Non-invasive Prenatal Gene Diagnosis: Progress through Cell-free Fetal DNA and RNA in Maternal Plasma and Urine

Non-invasive prenatal gene diagnosis has been developed rapidly in the recent years, and numerous medical researchers are focusing on it. Such techniques could not only achieve prenatal diagnosis accurately, but also prevent tangential illness in fetuses and thus, reduce the incidence of diseases. Moreover, it is non-invasive prenatal gene diagnosis that prevents potential threaten and danger to both mothers and fetuses. Therefore, it is welcomed by clinical gynecologist and obstetrian, researchers of medical genetics, and especially, pregnancies. This review article touches briefly on the advanced development of using cell-free DNA, RNA in maternal plasma and urine for non-invasive prenatal gene diagnosis.

Promoting the production of challenging proteins via induced expression in CHO cells and modified cell-free lysates harboring T7 RNA polymerase and mutant eIF2α

Chinese hamster ovary(CHO)cells are crucial in biopharmaceutical production due to their scalability and capacity for human-like post-translational modifications.However,toxic proteins and membrane proteins are often difficult-to-express in living cells.Alternatively,cell-free protein synthesis can be employed.This study explores innovative strategies for enhancing the production of challenging proteins through the modification of CHO cells by investigating both,cell-based and cell-free approaches.A major result in our study involves the integration of a mutant eIF2 translation initiation factor and T7 RNA polymerase into CHO cell lysates for cell-free protein synthesis.This resulted in elevated yields,while eliminating the necessity for exogenous additions during cell-free production,thereby substantially enhancing efficiency.Additionally,we explore the potential of the Rosa26 genomic site for the integration of T7 RNA polymerase and cell-based tetracycline-controlled protein expression.These findings provide promising advancements in bioproduction technologies,offering flexibility to switch between cell-free and cell-based protein production as needed.

Partial isolation and identification of hepatic stimulator substance mRNA extracted from human fetal liver

IM To partially isolate and identify hepatic stimulator substance mRNA from human fetal liver tissues.METHODS The poly (A)mRNA was extracted from human fetal liver tissues of 4-5 month gestation, fractionated by size on sucrose gradient centrifugation, translated into protein from each fraction in vitro and then its products were tested for HSS activity.RESULTS Twentytwo 500μg total RNA was obtained from human fetal liver tissues and pooled. mRNA of 420μg was yielded, processed by oligo(dT)cellulose column chromatography, then was sizefractionated by ultracentrifution on a continuous sucrose density gradient (5%-25%), and separated into 18 fractions. Translated products of mRNA in fraction 8 and 9 could produce a twofold increase in the incorporation of 3HTdR into DNA of SMMC7721 hepatoma cells and in a heatresistant and organspecific way.CONCLUSION The partially purified HSS mRNA was obtained and this would facilitate the cloning of HSS using expression vectors.

Two approaches for calculating female fetal DNA fraction in noninvasive prenatal testing based on size analysis of maternal DNA fragments

The concentration of cell-free fetal DNA fragments should be detected before noninvasive prenatal testing(NIPT).The fetal DNA molecules have significant clinical potential in determining the overall performance of NIPT and clinical interpretation.It is important to measure fetal DNA fraction before NIPT.However,there is still little research on how to calculate the concentration of female fetuses.Two estimation approaches were proposed to calculate fetal DNA fraction,including the fragments size-based approach,aneuploid-based approach,which are all approaches based on chromosome segments.Based on high-throughput sequencing data,two approaches to calculate the DNA fraction of male fetuses were tested and obtained the experiment values,which were close to the actual values.The correlation coefficient of fragments size-based approach was 0.9243(P<0.0001)and the aneuploid-based approach reached 0.9339(P<0.0001).We calculated the concentration of female fetuses and obtained remarkable experimental results.We came up with two approaches for calculating the fetal DNA fraction of female fetuses.It provides an important theoretical basis for the detection of female fetal concentration in future clinical diagnosis.

Universal RNA editing in a human liver at the fetal stage

It is known that RNA editing occurs in human cells, which can change the information transmission from DNA to RNA and proteins. Most previous studies have focused on editing of the mRNAs. Here we reported that several kinds of RNAs, including miRNA, rRNA, mRNA, miscRNA and unknown RNA, exhibited base editing in a human fetal liver. Several editing types are displayed. Our data reveals that RNA editing may occur in different species of RNAs.

胎儿游离RNA在产前诊断中的研究进展

传统产前诊断依靠有创性操作,具有潜在风险。目前无创性产前诊断方法集中于从母体血液中分离胎儿细胞或从血清或血浆中提取胎儿游离DNA,二者在临床的应用尚存不足。近年,妊娠妇女血循环中游离胎儿RNA的发现和应用,是无创性产前诊断领域的突破。已经证实,游离胎儿RNA分子主要来源于胎盘,在母体血循环中比较稳定,易于检测。同时,由于其无需依赖于胎儿性别或是父源性多态性位点,临床应用领域优于胎儿游离DNA;结合对分子标记物的定量检测方法更适用于产前诊断领域,并已取得初步成果。预测其具有很好的应用前景。

孕妇血中胎儿微小RNA等核酸在唐氏综合征无创产前诊断中的应用

唐氏综合征是最常见的染色体非整倍体遗传病,出生干预是预防该病的有效措施。传统的产前诊断具有创伤等缺陷,无创产前诊断是未来发展的需求。孕妇血胎儿细胞、胎儿游离DNA、胎儿游离RNA及胎儿游离microRNA的分析是新近发展的4种唐氏综合征无创产前诊断技术。母血胎儿游离miRNA有足够的稳定性、特异性和准确性,是最具临床应用价值的生物标志物。

质粒载体表达抑制猪瘟病毒shRNA在猪胚胎成纤维细胞上的复制

以质粒pSilencer3.1 Hygro为基础,构建了针对猪瘟病毒Npro基因和NS4A基因mRNA的siRNA双表达载体,转染猪胚胎成纤维细胞后,在潮霉素B的筛选压力下,获得5株稳定整合shRNA表达盒的猪胚胎成纤维细胞克隆。以100TCID50的CSFV分别感染96孔板内的上述细胞克隆,72 h后对感染细胞克隆进行间接免疫荧光分析及子代病毒滴度检测,结果显示,在所获得的5株细胞克隆中,有3株细胞上猪瘟病毒的增殖明显降低,表明所构建siRNA双表达载体转录产生的siRNA可以有效抑制CSFV的复制。本试验为研究猪瘟病毒的防治和通过RNAi建立猪的抗病育种提供了新的方法。

基于游离RNA的无创性产前诊断研究进展

孕妇血浆血清中游离RNA作为一种新的胎儿遗传信息材料给无创性产前诊断带来了新的契机。已经证实,游离胎儿RNA分子主要来源于胎盘,且稳定存在于母体血循环中,易于检测。同时,其由于无需依赖胎儿性别或父源性多态性位点而具有十分明显的应用优势。目前,已经有研究将血浆血清中游离RNA与胎儿非整倍体、宫内发育迟缓以及子痫前期等妊娠相关疾病联系起来,并且取得了十分可喜的研究成果。

逆转录病毒载体RNAi技术抑制猪瘟病毒在猪胚胎成纤维细胞的增殖

利用针对猪瘟病毒(Classical swine fever virus,CSFV)石门株Npro基因mRNA的shRNA逆转录病毒表达载体,转导猪胚胎成纤维细胞,在G418的筛选压力下,获得7个稳定整合shRNA表达构件的猪胚胎成纤维细胞株。以100TCID50的CSFV分别感染96孔板内的上述细胞克隆,72h后以对感染细胞克隆进行间接免疫荧光分析及子代病毒滴度检测,结果显示,在所获得的7个抗性细胞株中,有3株细胞上猪瘟病毒的增殖显著降低,表明所构建的针对猪瘟病毒Npro基因mRNA的shRNA逆转录病毒整合细胞基因组后转录产生的siRNA可以有效抑制CSFV的复制。

孕妇外周血胎儿ε血红蛋白基因mRNA的表达及与孕龄的相关性

目的 探讨孕妇外周血中胎儿ε血红蛋白基因mRNA的表达水平及其与孕龄的相关性.方法 选择2008年10月—2009年11月在潍坊市人民医院行产前检查的7～16周正常单胎妊娠孕妇30例,常规方法提取全血总RNA,采用逆转录聚合酶链式反应（RT-PCR）法扩增胎儿ε血红蛋白基因片段,琼脂糖凝胶电泳,并扫描成像,用ImageJ软件对电泳条带进行图像分析,测定电泳条带的灰度值,计算孕妇外周血中胎儿ε血红蛋白基因mRNA的浓度,并分析其与孕龄之间的相关性.结果 30例样品RNA质量浓度值为0.98～3.19 g/L,平均（1.87±0.76） g/L;光密度平均比值为1.79,7例阳性,23例阴性.孕妇外周血中胎儿ε血红蛋白基因mRNA浓度为0.537～1.790 μg/ml,中位数为1.241 μg/ml.孕妇外周血中胎儿ε血红蛋白基因mRNA的浓度与孕龄呈负相关（r=-0.750,P=0.026）.结论 孕妇外周血中存在胎儿ε血红蛋白基因mRNA且其含量在7～16周与孕龄呈负相关,利用孕妇外周血中胎儿RNA进行无创性产前诊断具有可行性.

山羊myostatin基因慢病毒RNA干扰载体的构建及效果验证

myostatin基因是肌肉生长和发育的关键负调控因子之一,该基因突变或失活的物种都呈现肌肉增大的双肌表型,暗示在家畜中使该基因失活可以增加其产肉量。为了进一步揭示myostatin在山羊胎儿成纤维细胞中的生物学功能以及制备可有效失活myostatin基因的工具,将筛选的myostatin基因潜在RNA干扰靶位点连接到慢病毒干扰载体的转移质粒中,构建慢病毒介导的RNA干扰载体,验证其干扰效果。结果表明,干扰序列与转移质粒连接正确,成功构建myostatin基因慢病毒干扰载体,慢病毒三质粒共转293T细胞,获得的慢病毒颗粒可高效转染山羊胎儿成纤维细胞,Real-time PCR检测发现慢病毒干扰载体Lv322有效减少山羊胎儿成纤维细胞中myostatin基因mRNA 75%的表达(P<0.01),Western blotting检测显示myostatin蛋白则有效降低了94%(P<0.01)。本试验为研究myostatin基因的功能及制备失活转基因动物提供有效工具以及理论基础。

人多药耐药基因mdr1野生型mRNA转导脐血干细胞提高其对化疗耐药性的体外研究

目的:将人类多药耐药基因(mdr1)全长野生型mRNA 转导至人脐带血干细胞,分析其P糖蛋白的表达及干细胞的耐药性。方法: 通过体外转录方法经人野生型mdr1 基因全长cDNA制备相应的mRNA, 经脂质体介导转染人造血干细胞,采用RT- PCR 和Western blot 检测转染效果,流式细胞仪检测蛋白表达、体外培养及细胞活率计数观察耐药性。结果: 转染后P糖蛋白表达丰度为9 .05%,对照组为1. 01%; 转染后具有Rhodamin 123 排泌功能的细胞占98 .72%, 对照组为66. 83%;转染后细胞耐药性明显增强,P=0 .002。结论: 通过脂质体介导,将人类mdr1基因野生型全长mRNA转导入脐血干细胞,增加了P糖蛋白的表达,提高了细胞的耐药性。该系细胞可用于缓解和治疗恶性肿瘤化疗带来的骨髓抑制。

孕妇血浆中胎儿游离RNA与先兆子痫的研究进展

母血中胎儿游离RNA作为一种新的生物学标志物,已经给无创产前诊断带来了广阔的前景。胎儿游离RNA主要来源于胎盘,其在母血中稳定、易检出且不受胎儿性别的影响,具有很强的应用优势。先兆子痫、胎儿非整倍体、先兆流产等疾病与胎儿游离RNA关系的研究已经取得了瞩目的成果。

Blocking effects of siRNA on VEGF expression in human colorectal cancer cells

AIM:To investigate the expression of vascular endothelial cell growth factor (VEGF) and its receptors Fmslike tyrosine kinase 1 (FLT-1) and fetal liver kinase 1 (FLK-1) in colorectal carcinoma (CRC),and the blocking effects of small interfering RNAs (siRNAs) on VEGF expression in human colorectal cancer HCT116 cells.METHODS:Immunohistochemical staining for VEGF,FLT-1 and FLK-1 proteins was performed in 82 cases of CRC and 14 normal colorectal mucosae.A siRNA targeting VEGF was synthesized and transfected into HCT116 cells using lipofectamine 2000.Immunocytochemical staining and Western blotting analyses were performed to detect the expression of VEGF protein.The suppressive effect of the siRNA on cell proliferation was detected using the 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltertrazolium bromide (MTT) assay.Cellular apoptosis was detected using flow cytometry (FCM).RESULTS:The expression of VEGF,FLT-1 and FLK-1 in tumor tissues was significantly higher than that in normal tissues (P=0.008,P=0.000,P=0.000).The expression of VEGF was positively correlated with both lymph node metastasis and clinical stage (P=0.009 and P=0.025,respectively).Immunocytochemistry showed that the expression of VEGF was weakly positive and Western blotting indicated a significant reduction in VEGF-siRNA cell protein levels.VEGF-siRNA cell growth inhibition was assessed by the MTT assay,and the tumor cell proliferation rate was significantly different at 24,48,and 72 h after transfection.FCM results showed that the VEGF-siRNA group had an apparent aneuploid peak.CONCLUSION:VEGF,FLT-1 and FLK-1 are associated with colorectal carcinogenesis.siRNA silencing of the VEGF gene suppresses proliferation,and induces apoptosis in HCT116 cells.The results suggest that VEGF may be a new gene therapy target for colorectal cancer.

核内RNA对AFP基因表达的影响

本文介绍介导正常 Wistar 大鼠肝细胞的 SnRNA 和同种动物的tRNA 对大鼠肝癌细胞 DNA 复制、RNA 转录和蛋白质合成的影响,其影响与介导物有剂量相关,采用间接免疫荧光法和放射免疫沉淀法检测了 SnRNA 对肝癌细胞AFP 合成的影响。对放射免疫沉淀法的步骤作了改进,如用~3H-亮氨酸和~3H-酪氨酸双标记以及加入未被标记的载体等等。本实验获得满意的结果。

Noninvasive Prenatal Testing for Fetal Chromosomal Abnormalities Using Massively Parallel Sequencing: Clinical Experience from 7910 Korean Pregnancies

Objective: The purpose of this study is to review the clinical experience and performance of noninvasive prenatal testing (NIPT) method, using cell-free DNAto detect chromosomes 21, 18, 13, X, and Y abnormalities in over 7910 clinical samples from South Korean population. Method: Pregnant women between 1st of November 2015 to 18th of February 2018, with obstetric clinical findings participated in the study. NIPT was performed based on masivelly parallel sequencing with 0.3× low coverage paired-end sequencing using cell-free DNA in maternal plasma. Further invasive prenatal testing was recommended for pregnant women with positive NIPT results. Results: Of the total 7910 participants, 7890 (99.75%) were tested for NIPT and the remaining 20 (0.25%) were below the Quality Control (QC) standards. T13, T18, XXX, XXY and XYY had 100% of sensitivity, specificity, positive predictive values (PPV) and accuracy. The overall sensitivity was 100% and specificity, PPV and accuracy of all chromosomal abnormalities with further validation were 99.92%, 94.25%, and, 99.92% respectively. Conclusion: Our NIPT results showed high positive predictive value for the detection of autosomal trisomies and sex chromosome aneuploidies in our sample cohort.

Circular RNA and its potential as prostate cancer biomarkers

Advancing knowledge of the transcriptome has revealed that circular RNAs(circRNAs)are widely expressed and evolutionarily conserved molecules that may serve relevant biological roles.More interesting is the accumulating evidence which demonstrates the implication of circRNAs in diseases,especially cancers.This revelation has helped to form the rationale for many studies exploring their utility as clinical biomarkers.CircRNAs are highly stable due to their unique structures,exhibit some tissue specificity,and are enriched in exosomes,which facilitate their detection in a range of body fluids.These properties make circRNAs ideal candidates for biomarker development in many diseases.This review will outline the discovery,biogenesis,and proposed functions of circRNAs.

Prenatal diagnosis of Down syndrome using cell-free fetal DNA in amniotic fluid by quantitative fluorescent polymersase chain reaction

Backgroud Amniotic fluid （AF） supernatant contains cell-free fetal DNA （cffDNA） fragments.This study attempted to take advantage of cffDNA as a new material for prenatal diagnosis,which could be combined with simple quantitative fluorescent polymerase chain reaction （QF-PCR） to provide an ancillary method for the prenatal diagnosis of trisomy 21 syndrome.Methods AF supernatant samples were obtained from 27 women carrying euploid fetuses and 28 women carrying aneuploid fetuses with known cytogenetic karyotypes.Peripheral blood samples of the parents were collected at the same time.Short tandem repeat （STR） fragments on chromosome 21 were amplified by QF-PCR.Fetal condition and the parental source of the extra chromosome could be determined by the STR peaks.Results The sensitivity of the assay for the aneuploid was 93％ （26/28; confidence interval,CI：77％-98％） and the specificity was 100％ （26/26; CI：88％-100％）.The determination rate of the origin of the extra chromosome was 69％.The sensitivity and the specificity of the assay in the euploid were 100％ （27/27）.Conclusions Trisomy 21 can be prenatally diagnosed by the QF-PCR method in AF supernatant.This karyotype analysis method greatly reduces the requirement for the specimen size.It will be a benefit for early amniocentesis and could avoid pregnancy complications.The method may become an ancillary method for prenatal diagnosis of trisomy 21.

胎儿骨髓间质干细胞体外诱导为神经细胞的初步实验研究

目的 :体外定向诱导胎儿骨髓间质干细胞 (MSC)分化为神经细胞。方法 :采用Ficoll淋巴细胞分离液分离MSC ,体外扩增、传代 ,采用 β 巯基乙醇等诱导剂诱导MSC分化为神经细胞 ,免疫组化鉴定神经丝蛋白(NF2 0 0 )、神经元烯醇化酶 (NSE)、胶质纤维酸性蛋白 (GFAP) ,并且用RT PCR鉴定NF2 0 0、NSE和GFAP的mR NA表达情况。结果 :胎儿骨髓MSC在体外扩增第 5代以后 ,经诱导后 ,形态学上可呈现神经细胞形态特征 ;免疫组化显示诱导出的神经细胞NF2 0 0、NSE和GFAP均有阳性表达 ;RT PCR也显示诱导后表达量明显增加。结论 :胎儿骨髓MSC在体外可以分化为神经细胞。