沉默XT-I基因阻抑蛋白多糖合成与唾液腺多形性腺瘤侵袭性的抑制  

The inhibited effect of proteoglycans by XT-I gene silenced on the invasive activity of salivary pleomorphic adenoma

作　　者：史小烁 刘慧娟[2] 王洁[2] 李廷 石宏[2] 张艳宁[2] SHI Xiaoshuo;LIU Huijuan;WANG Jie;LI Ting;SHI Hong;ZHANG Yanning(Department of Oral Pathology,College and Hospital of Stomatology,Key Laboratory of Stomatology,Hebei Medical University,Shijiazhuang 050017)

机构地区：[1]河北省人民医院口腔科,石家庄050017 [2]河北医科大学口腔医院·口腔医学院,河北省口腔医学重点实验室

出　　处：《现代口腔医学杂志》2024年第3期168-177,共10页Journal of Modern Stomatology

基　　金：国家自然科学基金面上项目(81170976);河北省自然科学基金项目(2009001046)。

摘　　要：目的探讨唾液腺多形性腺瘤(pleomorphic adenoma,PA)中蛋白多糖(proteoglycans,PGs)阻抑后,对肿瘤侵袭性生物学行为的抑制。方法(1)构建靶向沉默木糖基转移酶基因-I(xylosyltransferase-1,XT-1)的质粒载体shRNA-WJ4。(2)留取1例腮腺原发性多形性腺瘤标本,进行组织学和免疫组织化学染色,采用鼠抗人Hyaluronan synthase 2(HAS2)检测透明质酸,鼠抗人Heparan Sulfate Proteoglycan检测硫酸乙酰肝素。(3)原代细胞培养,采用兔抗人calponin,鼠抗人S-100蛋白和CK7单克隆抗体鉴定细胞。(4)脂质体转染培养的PA细胞,沉默XT-I基因。实验分三组,PA组(空白组)、PA-HK组(空载体组)、PA-WJ4组(沉默组),MTT法检测三组细胞的生长情况。(5)采用Real-Time PCR技术,检测基因沉默后XT-I基因的表达。(6)应用Blyscan Assay Kit试剂盒检测基因沉默后,PGs合成分泌的改变。(7)应用Transwell小室法检测基因沉默后,肿瘤细胞侵袭能力的改变。(8)统计学分析应用SPSS13.0统计分析软件,Excel软件导出数据,生成柱形图。结果(1)成功构建靶向沉默木糖基转移酶基因-I(XT-1)的质粒载体shRNA-WJ4。(2)唾液腺PA黏液软骨样组织中,富含透明质酸和硫酸乙酰肝素。(3)唾液腺PA原代培养5~7天,细胞从组织块周围爬出。培养的PA细胞鉴定发现,肿瘤性肌上皮细胞抗calponin阳性、抗S-100阳性;肿瘤性腺上皮细胞CK7阳性。(4)ShRNA-WJ4成功转染PA细胞后,表达绿色荧光蛋白(green fluorescent protein,GFP),转染效率44.2%。MTT法检测显示,沉默组PA细胞生长缓慢。(5)Real-Time PCR检测沉默组PA细胞,XT-I基因mRNA沉默效率为28.0%。(6)应用Blyscan Assay Kit试剂盒,检测基因沉默48 h后,沉默组PA细胞PGs合成分泌降低27.20%。(7)Transwell侵袭试验显示,沉默组PA组细胞穿过微孔膜到达下层的细胞数量为23.83±2.93个,较空载体组(64.50±3.94)和空白组(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,Objective To inhibit the biosynthesis of proteoglycans(PGs)in salivary pleomorphic adenoma(PA),and to observe the inhibition of invasion activity of PA cells.Methods①Xylosyltransferase-1(XT-I)expression vector shRNA-WJ4 and shRNA-HK(negative control)was constructed.②The sample was obtained from a patient with a parotid pleomorphic adenoma resection in Department of Oral Maxillofacial Surgery,Hospital and College of Stomatology,Hebei Medical University.Histologic stain and immunohistochemical stain against Hyaluronan synthase 2(HAS2)and Heparan Sulfate Proteoglycan were performed.③Primary culture of salivary PA was adopted,and PA cells were cultured in RPMI 1640 medium containing 20%fetal bovine serum.The second generation of PA cells was identified by immunocytochemical stain against calponin,and S-100 protein as well as CK7.④The XT-I gene expression of PA was silenced by transfection of constructed XT-I expression vector shRNA-WJ4 and shRNA-HK(empty-vector control)into PA cells by LipofectameneTM2000.Three groups were included,group PA(blank control),group PA-HK(empty-vector control),group PA-WJ4(XT-I expression vector),and MTT was used to detect the growth of 3 groups.⑤The expression of the mRNA level of XT-I in 3 groups was detected by the Real-Time PCR technology.⑥The content of PGs from 3 groups was detected by Blyscan Assay Kit.⑦Matrigel invasion assay was used to observe the inhibited effect of PGs on the invasive activity of PA cells by silencing the XT-I gene.⑧The statistical analysis was performed by SPSS 13.0 software.And data were exported to generate the column chart by Excel software.Results①Silenced XT-I expression vector shRNA-WJ4 and shRNA-HK(empty-vector control)was constructed successfully.②Hyaluronic acid and heparan sulfate were observed richly in the mucoid-,myxoid-and chondroid-type tissues of salivary PA.③After 5~7 days of primary culture,short spindle-shaped or polygonal PA cells emigrated from the tissue.Immunocytochemical stain showed anti-calponin,S-100 positive i

关 键 词：多形性腺瘤 蛋白多糖 RNA干扰 木糖基转移酶基因-I 侵袭性 

分 类 号：R739.8[医药卫生—肿瘤]