【作者】 李淳；

【导师】 温博贵；

【作者基本信息】 汕头大学 ， 病理学与病理生理学， 2005， 博士

【摘要】 目的异型性是肿瘤的形态特点,特别是恶性肿瘤细胞核。肿瘤细胞核的异型性是指肿瘤细胞核的形态与其来源的正常细胞核的形态存在差异。核形态主要由核被膜,核内容物(染色质、核基质、RNA、蛋白质等)的组成及分布所决定。核基质(nuclear matrix NM)是真核细胞核中存在的网状结构,它在细胞核的形态结构形成中起重要作用。本研究通过①观察癌与正常组织共有的核基质蛋白laminB1和NuMA在食管癌及正常食管粘膜上皮中的表达:②了解正常食管粘膜和食管癌核基质与端粒DNA结合的情况;初步探讨核基质蛋白在肿瘤细胞核异型性改变中的作用和意义。材料与方法 (1)材料:①收集经病理确诊的手术切除新鲜食管鳞癌标本30例,每例取癌组织及远端切缘粘膜组织各一式二份,一份做连续冰冻切片,供原位核基质制备、免疫组化检测用;另一份组织冻存于-70。C,供Western blot、Southern blot、RT-PCR、端粒酶活性检测;②培养食管癌细胞EC109,供免疫荧光-激光共聚焦显微镜观测。(2)应用改良的硫酸铵法制备冰冻切片组织原位核基质。(3)应用免疫组化方法检测正常食管粘膜上皮及食管鳞癌组织原位核基质制备前后核基质蛋白laminB1、NuMA的表达。(4)应用免疫荧光-激光共聚焦显微镜观测食管癌EC109细胞原位核基质制备前后核基质蛋白laminB1、NuMA的表达。(5)应用硫酸铵法制备正常食管粘膜及食管鳞癌组织核基质蛋白,Western blot检测核基质蛋白中laminBl、NuMA的表达。(6)制备核基质-DNA结合物、提取结合于核基质的DNA,用端粒重复序列探更多还原

【Abstract】 Objective Atypia is morphologic characterized in tumor, especially in malignant nucleus. Nuclear atypia means that morphology of tumor nucleus is different from normal nucleus. The morphology of nucleus was effected by compose of nuclear envelope and inner nuclear substance (included chromatin, RNA, nuclear matrix and protein) and those substance distributed. Nuclear matrix is a nuclear framework structure in eukaryotic nucleus. It is important for nuclear structural formation. To investigate the nuclear matrix effect on tumor nuclear morphology and its significance, we investigated follow as: (1) to examine the expression of the common nuclear matrix protein laminBl and NuMA in normal esophageal epithelia and in esophageal squamous cell carcinoma (ESCC). (2) To examine interaction between telomeric DNA and nuclear matrix in normal epithelia and in ESCC.Materials and methods (1) materials: (1)30 cases of fresh ESCC specimens surgically resected were collected, which have pathologically proved to be of esophageal squamous cell carcinoma nature. 2 samples ( mucosae of surgical up-edge and cancer lesions) were taken from each specimen. Each sample was divided into two parts. One half was made continuously frozen section for preparing nuclear matrix in situ and for immunohistochemical detecting. The other half was stored at -70℃ and for detecting Western blot, Southern blot, RT-PCR, and telomerase activity. (2) EC109 esophageal cells were cultured for immunofluorescence-confocal examination.(2) Prepared frozen section nuclear matrix in situ using the modified ammonium sulfate method.(3) Detected the expression of laminBl and NuMA proteins before and after prepared nuclear matrix in situ in normal epithelia and ESCC using immunohistochemical-method.(4) Detected the expression of laminBl and NuMA proteins before and after prepared nuclear matrix in situ in EC 109 cultured cells using immunofluorescence-confocal method.(5) Prepared nuclear matrix protein using ammonium sulfate method and detected the expression of laminBl, NuMA proteins in normal mucosa and ESCC using Western blot.(6) Prepared telomeric DNA-nuclear matrix complex, extracted DNA from nuclear matrix, and detected telomeric DNA fragments attached to the nuclear matrix using Southern blot with (TTAGGG)n probe and semi-quantitative analyzed.(7) Extracted ESCC tissue genomic DNA, digested with restriction enzyme Hinf I and Rsa I (never cut telomere repeated fragments), and detected telomere Terminal Restriction Fragment (TRF) using Southern blot with (TTAGGG)n probe. Image analysized and calculated the mean TRF length(8) Detected telomerase activity in ESCC using TRAP (telomeric repeat amplification protocal)-silver staining assay.(9) Detected hTERT mRNA transcription in ESCC using RT-PCR assay.(10) Detected the expression of hTERT protein in ESCC using immunohistochemical-method.Results (1) The architecture of tissue was retained in nuclear matrix of frozen sections in situ. Nuclear matrix showed less intense staining material (Hematoxylin stained) with reticular-like. Feulgen stained DNA was lightly positive signal and histone HI was negative staining.(2) Immunohistochemical observation of laminBl protein: laminBl positive signal was brown, located in nucleus. The positive rate of normal epithelia, nuclear matrix of normal epithelia, cancer, and nuclear matrix of cancer were 93.3%, 86.7%, 96.7% and86.7% respectively. The difference of positive rate was found no significant (x2=2.702, P>0.05) among normal epithelia, nuclear matrix of normal epithelia, cancer and nuclear matrix of cancer. In normal epithelia, the expression level of laminBl diminished from basal cells to granular cells. In nuclear matrix of normal epithelia, the expression level decreased and most positive cells located in basal cells. Most positive cells showed whole nuclear staining. The different of expression level between normal epithelia and nuclear matrix of normal epithelia was highly significant ( x 2=9.041, PO.01). In ESCC, expression of laminBl showed no regular staining patterns. In nuclear matrix of cancer tissue, the expression level of laminBl reduced and showed a rim at the nuclear periphery positive signal. The difference of expression level between cancer and nuclear matrix of cancer was significant ( x 2=4.176, PO.05). The difference of expression level between normal epithelia and cancer was no significant ( x 2=1.707, P>0.05). the expression level in nuclear matrix of cancer was higher than in nuclear matrix of normal epithelia, and the difference between them was significant (x2=5.042, PO.05).(3) Immunohistochemical observation of NuMA protein: NuMA positive signal was brown, located in nucleus. The positive rate of normal epithelia, nuclear matrix of normal epithelia, cancer, and nuclear matrix of cancer were 96.7%, 90.0%, 93.3% and 86.7% respectively. The difference of positive rate was found no significant (x2=2.182, P>0.05) among normal epithelia, nuclear matrix of normal epithelia, cancer and nuclear matrix of cancer. NuMA protein expressed in most epithelial cells and cancer cells. The expression level was reduced in nuclear matrix of normal epithelia and nuclear matrix of cancer. The difference of expression level between normal epithelia and nuclear matrix of epithelia was significant ( x 2=6.103, P<0.05), while the difference between cancer and nuclear matrix of cancer was significant ( x 2=5.417, P>0.05). The difference of expression level between normal epithelia and cancer was no significant (x2=3.766, P>0.05). The difference of expression level between nuclear matrix of normal epithelia and nuclear matrix of cancer was highly significant (x2=6.839, PO.01).(4) Localization of laminBl and NuMA protein revealed byimmunofluorescence-confocal microscope: laminBl protein positive signal was green fluorescence, located in EC 109 nucleus. The positive signal of laminBl in nuclear matrix was decreased and showed a rim at nuclear periphery. NuMA protein positive signal was green fluorescence, located in EC 109 nucleus. The positive signal of NuMA in nuclear matrix was decreased and most nuclear showed blue fluorescence.(5) Western blot analysis: LaminBl protein positive staining showed a 70kd band. NuMA protein positive staining showed a 228kd band. The positive rate of laminBl in normal mucosa nuclear matrix and cancer nuclear matrix was 93.3%, 90% respectively. The difference of laminB 1 positive rate between normal and cancer was no significant ( x 2=0.185, P>0.05). The positive rate of NuMA in normal mucosa nulear matrix and cancer nuclear matrix were same of 90%. But the bands of laminBl and NuMA in normal mucosae nuclear matrix were weaker staining than in cancer nuclear matrix.(6) Semi-quantitative analysis of telomeric DNA associated with nuclear matrix:The relative quantity of telomeric DNA associated with nuclear matrix was 2.046 + 0.505 ( x±s) in normal mucosae, and 3.414 + 0.544 ( x + s) in cancer. The difference between normal mucosae and cancer was highly significant (t=l0.093, P<0.01).The mean length of TRF in 30 cases of ESCC was 7.02Kb ±1.62 ( x + s) .The relative quantity of telomere-nuclear matrix associated fragment had no correlation with TRF in cancerous lesion (t=0.543, P>0.05).(7) Relationship between telomere DNA-nuclear matrix association and telomerase:The telomerase activity detected rate was 80% in ESCC. The relative quantity of telomeric DNA-nuclear matrix association fragment in cases of telomerase activity was higher than in cases of no telomerase activity. The difference between them was significant (t=2.679, P<0.05).The positive rate of hTERT mRNA expression was 65.0% in ESCC. The relative quantity of telomeric DNA-nuclear matrix association fragment in cases of positive expression was higher than in cases of negative expression. The difference between them was significant (t=2.941, PO.05).The positive rate of hTERT protein expression was 70.0% in ESCC. The relative quantity of telomeric DNA-nuclear matrix association fragment in cases of positive staining was higher than in cases of negative staining. The difference between them was significant (t=2.941, PO.05). Conclusions:(1) laminBl and NuMA proteins were expressed in most normal esophageal epithelia and esophageal squamous cell carcinoma.(2) In nuclear matrix of normal epithelia and cancerous lesions, laminB 1 and NuMA proteins were decreased. It suggested that some laminB K NuMA protein were soluble and some were insoluble. Soluble protein was extracted in the process of prepared nuclear matrix.(3) The expression level of laminBl > NuMA protein in nuclear matrix of cancerour lesion was higher than in nuclear matrix of normal epithelia. The expression of laminBl protein showed a rim at nuclear periphery in most nuclear matrix of cancer(4) Telomeric DNA-nuclear matrix association fragment in ESCC was more than in normal mucosae.(5) In ESCC, telomere was short, and the detected rate of telomerase activity was high. The telomeric DNA attached to nuclear matrix had correlation with telomerase activity, hTERT mRNA transcription, and hTERT protein expression.更多还原