人TIMP-2基因真核表达质粒的构建及其表达研究

目的构建人T IM P-2基因真核表达质粒,使其在真核细胞中稳定表达。方法利用RT-PCR方法获得人T IM P-2基因cDNA,以pcDNA 3为载体构建真核表达质粒pcDNA 3/T IM P-2,采用脂质体介导基因转染技术将重组质粒DNA导入CHO细胞中,加入G 418对转染细胞进行筛选获得稳定转染细胞,采用W estern b lot对重组质粒的表达进行检测。结果酶切及测序结果显示重组质粒pcDNA 3/T IM P-2构建正确,在转化细胞中检测到人T IM P-2的表达。结论成功构建人T IM P-2真核表达质粒并获得稳定表达人T IM P-2的CHO细胞株。     

Significance of the Detection of Epstein—Barr Virus DNA In Lymph Nodes in Patients with Hodgkin's Disease

Epstein-Barr virus (EBV) DNA is frequently identified in benign and malignant lymphoproliferative conditions. As shown by in situ hybridization studies viral DNA is localized within malignant cells as well as benign lymphocytes. Clonal and nonclonal EBV genomes are present in Hodgkin's disease (HD), lymphomas of the immunocompromised host and reactive lymph node hyperplasia. Lytic infection with formation of linear genomes is observed in the same conditions but appears to be infrequent in HD as shown by quantitation of mRNA coding for viral capsid antigen. Expression of the oncogene LMP (latent membrane protein) is seen in Sternberg-Reed (SR) cells and immunoblasts of AIDS-related lymphoma and infectious mononucleosis (IM). In HD, the region of the BNLF1 oncogene coding for the amino terminal and transmembrane domains (associated with oncogenic function) of LMP appears to be homogeneous whereas the region coding for the intracytoplasmic (carboxy terminal) domain of LMP is heterogeneous. Cytological similarities between SR cells and immunoblasts of IM and AIDS-related lymphomas are consistent with the hypothesis that the BNLF1 oncogene is one possible inducer of morphological features of SR cells. Whether chromosomal integration of EBV DNA is an important factor in activation of such a transforming activity remains to be elucidated. EBV DNA positive and negative HD cases with numerous SR cells lack significant mRNA expression of the two recombinase activating genes (RAG-1 and RAG-2). Therefore the SR cells appear to be derived from lymphocytes beyond the pre-B-cell or common thymocyte stage which may or may not subsequently become infected by EBV.     

前列腺特异性抗原启动子增强子调控重组质粒的构建及鉴定

目的:利用前列腺特异性抗原(pros-tate-specific antigen,PSA)组织特异性表达的特点,克隆出其上游增强子(prostate-specific antigen enhancer,PSAE)和启动子(prostate-specificantigen promoter,PSAP)片断,并对其表达效率和组织特异性表达进行初步研究。方法:从前列腺癌组织提取基因组DNA,并采用PCR方法分别扩增PSA增强子和启动子序列;利用报告基因pEGFP-1,分别构建含不同调控序列的表达载体;脂质体介导基因转染不同细胞并观察绿色荧光蛋白(GFP)的表达情况。结果:成功构建质粒pPSAE-EGFP、pPSAP-EGFP和pPSAE-PS-AP-EGFP;转染结果显示,pPSAE-PSAP-EGFP在前列腺癌细胞PC-3中的荧光强度明显高于pPSAP-EGFP,pPSAE-EGFP同样观察到荧光表达,说明PSA增强子能显著提高PSA启动子的转录能力,并具有单独调控基因表达的能力。结论:PSA增强子和启动子的共同调控可以提高基因表达的有效性和细胞特异性,为前列腺癌的临床基因治疗提供实验依据。     

Enhanced expression of anti-apoptotic proteins in human papillomavirus–immortalized and cigarette smoke condensate–transformed human endocervical cells: Correlation with resistance to apoptosis induced by DNA damage

Apoptosis plays an important role in various biological processes including embryogenesis, differentiation, homeostasis, and oncogenesis. We have developed a system composed of primary human endocervical cells (HEN), HEN immortalized by human papillomavirus (HPV) type 16, and their counterparts subsequently malignantly transformed by cigarette smoke condensate (CSC). To understand the role of apoptosis in the multistep oncogenesis of human cervical cells, we examined the expression of apoptosis-associated proteins in our in vitro model system. The results showed no significant difference in the levels of apoptosis-inducing proteins bak and bax among all the cell types examined. On the other hand, the levels of apoptosis-inhibiting proteins bcl-2, bcl-x_L and BAG-1 increased progressively after immortalization and transformation. The p53 protein level decreased in the HPV16-immortalized HEN and increased in one of two lines of the CSC-transformed HEN. Further, the increased levels of apoptosis-inhibiting proteins in the HPV16-immortalized and the CSC-transformed HEN correlated with progressively increased resistance of these cells to apoptosis induced by staurosporine or cisplatin. This study provided the first evidence that overexpression of apoptosis-inhibiting proteins is important for both multistep oncogenesis and resistance of human endocervical cells to apoptosis induced by DNA-damaging reagents. Mol. Carcinog. 22:95–101, 1998. © 1998 Wiley-Liss, Inc.