TWI727412B - Method for determining the risk of developing preeclampsia - Google Patents
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Abstract
Description
本發明係關於用於判定懷孕女性個體的子癇前期發生風險的方法。本發明另關於用於判定懷孕女性個體的子癇前期發生風險的試劑或試劑盒。The present invention relates to a method for determining the risk of preeclampsia in a pregnant female individual. The present invention also relates to reagents or kits for determining the risk of preeclampsia in a pregnant female individual.
子癇前期（preeclampsia）是一種妊娠疾病，在妊娠20周後開始，其特徵在於高血壓的出現，並且通常伴隨尿中蛋白質的存在超過2倍，或在24小時內累積超過5克（蛋白尿）、全身性水腫、噁心、嘔吐、視力模糊、體重突然增加等。如果診斷後未積極治療，除了胎兒發育不良，還可能導致胎盤提早破裂，從而導致胎兒死亡。Preeclampsia (preeclampsia) is a pregnancy disorder that begins after 20 weeks of pregnancy and is characterized by the appearance of high blood pressure, and is usually accompanied by the presence of more than 2 times the protein in the urine, or the accumulation of more than 5 grams in 24 hours (proteinuria) , Systemic edema, nausea, vomiting, blurred vision, sudden weight gain, etc. If not actively treated after diagnosis, in addition to fetal dysplasia, it may also cause premature rupture of the placenta, leading to fetal death.
Yang等人通過「次世代定序技術（next generation sequencing technology）」，發現4位有子癇前期孕婦血清中的迴圈miR-223的表達量低於1位正常孕婦的對照組的表達量（Yang等人，Clinica chimica acta 412.23（2011）：2167-2173）。Yang等人的結果並未區分miR-223-3p 和miR-223-5p。Yang et al. used "next generation sequencing technology" and found that the expression of miR-223 in the serum of 4 pregnant women with preeclampsia was lower than that of a control group of normal pregnant women (Yang Et al., Clinica chimica acta 412.23 (2011): 2167-2173). The results of Yang et al. did not distinguish between miR-223-3p and miR-223-5p.
在本發明中意外地發現，與相同胎齡的正常懷孕女性相比，亞裔孕婦於子癇前期第三孕期的miR-223-3p的血清濃度較高，以及亞裔孕婦於子癇前期第一孕期的miR-223-3p的血清濃度較低。In the present invention, it was unexpectedly discovered that compared with normal pregnant women of the same gestational age, the serum concentration of miR-223-3p in the third trimester of preeclampsia was higher in Asian pregnant women, and in the first trimester of preeclampsia. The serum concentration of miR-223-3p is lower.
因此，在一方面，本發明提供一種檢測miR-223-3p的試劑在製備用於判定懷孕女性個體的子癇前期發生風險的試劑或試劑盒中的用途。Therefore, in one aspect, the present invention provides the use of a reagent for detecting miR-223-3p in the preparation of a reagent or kit for determining the risk of preeclampsia in a pregnant female individual.
根據本發明，該血清樣本收集自該懷孕女性個體的第一到第三孕期期間。According to the present invention, the serum sample is collected from the first to third trimester of the pregnant female individual.
另一方面，本發明提供用於判定在懷孕女性個體的子癇前期發生風險的方法，包含檢測懷孕女性個體血清樣本中miR-223-3p的濃度，其中，針對第三孕期的懷孕女性，相對於對照組的較高濃度表示該懷孕女性個體處於子癇前期發生風險；其中，針對第一孕期的懷孕女性，相對於對照組的較低濃度表示該懷孕女性個體處於子癇前期發生風險。On the other hand, the present invention provides a method for determining the risk of preeclampsia in a pregnant female individual, comprising detecting the concentration of miR-223-3p in a serum sample of the pregnant female individual, wherein the concentration of miR-223-3p in the pregnant female during the third trimester is relative to A higher concentration in the control group indicates that the pregnant female individual is at risk of preeclampsia; among them, for pregnant women in the first trimester, a lower concentration relative to the control group indicates that the pregnant female individual is at risk of preeclampsia.
較佳地，懷孕女性個體為亞裔。Preferably, the pregnant female individual is Asian.
根據本發明的某些具體實施例，在懷孕女性個體的第一到第三孕期（trimester）期間收集血清樣本。According to certain embodiments of the present invention, serum samples are collected during the first to third trimesters of the pregnant female individual.
根據本發明的某些具體實施例，對照組為正常懷孕女性個體的血清樣本中miR-223-3p的平均濃度。According to some specific embodiments of the present invention, the control group is the average concentration of miR-223-3p in serum samples of normal pregnant female individuals.
應瞭解先前的一般描述及以下的詳述兩者皆僅為例示性及解釋性的且並不限制本發明。It should be understood that the previous general description and the following detailed description are both illustrative and explanatory and do not limit the present invention.
在一方面，本發明提供一種檢測miR-223-3p的試劑在製備用於判定懷孕女性個體的子癇前期發生風險的試劑或試劑盒中的用途。In one aspect, the present invention provides the use of a reagent for detecting miR-223-3p in the preparation of a reagent or kit for determining the risk of preeclampsia in a pregnant female individual.
根據本發明，該miR-223-3p的試劑用於檢測該懷孕女性個體的血清樣本中的miR-223-3p的濃度，其中，針對第三孕期的懷孕女性，相對於對照組的較高濃度表示該懷孕女性個體處於子癇前期發生風險；其中，針對第一孕期的懷孕女性，相對於對照組的較低濃度表示該懷孕女性個體處於子癇前期發生風險。According to the present invention, the miR-223-3p reagent is used to detect the concentration of miR-223-3p in the serum sample of the pregnant female individual, wherein the concentration of miR-223-3p in the pregnant female during the third trimester is higher than that of the control group. It means that the pregnant female individual is at risk of preeclampsia; among them, for pregnant women in the first trimester, a lower concentration relative to the control group indicates that the pregnant female individual is at risk of preeclampsia.
所述檢測miR-223-3p的試劑包括但不限於可檢測miR-223-3p的寡核苷酸引物。The reagents for detecting miR-223-3p include, but are not limited to, oligonucleotide primers that can detect miR-223-3p.
另一方面，本發明提供用於判定在懷孕女性個體的子癇前期發生風險的方法，包含檢測懷孕女性個體血清樣本中miR-223-3p的濃度，其中，針對第三孕期的懷孕女性，相對於對照組的較高濃度表示該懷孕女性個體處於子癇前期發生風險；其中，針對第一孕期的懷孕女性，相對於對照組的較低濃度表示該懷孕女性個體處於子癇前期發生風險。On the other hand, the present invention provides a method for determining the risk of preeclampsia in a pregnant female individual, comprising detecting the concentration of miR-223-3p in a serum sample of the pregnant female individual, wherein the concentration of miR-223-3p in the pregnant female during the third trimester is relative to A higher concentration in the control group indicates that the pregnant female individual is at risk of preeclampsia; among them, for pregnant women in the first trimester, a lower concentration relative to the control group indicates that the pregnant female individual is at risk of preeclampsia.
根據本發明的某些較佳具體實施例，所述懷孕女性個體為亞裔。According to some preferred embodiments of the present invention, the pregnant female individual is Asian.
本文中所述的亞裔的個體特別是指東亞裔，包括但不限於中國、印度、日本、韓國、馬來西亞、新加坡、中國臺灣及越南的個體。The individuals of Asian descent referred to herein particularly refer to individuals of Eastern Asian descent, including but not limited to individuals of China, India, Japan, Korea, Malaysia, Singapore, Taiwan, and Vietnam.
根據本發明，所述血清樣本可在所述懷孕女性個體的第一到第三孕期期間收集。According to the present invention, the serum sample may be collected during the first to third trimesters of the pregnant female individual.
根據本發明的某些具體實施例，所述對照組是在懷孕的相應孕期中正常懷孕女性個體的血清樣本中miR-223-3p的平均濃度。According to some specific embodiments of the present invention, the control group is the average concentration of miR-223-3p in serum samples of normal pregnant female individuals during the corresponding pregnancy period of pregnancy.
在本發明的某些具體實施例中，所述方法進一步包含比較所述懷孕女性個體的血清樣本中miR-223-3p的濃度與相應孕期中正常懷孕女性個體的血清樣本中miR-223-3p的平均濃度。In some specific embodiments of the present invention, the method further comprises comparing the concentration of miR-223-3p in the serum sample of the pregnant female individual with miR-223-3p in the serum sample of a normal pregnant female individual during the corresponding pregnancy period. The average concentration.
本文中所使用的「較高濃度」是指比相應的正常濃度高至少5%、10%、15%、20%、25%或30%的濃度。As used herein, "higher concentration" refers to a concentration that is at least 5%, 10%, 15%, 20%, 25%, or 30% higher than the corresponding normal concentration.
本文中所使用的「較低濃度」是指比相應的正常濃度低至少5%、10%、15%、20%、25%或30%的濃度。As used herein, "lower concentration" refers to a concentration that is at least 5%, 10%, 15%, 20%, 25%, or 30% lower than the corresponding normal concentration.
本發明通過以下實施例進一步說明，其提供作為例示的用途而不限制本發明。The invention is further illustrated by the following examples, which are provided as illustrative uses without limiting the invention.
實施例1：Example 1:
正常與子癇前期婦女的血液樣本在中國臺灣三軍總醫院收集，在懷孕的第一、第二、及第三孕期時進行常規產前檢查。通過離心（4℃，500g，20分鐘）從血液中分離血清。微小RNA(miRNA)樣本從血清中萃取出，並加入cel-miR-39作為一加入對照組（spike-in control）。miR-223-3p的濃度使用寡核苷酸5'-GTGCAGGGTCCGAGGT-3'（SEQ ID NO: 1）作為莖環引物（stem-loop primer）（miR-223-3p特異性）、寡核苷酸5'-TGAGAAGCCCAGACCAACTT-3'（SEQ ID NO: 2）作為正向引物、以及寡核苷酸5'-CCATCCTTCCAGGATTTGAA-3'（SEQ ID NO: 3）作為反向引物而通過RT-PCR而測定。子癇前期被定義為新的持續舒張壓高於90毫米汞柱(mmHg)，以及在不存在尿道感染的情況下，具有500毫克(mg)/24小時尿液樣本的新的持續蛋白尿。結果如圖1和圖2A-2B所示，圖2A-2B之結果另可見於表1。Blood samples of normal and preeclamptic women were collected at the General Hospital of Taiwan's Armed Forces, and routine prenatal examinations were performed during the first, second, and third trimesters of pregnancy. The serum is separated from the blood by centrifugation (4°C, 500g, 20 minutes). MicroRNA (miRNA) samples were extracted from the serum and added to cel-miR-39 as a spike-in control. The concentration of miR-223-3p uses oligonucleotide 5'-GTGCAGGGTCCGAGGT-3' (SEQ ID NO: 1) as stem-loop primer (miR-223-3p specific), oligonucleotide 5'-TGAGAAGCCCAGACCAACTT-3' (SEQ ID NO: 2) was used as a forward primer, and oligonucleotide 5'-CCATCCTTCCAGGATTTGAA-3' (SEQ ID NO: 3) was used as a reverse primer to be measured by RT-PCR. Preeclampsia is defined as a new sustained diastolic blood pressure higher than 90 millimeters of mercury (mmHg), and a new sustained proteinuria with a 500 milligram (mg)/24 hour urine sample in the absence of urinary tract infection. The results are shown in Figures 1 and 2A-2B, and the results of Figures 2A-2B can be seen in Table 1.
依據圖1，子癇前期第一孕期之懷孕婦女的血清中的miR-223-3p濃度低於正常第一孕期之懷孕婦女；但子癇前期第三孕期之懷孕婦女的血清中的miR-223-3p濃度高於正常第三孕期之懷孕婦女。According to Figure 1, the concentration of miR-223-3p in the serum of pregnant women in the first trimester of preeclampsia is lower than that of normal pregnant women in the first trimester; but the miR-223-3p in the serum of pregnant women in the third trimester of preeclampsia Pregnant women whose concentration is higher than normal in the third trimester.
因此，若樣本為第一孕期之孕婦之血清，其miR-223-3p濃度低於正常值時，可能具有子癇前期的風險。Therefore, if the sample is the serum of a pregnant woman in the first trimester, and the miR-223-3p concentration is lower than the normal value, there may be a risk of preeclampsia.
相對地，若樣本為第三孕期之孕婦之血清，其miR-223-3p濃度高於正常值時，可能具有子癇前期的風險。In contrast, if the sample is the serum of a pregnant woman in the third trimester, and the miR-223-3p concentration is higher than the normal value, there may be a risk of preeclampsia.
另依據圖2A和圖2B，可得知本發明所提供的子癇前期發生風險的試劑或試劑盒，可達成優異的特異性和敏感性。若為第一孕期，p值小於0.05而AUC為0.766；若為第三孕期，p值小於0.0001而AUC為0.808。
表1
習知技術者將理解可對上述具體實施例進行改變而不背離其廣泛的發明概念。因此，應當理解的是，本發明不限於所揭示的特定具體實施例，而是意在涵蓋在如隨附的請求項所界定的本發明的精神與範疇內的修改。Those skilled in the art will understand that changes can be made to the specific embodiments described above without departing from their broad inventive concept. Therefore, it should be understood that the present invention is not limited to the specific embodiments disclosed, but is intended to cover modifications within the spirit and scope of the present invention as defined by the appended claims.
本發明先前的概述以及以下詳述在配合隨附附圖閱讀時得以更佳地被瞭解。為說明本發明，在附圖中顯示目前較佳實施例。The previous summary of the present invention and the following detailed description can be better understood when read with the accompanying drawings. To illustrate the present invention, the presently preferred embodiments are shown in the drawings.
圖1揭露正常與子癇前期懷孕婦女之間的血清中miR-223-3p濃度的比較(正常1st，正常2nd，正常3rd：正常第一孕期，正常第二孕期，正常第三孕期；PE 1st，PE 2nd，PE 3rd：子癇前期第一孕期，子癇前期第二孕期，子癇前期第三孕期)(*P>0.05，****P>0.0001 表示PE組和其他組別之間的統計結果，統計方法為曼-懷特尼檢定(Mann-Whitney test))。Figure 1 reveals the comparison of serum miR-223-3p concentration between pregnant women with normal and preeclampsia (normal 1st, normal 2nd, normal 3rd: normal first trimester, normal second trimester, normal third trimester; PE 1st, PE 2nd, PE 3rd: the first trimester of preeclampsia, the second trimester of preeclampsia, the third trimester of preeclampsia) (*P>0.05, ****P>0.0001 indicates the statistical results between the PE group and other groups, The statistical method is the Mann-Whitney test (Mann-Whitney test).
圖2揭露使用本發明之檢測試劑檢測(2A)第一孕期和(2B)第三孕期之懷孕婦女的血清中miR-223-3p濃度，統計後以接收器操作特性(ROC)曲線表示(AUC：Area Under Curve)。Figure 2 discloses the use of the detection reagent of the present invention to detect the concentration of miR-223-3p in the serum of pregnant women in (2A) the first trimester and (2B) the third trimester, and the results are statistically expressed as receiver operating characteristic (ROC) curve (AUC) : Area Under Curve).
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