唾液腺多形性腺瘤蛋白多糖阻抑与肿瘤侵袭性生物学行为的关系探讨
发布时间：2018-04-20 08:09
本文选题：唾液腺 + 唾液腺多形性腺瘤 ； 参考：《河北医科大学》2014年硕士论文
【摘要】：目的: 唾液腺多形性腺瘤（salivary pleomorphic adenoma，SPA）是最常见的唾液腺肿瘤，约占唾液腺上皮性肿瘤的50%以上。SPA是源于上皮病变的良性肿瘤，但具有交界性肿瘤的特征，“好种植、易复发”，多次复发可以癌变，给临床治疗带来极大困难。研究该肿瘤的生物学特性对其治疗和预后有重要意义。 唾液腺多形性腺瘤由肿瘤性肌上皮细胞（neoplastic myoepithelialcells， NMCs）和腺上皮细胞构成。 NMCs具有分泌合成蛋白多糖（proteoglycans，PGs）的功能，通过胞吐作用将PGs分泌致细胞外基质，为SPA的黏液软骨样区域提供细胞来源。 PGs是一类具有复杂结构的大分子糖蛋白,由核心蛋白（core protein）和连接其上的氨基聚糖（glocosaminoglycans，GAGs）侧链构成，与弹性纤维、胶原纤维及糖蛋白一起构成细胞外基质，是细胞外基质的主要组成成分。PGs参与机体各项生理功能和物质代谢过程,对细胞的生长、增殖、细胞间的信号传导、相互作用以及肿瘤细胞的粘附、转移、侵袭等生物行为有重要影响。 PGs是在木糖基转移酶（xylosyltransferase，XT）的催化下进行合成的。目前已知的XT有两种，即XT-I和XT-II。XT-I是PGs合成的效率-限制阶段，对PGs的合成起至关重要的作用。XT-II是XT-I的一个亚型。二者的氨基酸的相似度小于55%，同样参与木糖基转移酶的合成。 RNA干扰（RNA interference，RNAi）是目前应用较广的一种基因研究技术。其核心是将与靶基因的mRNA对应的正义RNA和反义RNA组成的双链RNA导入细胞，特异性地使细胞中的mRNA发生降解，从而使相应的基因得到沉默。 本实验拟通过RNAi技术，沉默SPA细胞中指导PGs合成的关键起始酶XT-I，阻断PGs的生物合成。并通过肿瘤细胞体外侵袭实验，检测阻抑PGs合成、分泌后，SPA细胞侵袭性生物学行为的变化，为了解SPA侵袭性生物学行为的机制提供新的理论依据，并为SPA的临床治疗开创新的途径。 方法:1细胞培养 组织标本取自河北医科大学口腔医院口腔颌面外科一患者右侧腮腺原发肿瘤。采用组织块贴壁培养法，培养SPA细胞。培养液选用含20%胎牛血清的RPMI1640培养液，EDTA-胰酶消化液消化传代。2细胞鉴定 SPA细胞传至第2代，采用兔抗人calponin单克隆抗体，鼠抗人S-100蛋白单克隆抗体，鼠抗人角蛋白8（cytokeratin8，，CK8）单克隆抗体，进行免疫细胞化学鉴定。3细胞转染及分组 通过脂质体转染SPA细胞，沉默其XT-I基因的表达。将阻抑XT-I的真核表达载体shRNA-WJ4转染SPA细胞，命名为SPA-WJ4（沉默组）；将空载体shRNA-HK转染SPA细胞，命名为SPA-HK（空载体组）；将未转染shRNA真核载体的SPA细胞组，命名为SPA（未转染组）。4沉默效率的检测 采用Real-Time PCR技术，从mRNA水平检测基因沉默后SPA细胞中XT-I基因的表达。5PGs含量测定 基因转染48h后收集细胞，应用Blyscan Assay Kit试剂盒检测基因沉默后三组SPA细胞PGs合成分泌的改变，并进行统计学分析。6细胞侵袭实验 应用Transwell小室法检测基因沉默后细胞侵袭能力的改变。 结果:1细胞培养 SPA组织块原代培养5~7天，有细胞从组织块周围爬出。细胞呈短梭形，多角形，核大而居中。细胞呈多边形镶嵌排列。2细胞鉴定 采用第2代细胞进行免疫细胞化学染色技术鉴定。SPA细胞抗S-100呈阳性反应；抗calponin呈阳性反应；抗CK8呈阳性反应。3细胞转染及筛选 ShRNA-WJ4成功转染SPA细胞，转染后，细胞稳定表达绿色荧光蛋白（green fluorescent protein，GFP），转染效率44.2%。4沉默效率的检测 基因转染48h后收集细胞，进行Real-Time PCR实时定量检测shRNA-WJ4细胞XT-I基因mRNA沉默效率为28.0%。5PGs含量测定 应用Blyscan Assay Kit试剂盒检测基因沉默48h后三组细胞的PGs含量，其中SPA细胞PGs合成分泌降低27.20%。6细胞侵袭实验 Transwell侵袭试验显示:SPA-WJ4组细胞穿过微孔膜到达下层的细胞数量为23.83±2.93个，较SPA-HK（64.50±3.94）和SPA（67.50±2.35）细胞明显减少（P＜0.01），侵袭抑制率为64.70%。 结论: 靶向沉默XT-I基因，能有效阻抑SPA的PGs分泌，并有效抑制肿瘤细胞侵袭性生物学行为。
[Abstract]:Purpose :
The salivary gland polymorphic adenoma ( SPA ) is the most common salivary gland tumor , which accounts for more than 50 % of the salivary gland epithelial tumors . SPA is a benign tumor derived from epithelial lesions , but has the characteristics of borderline tumors .
The salivary gland morphic adenoma is composed of neoplastic myoepithelial cells ( NMCs ) and glandular epithelial cells . NMCs has the function of secreting synthetic protein polysaccharide ( PGs ) , which can secrete PGs into extracellular matrix through exocytosis , and provide cell source for the mucus cartilage - like area of SPA .
PGs is a kind of macromolecular glycoprotein with complex structure , which is composed of core protein and amino - glycans attached to it . It forms extracellular matrix with elastic fiber , collagen fiber and glycoprotein . It is the main component of extracellular matrix . PGs are involved in physiological function and substance metabolism process of organism . PGs have important influence on cell growth , proliferation , signal transduction , interaction and tumor cell adhesion , metastasis and invasion .
PGs are synthesized under the catalysis of xylosyltransferase ( XT ) . It is known that XT - I and XT - II . XT - I are the efficiency - limiting stages of PGs synthesis . XT - II is a subtype of XT - I . The similarity of amino acids is less than 55 % , which is also involved in the synthesis of xylosyltransferase .
RNA interference ( RNAi ) is a widely used gene research technique . The core is to introduce double - stranded RNA composed of the sense RNA and antisense RNA corresponding to mRNA of the target gene into the cell , specifically , the mRNA in the cell is degraded , so that the corresponding gene is silenced .
In this experiment , the key starting enzyme XT - I of PGs synthesis is guided in silence SPA cells by RNAi technology , and the biosynthesis of PGs is blocked . Through the in vitro invasion experiment of tumor cells , the changes of the invasive biological behavior of PGs are detected , which provides a new theoretical basis for understanding the mechanism of SPA invasive biological behavior , and provides a new approach for the clinical treatment of SPA .
Method : 1 Cell culture
Tissue samples were taken from primary tumors of the right parotid gland of the oral and maxillofacial surgery in the oral and maxillofacial surgery of Hebei Medical University . SPA cells were cultured by tissue patch culture method . The culture fluid was digested with RPMI1640 medium containing 20 % fetal bovine serum and digested with EDTA - trypsin digestive juice . The cells were identified .
SPA cells were transferred to the 2nd generation , monoclonal antibodies against human monoclonal antibodies against human calprotectin , monoclonal antibodies against human S - 100 protein , monoclonal antibodies against human keratin 8 ( CK8 ) were used to perform chemical identification of immune cells . Cells were transfected and grouped .
The eukaryotic expression vector shRNA - WJ4 was transfected into SPA cells and named SPA - WJ4 ( silent group ) .
The empty vector shRNA - HK was transfected into SPA cells , named SPA - HK ( empty vector group ) .
SPA cells transfected with shRNA eukaryotic vectors , designated as SPA ( untransfected group ) . 4 Detection of silencing efficiency
Expression of XT - I gene in SPA cells after gene silencing by Real - Time PCR and determination of 5PGs in SPA cells
After 48 hours of gene transfection , the cells were collected , and the changes of PGs synthesis secretion of three groups of SPA cells after gene silencing were detected by Blyscan Assay Kit kit .
Transwell cell method was used to detect the changes of cell invasion ability after gene silencing .
Results : 1 Cell culture
SPA tissue blocks were cultured in primary culture for 5 - 7 days , and the cells were isolated from the surrounding tissues .
Immunocytochemistry staining was carried out using the second generation of cells . SPA cells showed positive reaction against S - 100 .
The anti - calpain showed positive reaction .
Anti - CK8 positive reaction . Cells transfected and screened
ShRNA - WJ4 was successfully transfected into SPA cells . After transfection , the cells stably expressed green fluorescent protein ( GFP ) . The transfection efficiency was 44.2 % .
After 48 hours of gene transfection , the cells were collected and real - time PCR was performed to quantitatively detect the mRNA silencing efficiency of the shRNA - WJ4 cell XT - I gene and the mRNA silencing efficiency of the shRNA - WJ4 cell XT - I gene was 28 . 0 % . 5 PGs assay was performed .
搴旂敤Blyscan Assay Kit璇曞墏鐩掓娴嬪熀鍥犳矇榛
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