【作者】 王立平；

【导师】 李红钢；

【作者基本信息】 华中科技大学 ， 妇产科学， 2012， 硕士

【摘要】 背景与目的：本小组在前期研究中，已发现绝大部分精浆游离mRNA（cell-freeseminalmRNA,cfs-mRNA）存在于精浆小体中，从而保护不被精浆RNA酶降解。由于精浆小体直径大部分在0.10μm以上，本实验拟采用0.10μm滤膜对精浆标本微过滤（microfiltration,MF）截留大部分精浆小体，用于提取cfs-mRNA，检测这种基于存在形式的提取方法的提取量，同时也是对cfs-mRNA的存在形式进行证实性研究。方法：收集精液参数正常的男性精液标本，用0.10μm滤膜过滤后用TrizolLSReagent提取cfs-mRNA，通过荧光定量RT-PCR，扩增管家基因β-actin（ACTB）、睾丸组织特异基因DDX4、PRM2、附睾特异性表达基因WFDC9、前列腺特异基因TGM4、精囊腺特异基因SERPINA5，与不过滤直接应用TrizolLSReagent提取cfs-mRNA比较，分析0.10μm滤膜对cfs-mRNA提取含量的影响。结果：将正常男性精浆标本通过0.10μm滤膜后再采用TrizolLSReagent提取cfs-mRNA，与未过滤提取的mRNA比较，分别有93.9%±15.7%（Mean±SE）ACTB、99.1%±12.5%DDX4、76.9%±18.5%PRM2、88.1%±29.6%WFDC9、150.2±73.6%TGM4、98.6%±17.8%SERPINA5被提取，各基因水平均无明显降低（P>0.05,n=6,Paird-Samplest-test)。说明大部分含cfs-mRNA的精浆小体被0.1μm滤膜截留下来，滤膜上精浆小体内cfs-mRNA足以稳定被检测出。结论：利用0.10μm滤膜对精浆小体进行富集，进而再提取cfs-mRNA，这种基于存在形式的提取方法，不会对cfs-mRNA量产生明显降低，因此可有效的提取cfs-mRNA。且本实验结果与前期课题组对通过精浆小体的获取及过滤实验等结果一致，表明大部分精浆小体不能通过0.10μm滤膜，同时证实cfs-mRNA主要被包裹在精浆小体中。背景与目的：以上实验表明，基于存在形式的提取所获得的cfs-mRNA产量无明显损失，且操作简便，具有可行性。另一方面，精浆成分复杂，经滤膜过滤后，应可以避免一些成分对提取RNA的影响，而提高RNA的质量。本部分实验主要分析经滤膜过滤后所提取的cfs-mRNA的纯度和DNA污染等。方法：分别通过未过滤直接用TrizolLSReagent提取方法，和微过滤（microfiltration,MF,直径0.1μm）后TrizolLSReagent对留于滤膜上的精浆小体进行裂解提取的方法，提取cfs-mRNA。通过分光光度计测定两种方法提取的mRNA纯度（OD260/280）；通过毛细血管电泳对所提取的RNA片段分布进行分析；荧光定量PCR扩增DDX4，定量检测两种方法提取的cfs-mRNA中基因组DNA的残留量。提取滤膜上及滤液中的DNA，荧光RT-PCR定量检测DNA残留量，并与精浆DNA总量进行比较。结果：联合应用0.1μm滤膜和TrizolLSReagent的提取方法所提取的RNA，OD260/280为1.828±0.021（n=6），与TrizolLSReagent提取的RNA（1.570±0.042）相比，纯度明显提高（Ｐ<0.05,Paird-Samplest-test）。毛细管电泳片段分布说明微过滤后500nt以上的cfs-mRNA较未过滤组相对量有所提高。荧光定量PCR检测RNA中DNA浓度为15.5±10.3ng/ml，与未过滤精浆提取RNA中DNA（浓度216.4±95.2ng/ml）相比，残留量明显减少（Ｐ<0.05）。且通过对滤膜上和滤液中精浆DNA提取发现，DNA绝大部分通过0.1μm滤膜存在于过滤后的滤液中。将未过滤精浆中的DNA量设为1，微过滤后滤膜的DNA量仅为14.4%，而滤液DNA量为未过滤精浆组的162.6%。结论：联合应用0.10μm滤膜和TrizolLSReagent可高效富集cfs-mRNA，提取效果明显改善。0.10μm滤膜过滤精浆标本时大量DNA通过，从而更有利于提取滤膜之上游离mRNA，获得的mRNA纯度更高、DNA残留量更少。该方法可作为可靠cfs-mRNA提取技术，为后续microarray、northern-blot、RT-PCR等技术需求提供较好的方法基础。背景与目的：本小组前期研究中，已发现绝大部分精浆游离microRNA（cell-freeseminalmicroRNA,cfs-miRNA）并非和mRNA一样存在于小体中，而主要可能以蛋白复合物形式存在，且这种复合物颗粒直径小于0.10μm。因此本实验旨在将精浆通过微过滤（microfiltration,MF,直径0.10μm）与超滤（ultrafiltraton,UF,分子量30kDa）双重过滤后，对蛋白浓缩液进行游离microRNA的提取，检测这种基于cfs-miRNA存在形式的提取量。同时是对cfs-miRNA存在形式的证实性研究。方法：收集精液参数正常的男性精液标本，应用0.10μm滤膜微过滤精浆，将滤液通过30kDa蛋白超滤，对蛋白进行浓缩后提取cfs-miRNA，通过荧光定量RT-PCR扩增各生殖腺体特异表达或丰度较高的miR-30d、miR-93、miR-202、miR-514、miR-891b、miR-892a、miR-141、miR-221，与应用TrizolLSReagent提取方法比较，分析0.10μm滤膜微过滤和蛋白超滤对cfs-miRNA提取含量的影响。结果：将正常男性精浆标本通过0.10μm滤膜后，对滤液进行蛋白浓缩，再采用TrizolLSReagent提取cfs-miRNA，与未过滤直接提取方法比较，各microRNA水平均无明显降低，分别有119.1%±57.2%miR-30d，165.1%±62.3%miR-93，108.6%±23.3%miR-202，126.7%±45.4%miR-514，163.4%±88.0%miR-891b，142.1%±54.6%miR-892a，129.7%±59.9%miR-141，94.9%±13.2%miR-221被提取出。表明cfs-miRNA颗粒直径小于0.10μm，可被30kDa孔径大小的蛋白浓缩柱截留以及浓缩后，microRNA足以被稳定检测。结论：通过超滤（分子量30kDa）蛋白浓缩离心可将含cfs-miRNA的蛋白复合颗粒富集，通过该方法提取的cfs-miRNA无明显降低（Ｐ>0.05），此种基于其存在形式的方法，可有效的提取cfs-miRNA。同时也证实了cfs-miRNA确实以蛋白形式存在，且蛋白复合颗粒直径小于0.10μm。背景与目的：从本部分实验一的研究中，我们发现基于microRNA存在形式，利用微过滤以及超滤对蛋白浓缩后，对蛋白浓缩液提取的cfs-miRNA的量无明显降低。那么，这种方法是否会提高miRNA的质量呢？本部分实验将对其质量改善与否进行进一步检测。方法：将精液参数正常的男性精浆，通过0.10μm滤膜微过滤，取滤液进行蛋白超滤离心管离心，对存于离心管膜上的蛋白浓缩液通过TrizolLSReagent裂解提取cfs-miRNA，采用毛细血管电泳对所提取RNA进行片段分布的分析；通过荧光定量PCR扩增DDX4，定量检测提取的cfs-miRNA中DNA基因组的残留量，与采用TrizolLSReagent提取精浆游离总RNA中DNA基因组残留量进行比较。且对超滤离心管膜上蛋白浓缩液及离心管滤液中的精浆标本进行DNA提取，荧光定量PCR定量检测DNA（DDX4）量，与未做任何处理提取的精浆DNA总量进行比较，分析超滤离心管对DNA的影响。结果：对超滤离心管中和超滤液中精浆DNA提取发现，DNA与cfs-miRNA一样，大部分存在于超滤离心管中。将未过滤精浆提取DNA量设为1，则超过滤后滤液中提取DNA量为0.8%，超滤管中DNA量为237.0%。尽管如此，我们通过微过滤以及超滤后提取cfs-miRNA后，荧光定量PCR检测RNA中DNA浓度为176.5±110.4ng/ml，与未过滤RNA中DNA（浓度281.9±159.9ng/ml）相比，残留量仍明显减少。毛细管电泳片段分布分析提示超滤后，小于200nt的RNA相对量有所提高。结论：联合应用0.10μm滤膜微过滤和超滤离心管可高效富集cfs-miRNA，获得miRNA中DNA基因组残留量明显减少，提取效果明显改善。该方法可作为可靠cfs-miRNA提取技术，为后续研究提供可靠基础。更多还原

【Abstract】 Background and Objective:In our previous research, it has been found that human cell-free seminal mRNAs (cfs-mRNAs) are substantially present in the seminal microvesicles (SMVs), which protecting seminal plasma mRNA against seminal ribonuclease activity. Since the diameter of most SMVs are above0.10μm, in the present study a microfilter (0.10μm) was used to collect SMVs for cfs-mRNA recovery, aiming to develop a method for cfs-mRNA isolation based on its existing form.Methods:SMVs were collected from semen of normozoospermic individuals by the microfiltration (MF) of the0.10μm filter and subjected to the Trizol LS reagent (Invitrogen) procedure to extract cfs-mRNA. Cfs-mRNA quantity was assessed by real time RT-PCR targeting transcripts of a house keeping gene (β-actin, ACTB), testis specific genes (DEAD box polypeptide4, DDX4;Protamine2, PRM2), an epididymis specific gene (WAP-type four-disulfide core domain9, WFDC9), a prostate specific gene (serpin peptidase inhibitor, clade A, member5, SERPINA5), a seminal vesicle specific gene (transglutaminase4, TGM4). Quantities of cfs-mRNAs were compared with single Trizol LS reagent procedure without0.10μm filtration, to analyze the impact of0.10μm membrane to the yield of cfs-mRNA. Results:Comparing with the cfsRNA extracted from the unfiltered seminal plasma,93.9%15.7%(Mean±SE) ACTB,99.1%±12.5%DDX4,76.9%±18.5%PRM2,88.1%±29.6%WFDC9,150.2%±73.6%TGM4,98.6%±17.8%SERPINA5were recovered from the membrane of the0.10μm microfilter.The yield of cfs-mRNAs recovered from the membrane was comparable with total amounts in seminal plasma (P>0.05, n=6, Paird-Samples/-test).Conclusion:The extraction method based on the presence forms,utilizing0.10μm filter membrane to enrich the SMVs and then extracting mRNAs, does not result in a significantly reduce of mRNAs yield. Also, the experimental results are consistent with our previous study about the SMVs and cfs-mRNAs. Background and Objective:The above RNA extraction method based on the presence forms does not result in a significantly reduce of mRNAs yield and is easy to operate. We further presumed that it should improve the purity of the mRNA. The present experiment aimed to analyze the purity of cfs-mRNA, and DNA contamination.Methods:The purity of the extracted RNA was measured by a spectrophotometer (OD260/280). And the distribution of RNA fragments was analyzed by capillary electrophoresis. The residual amount of DNA in cfs-mRNAs was assessed by quantitative PCR (qPCR) targeting DDX4DNA. Also, we detected the amount of the filtrate and filter DNA to analyze the impact of the membranes for DNA.Results:Compared with cfsRNA extracted from the unfiltered seminal plasma, the OD260/280value of our new method (1.828±0.051, n=6) increased significantly (P<0.05, Paird-Samples t-test). Capillary electrophoresis showed that in microfiltration group the proportion cfsRNA above500nt increased than unfiltered group. Determined by qPCR, DNA residues in cfs-mRNA significantly reduced in the new method. The DNA concentration in RNA of microfiltration group was15.5±10.3ng/ml, whearas in the unfiltered group was216.4±95.2ng/ml. DNA substantially present in the filtrate after microfiltration through0.10μm membranes in the process of DNA extraction from the filtrate and filtration. Yield of DNA was set to1in the unfiltered seminal plasma. Relative to the unfiltered group, the proportion of DNA amount in the MF membrane was14.4%and162.6%in the filtrate.Conclusion:The0.10μm filter membrane can efficiently enrich the SMVs. DNA residues are reduced and purity is improved apparently. And large amounts of DNA can be filtered through0.10μm membrane. This is more conducive to the extraction of the cfs-mRNA on the filter. Background and Objective:In the previous study, the vast majority of human cell-free seminal microRNA (cfs-miRNA) was found to be existed in the form of protein complexes, which is less than0.10μm in diameter. Therefore, the present experiment aimed to extract cfs-miRNA from protein complexes which is enriched from the filtrate of the0.10μm microfiltration by ultrafiltration (UF).Methods:Seminal plasma was filtered through the0.10μm microfilter. And the filtrate was centrifuged by a30kDa protein ultrafiltration(UF) tube. Then cfs-miRNA was recovered from the protein concentrate. Quantity of cfs-miRNA was assessed by real time RT-PCR targeting miRNAs with high abundance in semen (miR-30d, miR-93), or with specific expression in the male reproductive organs (miR-202, miR-514, miR-891b, miR-892a, miR-141, miR-221), and compared with their amounts in total cfsRNA which was extracted from seminal plasma by Trizol LS Reagent.Results:Compared with miRNA levels in total cfsRNA extracted from seminal plasma, the relative yield of cfs-microRNAs recovered from the protein concentrate were119.1%±57.2%(miR-30d),165.1%±62.3%(miR-93),108.6%±23.3%(miR-202),126.7%±45.4%(miR-514),163.4%±88.0%(miR-891b),142.1%± 54.6%(miR-892a),129.7%±59.9%(miR-141),94.9%±13.2%(miR-221). No significant difference was observed in miRNA levels between the total cfsRNA and the cfs-miRNA recovered from the protein concentrate (P>0.05, Paired-Samples t-test).Conclusion:30kDa protein concentrate centrifugal can enrich the protein complex particles containing cfs-miRNAs. The method based on the cfs-miRNA existing form can effectively extract cell-free miRNA from human seminal plasma. Background and Objective:From the data of above experiment, we find that the extracted miRNAs in the protein concentrate have no significant decrease. They are enriched based on their existing forms with the use of ultrafiltration centrifuge tube for concentrating protein, indicating the feasibility of this extraction method. Whether this method, which is able to enrich and extract microRNA/protein complexes, can improve the quality of the miRNA? The present experiment determined the RNA quality of this protocol.Methods:The distribution of RNA fragments was determined by capillary electrophoresis. The residual amount of DNA in cfs-miRNA was assessed by qPCR targeting DDX4introns. Also, the DNA amount in protein concentrate and the filtrate was quantified by qPCR. Results:Similar with cfs-miRNA, most genomic DNA was present in protein concentrate, and little amount DNA passed the ultrafilter. Yield of DNA was set to1in the unfiltered seminal plasma. Relative to the unfiltered group, the proportion of DNA amount in the UF tube was237.0%and0.8%in the filtrate. However, DNA residues in cfs-miRNA extracted by our new method (176.5±110.4ng/ml) were significantly reduced compared with the DNA concentration (281.9±159.9ng/ml) in total cfsRNA isolated from the unfiltered seminal plasma. Capillary electrophoresis showed that in ultrafiltration group the proportion cfsRNA below200nt increased than unfiltered group.Conclusion:The30kDa ultrafiltration centrifuge tube can efficiently enrich the miRNA/protein complexes. DNA residues were reduced and purity is improved apparently.更多还原