後腎主要是由兩個部分：後腎間葉組織(metanephric mesenchyme) 和輸尿管芽(ureteric bud)交互作用而形成將來的成熟腎臟。在後腎轉化為成熟腎臟的期間，根據文獻，有很多生長的訊息、化學物質或萃取物，在不同的階段與不同的位置上作表達，藉以維持後腎間葉組織(metanephric mesenchyme) 和輸尿管芽(ureteric bud) 的正常互動，並且促進胚胎腎臟的成熟，例如： BMP-7、LIF、Pituitary extract、oncostatin M、GDNF、EGR-alpha、Wnts、FGFs等，扮演著促進生長，抑制拮抗，細胞間交互作用等等調控機制，來完成腎臟的生長與發育。
有許多的研究指出，母親糖尿病 (maternal diabetes) 會導致胎兒許多器官之生長發育異常或缺損，就人類而言，infants of diabetic mothers比起正常新生兒，高出3 ~ 4倍的機率會導致先天性的異常或缺損。在腎臟發育方面，由臨床觀察經驗，可見到先天性腎臟發育不全，馬蹄腎，先天小腎等。
我們設計了一個動物實驗，以藥物streptozotocin (STZ) 腹腔注射的方式來誘導C57BL/6J實驗小鼠成為糖尿病小鼠，並且讓這樣的小鼠交配懷孕藉此方式來建立一個母體高血糖與胎兒腎臟發育缺損與異常的關聯模式，由gross observation，histological observation，基因與分子生物醫學等各個方面，去瞭解某些特定基因與腎臟發育結構上，因母體高血糖的影響而造成腎臟生長發育異常的相關性。
結果中發現糖尿病小鼠胚胎的確存有腎臟發育異常的情形，其中包含腎臟單側發育（renal agenesis）及雙套泌尿系統（duplication of collecting system）; 組織學的觀察則是可見到腎元(nephrons)數目較正常對照組明顯減少，以及在腎臟腎小管與collect tubular system部分，可見到明顯的renal tubular detachment的情形；分子生物學的部分則是發現Wnt11、FGFs、Cyclins的基因表達有partial inhibition的情形，且GDNF與 EGRa在糖尿病母鼠的胚胎腎臟發育過程中有complete block的現象。根據以上的結果可初步的看見母體高血糖對胎兒腎臟發育的影響，這將有助於吾人由分子細胞學，基因遺傳學，以及器官發育等等之層次，來瞭解母體糖尿病對胎兒腎臟生長發育之影響與衝擊，並作為日後分子醫學及基因治療之先驅。

Mammalian kidney development is divided into three stage: pronephros, mesonephros, and metanephros. Metanephros stage starts since gestational age of 5th weeks of human being and embryonic 10.5 to 11 day of mice. Metanephros is precursor of the adult mammalian kidney. According to previous experiments and studies, some of the genes play the roles of growth signals to promote kidney development: including FGF-2, BMP-7, LIF, Wnts, pituitary extract oncostain M etc. These genes control and adjust the growth of kidneys at specific regions, and at specific different period.
According to the clinic experiences and animal model findings, maternal diseases will induce fetal growth defects and development abnormalities. In human, infants of diabetic mothers reveal 3- to 4-fold incidence of congenital abnormalities. In renal diabetic embryopathy, by clinical observation experience, can see congenital renal agenesis, horseshoe kidney, congenital small kidney, etc.
We had established a mice animal model to confirm the relationship of maternal hyperglycemia and defects/abnormalies of fetal kidney development. Our female mice, C57BL/6J, had been induced to become diabetic mice by intra- peritoneal injection of streptozotocin. These treated mice had been sacrificed after pregnancy, then fetuses was harvested for kidney observation and experiment. The structure abnormalities including renal agenesis and duplication of collecting system were found via gross observation. Microscopic observation disclosed nephron number decreasing and renal tubular detachment.
In molecular study, through RT-PCR analysis, we found that some gene expressions were partial inhibited at fetal kidneys of DM maternal mice (included Wnt11, FGFs, and Cyclins). Otherwise, GDNF and EGR-alpha expressions were blocked completely. All of our findings provide a new aspect of kidney development of diabetic embryopathies in the levels of molecular biology, gene science, and the organ structural development, etc. Furthermore, our study regarded as preliminary experience of molecule medical science and gene therapy of renal diabetic embryopathy in the future.

目錄
中文摘要1
英文摘要2
第一章、緒論3
第一節、腎臟的發育3
第二節、 腎臟發育過程的訊息因子3
第三節、訊息因子個論4
第四節、建立一個由STZ所導致的母鼠高血糖模式7
第五節、 母體高血糖和腎臟發育異常的觀察8
第六節、腎臟超音波迴音強度的臨床應用9
第七節、研究目的9
第二章、研究方法與材料10
第一節、實驗步驟10
第二節、實驗儀器20
第三章、結果21
第一節、誘發高血糖母鼠的統計比例21
第二節、高血糖母鼠的觀察21
第三節、高血糖母鼠之胎鼠的外觀觀察22
第四節、高血糖母鼠之胎鼠的體重、體長和腎臟重量與對照組的比較22
第五節、高血糖母鼠之胎鼠的組織學結果22
第六節、高血糖母鼠之胎鼠的細胞凋亡和增生的觀察23
第七節、建立胎鼠腎臟細胞的初級培養23
第八節、高血糖母鼠之胎鼠與仔鼠的分子生物學結果24
第九節、以FISH觀察GDNF、EGR1和EGRa的表達型態25
第十節、人類腎臟超音波迴音強度的比較26
第四章、 討論27
第一節、胚胎收集的時間點設定及限制27
第二節、STZ注射劑量和血糖反應的控制27
第三節、胎鼠和仔鼠的先天性畸形28
第四節、胎鼠和仔鼠的體重、體長和腎臟重量的異常變化28
第五節、胚胎腎臟細胞的初級培養方式29
第六節、分生層次的基因選擇29
第七節、GDNF、EGR1、EGRα的相關性30
第八節、腎臟超音波迴音強度與人類糖尿病妊娠子代腎臟發育的關連30
第九節、未來實驗方向與展望31
參考資料32
表格
表一、高血糖母鼠之胎鼠與仔鼠的致死率比較37
表二、引子一覽表38
圖片
圖一、小鼠胚胎腎臟發育過程39
圖二、哺乳類腎臟發育過程的相互作用40
圖三、GDNF-RET的訊息分子作用途徑41
圖四、EGRa和Tieg1的 mRNA Map42
圖五、建立高血糖母鼠的模式流程43
圖六、高血糖母鼠過期妊娠及其胎鼠卡於產道之中44
圖七、高血糖母鼠和正常血糖母鼠泌尿道系統的比較45
圖八、高血糖母鼠妊娠天數過長所取出的仔鼠和正常血糖的仔鼠比較46
圖九、高血糖母鼠所取出的E17d的胎鼠和正常血糖的胎鼠比較47
圖十、高血糖母鼠所取出的E18d的胎鼠和正常血糖的胎鼠比較48
圖十一、高血糖母鼠和正常血糖母鼠所導致小鼠體重差異的比較49
圖十二、高血糖母鼠和正常血糖母鼠所導致小鼠體長差異的比較50
圖十三、高血糖母鼠和正常血糖母鼠所導致小鼠腎臟重量差異的比較51
圖十四、高血糖母鼠所產下的E16d的胎鼠其glomeruli異常(100X)52
圖十五、高血糖母鼠所產下的E16d的胎鼠其glomeruli異常(200X)53
圖十六、高血糖母鼠所產下的E17d的胎鼠其plevis異常(200X)54
圖十七、高血糖母鼠所產下的新生仔鼠glomeruli及tubular異常(100X)55
圖十八、BrdU assay在E18d胎鼠腎臟的比較56
圖十九、TUNEL assay在E17d胎鼠腎臟medulla的比較57
圖二十、TUNEL assay在NB1d新生仔鼠腎臟medulla和cortex的比較58
圖二十一、TUNEL assay的統計圖表59
圖二十二、初級培養的人類和小鼠胚胎腎絲球細胞型態比較60
圖二十三、初級培養的小鼠胚胎腎絲球細胞型態(對照組61
圖二十四、初級培養的小鼠胚胎腎小管細胞62
圖二十五、初級培養的小鼠胚胎腎絲球細胞型態(實驗組) 63
圖二十六、利用RT-PCR來偵測FGF10和Cdh1的表達64
圖二十七、利用RT-PCR來偵測GDNF、EGR1、Wnt11和FGF8的表達65
圖二十八、利用RT-PCR來偵測EGRa和Tieg1的表達66
圖二十九、利用RT-PCR來偵測Cyclin A和Cyclin D的表達67
圖三十、以GDNF Probe進行偵測(FISH)，在E17d小鼠腎臟進行觀察68
圖三十一、以GDNF Probe進行偵測(FISH)，在E18d小鼠腎臟進行觀察69
圖三十二、以GDNF Probe進行偵測(FISH)，在NB1d仔鼠腎臟進行觀察70
圖三十三、以EGR1 Probe進行偵測(FISH)，在E17d小鼠腎臟進行觀察71
圖三十四、以EGR1 Probe進行偵測(FISH)，在E18d小鼠腎臟進行觀察72
圖三十五、以EGR1 Probe進行偵測(FISH)，在NB1d小鼠腎臟進行觀察73
圖三十六、以EGRa和Tieg1 Probe進行偵測(FISH)，E18d小鼠腎臟進行觀察74
圖三十七、EGRa和Tieg1 Probe進行偵測(FISH)，NB1d小鼠腎臟進行觀察75
圖三十八、實驗組與對照組的孩童的腎臟/肝臟超音波迴音強度比值統計76
圖三十九、實驗組與對照組的孩童的腎臟/肝臟超音波迴音強度比值圖片77
附錄一 中華民國斐陶斐榮譽學會榮譽會員證書78
附錄二 財團法人彰化基督教醫院臨床試驗中心受訓合格證書79
附錄三 財團法人彰化基督教醫院動物實驗計畫審查證明書80
附錄四 財團法人彰化基督教醫院動物實驗計畫實驗動物追加、展期證明書81

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