本文選題：唾液腺 + 唾液腺多形性腺瘤��； 參考：《河北醫(yī)科大學(xué)》2014年碩士論文

【摘要】：目的: 唾液腺多形性腺瘤（salivary pleomorphic adenoma，SPA）是最常見的唾液腺腫瘤，約占唾液腺上皮性腫瘤的50%以上。SPA是源于上皮病變的良性腫瘤，但具有交界性腫瘤的特征，“好種植、易復(fù)發(fā)”，多次復(fù)發(fā)可以癌變，給臨床治療帶來(lái)極大困難。研究該腫瘤的生物學(xué)特性對(duì)其治療和預(yù)后有重要意義。 唾液腺多形性腺瘤由腫瘤性肌上皮細(xì)胞（neoplastic myoepithelialcells， NMCs）和腺上皮細(xì)胞構(gòu)成。 NMCs具有分泌合成蛋白多糖（proteoglycans，PGs）的功能，通過(guò)胞吐作用將PGs分泌致細(xì)胞外基質(zhì)，為SPA的黏液軟骨樣區(qū)域提供細(xì)胞來(lái)源。 PGs是一類具有復(fù)雜結(jié)構(gòu)的大分子糖蛋白,由核心蛋白（core protein）和連接其上的氨基聚糖（glocosaminoglycans，GAGs）側(cè)鏈構(gòu)成，與彈性纖維、膠原纖維及糖蛋白一起構(gòu)成細(xì)胞外基質(zhì)，是細(xì)胞外基質(zhì)的主要組成成分。PGs參與機(jī)體各項(xiàng)生理功能和物質(zhì)代謝過(guò)程,對(duì)細(xì)胞的生長(zhǎng)、增殖、細(xì)胞間的信號(hào)傳導(dǎo)、相互作用以及腫瘤細(xì)胞的粘附、轉(zhuǎn)移、侵襲等生物行為有重要影響。 PGs是在木糖基轉(zhuǎn)移酶（xylosyltransferase，XT）的催化下進(jìn)行合成的。目前已知的XT有兩種，即XT-I和XT-II。XT-I是PGs合成的效率-限制階段，對(duì)PGs的合成起至關(guān)重要的作用。XT-II是XT-I的一個(gè)亞型。二者的氨基酸的相似度小于55%，同樣參與木糖基轉(zhuǎn)移酶的合成。 RNA干擾（RNA interference，RNAi）是目前應(yīng)用較廣的一種基因研究技術(shù)。其核心是將與靶基因的mRNA對(duì)應(yīng)的正義RNA和反義RNA組成的雙鏈RNA導(dǎo)入細(xì)胞，特異性地使細(xì)胞中的mRNA發(fā)生降解，從而使相應(yīng)的基因得到沉默。 本實(shí)驗(yàn)擬通過(guò)RNAi技術(shù)，沉默SPA細(xì)胞中指導(dǎo)PGs合成的關(guān)鍵起始酶XT-I，阻斷PGs的生物合成。并通過(guò)腫瘤細(xì)胞體外侵襲實(shí)驗(yàn)，檢測(cè)阻抑PGs合成、分泌后，SPA細(xì)胞侵襲性生物學(xué)行為的變化，為了解SPA侵襲性生物學(xué)行為的機(jī)制提供新的理論依據(jù)，并為SPA的臨床治療開創(chuàng)新的途徑。 方法:1細(xì)胞培養(yǎng) 組織標(biāo)本取自河北醫(yī)科大學(xué)口腔醫(yī)院口腔頜面外科一患者右側(cè)腮腺原發(fā)腫瘤。采用組織塊貼壁培養(yǎng)法，培養(yǎng)SPA細(xì)胞。培養(yǎng)液選用含20%胎牛血清的RPMI1640培養(yǎng)液，EDTA-胰酶消化液消化傳代。2細(xì)胞鑒定 SPA細(xì)胞傳至第2代，采用兔抗人calponin單克隆抗體，鼠抗人S-100蛋白單克隆抗體，鼠抗人角蛋白8（cytokeratin8，，CK8）單克隆抗體，進(jìn)行免疫細(xì)胞化學(xué)鑒定。3細(xì)胞轉(zhuǎn)染及分組 通過(guò)脂質(zhì)體轉(zhuǎn)染SPA細(xì)胞，沉默其XT-I基因的表達(dá)。將阻抑XT-I的真核表達(dá)載體shRNA-WJ4轉(zhuǎn)染SPA細(xì)胞，命名為SPA-WJ4（沉默組）；將空載體shRNA-HK轉(zhuǎn)染SPA細(xì)胞，命名為SPA-HK（空載體組）；將未轉(zhuǎn)染shRNA真核載體的SPA細(xì)胞組，命名為SPA（未轉(zhuǎn)染組）。4沉默效率的檢測(cè) 采用Real-Time PCR技術(shù)，從mRNA水平檢測(cè)基因沉默后SPA細(xì)胞中XT-I基因的表達(dá)。5PGs含量測(cè)定 基因轉(zhuǎn)染48h后收集細(xì)胞，應(yīng)用Blyscan Assay Kit試劑盒檢測(cè)基因沉默后三組SPA細(xì)胞PGs合成分泌的改變，并進(jìn)行統(tǒng)計(jì)學(xué)分析。6細(xì)胞侵襲實(shí)驗(yàn) 應(yīng)用Transwell小室法檢測(cè)基因沉默后細(xì)胞侵襲能力的改變。 結(jié)果:1細(xì)胞培養(yǎng) SPA組織塊原代培養(yǎng)5~7天，有細(xì)胞從組織塊周圍爬出。細(xì)胞呈短梭形，多角形，核大而居中。細(xì)胞呈多邊形鑲嵌排列。2細(xì)胞鑒定 采用第2代細(xì)胞進(jìn)行免疫細(xì)胞化學(xué)染色技術(shù)鑒定。SPA細(xì)胞抗S-100呈陽(yáng)性反應(yīng)；抗calponin呈陽(yáng)性反應(yīng)；抗CK8呈陽(yáng)性反應(yīng)。3細(xì)胞轉(zhuǎn)染及篩選 ShRNA-WJ4成功轉(zhuǎn)染SPA細(xì)胞，轉(zhuǎn)染后，細(xì)胞穩(wěn)定表達(dá)綠色熒光蛋白（green fluorescent protein，GFP），轉(zhuǎn)染效率44.2%。4沉默效率的檢測(cè) 基因轉(zhuǎn)染48h后收集細(xì)胞，進(jìn)行Real-Time PCR實(shí)時(shí)定量檢測(cè)shRNA-WJ4細(xì)胞XT-I基因mRNA沉默效率為28.0%。5PGs含量測(cè)定 應(yīng)用Blyscan Assay Kit試劑盒檢測(cè)基因沉默48h后三組細(xì)胞的PGs含量，其中SPA細(xì)胞PGs合成分泌降低27.20%。6細(xì)胞侵襲實(shí)驗(yàn) Transwell侵襲試驗(yàn)顯示:SPA-WJ4組細(xì)胞穿過(guò)微孔膜到達(dá)下層的細(xì)胞數(shù)量為23.83±2.93個(gè)，較SPA-HK（64.50±3.94）和SPA（67.50±2.35）細(xì)胞明顯減少（P＜0.01），侵襲抑制率為64.70%。 結(jié)論: 靶向沉默XT-I基因，能有效阻抑SPA的PGs分泌，并有效抑制腫瘤細(xì)胞侵襲性生物學(xué)行為。
[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嫻嬪熀鍥犳矇榛

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