| Laryngeal carcinoma is a common ENT malignant tumor with very high morbidityrate, which accounts for 1% to 2% of the human tumors and 11% to 22 % of ENTmalignant tumors in China. Surgery and radiotherapy are often adopted for the laryngealcarcinoma in early stage. In the cases of middle and advanced stage, surgery is used asthe predominant therapy of comprehensive treatment. Since 1980s the treatment of thelaryngeal carcinoma has made rapid progress. Operation, radiotherapy, chemotherapyand the application of the immunization therapy have greatly raised the survival rate oflaryngeal carcinoma patients by five years. However, those therapies can cause high rateof disability and complications as well as many serious toxic and side effects.Recurrence and metastasis are still the major death cause of laryngeal carcinoma. Itseems that the treatment with the aim of raising the survival rate beyond 5 years hascome to a bottleneck stage, hard to make any breakthrough progress. Therefore, seekingfor new simple and effective therapy with little side effects has become the researchfocus for ENT doctors. Especially when the tumor is at an early stage or the tumor issmall after the treatment, the proper treatment to sub-clinical tumors is the key to avoidthe recurrence and metastasis of malignant tumors and the key to raise the survival ratebeyond 5 years. The treatment to the sub-clinical tumors after surgery and radiotherapy is a keystep that affects the prognosis of malignant tumors and is a hot topic for research in thisyear. Biotherapy is a new therapy by applying modem biological technique and itsproducts to tumor treatment, which achieves anti-tumor effect by activating the innateprevention mechanism of the host or by depending on the material with high pertinencyand strong targeting characteristic that are produced naturally or through geneticengineering. With the deepening into the research of the molecular mechanism oftumors and the development of bio-tech, biotherapy has become the fourth mode ofcomprehensive therapy of tumors. Biotherapy of malignant tumors mainly includesgene therapy, vaccination therapy and biological responses modifier therapy, amongwhich gene therapy is a direct and effective therapy which not only can reverse themalignant phenotype of malignant tumor cells, inhibit the tumor growth, but also canrepair damaged cells and get rid of the initiative factors of mutation. Significantprogress has been made in clinical therapy. However, traditional tumor gene therapy hasthe shortcoming of low transfection efficiency, poor targeting, and low anti-oncogeneexpression.To solve the above problems, RNAi technique has been applied to experimentalstudies of malignant tumor gene therapy. RNAi technique is based on the alkali repairprinciple, which makes use of the short piece of RNA (siRNA) to combine to targetgene's mRNA to silence or terminate the expression of oncogene, making the tumor cellto divide toward maturity or inducing the cell apoptosis, so as to achieve the aim oftreatment. RNAi aims at changing tumor-control genes at nucleic acids level, which ismore specific and has less side effects and promises to become a new way for genetherapy.Survivin is a new member of the newly-discovered IAP family, which is the mostpowerful inhibitor of apoptosis identified so far. It has unique characteristic andstructure different from other members of IAP family. It is not expressed in the tissuesof normal adults but is selectively expressed in the tissue of malignant tumors and isclosely related to the proliferation and the infiltration and metastasis of tumor cells. This unique expression characteristic and its close relation with the development ofmalignant tumors make it become a new target for the diagnosis and treatment ofmalignant tumors.Objectives:1. To find the expression of survivin in laryngeal carcinoma and its relation with thegrowth and metastasis of laryngeal carcinoma.2. To observe the growth of laryngeal cancer cells after the survivin has been knockedout by RNAi technique.3. To study the effect of RNAi on living laryngeal carcinoma cells.4. To study the safety of the application of RNAi on living body.Design of the experiment:Detect the clinical specimens of laryngeal squamous cell carcinoma byimmunohistochemical method. Analyze the relationship between the expression ofsurvivin and other apoptosis-related genes and the clinical characteristics of laryngealcarcinoma so as to study the roles they play in the growth and development of laryngealcarcinoma. Establish siRNA with survivin as target gene and plasmid as carrier, whichis imbed by lipidosome and transinfected with laryngeal carcinoma so as to observe thegrowth of cells and the survivin expression and the apoptosis. Establish Hep-2 nudemice model and admistrate medicine through veins. Observe the growth of the tumorand the survivin expression and apoptosis.Methods:1. The expression of survivin caspase-3, bcl-2, bax and p53 in human laryngealsquamous cell carcinomas was detected by immunohistochemical method.2. The expression of survinvi protein in Hep-2 cells of laryngeal carcinoma wasobserved through cell immunocytochemical mehthod.3. Western-blotting method was used to examine the influence of survivin siRNAtransfection on the survivin protein level in Hep-2 cells.4. RT-PCR method was employed to examine the influence of survivin siRNA on theexpression of survivin mRNA at different concentration and after different time duration.5. MTT method was adopted to investigate the proliferation of the Hep-2 cells after thetransfection of survivin siRNA and after the treatment with survivin siRNA andpaclitaxel at different concentration.6. Cell cycle was detected by flow cytometry in the control group and the treatmentgroups after Hep-2 cells were interfered by paclitaxel, siRNA and paclitaxel +siRNArespectively.7. After the nude mice tumor model was formed, 15 BALB/c 4-6-week-old female nudemice were randomly distributed to A,B,C group after Hep-2 had been injected into theback side of their front legs. The three groups were treated with PBS, paclitaxel(5μg/ml), and paclitaxel + siRNA(50nm/L,) respectively. 200μl of PBS, paclitaxel andpaclitaxel + siRNA were transfused into the mice's abdominal cavity every 3 days for15 days. The mice were kept in pathogen-free environment and checked every day.8. Immunohistochemical technique was used to examine the expression of survivinprotein in nude mice tumor tissue in control group and treatment group.9. TUNEL was used to detect the apoptosis cells in control group and treatment group.10. After the treatment, the nude mice in each group were checked for any changes intheir appetite, state, blood routine, and liver and kidney function. The heart, liver, spleen,kidney and other major organs were examined under light microscopes so as to look forany signs of poisonous and side effects after treatment.11. Statistical software of SPSS13.0 and sas9.0 were applied to the statistical analysis.x~2 test and Fisher test and Spearman correlation analysis were used to analyze thecategorical variables and numerical variables were expressed in form of ((?)±s).Single factor variance analysis (ANOVA) was used for mean comparison amongmultiple samples and t-test was used for mean comparison between two smaples. Thesignificant level was P=0.05.Results:1. The immunohistochemical results showed: that the expression of survivn, bcl—2, and p53 in were signicantly elevated in cancer group, while the expression of caspase—3and bax markedly declined in cancer group. The abnormal expression was closelyrelated to laryngeal squamous cell carcinomas and its tumor metastasis phenotype.2. The immunocytochemistry result exhibited that survivin showed high expression inHep-2 cells.3. The western-blotting analysis of transfected Hep-2 cells after 24h, 48h and 72hmanifested that the level of survivin protein was delined with time by survivin siRNA.4 RT-PCR results showed survivin siRNA of different concentration and afterdifferent time duration all exert some effect on mRNA.. With the prolonging of time andthe increase of concentration, the expression of survivin mRNA gradually decreased.5 MTT showed that there was significant difference (P＜0.05) of the inhibition of SDHenzyme activity in Hep-2 cell after survivin siRNA transfection between the controlgroup and the treatment group at different concentration and after different timeduration. After the use ofpaclitaxel, the inhibiting effect on SDH activity of Hep-2 cellsbecame more significant (P＜0.01), which indicated that the two had a synergisticeffect6 48hs after the cells were treated with paclitaxel, siRNA, and paclitaxel+siRNArespectively, the percentage of S stage ceils in each group were in the follwing order:contrast group, paclitaxel group and paclitaxel+siRNA group. The declining trend wassignificant (P＜0.05), and the effect of paclitaxel+siRNA group was most significant.7 Flow cytometer was used to examine the apoptosis in control group and the treatmentgroups. The proportion of apoptosis between the contrast group and the treatment groupswas significantly different and the differences among the treatment groups were alsosignificant. (P＜0.05).8. Before the treatment, the nude mice weight and the tumor size in the control group,paclitaxel group, siRNA group and paclitaxel+siRNA group were of uniformity. Afterthe treatment, significant difference was found between the control group and othergroups (p＜0.05). The nude mice tumor size in each group increased gradually. Thetumor of the control group grew limitlessly. Statistical analysis showed that the tumor size in each treatment group became significantly smaller than that of control groupafter the treatment (p＜0.05). Compared with other treatment groups, the shrinking ofthe tumor size in paclitaxel+siRNA group was most significant (p＜0.05).9. Immunohistochemical results: of nude mice tumor tissues showed high expression ofsurvivin protein in the control group and significant weak expression of survivin proteinin the treatment groups (p＜0.05). The weakening of the expression of survivin proteinwas most significant in paclitaxel+siRNA group (P＜0.05).10. The tumor tissue were made to slice and were detected by TUNEL. A great deal ofpositive cells could be found in the tissue, while few positive cells in the control tumortissue. There were significant difference among the groups (P＜0.05)11. In terms of the poisonous and side effects after the treatment, it was found that thenude mice had normal appetite and were in good state. No significant difference wasfound about the blood routine and liver and kidney functions. No abmormal changeswas noticed about the nude mice's heart, liver, spleen, kidney and other major organsunder light microscope.Conclusion:1. In the human tissue of laryngeal squamous cell carcinomas, the expression ofsurvivin, bcl-2 and p53 increases, while the expression of caspase-3 and bax dereases.The abnormal expression is closely related to laryngeal squamous cell carcinomas andits tumor metastasis phenotype.2. Survivn siRNA has significant inhibition effects on the proliferation of the Hep-2cells and expression of survivn protein and m RNA and can induce apoptosis.3. Nude mice neoplasm model of Hep-2 cells were set up in this experiment. It isconfirmed that survivn siRNA can promote the apoptosis of tumor cells and inhibit thetumor growth in nude mice.4. When survivin siRNA was used in combination with paclitaxel, the two had asynergistic effect, acting jointly on the microtubule system which plays an importantrole in the development of cells towards proliferation, so as to inhibit the proliferation of cells more completely and make them develop towards apoptosis.5. When survivin siRNA was used to mice, no evident side and toxic effects was noticed.Nomal grwoth was not affected and no harm was done to vital organs.
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