细胞微泡内的DNA组成与功能及其在动脉粥样硬化中的作用研究
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摘要
背景：
细胞间通讯可以确保组织内不同细胞的功能相互协调。细胞间通讯包括可溶性因子、隧道纳米管和细胞微泡等，它们都可以在不同细胞间水平传递细胞表面分子或细胞质。微泡是一种大多数细胞在生理或病理状态下都会分泌的圆形的质膜片断，包括外泌体和微粒或脱落小泡[1,2]。目前，对微泡的生物学功能了解甚少，有研究认为它主要参与细胞分泌、免疫调节、凝血、细胞间通讯等生理或病理过程[3-5]。
微泡是一种亚细胞结构，其生成、丰度、大小、内含物和生物学功能等均存在异质性。微泡内含蛋白质、mRNA、miRNA等多种生物大分子[6-9]，表面还携带母体细胞来源的的抗原、脂质等，这些均有利于其在循环中与受体细胞融合，水平运输外源的蛋白质和核酸[10-13]，参与细胞间通讯并调节受体细胞的生物学功能[8,9,14,15]。此外，有研究报道微泡介导的细胞间通讯在动脉粥样硬化的形成中扮演了重要角色[15]。
最近的研究发现微泡内含线粒体DNA和染色体DNA[16,17]。Waldenstrom等[17]进一步报道了在心肌细胞微泡内存在的染色体DNA可以转移至受体细胞的细胞质和细胞核内，但这些转移的微泡DNA是否具有功能并不清楚。本研究旨在全面分析微泡DNA的组成、深入探索其在细胞间通讯中的生物学功能和生理意义并进一步阐明其在动脉粥样硬化形成中作用和具体机制。
方法：
1、根据之前的报道通过多步离心法分离人血浆或细胞上清液中的微泡，并应用透射电镜、免疫印迹和流式细胞术等多种方法进行鉴定[8,9,15]。
2、通过荧光激活细胞分选器、微流控技术、琼脂糖凝胶电泳、高通量Solexa测序和PCR等方法分析血浆和细胞上清液中微泡内DNA的特征。
3、为进一步探索微泡DNA的转移与功能，我们将过表达AT1受体的HEK293细胞或平滑肌细胞分泌的微泡DNA转移至未转染的HEK293细胞中，通过PCR、免疫印迹等方法检测受体细胞中AT1的mRNA和蛋白水平。为了揭示转移的微泡DNA转录的机制，我们通过激光共聚焦的方法观察受体细胞中内源性的转录因子NF-κB与微泡转移的AT1DNA的作用关系。
4、平滑肌细胞与HEK293细胞的研究对于证实转移的微泡DNA转录进而增加相应mRNA表达水平仅提供了间接的证据。为克服这一局限，我们将K562细胞微泡中的BCR/ABL融合基因转移至HEK293细胞中，优势在于HEK293细胞中并不存在BCR/ABL融合基因，通过PCR、免疫印迹等方法检测受体细胞中BCR/ABL融合基因的mRNA和蛋白水平。为深入探索微泡中BCR/ABL融合基因的生理意义，我们将其转移至正常人外周血来源的中性粒细胞中，通过双色荧光原位杂交技术检测中性粒细胞中BCR/ABL融合基因的表型。
5、为深入探索微泡DNA在动脉粥样硬化形成中的作用与机制，我们应用高通量的Solexa技术对冠心病人群与非冠心病人群血浆微泡DNA基因拷贝数的差异表达进行了初筛，然后用定量PCR进行验证。此外，我们应用PCR和免疫印迹的方法对上述两个人群中的白细胞内的SRY基因拷贝数、mRNA和蛋白水平也进行了比较。进一步地，我们观察了两个人群中白细胞粘附因子的表达差异。
结果：
1、透射电镜显示微泡呈现圆形双层小泡结构，大小30-1000nm之间，与之前报道结果一致[8,9,15]。我们流式细胞术进一步检测分析了大于200nm的微泡颗粒[18]。免疫印迹法也证实在微泡中存CD63、AGO2、TSG101、Flotillin-1和HSP70等标志蛋白。
2、荧光激活细胞分选器、微流控技术、琼脂糖凝胶电泳和PCR等多种方法均检测到在人血浆或平滑肌细胞培养上清液中分离的微泡内含DNA。这种DNA中包含双链DNA，长度在1-20kb之间，大多数在17kb左右。高通量Solexa测序技术也发现人血浆微泡中至少存在16434种基因组DNA。
3、通过PCR测序方法证实从稳定过表达AT1蛋白的HEK293细胞(AT1-HEK293细胞)培养上清液中分离的微泡中存在AT1-EGFP DNA，而平滑肌细胞培养上清液中分离的微泡中存在AT1DNA。将AT1-HEK293细胞或平滑肌细胞微泡与HEK293细胞共培养后，免疫荧光研究提供了微泡DNA转移至受体细胞核周及核内的直接证据。而且，转移的AT1DNA还可以与其转录因子NF-κB结合，启动该DNA转录的同时增加AT1mRNA和蛋白表达。另外，这些新生成的AT1mRNA和蛋白可以显著地被放线菌素D所抑制。
4、K562细胞微泡与HEK293细胞共培养后使受体细胞表达BCR/ABL融合基因的mRNA和蛋白，而且这种外源性的表达可以被同时孵育的放线菌素D所阻断。应用双色荧光原位杂交技术从基因组水平也检测出20%的中性粒细胞在孵育了K562细胞微泡后表达BCR/ABL融合基因。
5、应用高通量Solexa测序和定量PCR，我们发现冠心病患者血浆微泡中SRY DNA的基因拷贝数显著高于非冠心病人群。同时，我们也检测出冠心病患者外周血白细胞中SRY的基因拷贝数、mRNA和蛋白水平亦显著高于正常者。更重要地是，这些白细胞中粘附因子CD11a表达显著增强，提示微泡在细胞间水平转移SRY基因并可能通过上调SRY蛋白的表达而增强白细胞的粘附功能。
结论：
总之，母体细胞来源的微泡DNA可以传递至受体细胞并且增加受体细胞相应基因编码的mRNA和蛋白水平，进而影响受体细胞的生物学功能。此外，微泡转移的SRY基因在动脉粥样硬化形成中扮演了重要角色。微泡介导的DNA转移提供了一种在细胞间基因传递的新方法和信号传导的新途径。这些发现将有助于探索疾病发生的新机制和治疗的新措施。
Background：
Cell-to-cell communication is required to guarantee proper coordination amongdifferent cell types within tissues. There are multiple types of intercellular communication,including soluble factors, tunneling nanotubules, and extracellular vesicles (EVs), whichallow the transfer of surface molecules or cytoplasmic components from one cell to another.EVs are circular plasma membrane fragments that include exosomes and microparticles orshed vesicles，which are shed from almost all cell types under both physiological andpathological conditions[1,2]. The biological function of EVs is poorly understood, but mayinclude secretion, immunomodulation, coagulation, and intercellular communication[3-5].
EVs may vary in their formation, abundance, size, and composition, but they oftencontain abundant molecules, which include functional transmembrane and cytosol proteins,message RNA (mRNA), and microRNA (miRNA)[6-9]. The components in EVs could betransferred from one cell to another by endocytosis or fusion with the recipient cell[10-13].Importantly, the transferred components in EVs are functional and can regulate thebiological functions of the recipient cells[8,9,14,15]. Moreover, The EV-mediatedintercellular communication plays an important role in pathogenesis of atherosclerosis[15].
Recent studies have shown that both mitochondrial DNA (mtDNA) and chromosomalDNA were found in EVs[16,17]. Waldenstrom et al.[17]reported that chromosomal DNAsequences in EVs from cardiomyocytes could be transferred to the cytosol or nuclei oftarget cells. However, whether the transferred EV DNAs are functional or not is unclear. Inthis study, we investigated the function and potential mechanisms of transferrable EVgenomic DNAs (gDNAs) in the recipient cells. Finally, we also elucidated the critical roleof EV DNA that plays in pathogenesis of atherosclerosis.
Methods：
1. EVs from human plasma or cell culture supernatants were isolated through a seriesof ultracentrifugation steps as previously described[8,9,15]. To ensure that the EVs werecorrectly identified, electron microscopic, immunoblotting, and flow cytometry (FCM)analyses were used.
2. We examined the characteristics of DNA in EVs derived from human plasma andsupernatants of vascular smooth muscle cells (VSMCs) in culture by fluorescence-activatedcell sorter (FACS), high performance liquid chromatography (HPLC), agarose gelelectrophoresis, Solexa sequencing and polymerase chain reaction (PCR).
3. To determine the transportability and functionality of the DNA fragments in EVs,we studied the transport of gDNAs (AT1receptor DNA) in EVs from AT1receptortransfected-HEK293cells or VSMCs to non-transfected HEK293cells by PCR andimmunoblotting. To uncover the underlying mechanisms leading to the transcription oftransferred EV DNA, we investigated the effect of endogenous NF-κB in recipient cells, apromoter of the AT1receptor gene, on transferred DNAs including AT1receptor DNA byimmunofluorescence using laser confocal microscopy.
4. The VSMC and HEK293cell studies only provided indirect evidence that theincreased AT1mRNA is due to the incorporation of DNA from EVs. To overcome thislimitation, we studied the transport of the BCR/ABL hybrid gene in EVs from K562cells toHEK293cells, which is not normally expressed in HEK293cells. To determine thepathophysiological significance of transferred gDNAs in EVs between cells, we examinedthe transfer of BCR/ABL hybrid gene in EVs from K562cells to normal human neutrophilsisolated from human peripheral blood by dual-color fluorescence in-situ hybridization(D-FISH).
5. To investigate the critical role of EV DNA that plays in pathogenesis ofatherosclerosis, we screened out EV DNA fragments with differential gene copy numbers(GCNs) in plasma from patients with coronary artery disease (CAD) compared to controlhealthy subjects by Solexa sequencing. Then, we validated the screening EV DNAfragments in plasma by quantitative PCR. We also compared the levels of SRY GCNs,mRNA, and protein in WBCs between two groups by PCR and immunoblotting. Finally, wetransferred sex-determining region Y (SRY) gene-containing EVs or plasmids to monocytes or human umbilical vein endothelial cells (HUVECs) and observed adhesion functionbetween the two cells.
Results：
1. The electron micrographs of the EVs revealed rounded double-layer membranousvesicles of approximately30-1000nm in size, similar to previously described EVs[8,9,15].EVs more than200nm in size were confirmed by FCM analysis as this technology isunable to distinguish or enumerate single particles below200nm in size[18].Immunoblotting showed the presence of CD63, argonaute2(AGO2), tumor susceptibilitygene101(TSG101), Flotillin-1, and heat shock protein70(HSP70) proteins, commonlyused markers for EVs.
2. Substantial amounts of DNA were detected by FACS, HPLC, agarose gelelectrophoresis, and PCR in EVs derived from human plasma and supernatants of VSMCsin culture. We found that EVs contained double-strand DNA (dsDNA) ranging in size from1to20kb, but mostly around17kb. Furtherly, Solexa sequencing indicated the presence ofat least16434genomic DNA (gDNA) fragments in the EVs from human plasma.
3. We found the presence of AT1-EGFP DNA in EVs from AT1-HEK293cells thatstably expressed AT1protein by PCR and sequencing. After incubation of HEK293cellswith EVs from AT1-HEK293cells or VSMCs, immunofluorescence study showed directevidence that EV DNAs could be transferred into the recipient cells and localize to andinside the nuclear membrane. As an endogenous promoter of the AT1receptor, NF-κB,could be recruited to the transferred DNAs in the nucleus, and increased the transcription ofAT1receptor in the recipient cells. We also found the de-novo mRNA and protein of AT1receptor could be significantly inhibited by actinomycin D.
4. Incubating HEK293cells with EVs from K562cells resulted in the expression ofBCR/ABL hybrid gene mRNA and protein in the recipient HEK293cells. Moreover, thisheterologous expression of BCR/ABL hybrid gene in the HEK293cells was prevented bythe concurrent incubation with actinomycin D. Using a dual-color fluorescence in situhybridization (D-FISH) method, we found that incubation of normal human neutrophilswith EVs from K562cells resulted in the expression of BCR/ABL hybrid gene in20%ofthe neutrophils by genomic analysis.
5. Using Solexa sequencing followed by quantitative PCR, the GCNs of SRY DNA in EVs from plasma were significantly higher in patients with CAD than those in controlhealthy subjects. We also found the levels of SRY GCNs, mRNA, and protein in WBCswere higher in patients with CAD than those in control healthy subjects. Importantly,Transferred SRY gene-containing EVs or plasmids to monocytes and HUVECs enhancedadhesion function between the two cells
Conclusions：
In conclusion, we have shown that EV DNAs, which could be delivered from one cellto another, can increase the gDNA-coding mRNA and protein expressions in the recipientcells, and affect the physiological function in the recipient cells. Furtherly, we have foundSRY gene-containing EVs may play an critical role in atherosclerosis. EV-mediated transferof gDNAs may represent a new method of gene delivery and novel way of signaltransduction among cells. These findings would help in the discovery of novel mechanismsof cardiovascular disease and development of new therapeutic strategies.
细胞间通讯可以确保组织内不同细胞的功能相互协调。细胞间通讯包括可溶性因子、隧道纳米管和细胞微泡等，它们都可以在不同细胞间水平传递细胞表面分子或细胞质。微泡是一种大多数细胞在生理或病理状态下都会分泌的圆形的质膜片断，包括外泌体和微粒或脱落小泡[1,2]。目前，对微泡的生物学功能了解甚少，有研究认为它主要参与细胞分泌、免疫调节、凝血、细胞间通讯等生理或病理过程[3-5]。
微泡是一种亚细胞结构，其生成、丰度、大小、内含物和生物学功能等均存在异质性。微泡内含蛋白质、mRNA、miRNA等多种生物大分子[6-9]，表面还携带母体细胞来源的的抗原、脂质等，这些均有利于其在循环中与受体细胞融合，水平运输外源的蛋白质和核酸[10-13]，参与细胞间通讯并调节受体细胞的生物学功能[8,9,14,15]。此外，有研究报道微泡介导的细胞间通讯在动脉粥样硬化的形成中扮演了重要角色[15]。
最近的研究发现微泡内含线粒体DNA和染色体DNA[16,17]。Waldenstrom等[17]进一步报道了在心肌细胞微泡内存在的染色体DNA可以转移至受体细胞的细胞质和细胞核内，但这些转移的微泡DNA是否具有功能并不清楚。本研究旨在全面分析微泡DNA的组成、深入探索其在细胞间通讯中的生物学功能和生理意义并进一步阐明其在动脉粥样硬化形成中作用和具体机制。
方法：
1、根据之前的报道通过多步离心法分离人血浆或细胞上清液中的微泡，并应用透射电镜、免疫印迹和流式细胞术等多种方法进行鉴定[8,9,15]。
2、通过荧光激活细胞分选器、微流控技术、琼脂糖凝胶电泳、高通量Solexa测序和PCR等方法分析血浆和细胞上清液中微泡内DNA的特征。
3、为进一步探索微泡DNA的转移与功能，我们将过表达AT1受体的HEK293细胞或平滑肌细胞分泌的微泡DNA转移至未转染的HEK293细胞中，通过PCR、免疫印迹等方法检测受体细胞中AT1的mRNA和蛋白水平。为了揭示转移的微泡DNA转录的机制，我们通过激光共聚焦的方法观察受体细胞中内源性的转录因子NF-κB与微泡转移的AT1DNA的作用关系。
4、平滑肌细胞与HEK293细胞的研究对于证实转移的微泡DNA转录进而增加相应mRNA表达水平仅提供了间接的证据。为克服这一局限，我们将K562细胞微泡中的BCR/ABL融合基因转移至HEK293细胞中，优势在于HEK293细胞中并不存在BCR/ABL融合基因，通过PCR、免疫印迹等方法检测受体细胞中BCR/ABL融合基因的mRNA和蛋白水平。为深入探索微泡中BCR/ABL融合基因的生理意义，我们将其转移至正常人外周血来源的中性粒细胞中，通过双色荧光原位杂交技术检测中性粒细胞中BCR/ABL融合基因的表型。
5、为深入探索微泡DNA在动脉粥样硬化形成中的作用与机制，我们应用高通量的Solexa技术对冠心病人群与非冠心病人群血浆微泡DNA基因拷贝数的差异表达进行了初筛，然后用定量PCR进行验证。此外，我们应用PCR和免疫印迹的方法对上述两个人群中的白细胞内的SRY基因拷贝数、mRNA和蛋白水平也进行了比较。进一步地，我们观察了两个人群中白细胞粘附因子的表达差异。
结果：
1、透射电镜显示微泡呈现圆形双层小泡结构，大小30-1000nm之间，与之前报道结果一致[8,9,15]。我们流式细胞术进一步检测分析了大于200nm的微泡颗粒[18]。免疫印迹法也证实在微泡中存CD63、AGO2、TSG101、Flotillin-1和HSP70等标志蛋白。
2、荧光激活细胞分选器、微流控技术、琼脂糖凝胶电泳和PCR等多种方法均检测到在人血浆或平滑肌细胞培养上清液中分离的微泡内含DNA。这种DNA中包含双链DNA，长度在1-20kb之间，大多数在17kb左右。高通量Solexa测序技术也发现人血浆微泡中至少存在16434种基因组DNA。
3、通过PCR测序方法证实从稳定过表达AT1蛋白的HEK293细胞(AT1-HEK293细胞)培养上清液中分离的微泡中存在AT1-EGFP DNA，而平滑肌细胞培养上清液中分离的微泡中存在AT1DNA。将AT1-HEK293细胞或平滑肌细胞微泡与HEK293细胞共培养后，免疫荧光研究提供了微泡DNA转移至受体细胞核周及核内的直接证据。而且，转移的AT1DNA还可以与其转录因子NF-κB结合，启动该DNA转录的同时增加AT1mRNA和蛋白表达。另外，这些新生成的AT1mRNA和蛋白可以显著地被放线菌素D所抑制。
4、K562细胞微泡与HEK293细胞共培养后使受体细胞表达BCR/ABL融合基因的mRNA和蛋白，而且这种外源性的表达可以被同时孵育的放线菌素D所阻断。应用双色荧光原位杂交技术从基因组水平也检测出20%的中性粒细胞在孵育了K562细胞微泡后表达BCR/ABL融合基因。
5、应用高通量Solexa测序和定量PCR，我们发现冠心病患者血浆微泡中SRY DNA的基因拷贝数显著高于非冠心病人群。同时，我们也检测出冠心病患者外周血白细胞中SRY的基因拷贝数、mRNA和蛋白水平亦显著高于正常者。更重要地是，这些白细胞中粘附因子CD11a表达显著增强，提示微泡在细胞间水平转移SRY基因并可能通过上调SRY蛋白的表达而增强白细胞的粘附功能。
结论：
总之，母体细胞来源的微泡DNA可以传递至受体细胞并且增加受体细胞相应基因编码的mRNA和蛋白水平，进而影响受体细胞的生物学功能。此外，微泡转移的SRY基因在动脉粥样硬化形成中扮演了重要角色。微泡介导的DNA转移提供了一种在细胞间基因传递的新方法和信号传导的新途径。这些发现将有助于探索疾病发生的新机制和治疗的新措施。
Background：
Cell-to-cell communication is required to guarantee proper coordination amongdifferent cell types within tissues. There are multiple types of intercellular communication,including soluble factors, tunneling nanotubules, and extracellular vesicles (EVs), whichallow the transfer of surface molecules or cytoplasmic components from one cell to another.EVs are circular plasma membrane fragments that include exosomes and microparticles orshed vesicles，which are shed from almost all cell types under both physiological andpathological conditions[1,2]. The biological function of EVs is poorly understood, but mayinclude secretion, immunomodulation, coagulation, and intercellular communication[3-5].
EVs may vary in their formation, abundance, size, and composition, but they oftencontain abundant molecules, which include functional transmembrane and cytosol proteins,message RNA (mRNA), and microRNA (miRNA)[6-9]. The components in EVs could betransferred from one cell to another by endocytosis or fusion with the recipient cell[10-13].Importantly, the transferred components in EVs are functional and can regulate thebiological functions of the recipient cells[8,9,14,15]. Moreover, The EV-mediatedintercellular communication plays an important role in pathogenesis of atherosclerosis[15].
Recent studies have shown that both mitochondrial DNA (mtDNA) and chromosomalDNA were found in EVs[16,17]. Waldenstrom et al.[17]reported that chromosomal DNAsequences in EVs from cardiomyocytes could be transferred to the cytosol or nuclei oftarget cells. However, whether the transferred EV DNAs are functional or not is unclear. Inthis study, we investigated the function and potential mechanisms of transferrable EVgenomic DNAs (gDNAs) in the recipient cells. Finally, we also elucidated the critical roleof EV DNA that plays in pathogenesis of atherosclerosis.
Methods：
1. EVs from human plasma or cell culture supernatants were isolated through a seriesof ultracentrifugation steps as previously described[8,9,15]. To ensure that the EVs werecorrectly identified, electron microscopic, immunoblotting, and flow cytometry (FCM)analyses were used.
2. We examined the characteristics of DNA in EVs derived from human plasma andsupernatants of vascular smooth muscle cells (VSMCs) in culture by fluorescence-activatedcell sorter (FACS), high performance liquid chromatography (HPLC), agarose gelelectrophoresis, Solexa sequencing and polymerase chain reaction (PCR).
3. To determine the transportability and functionality of the DNA fragments in EVs,we studied the transport of gDNAs (AT1receptor DNA) in EVs from AT1receptortransfected-HEK293cells or VSMCs to non-transfected HEK293cells by PCR andimmunoblotting. To uncover the underlying mechanisms leading to the transcription oftransferred EV DNA, we investigated the effect of endogenous NF-κB in recipient cells, apromoter of the AT1receptor gene, on transferred DNAs including AT1receptor DNA byimmunofluorescence using laser confocal microscopy.
4. The VSMC and HEK293cell studies only provided indirect evidence that theincreased AT1mRNA is due to the incorporation of DNA from EVs. To overcome thislimitation, we studied the transport of the BCR/ABL hybrid gene in EVs from K562cells toHEK293cells, which is not normally expressed in HEK293cells. To determine thepathophysiological significance of transferred gDNAs in EVs between cells, we examinedthe transfer of BCR/ABL hybrid gene in EVs from K562cells to normal human neutrophilsisolated from human peripheral blood by dual-color fluorescence in-situ hybridization(D-FISH).
5. To investigate the critical role of EV DNA that plays in pathogenesis ofatherosclerosis, we screened out EV DNA fragments with differential gene copy numbers(GCNs) in plasma from patients with coronary artery disease (CAD) compared to controlhealthy subjects by Solexa sequencing. Then, we validated the screening EV DNAfragments in plasma by quantitative PCR. We also compared the levels of SRY GCNs,mRNA, and protein in WBCs between two groups by PCR and immunoblotting. Finally, wetransferred sex-determining region Y (SRY) gene-containing EVs or plasmids to monocytes or human umbilical vein endothelial cells (HUVECs) and observed adhesion functionbetween the two cells.
Results：
1. The electron micrographs of the EVs revealed rounded double-layer membranousvesicles of approximately30-1000nm in size, similar to previously described EVs[8,9,15].EVs more than200nm in size were confirmed by FCM analysis as this technology isunable to distinguish or enumerate single particles below200nm in size[18].Immunoblotting showed the presence of CD63, argonaute2(AGO2), tumor susceptibilitygene101(TSG101), Flotillin-1, and heat shock protein70(HSP70) proteins, commonlyused markers for EVs.
2. Substantial amounts of DNA were detected by FACS, HPLC, agarose gelelectrophoresis, and PCR in EVs derived from human plasma and supernatants of VSMCsin culture. We found that EVs contained double-strand DNA (dsDNA) ranging in size from1to20kb, but mostly around17kb. Furtherly, Solexa sequencing indicated the presence ofat least16434genomic DNA (gDNA) fragments in the EVs from human plasma.
3. We found the presence of AT1-EGFP DNA in EVs from AT1-HEK293cells thatstably expressed AT1protein by PCR and sequencing. After incubation of HEK293cellswith EVs from AT1-HEK293cells or VSMCs, immunofluorescence study showed directevidence that EV DNAs could be transferred into the recipient cells and localize to andinside the nuclear membrane. As an endogenous promoter of the AT1receptor, NF-κB,could be recruited to the transferred DNAs in the nucleus, and increased the transcription ofAT1receptor in the recipient cells. We also found the de-novo mRNA and protein of AT1receptor could be significantly inhibited by actinomycin D.
4. Incubating HEK293cells with EVs from K562cells resulted in the expression ofBCR/ABL hybrid gene mRNA and protein in the recipient HEK293cells. Moreover, thisheterologous expression of BCR/ABL hybrid gene in the HEK293cells was prevented bythe concurrent incubation with actinomycin D. Using a dual-color fluorescence in situhybridization (D-FISH) method, we found that incubation of normal human neutrophilswith EVs from K562cells resulted in the expression of BCR/ABL hybrid gene in20%ofthe neutrophils by genomic analysis.
5. Using Solexa sequencing followed by quantitative PCR, the GCNs of SRY DNA in EVs from plasma were significantly higher in patients with CAD than those in controlhealthy subjects. We also found the levels of SRY GCNs, mRNA, and protein in WBCswere higher in patients with CAD than those in control healthy subjects. Importantly,Transferred SRY gene-containing EVs or plasmids to monocytes and HUVECs enhancedadhesion function between the two cells
Conclusions：
In conclusion, we have shown that EV DNAs, which could be delivered from one cellto another, can increase the gDNA-coding mRNA and protein expressions in the recipientcells, and affect the physiological function in the recipient cells. Furtherly, we have foundSRY gene-containing EVs may play an critical role in atherosclerosis. EV-mediated transferof gDNAs may represent a new method of gene delivery and novel way of signaltransduction among cells. These findings would help in the discovery of novel mechanismsof cardiovascular disease and development of new therapeutic strategies.
引文
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