臺灣博碩士論文加值系統

RhD抗原是血庫作業中的重要血型系統之一，在臨床上有可能會造成溶血性輸血反應或新生兒溶血疾病。RHD基因的變異會造成RhD抗原表現的多樣性，並且影響其誘發抗體的能力。在所謂Rh陰性的人當中，有極少數是屬於Del，其紅血球表面D抗原的含量非常微弱，約小於正常人的5-10%，在臨床上，需經由吸附沖出試驗才可偵測到含量極低的D抗原。根據文獻顯示，在Rh陰性的東方人之中，約有10-30%是屬於Del血型，而其基因型幾乎可以說100%都是RHD(G1227A)的變異。本研究主要欲探討台灣地區apparent D negative血型的RHD基因分型，因此我們利用sequence-specific primers polymerase chain reaction (SSP-PCR)、RHD(G1227A) real-time polymerase chain reaction、polymerase chain reaction-based methods以及RhD zygosity test針對從林口長庚醫院所收集到的44隻Rh陰性檢體來探討其RHD genome。實驗結果顯示，在apparent D negative的個體當中，18.2%是屬於Del型，且其基因型均為RHD(G1227A)，而在Del血型之中，有25%是屬於homozygous alleles；至於剩下的81.8%則是屬於true D negative，其基因型均為RHD gene total deletion。

RhD antigen is one of the important antigen systems in blood transfusion services, and it could cause haemolytic reaction or haemolytic disease of newborns. The Del which is only detected by adsorption-elution of anti-D expresses a very low density of RhD antigen. In East Asian, there is approximately 10-30% of the Del among the D negative individuals. In our study, forty-four RhD negative blood samples which were collected from the blood bank of the LinKou Chang Gung Memorial Hospital were analyzed by sequence specific primer-polymerase chain reaction (SSP-PCR) for the ten exons of RHD gene. We found that there were eight specimens revealing all the ten RHD exons, and thirty-six samples didn’t represent all exons. Afterwards, the eight samples were tested by real-time PCR for the exon 9 of RHD gene and we identified their Tm. Among the eight PCR products, all of them had a characteristic 1227G→A mutation, which meant that they were belong to the Del genotype. We also checked their RHD gene by four PCR-based methods, and analyzed the RhD zygosity. During apparent RhD negative individuals, the result suggested that there was 18.2% of the Del whose genotype was RHD(G1227A), and there was 81.8% of the true D negative whose genotype was RHD gene total deletion. By the way, we found that there was 25% homozygous alleles among the Del people in our experiments.

指導教授推薦書……………………………………………………… i
口試委員會審定書…………………………………………………… ii
授權書………………………………………………………………… iii
誌謝…………………………………………………………………… iv
中文摘要……………………………………………………………… v
英文摘要……………………………………………………………… vi
目錄…………………………………………………………………… vii
Introduction…………………………..…………………………………. 1
Rh blood system………….…………………………………...…… 1
Clinical tests of Rh blood system………………………..………… 3
The importance of Rh blood system…………………………..…... 4
RH gene…………………………………………………...……….. 4
Reaserch goal…………………………………………………….…….. 7
Materials and methods…………………………………………………. 8
Materials..………………………………….……………………… 8
DNA extraction……………………………………………….…... 10
Sequence-specific primers polymerase chain reaction……………. 10
Polymerase chain reaction-based methods………………….…….. 11
PCR purification………………………………………………….. 11
Enzyme digestion……………………………………………….… 12
RHD 1227G→A real-time polymerase chain reaction…………… 12
RhD zygosity test…………………………………………………. 12
Results………………………………………...….……………………. 13
Analyze the molecular basis of RhD positive in Taiwa………...… 13
Analyze the molecular basis of apparent D negative in Taiwan…. 13
Analyze the RHD(G1227A)…….………………………………... 14
Analysis of RHD gene by PCR-based RhD typing methods……... 14
RhD zygosity test…………………………………………………. 15
Discussion………………………………………………………………. 16
Reference…………………………………………….…………….…… 19
Tables…………………………………………………..………………. 25
Figures…….…………………………………………………………… 34
Appendix………………………………………………………………. 45

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