Study Of ATRA Enhancing The Bystander Effect Of HSV-TK/GCV Gene System On Bladder Cancer

Background: Bladder cancer is one of the most common malignant in urinal system. The curative effect is reduced for the high recur rate after surgery and unsensitivity to the chemical therapy. It is one of the most important strategies of gene therapies to transfect HSV-TK into urinal bladder cancers at present, which shows preferable research value and clinical applying foreground. Many studies have confirmed that the "bystander effect", that the positive-affected cells can induce the negative-affected cells apoptosis is an important approach. It and the efficiency of transfection are regarded as the two key elements of successful suicide gene therapy. More and more attention was paid to the bystander effect for the efficiency of transfection is not satisfactory at present. It is significant to probe into how to improve the bystander effect. Gap junction intercellular communication is an important mechanism of bystander effect. The connect channel that formed by the connexon between the adjacent cells is the basic construction of GJIC. The expression of tissue special connexins is abnormal, and the GJIC function is reduced in many tumors. These can explain why the bystander effect of suicide gene is not very evident in many tumor therapies. Many studies overseas have approved that the connexin26 is an important connectin in the transitional epithelium cells of the urinal tracts. It acts as an important role on controlling the proliferation and differentiation of cells. Abnormal expression of Cx26 is related to the tumors accruing and development of bladder cancer. It is reasonable to enhance the bystander effect of HSV-TK gene therapy by increasing the expression of connexins or improving the function of GJIC.All trans retinoic acid (ATRA) is manual ramification of Vitimin A, which is a good modulation medium of the GJIC function and performs different effect on different organ cells. It is rarely reported whether the ATRA can modulate the GJIC function of bladder cancer cells up to now.Object of the experiment: 1,We are going to study the expression of Cx26 in bladder transitional cell cancers and its biological meaning. 2,To observe if ATRA can enhance the bystander effect of HSV-TK/GCV on BIU-87 cells in vitro. And discuss whether the mechanism is associated to the role of improving the expression of Cx26 and the function of GJIC. We offered the theory evidence of combining using ATRA and HSV-TK/GCV gene system. The methods and the results:1,We evaluated the expression of Cx26 by immunohistochemistry in 15 normal bladder tissues, 35 transitional cell cancer tissues and the BIU-87 cell line . The expression of Cx26 was intensive in transitional epithelium cells in normal tissues. It was clearly located on the plasma membrane, but faint or negative in 68.3% of the cancer tissues. It was abnormally located in cytoplasm or nucleolus in 5 cancer specimens. The expression of Cx26 was reduced in early highly differentiated bladder cancers, and was related to the malignant type. Cx26 is hardly expressed in BIU-87 cells. At the same time, the mRNA of 6 fresh normal epithelium tissues and 6 fresh bladder cancers were evaluated by semi-quantity- RT-PCR, we fount that the mRNA of Cx26 was decreased in tumors(p<0.05).2,We observed the growth of BIU-87 cells after ATRA therapy by MTT in vitro.We evaluated the GJIC of cells by scrap loading dye transfer technology, and observed the expression of Cx26 protein by immunocytochemistry and the level of its mRNA by semi-quantity-RT-PCR in BIU-87 cells after ATRA therapy. We found that the ATRA can not only restrain the growth of BIU-87 cells and induce its apoptosis but also improve the GJIC function and increase the expression of Cx26 .The effect was related to the concentration of ATRA.3,We evaluated the sensitivity of BIU-87 cells to the HSV-TK/GCV after ATRA therapy, and evaluated whether ATRA can enhance the bystander effect of HSV-TK gene. The BIU-87-TK(+) cells and the BIU-87-TK(-) cells were cocultivated by different rations in different groups...