CKLF基因与CKLFSF1基因间的顺式调控元件

目的：探讨CKLF基因与CKLFSF1基因间序列(CCS)的调控作用。方法：运用PCR技术扩增CCS，并将此片段插入含有荧光素酶(luriferase)报告基因载体pGL3-basic，以及pGL3-SV40启动子中，构建受其调控的荧光素酶报告基因载体。应用脂质体介导基因转染技术，将4种重组质粒转染Hela细胞，进行瞬时表达分析。结果：在pGL3-basic和pGL3-basic-CCS质粒中的报告基因luciferase均无表达，但将CCS片段插入至promoter上游后，荧光素酶活性增加近1倍。结论：CKLF与CKLFSF1基因间的序列不具有启动子活性，但该序列中却可能存在调控其下游基因表达的顺式增强子元件。     

重型肝炎相关的人纤维介素基因启动子的克隆及功能分析

目的明确在病毒蛋白HBc和HBx作用下,hfgl2基因5′端非编码区对转录激活起重要作用的转录调控序列。方法运用基因重组的方法,构建一系列5′端缺失而保留共同3′端的hfgl2基因启动子虫荧光素酶报告质粒,将其分别与病毒蛋白HBc和HBx真核表达质粒共转染CHO细胞和HepG2细胞,检测各组细胞的相对荧光素酶活性。结果酶切鉴定以及DNA测序等证实一系列hfgl2基因启动子虫荧光素酶报告基因质粒构建成功,系列启动子缺失试验证实:在hfgl2基因启动子-817位至-467位(相对于转录起始点)之间存在着激活该基因的调控序列。结论在HBV病毒蛋白HBc及HBx作用下,hfgt2基因的启动子区存在一个与其转录表达有关的调控序列,探讨了重型肝炎相关的hfgl2基因高度表达的分子机制,为下一步研究相关的顺式作用元件及反式作用因子奠定了基础。     

人血红素加氧酶基因近端启动子区研究进展

分析血红素加氧酶(heme oxygenase,HO)基因mRNA加帽位点上游1416bp及mRNA5′端未翻译区24bp全长1.44kb片段。通过大片段缺失,发现与诱导剂相关的mRNA加帽位点近端上游121bp,可使基因的瞬时表达提高3～5倍,同时诱导作用发生在SV_(40)增强子元件位于启动子序列上游。另外在1.44kb片段间mRNA加帽位点上游有沉默子或负调控元件及NF-κB-和AP-2结合位点,而在1.44kb之外还有附加的诱导增强子元件。     

小鼠BCMA基因启动子活性研究

目的:探索B淋巴细胞成熟抗原(BCMA)基因5′上游序列的启动子活性, 为进一步研究BCMA基因的表达调控机制提供实验依据.方法:构建由BCMA基因5′上游-820～ 145、 -607～ 145、 -359～ 145、 -157～ 145、 -93～ 145 5个片段驱动的荧光素酶报告载体pGL3-B820、 pGL3-B607、 pGL3-B359、 pGL3-B157、 pGL3-B93, 通过转染J558L、 293T、 HeLa细胞检测荧光素酶的表达, 观察荧光素酶相对活性.结果:5种5′删除体的转录激活性由大到小为pGL3-B157>pGL3-B607>pGL3-B359>pGL3-B93>pGL3-B820.pGL3-B157在3种细胞中的转录激活能力为J558L>HeLa>293T, 且在J558L中的转录激活能力明显强于Hela和293T细胞.结论:BCMA基因5′上游序列"-820 bp～ 145 bp"具有启动子活性, 核心启动子可能位于-93～ 145区域中.     

人BAFF-R基因启动子活性的初步研究

目的以B淋巴细胞刺激因子受体BAFF-R基因5'上游区序列为研究对象,初步鉴定、分析该基因的启动子所在区域。方法克隆BAFF-R基因5'侧翼区1823 bp序列,构建8个含有不同长度启动子序列的荧光素酶报告基因表达质粒pGL3-B1～B8,将这8个序列缺失重组质粒与psv-β-gal质粒共转染细胞,检测荧光素酶的相对活性,确定启动子所在区域。结果8个重组质粒中pGL3-B2、pGL3-B3、pGL3-B5、pGL3-B6启动子的活性较低;pGL3-B7启动子的活性最强,pGL3-B8启动子活性最低。结论BAFF-R基因5'端-288～-430、-712～-868和-1420～-1562三个区段可能存在转录沉默子元件,-617～-712和-1277～-1420区段存在转录增强子元件,BAFF-R基因的核心启动子可能位于-1420～+261区域。     

人前列腺特异性膜抗原基因启动子/增强子表达载体的构建及组织特异性鉴定

目的：构建前列腺特异性表达的人前列腺特异膜抗原（PSMA）基因启动子/增强子表达载体，并进行组织特异性鉴定。 方法：从人外周血中提取基因组DNA，采用PCR技术分别扩增PSMA上游 1 175 bp 的启动子序列，及第三内含子中258 bp的增强子序列，将两个序列定向克隆至荧光素酶报告基因表达质粒pGL3-Basic，构建前列腺特异性表达载体pGL3-PSMP-PSME。将构建载体用脂质体分别转染前列腺癌PC-3M细胞株及4种非前列腺癌细胞株，48 h后通过检测荧光素酶表达活性，确定克隆的启动子及增强子的活性及其组织特异性表达活性。结果：构建的pGL3-PSMP-PSME质粒经酶切及DNA测序分析鉴定，证实克隆片段的大小、插入方向及其序列正确。细胞转染结果显示， pGL3-PSMP-PSME在PC-3M细胞株中表达活性明显高于其它4种非前列腺癌细胞株。结论：构建的前列腺表达载体具有较高的组织特异性, 为进一步研究PSMA调控序列驱动治疗基因进行前列腺癌的靶向生物治疗奠定了基础。     

CK13基因5′旁侧的初步功能分析

目的　探讨细胞角蛋白 13(cytokeratin13,CK13)基因表达调控的机理 ,研究 CK13基因 5′旁侧不同基序对其转录活性的影响。　方法　采用分子克隆结合报告基因分析的方法 ,构建 CK 13基因 5′旁侧 5 13bp内不同基序与氯霉素乙酰转移酶 (chloramphenicol acetyltransferase,CAT)报告基因增强子载体p CAT的重组体 ,通过脂质体介导的转染技术导入 He L a细胞 ,检测各报告基因载体 CAT的相对活性。　结果　 CK13基因 5′旁侧起始密码子 ATG上游 - nt.32 5～ - nt.2 0 7间 119bp中具有某种抑制子元件 ,-nt.2 0 6～ - nt.94间 113bp中具有某种增强子元件。　结论　 CK13基因 5′旁侧 5 13bp内存在促进及抑制CK13基因表达的反应元件 ,进一步定位这些顺式反应元件并研究与之相互作用的反式作用因子 ,可望阐明 CK13基因表达调控及组织特异性表达的详细机理。     

肝癌靶向性超抗原表达载体的构建与鉴定

目的：设计并构建受AFP基因顺式作用元件调控，并经减毒处理的肝癌特异性超抗原表达载体。方法：用新设计的引物作PCR扩增截短的AFP基因启动子和增强子、linker-CD80tm和SEA（D227A）。将上述片段与逆转录病毒载体pLXSN的多克隆位点连接，构建成为AFP基因顺式作用元件调控的肝癌特异性减毒超抗原表达载体[pLXSN SEA(D227A)-Linker-CD80tm]，并用软件对其开放读框进行分析。结果：成功克隆了截短的AFP基因启动子、增强子、linker-CD80tm和SEA（D227A）。将上述序列连接到逆转录病毒载体pLXSN的多克隆位点，酶切鉴定和DNA序列分析无误。结论：AFP基因转录启动元件修饰的SEA表达载体，在基因转录水平定量、定向、特异性调控强有力的细胞和体液免疫活化剂（SEA），为下一步将其作为基因疫苗治疗肝癌奠定了基础。     

人β-防御素-2基因5′-端转录调控序列分析

目的 :对在LPS、TNF α刺激下 ,人 β 防御素 2基因 5′ 端转录调控机制进行初步分析。 方法 :将HBD 2基因 5′ 端上游序列连接入无启动子的pEGFP 1质粒 ,构建了系列 5′端缺失的 pEGFP 1 /HBD 2报告质粒。pEGFP 1 /HBD 2质粒转染细胞后给予LPS、TNF α刺激 ,用RT PCR检测 pEGFP的mRNA浓度。 结果 :显示HBD 2基因上游 2 41段有较强的启动子活性 ;LPS、TNF α刺激后 ,即使上游序列缩短至 2 41位点时 ,报告基因 pEGFP的mRNA表达量仍明显增高。说明HBD 2基因上游 2 41区域含有LPS、TNF α刺激后激活的转录因子作用位点。计算机分析表明 2 3 2 / 2 2 2区域有一同源性极高的NF kB结合元件 ,提示NF kB结合元件可能调控HBD 2的增强表达     

人血管细胞中hCREG1基因启动子对血清饥饿发生应答

目的： 为揭示E1A 激活基因阻遏子（CREG1）在人血管平滑肌细胞(VSMCs)和人脐静脉内皮细胞（HUVECs）中表达的调控机制，构建人CREG1（hCREG1）启动子报告基因载体，探讨CREG1在不同血管细胞中的表达。方法: 在对hCREG1进行生物信息学分析的基础上，以人基因组DNA为模板，PCR方法扩增含有hCREG1基因上游-3 677 bp序列，构建了hCREG1 5′上游-3 677 bp、-2 310 bp和-945 bp序列片段，分别将这3个序列克隆到pMD18-T载体上并定向亚克隆到pEGFP-1报告基因载体-pEGFP-hCREG1-P3677、pEGFP-hCREG1-P2310和pEGFP-hCREG1-P945。将重组质粒瞬时转染VSMCs株HITASY和HUVECs，检测绿色荧光蛋白（GFP）的表达。结果: 经测序鉴定证实，3个报告基因载体中插入的PCR扩增序列均与hCREG1基因上游DNA序列完全匹配。瞬时转染体外培养的人VSMCs和HUVECs后，经荧光观察和蛋白质印迹（Western blotting）证实，细胞内存在GFP的表达，并且0.5%FBS培养的HITASY细胞中GFP表达较10%FBS培养的细胞中明显增加。HUVECs中的GFP表达较人VSMCs明显增加。结论: hCREG1基因启动子报告基因载体构建成功，核心启动子存在于5′上游序列的-945 bp-0 bp区域，为进一步研究hCREG1基因表达提供了条件。     

3-Hydroxy-3-methylglutaric aciduria

3-Hydroxy-3-methylglutaric aciduria was found in a newborn infant whose parents are first cousins. The patient presented at 5 days of life with hyperammonemia, hypoglycemia, and metabolic acidosis. There was no ketonuria. Diagnosis was made by analysis of the pattern of organic acids excreted in the urine. A profound deficiency in activity of 3-hydroxy-3-methylglutaryl-coenzyme A lyase was found in cultured skin fibroblasts. The parents had intermediate levels of enzyme activity.     

3-Hydroxy-3-Methylglutaric Aciduria

3-Hydroxy-3-methylglutaric aciduria was found in a newborn infant whose parents are first cousins. The patient presented at 5 days of life with hyperammonemia, hypoglecemia, and metabolic acidosis. There was no ketonuria. Diagnosis was made by analysis of the pattern of organic acids excreted in the urine. A profound deficiency in activity of 3-hydroxy-3-methylglutaryl-coenzyme A lyase was found in cultured skin fibroblasts. The parents had intermediate levels of enzyme activity.     

Fetal thymocyte potential for T cell receptor Vγ3-Jγ1 junctional modification

Junctional modifications of T cell receptor (TcR) and immunoglobulin (Ig) gene joining regions provide great diversity to respective protein repertoires. The addition of non-germ-line-encoded nucleotides (N-regions) in the V-Jγ junction is one such modification which is developmentally regulated, rarely evident in the fetal animal, but common in the adult. A question has recently arisen as to whether developmentally patterned N-region additions in V-Jγ joins are a reflection of T cell progenitors which are committed to particular types of rearrangement prior to the event, or of changing environmental influences on uncommitted cell populations. To address this question with regard to theVγ3-Jγ1 join, T cells were examined in the fetal thymic organ culture (FTOC), a system with which the environment of early progenitor cells could be deliberately altered. At various times following FTOC initiation, cells were isolated for examination by the polymerase chain reaction, cloning and sequencing. Vγ3-Jγl sequences within genomic DNA as well as cDNA were evaluated. Data from these studies revealed frequent N-region additions within V-Jγ joins among day 14 fetal thymocyte populations, a situation dissimilar from that in vivo. Also dissimilar from the in vivo situation was the degree of exonuclease activity evident in FTOC. The canonical Vγ3-Jγl join (a frequent junction lacking N-region addition) was recognized in all experiments, but was least common among DNA versus cDNA sequences. Results illustrate that early progenitor cell populations are not programmed to exclude junctional modifications from Vγ3-Jγ1 joins.     

γδ cells involved in contact sensitivity preferentially rearrange the Vγ3 region and require interleukin-7

Ptak and Askenase showed that both αβ and γδ cells are required for transfer of contact sensitivity (CS). This study confirms that day 4 immune cells depleted of γδ cells fail to transfer CS to trinitrochlorobenzene (TNP-Cl) systemically and demonstrates that administration of anti-γδ monoclonal antibodies (mAb) in vivo abolishes the CS reaction. Moreover, γδ cells accumulate at the antigen challenge site: these cells have the unusual phenotype CD8α⁺, CD8β^-, IL-4 R⁺ which we suggest is due to their state of activation. Following immunization with contact sensitizer on the skin, the absolute number of γδ cells increases in the regional lymph nodes with a peak at 4 days. Of the γδ cells, 80%, both in the lymph nodes of TNP-Cl-immune mice and accumulating at the antigen challenge site are Vγ3⁺. The γδ cells expressing Vγ3, which is characteristic of dendritic epithelial T cells (DETC), obtained 4 days after sensitization, proliferate in response to interleukin (IL)-7, but only poorly to IL-2 and IL-4. They also respond to concanavalin A and immobilized anti-γδ mAb, but not to haptens or heat-shocked syngeneic spleen cells. Furthermore, injection of mice with mAb to IL-7 inhibits accumulation of Vγ3⁺ cells both in the lymph nodes after skin sensitization and at the antigen-challenge site. Altogether, these results strongly support the view that DETC are related to, or the original source of, the γδ cells found in the lymph node after skin sensitization and at the site of challenge, and that IL-7 is implicated in these phenomena.     

Hypoxia Increases Serum Amyloid A3 (SAA3) in Differentiated 3T3-L1 Adipocytes

Hypoxia has been implicated as a possible cause of adipose tissue inflammation. Furthermore, the acute phase protein serum amyloid A (SAA) has been associated with the modulation of the adipogenic process, and it is well-known that obese individuals have increased levels of SAA. The effect of hypoxia in the expression and production of SAA was examined in murine 3T3-L1 adipocytes. Hypoxia leads to a substantial increase in SAA3 mRNA and protein level, apparently in a time-dependent manner (threefold in 48 h), in fully differentiated 3T3-L1, followed by reestablishment of gene expression to basal levels after 24 h of reoxygenation. Hypoxia-induced SAA may be one of the key molecules to the development of the inflammatory response in adipose tissue.     

γδ cells regulate autoimmunity

γδ cells are attractive candidates for mediators of autoimmune disease. They can expand in germ-free mice, probably through recognition of autoantigens, and γδ-cell-deficient mice, unlike mice deficient in αβ T cells or B cells, show no severe defects in the immune response to foreign antigen challenge. A capacity of γδ cells to effect or regulate tissue damage is also plausible, given their ready localization to tissues, and their myriad of effector functions. Added to this, attempts to reconstruct the physiological course of autoimmune diseases with only autoreactive αβ T cells seem invariably to fall short for lack of other unidentified players. γδ cells and their putative ligands have been linked to autoimmune conditions, and recent experiments confirm that γδ cells play a significant role in autoimmune disease in vivo.