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研究生:方俊能
研究生(外文):FANG CHUN NENG
論文名稱:應用以免疫組織化學法染Cytokeratin-7及以聚合酶鏈反應放大SY158基因之DNA片段於懷孕初期子宮頸的黏液中檢測胎兒狀態
論文名稱(外文):Detection of Fetal Cells and DNA from Transcervical Mucus Plug before First Trimester by Cytokeratin-7 immunohistochemistry, and PCR Amplification of DNA Sequence of SY158 Gene
指導教授:蕭長春
指導教授(外文):Hsiao Chang-Chun
學位類別:碩士
校院名稱:長庚大學
系所名稱:臨床醫學研究所
學門:醫藥衛生學門
學類:醫學學類
論文種類:學術論文
論文出版年:2006
畢業學年度:94
語文別:中文
論文頁數:58
中文關鍵詞: cytokeratin-7 胎兒細胞 免疫組織化學法 經子宮頸黏液 懷孕 聚合酶鏈反應 築巢式聚合酶鏈反應液 懷孕 聚合酶鏈反應 築巢式聚合酶鏈反應
外文關鍵詞:cytokeratin-7fetal cellsimmunohistochemistryPCRnested PCRtranscervical mucuspregnancy
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目的:最初於1971年報導在懷孕婦女的子宮頸內黏液內發現胎兒細胞。在懷孕最初三個月,於羊膜和絨毛膜融合之前,子宮腔是開放的。因此理論上胎兒細胞在界於7至13週時會由退化中的葉狀絨毛膜(chorion frondosum)脫落至子宮腔中而被捕捉於經子宮頸的黏液栓塞裡;它們可以經由免疫組織化學的方法而被發現,因此可能利用胎兒細胞做為遺傳診斷之用。我們也嘗試利用子宮頸抹片的細胞刷來取得懷孕子宮頸黏液,再利用聚合酶鏈反應(PCR)及築巢式聚合酶鏈反應(nested PCR)或的方法來偵測男性胎兒DNA,並作比較。

方法:在Cytokeratin-7免疫組織化學染色法的實驗中,有99件在懷孕7至13週時的孕婦,在終止懷孕前取樣經子宮頸的黏液栓塞,對其加以收集、固定、包埋、切片,並依照免疫組織化學的方法(immunohistochemistry),以cytokeratin-7(CK-7)的單株抗體加以染色。
在PCR及nested PCR放大SY158基因之DNA片段的實驗中,另共有56個懷孕7-12週的孕婦,進入本實驗。在笫一次產檢,或中止懷孕前利用子宮頸抹片刷取樣子宮頸黏液。將子宮頸黏液溶解,再萃取出DNA,再以兩對引子以SY158的基因上的DNA片斷,進行PCR及 nested PCR的擴增。

結果:在顯微鏡下所發現的滋胚層細胞,依據染色的結果以及其比照絨毛膜絨毛的組織相似性被區分成五類。這五類依序為:(1)每一切片上沒有陽性染色的細胞,(2)每一切片上有少於5個單獨的陽性染色的細胞,(3)每一切片上有多於5個單獨的陽性染色的細胞,(4)每一切片上有聚集呈團的陽性染色的細胞,(5)每一切片上有在組織學上類似於完整的或破碎斷裂的絨毛膜組織細胞。在71件品質良好能夠被檢測的樣本中,每件切片樣本被評估後且分類成三群,其結果為:32件(45.1%)分類為第一群(包含第(1)類)象徵陰性的結果;10件(14.1%)分類為第二群(第(2)類)象徵可能為陽性的結果;29件(40.8%)分類為第三群(包含第(3)類至第(5)類)象徵陽性的結果。
共有53個樣本含DNA,可以進行PCR及nested PCR放大SY158基因之DNA片段的擴增步驟。在我們的病人群,共有33個男胎及20個女胎。在第一次的PCR的結果,共有34個標本擴增出男性DNA片斷,19個標本則呈陰性結果。敏感度為84.8%,專一性70%,陽性預測率為82.4%,陰性預測率為73.7%。而 nested PCR的結果為,共有40個標本擴增出男性DNA片斷,13個標本則呈陰性結果。敏感度為93.9%,專一性55%,陽性預測率為77.5%,陰性預測率為84.6%。

結論:以cytokeratin-7免疫組織化學染色法,有59.2%可以在早期懷孕的經子宮頸黏液栓塞中,偵測得胎兒細胞。因此經由非侵入性的取樣方法以及現代的分子科技,產前遺傳診斷經由子宮頸黏液診斷單基因或染色體異常是有可能的。
由於胎兒細胞於母體子宮頸黏液的細胞數頗高,PCR或 nested PCR兩者在胎兒細胞於孕婦子宮頸黏液的偵測能力均有統計效力。但兩者的偵測率並無統計學上的差異。使用PCR便足以偵測孕婦子宮頸黏液中的胎兒細胞。
胎兒細胞確實存在於早期懷孕的子宮頸黏液,若利用合適的分子生物技術,則可以測得胎兒DNA或由母體細胞的背景中分離完整的胎兒細胞。經由完善的實驗設計及仔細的實驗對象的控制以避免受父、母親細胞來源的的汙染,則進行胎兒細胞染色體或基因變異的檢查是可行的。並值得進一步研究。
The presence of fetal cells in endocervical mucus of pregnant women was first reported in 1971. Uterine cavity is patent during the first trimester prior to fusion of amnion and chorion. Fetal cells are theoretically shed into the uterine cavity between 7-13 weeks from degenerating chorion frondosum, trapped in the transcervical mucus and can be identified by immunohistochemistry. We also tried to identify the presence of fetal male deoxyribo-nucleic acid (DNA) by transcervical cytobrush Pap smear from pregnant women in the first trimester by polymerase chain reaction (PCR) and nested PCR and compare the ability of accurate prediction rate between these two methods.

METHOD:
In the experiment of immunoreactive cytokeratin-7 staining, ninety-nine transcervical mucosal plugs from pregnant women between pregnant 7 and 13 weeks before abortion was collected, fixed, embedded, sectioned and stained with monoclonal antibody of cytokeratin-7 by immunohistochemistry. In the experiment of PCR and nested PCR to amplify DNA sequence of SY158 gene, another fifty-six pregnant patients were recruited into this program during 7-12 weeks of gestation. The transcervical mucus specimens were recruited and treated with mycolytic agent, DNA extraction, and the PCR then nested PCR to amplify the DNA sequence of SY158 gene.

RESULT:
In the experiment of immunoreactive cytokeratin-7 staining, the identification of trophoblasts on each slide was defined by 5 categories according to positive staining and histologically chorionic villous similarity under microscopic examination, as: none (1), less than 5 single positive-stained cells per-section (2), more than 5 single positive-stained cells per-section (3), clumps of positive-stained cells (4), and histological-like intact or fragmented chorionic villi (5). Among 71 samples qualified for analysis, individual slides were scored into 3 groups: 32(45.1%) are of group 1 (category 1) standing for negative result, 10(14.1%) are of group 2 (category 2) standing for possible positive result and 29(40.8%) are of group 3 of any combination of category 3-5 standing for positive result.
In the experiment of PCR and nested PCR to amplify DNA sequence of SY158 gene, there were 53 qualified specimens proceeded into the PCR and nested PCR procedure. There were 33 males and 20 females fetuses in our patients’ study group. The PCR result showed that the sensitivity was 84.8%, specificity 70.0%, positive predictive rate 82.4% and negative predictive rate 73.7%. The nested PCR results showed 93.9% as the sensitivity, specificity 55.0%, positive predictive rate 77.5% and negative predictive rate 84.6%. Both PCR and nested PCR method in sex prediction are statistically consistent with the true fetal sex of the transcervical samples. Additionally, the accuracy of fetal sex prediction was identical between these two methods.

CONCLUSION:
Fetal cells, identified by cytokeratin-7, can be found in over 59.2 % of early pregnant transcervical mucus by minimal invasive sampling method.
The PCR method is good enough to detect male fetal DNA in transcervical samples. Fetal cells do exist in the transcervical Pap smear samples in the early pregnancy. With the proper molecular techniques, it is possible to detect the fetal DNA and furthermore to identify and isolate the fetal cells from the background of maternal cells for prenatal genetic diagnosis.
Prenatal diagnosis of single-gene or chromosome disorders may be possible in pregnant transcervical mucus using modern molecular methods and deserves further study.
第一章 簡介……………………………………………………… 1
1.1 計畫背景及相關研究……………………………………… 1
1.2 計畫目標…………………………………………………… 6
第二章 實驗對象及使用之工具方法…………………………… 8
2.1在Cytokeratin-7免疫組織化學染色法的實驗中
2.1.1取樣對象及取樣前準備及檢查………………………… 8
2.1.2取樣方法及樣品前處理………………………………… 8
2.1.3實驗方法及步驟………………………………………… 8
2.1.4實驗結果觀察及判讀條件……………………………… 10
2.1.5統計方法之運用………………………………………… 11
2.2在PCR及nested PCR放大SY158基因之DNA片段的實驗中…11
2.2.1取樣對象及取樣前準備及檢查………………………… 11
2.2.2取樣方法及樣品前處理………………………………… 11
2.2.3實驗方法及步驟………………………………………… 12
2.2.4實驗結果觀察及判讀條件……………………………… 13
2.2.5統計方法之運用………………………………………… 13
第三章 結果……………………………………………………… 14
3.1在Cytokeratin-7免疫組織化學染色法的實驗中………………14
3.1.1樣品之週數大小分析………………………………… 14
3.1.2樣品之質量以及懷孕週數與懷孕特性之間之相關性分析……………………………………………………………… 14
3.1.3實驗結果判讀的條件標準……………………………… 14
3.1.4實驗結果與懷孕週數相關性之分析…………………… 15
3.1.4.1實驗結果為陽性之百分比與懷孕週數相關性之
分析………………………………………………… 15
3.1.4.2實驗結果為可能陽性的百分比與懷孕週數相關
性之分析…………………………………………… 16
3.1.4.3實驗結果為陰性百分比與懷孕週數相關性之分
析…………………………………………………… 16
3.2在PCR及nested PCR放大SY158基因之DNA片段的實驗中… 17
3.2.1樣品之週數大小分析………………………………… 17
3.2.2實驗結果判讀的條件標準…………………………… 17
3.2.3 PCR在子宮頸口黏液中偵測男性DNA的實驗結果與實際胎兒性別比較………………………………………… 18
3.2.4 nested PCR在子宮頸口黏液中偵測男性DNA的實驗結果與實際胎兒性別比較……………………………… 18
3.2.5兩種PCR的方法在子宮頸口黏液中偵測男性DNA的實驗結果相對實際胎兒性別的偵測準確度比較………… 19
第四章 討論及建議………………………………………………… 20
4.1在Cytokeratin-7免疫組織化學染色法的實驗中……………… 20
4.1.1取樣方式之改進…………………………………… 20
4.1.2單株抗體的選擇對實驗結果之影響……………… 20
4.1.3早期懷孕週數之取樣時機對實驗結果之影響…… 21
4.1.4不同方式之子宮頸樣品取樣術對實驗結果之影響.22
4.2在PCR及nested PCR放大SY158基因之DNA片段的實驗中…22
4.2.1早期懷孕之取樣條件對實驗結果之影響………… 23
4.2.2取樣後標本保存及處理對實驗結果之影響…………23
4.2.3兩種PCR方法對實驗結果之影響及分析…………… 24
4.3檢討與未來的改進……………………………………… 24
4.4臨床的運用……………………………………………… 25
第五章 結論………………………………………………………… 26
參考文獻……………………………………………………………… 28
附錄…………………………………………………………………… 31
表1. 檢體的臨床特性………………………………………… 32
表2. 兩個不同懷孕週數分組下,顯微鏡發現之不同胎兒
細胞以及黏膜品質之平均排序(mean rank)之相關
性統計…………………………………………………… 33
表3. 在不同懷孕週數下的不同顯微鏡發現之不同胎兒細
胞特性之百分比………………………………………… 34
表4. 在不同懷孕週數所做分組下所取得的不同胎兒細胞
偵測結果之百分比……………………………………… 35
表 5 在PCR及nested PCR實驗中檢體的懷孕週數分布…36
表6 胎兒性別與PCR及nested PCR之結果……………… 37 表 7 PCR的結果與實際胎兒性別之比較…………………… 38
表 8 Nested PCR的結果與實際胎兒性別之比較……………39
表 9 PCR 與Nested PCR的結果與實際胎兒性別預測準確度之比較………………………………………………………40
圖.1及說明…………………………………………………… 41
圖.2及說明…………………………………………………… 43
圖.3及說明…………………………………………………… 45
圖.4及說明…………………………………………………… 47
圖.5………………………………………………………………49
圖.6………………………………………………………………50
圖.7………………………………………………………………51
圖.8及說明………………………………………………………52
圖.9………………………………………………………………54
圖.10…………………………………………………………… 55
圖.11…………………………………………………………… 56
實驗步驟1………………………………………………………57
實驗步驟2………………………………………………………58
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