利用反义RNA探索外源基因丢失的分子机理

利用反义ＲＮＡ技术研究了调控ＰＡＲＰ酶基因的表达对外源基因整合稳定性的影响。将ＰＡＲＰ基因ｃＤＮＡ的部分序列反向插入到真核表达载体ｐＳＭＧ中,将重组质粒分别导入携带有外源基因的细胞中,地塞米松诱导反义ＰＡＲＰ基因的表达后,进行Ｓｏｕｔｈｅｒｎ杂交检测。结果表明,外源基因仍保留在基因组中,这意味着外源基因的丢失并不是由于单一ＰＡＲＰ酶活性降低所致。     

A trans-acting class II regulatory gene unlinked to the MHC controls expression of HLA class II genes

Class II (or Ia) antigens are highly polymorphic surface molecules which are essential for the cellular interactions involved in the immune response. In man, these antigens are encoded by a complex multigene family which is located in the major histocompatibility complex (MHC) and which comprises up to 12 distinct alpha- and beta-chain genes, coding for the HLA-DR, -DQ and -DP antigens. One form of congenital severe combined immunodeficiency (SCID) in man, which is generally lethal, is characterized by an absence of HLA-DR histocompatibility antigens on peripheral blood lymphocytes (HLA class II-deficient SCID). In these patients, as reported here, we have observed an absence of messenger RNA for the alpha- and beta-chains of HLA-DR, -DQ and -DP, indicating a global defect in the expression of all class II genes. Moreover, the lack of expression of HLA class II mRNAs could not be corrected by gamma-interferon, an inducer of class II gene expression in normal cells. Family studies have established that the genetic defect does not segregate with the MHC. We conclude, therefore, that the expression of the entire family of class II genes is normally controlled by a trans-acting class II regulatory gene which is unlinked to the MHC and which is affected in the patients. This gene controls a function or a product necessary for the action of gamma-interferon on class II genes.     

HLA class II induction in human islet cells by interferon-gamma plus tumour necrosis factor or lymphotoxin

HLA class II molecules are surface glycoproteins which are essential in the initiation of immune responses. It has been postulated that induction of class II in epithelial cells such as endocrine cells, which are normally class II negative, may result in autoimmunity. In type I diabetes, islet beta cells, the target of the autoimmune process, selectively express class II antigens. But in contrast to most other cell types, islet beta cells are not stimulated to express class II by interferon-gamma (IFN-gamma) and thus the conditions under which this induction occurs have been particularly elusive. The cytotoxins tumour necrosis factor (TNF) and lymphotoxin (LT) synergize with IFN-gamma in a number of activities. We report here that IFN-gamma in combination with either TNF or LT induces islet cell class II expression. This finding has important implications for the pathogenesis of type I diabetes and the understanding of the differential control of class II expression.     

Effects of antisense CENP-B on the proliferation of HeLa (Tet-off) cells

In order to study the change of the expression of centromere protein B (CENP-B) caused by antisense transfection, proto-eukaryotically expressed fused protein GST-CENP-B (65 ku) was injected into mouse, and a peculiar anti-CENP-B serum MaCenpB was collected. A strain of transfected HeLa Tet-off cell HaCb, which contains antisense CENP-B expressing vector pBI-EGFP-as-CenpB, was prepared. Northern blot and Western blot were used to analyze the repression of internal CENP-B in transfected cells. According to the growth curve, the proliferation of HeLa (Tet-off) is repressed by antisense CENP-B, and the multiplication time is prolonged for 32.81 h. The analysis of flow cytometry revealed that, compared with HeLa (Tet-off), the G₁ cell population of HaCb is increased (ΔG₁= 9%) while S fraction is decreased (ΔS = 11%), but the G₂/M phase is nearly unchanged (ΔG₂/M=3%). In the meanwhile, the mitotic index of HaCb declines greatly compared with that of HeLa (Tet-off). Immunofluorescence showed that the assembling of centromeres in HaCb cell is arrested. These results suggest that a normal expression of CENP-B may be necessary for cell proliferation.     

Specific recognition of staphylococcal enterotoxin A by human T cells bearing receptors with the V gamma 9 region

T cells bearing the alpha beta receptor can specifically react with target cells coated with staphylococcal enterotoxin and expressing major histocompatibility complex class II molecules; these responses depend on which variable region (V) of the receptor's beta-subunit is used. We have now examined whether a similar situation exists for human T cells bearing the gamma delta receptor. We found that reactivity to staphylococcal enterotoxin A is strictly dependent on the presence of the V gamma 9 variable region in the gamma delta T-cell receptor (TCR). These cytotoxic responses required the expression of HLA class II molecules by the target cell and could be inhibited by anti-gamma delta TCR and by anti-HLA-class-II monoclonal antibodies. In contrast to alpha beta TCR+ cell clones, no proliferative response of V gamma 9+ T-cell clones towards stimulator cells coated with enterotoxin A was observed in vitro. These results indicate that the gamma delta TCR repertoire might be influenced by enterotoxin A produced during staphylococcal infections in vivo. This could provide a molecular basis for the observation that V gamma 9+ T cells form the large majority of peripheral gamma delta TCR+ cells but only a small proportion of thymic gamma delta TCR+ cells.     

Effects of antisense CENP-Bon the proliferation of HeLa (Tet-off) cells

In order to study the change of the expression of centromere protein B (CENP-B) caused by anfisense tcansfection, proto-eukaryotically expressed fused protein GST-CENP-B (65 ku) was injected into mouse, and a peculiar anti-CENP-B serum MaCenpB was collected. A strain of transfected HeLa Tet-off cell HaCb, which contains antisense CENP-B expressing vector pBI-EGFP-as-CenpB, was prepared. Northern blot and Western blot were used to analyze the repression of internal CENP-B in transfected cells. According to the growth curve, the proliferation of HeLa (Tet-off) is repressed by antisense CENP-B, and the multiplication time is prolonged for 32.81 h. The analysis of flow cytometry revealed that, compared with HeLa (Tet-off), the G1 cell population of HaCb is increased (△G1 = 9%) while S fraction is decreased (△S = 11%), but the G2/M phase is nearly unchanged (△G2/M=3%). In the meanwhile, the mitotic index of HaCb declines greatly compared with that of HeLa (Tet-off). Immunofluorescence showed that the assembling of centromeres in HaCb cell is arrested. These results suggest that a normal expression of CENP-B may be necessary for cell proliferation.     

Functional expression of HLA-DP genes transfected into mouse fibroblasts

The HLA class II antigens are a highly polymorphic family of dimeric cell-surface glycoproteins, expressed predominantly on the surface of immunocompetent cells. They are intimately involved with the induction of the T-cell response to extrinsic antigen and are important predisposing factors for a wide spectrum of autoimmune diseases. We describe here the expression of a class II product from the HLA-DP (new WHO nomenclature, formerly SB) subregion after transfer of cloned genes into mouse fibroblasts. The transfected DP antigen is recognized by several HLA class II monoclonal antibodies and, though present in a mouse cell background, is able to function in the presentation of influenza antigen to cloned DP-restricted human T lymphocytes.     

肝癌相关基因MEF2D反义RNA的鉴定及分析

鉴定和分析肝癌细胞中新的天然反义RNA MEF2D-AS根据已建立的双链RNA文库筛选与肝癌相关的反义RNA.通过RT-qPCR验证其反义RNA的存在,采用cDNA末端快速扩增技术获得其全长序列,并检测HepG2细胞经5-Aza-dC处理前后MEF2D基因正反义链表达量的变化.结果显示,成功鉴定出1条全长为1 265bp的新的天然反义RNA,并预测其属于长链非编码RNA(long non-coding RNA,简称为lncRNA),命名为MEF2D-AS.经5-Aza-dC处理后,MEF2D-AS的表达量升高,而MEF2DmRNA的表达量降低.证实了MEF2D-AS的存在并预测其属于长链非编码RNA,且与MEF2D正反义RNA是反相关关系.此研究结果可为进一步探讨肝癌发生机制提供参考.     

Effect of PKC pathway on G1/S progression control in HeLa cells

The effect of PKC activity on G1/S progression in HeLa cells has been studied.The result shows that (ⅰ) PKC activity alteration in G1 phase affects G1/S progression in HeLa cells.It has been observed that G1/S progression is stimulated by PKC agonist TPA and inhibited by PKC inhibitor GF-109203X.(ⅱ) The expression of c-myc and c-jun is stimulated by TPA and inhibited by GF-109203X treatment in early G1 phase.(ⅲ) During G1/S progression,the expression of CyclinD1 is stimulated by TPA treatment and inhibited by GF-109203X treatment.There is no effect on the expression of CDK4.It is likely that PKC pathway regulates G1/S progression through regulating the expression of some early response genes and engine molecules in HeLa cells.     

反义PARP基因真核表达重组体的构建及其HeLa转化细胞系的建立

利用DNA体外重组技术将PARP基因cDNA部分序列反向克隆到真核表达载体pMAMneo上,构建成重组质粒pMAMneo－C1．4和pMAMneo－C0．3．将重组质粒pMAM－neo－C1．4转染HeLa细胞,经G418筛选,建成细胞系HeLa－C1．4－neo,Southern杂交结果表明,外源PARP基因cDNA部分序列已稳定整合到受体细胞基因组中,该细胞系的建立为研究聚ADP核糖基化作用在细胞内的功能打下了基础．     

Sequence polymorphism of HLA DR beta 1 alleles relating to T-cell-recognized determinants

HLA class II molecules are a highly polymorphic family of dimeric cell-surface proteins primarily involved in regulating T-cell responses to extrinsic antigens. To define regions of class II molecules involved in T-cell recognition, we have now compared sequences of three HLA DR beta cDNA clones obtained from cells that all express the same serologically defined determinants but differ in terms of T-cell-recognized specificities. The comparisons indicate that very few (one to four) nucleotides differ between what are almost certainly alleles of the DR beta 1 locus. All differences were in the first domain of the molecule and all localized to a region from amino acids 71-86. Because all differences were found only in this region of the molecule, and because DR alpha-chains seem to be relatively non-polymorphic, these positions in the DR beta-chain must have a major role in influencing T-cell recognition of the DR molecule.     

Effect of PKC pathway on G1/S progression control in HeLa cells

The effect of PKC activity on G₁/S progression in HeLa cells has been studied. The result shows that (ⅰ ) PKC activity alteration in G₁ phase affects G₁/S progression in HeLa cells. It has been observed that G₁/S progression is stimulated by PKC agonist TPA and inhibited by PKC inhibitor GF-109203X. ( ⅱ) The expression of c-myc and c-jun is stimulated by TPA and inhibited by GF-109203X treatment in early G₁ phase. (ⅲ ) During G₁/S progression, the expression of CyclinD₁ is stimulated by TPA treatment and inhibited by GF-109203X treatment. There is no effect on the expression of CDK4. It is likely that PKC pathway regulates G₁/S progression through regulating the expression of some early response genes and engine molecules in HeLa cells.     

Effects of STAT3 antisense RNA on IL-6-induced differentiation and growth arrest of Ml leukemia cells

To investigate the biological roles of STAT3 in the regulation of growth and differentiation of leukemia cells, we modified a murine myeloid leukemia cell line Ml with STAT3 antisense RNA. The effects of STAT3 antisense RNA on the growth arrest and terminal differentiation of Ml cells induced by interleukin 6 (IL-6) were determined. It was found that STAT3 antisense RNA blocked the activation of STAT3, and reduced the growth arrest and terminal differentiation of IL-6-induced Ml leukemia cells. These results indicate that STAT3 activation is a necessary process for IL-6-induced growth arrest of Ml cells and for the differentiation of Ml cells into macrophage.     

低表达长非编码RNA MALAT1对人宫颈癌HeLa细胞体外生长和迁移的影响

设计3个针对MALAT1不同靶序列的小干扰RNA(small interfering RNA, siRNA), 筛选出有效的siRNA序列, 设计并构建短发夹RNA(short hairpin RNA, shRNA)干扰质粒, 转染到HeLa细胞中, 构建低表达MALAT1的稳定细胞株.通过细胞的生长曲线和细胞划痕实验验证降低MALAT1对HeLa细胞增殖和迁移的影响.结果显示MALAT1表达水平在HeLa细胞中得到有效地降低, 低表达MALAT1的HeLa细胞生长速度和迁移速度减小.表明MALAT1在HeLa细胞中具有调控细胞增殖和迁移的能力