【作者】 包刚；

【导师】 王洁；

【作者基本信息】 河北医科大学 ， 病理学与病理生理学， 2014， 博士

【摘要】 目的:唾液腺腺样囊性癌（salivary adenoid cystic carcinoma, SACC）是最常见的唾液腺恶性肿瘤之一，侵袭性强，可转移，易复发，预后不佳，迄今尚无较理想的治疗措施。SACC中的肿瘤性肌上皮细胞（neoplastic myoepithelial cells, NMCs）可以分泌蛋白多糖（proteoglycans, PGs），构成肿瘤的细胞外基质（extracellular matrix, ECM）。在SACC中，过多的ECM逐渐堆积，形成囊腔样结构，从而呈现出SACC特有的组织学特点。富含PGs的ECM，构成了SACC细胞的大分子微环境（macromolecules microenvironment）。SACC细胞在这一微环境内生存，继而进展，出现增殖、分化、侵袭、转移、复发等一系列生物学行为。目前认为，PGs是SACC的形态学表现和生物学行为必不可少的物质基础，参与肿瘤的发生和发展过程。人木糖基转移酶（xylosyltransferase, XT）是人体PGs生物合成的起始酶和限速酶，催化此合成过程的效率—限制阶段（rate-limited step），它的活性真实反映PGs的生物合成率。人XT有两个亚型（isoform）：XT-I和XT-II，催化作用几乎相同，但是在人体不同细胞的表达有所差异。RNA干扰（RNA interference, RNAi）是目前最有效的基因沉默（genesilencing）技术，可以特异、高效地阻断特定基因的表达。腺病毒载体是将外源基因导入细胞内的常用媒介，同时表达一个以上目的基因的腺病毒载体，可同时发挥多个基因的功能，提高了腺病毒载体的利用率。本研究拟构建一个同时沉默XT-I和XT-II基因的质粒载体，即编码2条shRNA的干扰质粒，腺病毒包装，获得重组腺病毒rAd5-shRNA-WJ7＋WJ4；通过感染SACC-83细胞，干扰XT-I和XT-II的表达，阻抑PGs合成，检测其沉默效果；建立SACC-83的裸鼠移植瘤模型，瘤体内注射重组腺病毒，观察其对移植瘤凋亡的影响。旨在探讨SACC中XT-I和XT-II对PGs的调控作用，以及XT-I和XT-II共沉默对细胞凋亡的影响，并为唾液腺肌上皮性肿瘤的生物治疗提供更多的依据。方法：1构建分别靶向抑制人XT-I基因和人XT-II基因的两个重组质粒设计靶向抑制人XT-I基因的shRNA-WJ4和靶向抑制人XT-II基因的shRNA-WJ7的干扰位点和引物序列，合成shRNA-WJ4和shRNA-WJ7的单链目的基因片段，分别与质粒pGenesil-1.2和pGenesil-1.1的线性化载体连接，扩增质粒，酶切鉴定，确定获得重组质粒pGenesil-1.2-shRNA-WJ4和pGenesil-1.1-shRNA-WJ7。2构建同时靶向抑制人XT-I基因和人XT-II基因的共沉默质粒双酶切以上两个重组质粒，回收大小片段，WJ7大片段与WJ4小片段的连接，扩增质粒，酶切鉴定，测序验证共沉默质粒shRNA-WJ7＋WJ4。3重组腺病毒质粒的制备从质粒pGenesil上通过LR体外同源重组将shRNA-WJ7＋WJ4表达框转移至pGSadeno腺病毒表达载体上，扩增质粒，酶切鉴定。4包装共沉默重组腺病毒制备线性化腺病毒DNA，连接重组腺病毒质粒片段，转染HEK293细胞，放大培养共沉默重组腺病毒rAd5-shRNA-WJ7＋WJ4。5构建阴性对照重组腺病毒rAd5-shRNA-HK6重组腺病毒感染细胞培养SACC-83细胞，分3组：SACC-83-WJ7＋WJ4组（沉默组）、SACC-83-HK组（空载体组）、SACC-83组（未感染组），确定最佳MOI值为100，细胞感染48h的荧光最强，计算感染率。7检测感染48h后3组细胞XT-I和XT-I mRNA的表达TRIzol法提取细胞总RNA，测定RNA纯度、浓度、完整性，逆转录合成cDNA第一链，实时荧光定量PCR（Real-Time PCR）检测3组细胞XT-I和XT-I mRNA的相对表达量，计算沉默率。8检测感染48h后3组细胞培养液中PGs的含量用标准品建立浓度-吸光度标准曲线，生物染色法测定PGs含量，计算抑制率。9裸鼠移植瘤模型的建立及分组4周龄雌性BALB/c-nu裸小鼠32只，14～15g，无特定病原体（specificpathogen-free, SPF）条件饲养，每只于左侧背部接种SACC-83细胞2.5×106个／250μl；62d后，选取瘤体最长径在1cm以上的24只，随机数字表法分为3组：SACC-83-WJ7＋WJ4组（沉默组）、SACC-83-HK组（空载体组）、SACC-83组（未感染组），每组8只。10重组腺病毒裸鼠移植瘤内注射接种肿瘤细胞后62d，首次瘤内注射重组腺病毒，SACC-83-WJ7＋WJ4组（沉默组）注射共沉默腺病毒rAd5-shRNA-WJ7＋WJ4，SACC-83-HK组（空载体组）注射阴性对照腺病毒rAd5-shRNA-HK，SACC-83组（未感染组）注射PBS，每只4×109PFU／200μl，每周注射一次，共5次。11标本采集3组裸鼠首次注射腺病毒5周后断颈处死，分离移植瘤，测量体积，新鲜瘤组织立即分切为4份，1份80C冻存，3份用于以下实验。12流式细胞术样品制备和凋亡检测70%乙醇固定标本，剪碎法＋网搓法制备单细胞悬液，PI染色，流式细胞术检测3组样品的细胞凋亡情况。13HE染色样品制备和观察10%福尔马林固定标本、石蜡包埋、切片、HE染色、光学显微镜观察3组标本的形态学变化。14透射电镜超薄切片样品制备和观察标本分切至小于1mm3，4%戊二醛前固定、1%锇酸后固定、环氧树脂包埋、超薄切片、醋酸铀和柠檬酸铅双重染色30min、透射电镜观察细胞凋亡情况。结果：1分别靶向XT-I、XT-II和阴性对照的siRNA序列设计结果shRNA-WJ4CAGGCAGCCCATCAAACCTshRNA-WJ7CGTCTCCTCAAGGCCGTTTATshRNA-HK GACTTCATAAGGCGCATGC2酶切和琼脂糖凝胶电泳鉴定结果、测序验证结果均证明质粒正确。3重组腺病毒感染SACC-83细胞的效率重组腺病毒感染后48h，以表达绿色荧光的细胞作为成功感染的细胞，SACC-83-WJ7＋WJ4组（沉默组）与SACC-83-HK组（空载体组）的感染率分别为98.7%和99.1%。4Real-Time PCR检测XT-I和XT-II的沉默效果感染后48h，SACC-83-WJ7＋WJ4组（沉默组）XT-I和XT-II的mRNA相对表达量明显低于SACC-83-HK组（空载体组）与SACC-83组（未感染组），SACC-83-HK组与SACC-83组之间无差别。SACC-83-WJ7＋WJ4组XT-I和XT-II的沉默率分别为97.3%和88.0%。5XT-I和XT-II基因共沉默对SACC-83细胞PGs合成分泌的影响感染后48h，SACC-83-WJ7＋WJ4组（沉默组）细胞培养液中PGs含量明显低于SACC-83-HK组（空载体组）和SACC-83组（未感染组），抑制率为68.61%，SACC-83-HK组PGs未受到抑制。6裸鼠移植瘤最终体积首次注射腺病毒后5周，3组裸鼠移植瘤体积无统计学差异。7流式细胞术测定裸鼠移植瘤细胞凋亡率首次注射腺病毒后5周，3组裸鼠移植瘤细胞凋亡率无统计学差异。8光学显微镜观察裸鼠移植瘤形态学变化首次注射腺病毒后5周，光镜观察裸鼠移植瘤HE染色石蜡切片，3组间未见明显的形态学差异，均未观察到显著的细胞凋亡现象。9透射电镜观察裸鼠移植瘤细胞凋亡现象首次注射腺病毒后5周，SACC-83-WJ7＋WJ4组（沉默组）易见细胞凋亡现象，其余两组较少。结论：1重组腺病毒rAd5-shRNA-WJ7＋WJ4能够有效诱导SACC-83细胞XT-I和XT-II基因沉默，并阻抑PGs合成分泌。2裸鼠SACC-83移植瘤内注射沉默XT-I和XT-II基因5周，沉默组出现细胞凋亡，但无统计学意义。更多还原

【Abstract】 Objectives:Salivary adenoid cystic carcinoma (SACC) is one of the most commonsalivary malignant tumors with the tendency of invasion, metastasis, andrecurrence. More ideal therapeutic measures for this neoplasm of poorprognosis are desired yet.Proteoglycans (PGs) secreted from neoplastic myoepithelial cells (NMCs)in SACC contribute to extracellular matrix (ECM). Abundant PGs maketypical cribriform structures full with proteoglycans and provide nutrition andmacromolecules microenvironment for the biological behaviors of SACC,including surviving, proliferation, differentiation, invasion, metastasis, andrecurrence. Serving as an indispensible material basis of morphologicalcharateristics and biological behaviors, PGs are believed to be involved intumorigenesis and development of SACC.As the initiative and rate-limited enzyme of PGs biosynthesis in vivo,human xylosyltransferases (XT) catalysis the rate-limited step in thebiosynthesis. Accordingly, the activity of XT represents the biosynthetic rateof PGs literally. XT-I and XT-II, two isoforms of XT, with unequal expressionin various tissues, play an identical catalytic role.RNA interference (RNAi) is recently regarded as the most efficient genesilencing method for its high specificity and high performance as well inblocking particular target gene expression. Adenoviral vector is generallytaken as a common medium to deliver exogenous gene to intra-cellularcircumstance. In addition, an adenoviral vector expressing multiple targetgenes may maximize availability of a vetor.In present study, to silence XT-I and XT-II simultaneously, an interferingplasmid encoding two shRNAs was designed, and then the corresponding recombinant adenoviral vector rAd5-shRNA-WJ7+WJ4was constructed.After SACC-83cell infection by the recombinant adenoviral vector, XT-I andXT-II mRNA level and PGs content were detected to assess silencingeffectiveness. SACC-83tumor-burdened mice were established prior tointra-tumor injection of recombinant adenoviral vector and followingobservation for apoptosis. These experiments were undertaken to investigatethe regulation of XT-I and XT-II on PGs in SACC and the effect ofco-silencing on apoptosis, so as to provide more evidence for biotherapy ofsalivary myoepithelial tumors.Methods:1Construction of two recombinant interfering plasmids respectively targetinghuman XT-I gene and human XT-II geneThe interfering sites and primer sequences of plasmids namedshRNA-WJ4and shRNA-WJ7were designed and synthesized to suppresshuman XT-I gene and human XT-II gene, which was followed by respectiveconjunction to linearized vectors of pGenesil-1.2and pGenesil-1.1. Throughplasmid amplification and digestive identification, the obtaining ofrecombinant plasmids, pGenesil-1.2-shRNA-WJ4andpGenesil-1.1-shRNA-WJ7, were determined.2Construction of recombinant co-silencing plasmid simultaneously targetinghuman XT-I gene and human XT-II geneFollowing double digestion of both plasmids above, fragments wererecovered and connected. Plasmid amplification, digestive identification, andsequencing were performed to verify the recombinant co-silencing plasmid,pGenesil-1.1+2-shRNA-WJ7+WJ4.3Preparation of recombinant adenoviral plasmidThe expressing box shRNA-WJ7+WJ4was transferred from plasmidpGenesil to adenoviral expressing vector pGSadeno by LR homologousrecombination in vitro, plasmid amplification and digestive identification wasoperated then.4Enclosing of recombinant adenoviral vector HEK293cells were tranfected by the connector of linearized adenoviralDNA with recombinant adenoviral plasmid fragments, and co-silencingrecombinant adenoviral vector rAd5-shRNA-WJ7+WJ4was obtained andamplified.5Synthesis of recombinant adenoviral vector rAd5-shRNA-HK as negativecontrol6Infection of SACC-83cells by recombinant adenoviral vectorsSACC-83cells were cultivated and divided into3groups: groupSACC-83-WJ7+WJ4(silencing group), group SACC-83-HK (blank vectorgroup), and group SACC-83(non-infection group). The best MOI (100) andthe moment of the best fluorescence (48h after infection) were determined，and infection rate was calculated.7Detection of XT-I and XT-I mRNA level (48h after infection)Total RNA extraction by TRIzol was followed by determination of purity,concentration, and integrity. After RT-PCR and Real-Time PCR in turn, therelative quantity of XT-I and XT-I mRNA in all groups and then silencing ratewere acquired.8Determination of PGs content in culture media of all groups (48h afterinfection)Based on the standard curve of concentration-optical density usingstandard substance, PGs content was determined by bio-staining andinhibitory rate was measured.9Establishment of tumor-burdened mice and groupingThirty-two four-week-old female BALB/c-nu nude mice,14~15g, wereraised under SPF condition.250μl cell suspension containing2.5×106SACC-83cells was injected subcutaneously at the left back of each animal. Atthe62nd day after injection of cell suspension, twenty-four tumor-burdenedanimals with mass of long diameter more than1cm were internalized anddivided, in accordance with the random number table, into3groups of8miceeach: group SACC-83-WJ7+WJ4(silencing group), group SACC-83-HK(blank vector group), and group SACC-83(non-infection group). 10Intra-tumor injection of recombinant adenoviral vectorsThe sixty-second day after inoculation of tumor cells，the first intra-tumorinjection of recombinant adenoviral vectors was performed, includingco-silencing adenoviral vector rAd5-shRNA-WJ7+WJ4for groupSACC-83-WJ7+WJ4(silencing group), negative control adenoviral vectorrAd5-shRNA-HK for group SACC-83-HK (blank vector group), and PBS forgroup SACC-83(non-infection group).4×109PFU/200μl was injected foreach animal. Such injection was taken once a week for5times totally.11Sample collectionIn the fifth week after the first intra-tumor injection,3groups oftumor-burdened mice were killed. Each mass was separated and measured forthe volume. Following instant quartering dissection of fresh neoplastic tissue,a quarter was preserved at80C, the others were used in the experimentsbelow.12Sample preparation for flow cytometry (FCM) and detection of apoptosisby FCM70%Ethanol-fixed specimens were sheared and rubbed to obtain singlecell suspension, which underwent propidium iodide (PI) staining subsequently.apoptosis of three groups were detected by FCM.13Preparation and observation of formalin-fixed/paraffin-embeddedspecimensFormalin-fixed/paraffin-embedded specimens were prepared by ordinaryprocedures, sections were stained with hematoxylin and eosin (HE), and thenexamined microscopically to evaluate morphological changes.14Preparation and observation of ultrathin section for transmission electronmicroscopySpecimens less than1mm3were successively fixed in4%glutaraldehydeand1%osmic acid, embedded in epoxy resin, sectioned into ultrathin slices,stained by uranium acetate and lead citrate respectively30min, and finallyexamined by transmission electron microscope (TEM) to evaluate cell1apoptosis. Results：1Designed siRNA sequences respectively targeting XT-I, XT-II, and negativecontrolshRNA-WJ4CAGGCAGCCCATCAAACCTshRNA-WJ7CGTCTCCTCAAGGCCGTTTATshRNA-HK GACTTCATAAGGCGCATGC2Conbinant plasmids were proved correct by enzyme digestion, agarose gelelectrophoresis, and sequencing.3Infection efficiency of SACC-83cells by recombinant adenovirus48h after recombinant adenovirus infection, according to expression ofgreen fluorescent taken as successful infection, the infection rate of groupSACC-83-WJ7+WJ4(silencing group) and group SACC-83-HK (blank vectorgroup) was98.7%and99.1%respectively.4Detection of XT-I and XT-II silencing effect by Real-Time PCR48h after infection, the relative expression of XT-I mRNA and XT-IImRNA in group SACC-83-WJ7+WJ4(silencing group) considerablydecreased compared with those in group SACC-83-HK (blank vector group)and group SACC-83(non-infection group), no difference was found betweengroup SACC-83-HK (blank vector group) and group SACC-83(non-infectiongroup). Silencing rate of XT-I and XT-II in group SACC-83-WJ7+WJ4(silencing group) was97.3%and88.0%respectively.5Effect of SACC-83cell XT-I and XT-II gene co-silencing on PGs synthesisand secretion48h after infection, PGs content of culture media from groupSACC-83-WJ7+WJ4(silencing group) decreased compared with those fromgroup SACC-83-HK (blank vector group) and group SACC-83(non-infectiongroup), with the inhibitory rate of68.61%. PGs from group SACC-83-HK wasnot inhibited.6Final volumes of transplantation tumors from tumor-burdened miceFive weeks after the first injection of adenovirus, the final volumes oftransplanted tumors from three groups of tumor-burdened mice showed no statistical difference.7Determination of apoptosis rate of transplantation tumor cell by flowcytometry (FCM)Five weeks after the first injection of adenovirus, apoptotic rates oftransplanted tumor cells from three groups of tumor-burdened mice showed nostatistical difference.8Morphological observation of nude mice transplantation tumor by opticalmicroscopicFive weeks after the first injection of adenovirus, among all groups,neither remarkable morphological differences nor significant apoptosis werefound in HE-stained formalin-fixed/paraffin-embedded specimens oftransplantation tumors form all groups.9Evaluation of transplantation tumor cell apoptosis by transmission electronmicroscope (TEM)Five weeks after the first injection of adenovirus, apoptosis is easy to findin group SACC-83-WJ7+WJ4(silencing group), but relatively rare in theother two groups.Conclusions：1Recombinant adenovirus rAd5-shRNA-WJ7+WJ4can effectively induceXT-I and XT-II gene silencing and inhibit PGs synthesis and secretion inSACC-83cells.2Five weeks after intra-tumor injection to silence XT-I and XT-II gene, cellapoptosis arose in group SACC-83-WJ7+WJ4(silencing group), but withoutstatistical significance.更多还原