The Research Of Transgene Therapy For Salivary Adenoid Cystic Carcinoma

Traditional non-invasive therapy for tumor can kill tumor cells as well as normal cells at the same time due to lack of tumor-specific effect, and often lead to severe side effects. Gene therapy is a new treatment for tumors following traditional therapy such as chemo-therapy, radio-therapy and surgical treatment etc. The gene therapy means to modify tumor cells or immunity effect cells in vitro, and then transfuse these cells back to patients. Now there is increasing interests in this field. Many tumors including salivary tumors have been tested extensively with various gene therapies, among which resuming tumor cell apoptotic procedure is a promising strategy for tumor gene therapy.The mammal salivary glands (parotid gland and submandibular gland) become a excellent gene therapy target because of its anatomy characteristics:①through the orifice of salivary duct in mouth, we can inject all sorts of gene to salivary glands directly without trauma;②acinic cell and duct cell are monolayer ,which greatly increases the opportunity to touch gene transfer vector directly and promote transgenic expression efficiency;③there is intact envelope for human salivary glands, which can restrict vector diffusion so as to avoid side effects to other tissues;④Salivary glands are secretive and produce abundant proteins, which function as exocrine and endocrine. Nevertheless, finding targeting gene transfer vector without side effects is still an important goal for salivary gene therapy. We do many endeavors for it.Salivary cystic carcinoma(ACC)is a common salivary malignant tumor,which infiltrates facial nerve and metatasis earlier and often leads to obvious maxillofacial malformation after operation. It also reoccurs easily and is not sensitive to radio–therapy or chemo-therapy, so has a deep effect to patient's life. p16 and p21 are cell cycle correlated genes, which also the key points of cell cycles. p16 protein can suppress the CDK4, which in turn suppress the growth of cancer cells. But p21can bind with CDK4 and CDK6, low p21 protein can active the CDK to process the cell cycle, promote G1 stage to S stage. But high p21 protein can bind CDK competing against with cyclin, so suppress the activity of CDK and block cell cycle in G1,which induce the suppress of cell proliferation. mda-7/IL-24 is a new gene ,which suppress the cell proliferation independent p53,Rb and p16,which can induce the apoptosis of cancer cells but has no effect of normal cells.mda-7/IL-24 can also enhance the effect of radio-therapy or chemo-therapy. In this study, we took SACC as the target to test the gene therapy for it. This article is mainly divided into three parts: in the first part, we adopt the immunohistochemistry technique to detect the expression of p16 and p21 WAF1 protein at the salivary adenoid cystic carcinoma tissues. The result showed that p16 and the p21 WAF1 protein are low expression or have no expression in the salivary adenoid cystic carcinoma but high expression in normal salivary tissue. It hints that p16 and the p21 WAF1 possibly be the therapy target of salivary adenoid cystic carcinoma. In the second part we construct the adenovirus vectors mediated p16 and p21 gene to transfect SACC-83 cells, with the experimental control of transfected p16 or p21 alone, with nothing in the blank control. The result obviously represses that combined p16 and p21 gene can suppress the growth of SACC-83, but have no statistics learn meaning compare with the group transfected p16 alone. p21 has little suppressed effect of salivarys adenoid cystic carcinoma cells. The cell cycle examination discovers that G0-G1 cells are higher of the combined group,the group tranfected p16 alone and the group transfected p21 alone.It hints that p16 or p21 suppress SACC-83 cells by G1 stage block. The cells of combined group and the group transfected p16 alone are obviously apoptosis, but the group transfected p21 is not. It indicate that the p21 can only block the SACC-83 cells by G1 stage block but has no apoptosis. In the third part, we constructed the AdLTR2-EF1α-mda-7/IL-24 with the mda-7/ IL-24 gene which can induce apoptosis in cancer cells but has no effect in normal cells. Using the vector,we transfected mouse fibroblast L-929 and SACC-83 ,the RT-PCR can examine the gene expression of mda-7/IL-24 , The MTT assay shows that the mda-7/IL-24 group has no difference with the control in L-929, but has obviously different in SACC-83 cells. The soft agar assay also show the suppress effect of mda-7/IL-24. Especially the supernatant fluid of the mda-7/IL-24 trafected L-929 can also suppress the growth of SACC-83. The flow cytometry examination confirms mda-7/IL-24 can induce the salivary adenoid cystic carcinoma cells apoptosis. So these experiment results establish the base for our next experiment of these genes to animal research in vivo. Conclusions:1. The adenovirus can get high transgenic efficiency, which is suitable virus for transfection and the suitable titre has little influence on the cells. So it could be the gene vector safely.2. Immunohistochemistry detection confirmed that p16 and p21WAF1 have a weak expression or no expression in salivary adenoid cystic carcinoma.3. The expression of p16,p21 and mda-7/ IL-24 could be found in SACC-83 cells after transfected by adenovirus vectors.4. The combined transfection of p16 and p 21 can obviously suppress the growth of salivary adenoid cystic carcinoma cells. P21 can induce the cells blockage in G1 but not cause cells'apoptosis. The mda-7/ IL-24 can suppress the proliferation of SACC-83 but has little effect on L-929 cells. It can also induce the apoptosis of SACC-83 cells, and can suppress the proliferation of SACC as a secreting protein.