Empirical Study On Anti-tumor Peptide Of Tumstatin Treating Laryngeal Squamous Cell Carcinoma

IntroductionLaryngocarcinoma is one of common pate malignant tumor, nowadays, the morbility of laryngeal carcinoma has a tendency to grow obviously.There is great territory different in the morbility of laryngocarcinoma. The area of the maxlmum morbility is in the northeast of our contury, 5.7-7.6 percent of the sys. Carcinoma is confined to laryngeal, 7.9-35 percent of the otorhinolaryngeal carcinoma is confined to laryngeal. It is important to make sure the mechanisms of laryngeal carcinoma to help early diagnosis and treatment that can improve the quality of life and survival rate.As same as the other tumors, invasion of laryngocarcinoma is the patho course relating to multitude factors and many steps. At present, the most common treatment curing the laryngocarcinoma is operation, aimed by orthodox radiotherapy and chemotherapy. Nevertheless, this therapeutic efficacy is not good yet, especially, it affects patient existence quality.The angiogenesis are important in the formation, infiltration and displace of tumor, the tumor whose volume exceed 2-3mm³ need new vessels which can maintain nourishment supply and excrete metabolites, and also can provide transterred tunnel. Restraining new vessels make tumor cell rest and induce Apoptosis. Consequently the biological treatment of tumor which take the new vessels as target has become investigate hot spot. Tumstatin is a polypeptide fragment of NC1structural domain in typeâ…£collagenα3chain, which is composed of 244 pieces amino-acid residue, which relative molecular weight is 28KDa. Tumstatin was discovered as goodpasture antigen in 1980's.By 2000,Kamphaus found that tumstatin had two distinct anti-tumor domains.One was 19peptide composed of 185～203 amino acid. 19peptide had direct anti-tumor activity by inhibiting tumor cell proliferation and promoting tumor cell apoptosis.The other was T7 peptide including 74～98 amino acid.T7 peptide had indirect anti-tumor activity though anti-angiogenesis. Tumstatin is the fortest antiangiogenesis factor in present, its effect is more than 10 times as endodermis chalone. Its mechanism of action refrain FAK,pI3 kinase,PKB/AK+ activity by combiningαvβ3, induce integrin mediated CAP dependence protein synthetic route to be locked and refrain endotheliocyte growth selectively, so it has strongly anti-angiogenesis role. Therefore, treating tumour with tumstatin has become study hot all over the world.Tumstatin is hydrone protein, the price is of direct biosynthesis is too expensive, which is disadvantage of large scale application in clinic. Otherwise, it is not reported in the world if tumstatin can cure laryngeal carcinoma. The study aims at approaching the optimum condition that utilize bionic way to obtain tumstatin function peptide, furthermore definite its therapeutic action for larynx SC and possible mechanisms. This establishs rationale for clinical application of tumstatin.Methods1,Cleansing and Detecting 19 peptide of tumstatin: 19 peptide genetic engineering express bacterium are cultivated in containing acillin LB medium, A₆₀₀=0.5, adding IPTG induce express, collect thallus precipitate, weighing. Repeating freeze thawing and supersonic wave comminution to split thalline. Making use of proteinum cleating-affinity chromatography clean up 19 peptide. Bradford way measure 19 peptide protein concentration, high performance liquid phase method survey 19 peptide purity.2,MTT shade selection experiment detects level which different density 19 peptide restrain Hep-2 growth.3,Drawing auxodrome by Tiphulone method detects level which different time 19 peptide restrain Hep-2 growth.4,DNA ladder measure Hep-2 apoptofic information.5,Using electron microscope observe 19 peptide affected cell morphologic change. 6,Using apoptosis experiment (TUENL) measure apoptosis.7,Using flow cytometry measure cell aspection.8,Westem Blot detection: measure Bcl-2, Bax, cytopigment C and caspase-3 P20 subunit express.9,Raising athymic mouse, growing laryngocarcinoma Hep2 cell and making it growth, copying hairless mouse animal model of laryngocarcinoma.10,Animal experiment: the experimentation are proinjected with tumstatin around tumor everyday, the control group are injected with PBS. At treatment course these hairless mouse contine be raised till protocol time at lamina flow. In the treatment course, it is necessary that observing each hairless mouse mental state, activity, drink and eat, measuring those weight and tumor volume change.11,After putting hairless mouse to death, striping tumor, measuring net volume and weight. We take piece of them at-70℃refrigerator, and the other is made paraffin block forpre-emergency.12,Observing tumor fine structure change by electron microscope. Detecting tumor vessel growth by immunity.Result1,Cleansing and Detecting 19 peptide of tumstatin: The express weigh of 19 peptide fusion protein can reach to maximum weigh at temperature 28, IPTG density is 0.1～1.0mmol/L, guide time is within 6 hours. The density of 19 peptide is 0.26～0.44μg/μl. By high performance analysis of liquid phase, the 19 peptide purity of extracted depurant exceed percent 90.2,MTT shade selection experiment: Tumstatin can obviously depress Hep-2 cell of laryngocarcinoma growth, as adding density of tumstatin the persistence of laryngocarcinoma Hep-2 cell is lower.3,Growth curve: Tumstatin can obviously depress Hep-2 cell of laryngocarcinoma growth, living cell is taper as extending time.4,Hep-2 cell affected by tumstafin assume apoptosis on electron microscope.5,DNA ladder: The laryngocarcinoma Hep-2 cell take on typically step shape degrdn stripe band in the treatment group. 6,Apoptosis experiment: The laryngocarcinoma Hep-2 cell appear generous apoptosis after affected by tumstatin.7,Cell cycle measure: Compared with the control group, cell in G0-G1, G2-M stage obviously mult the treatment group, this explain that 19peptide can inhibit tumor cell proliferation and promot tumor cell apoptosis.8,Chick embryo allantocherion essay: We can find blood vessel quantity reduce, blood vessel thinningz.9,Westem blot result: Bax in endochylema and bioblast of Hep-2 cell express augment in the experiment group, Bcl-2 in endochylema express diminutus, cytochrome C in endochylema express augment obviously, cytochrome C in bioblast express diminutus obviously, p20 in endochylema express augment obviously.10,Tumstatin therapeutic efficacy for athymic mouse of anthropolaryngocarcinoma: There are significant deviation between two sets hairless mouse about its net measure, tumor weight, tumor volume and tumor weight/tumor volume. Anti-tumor rate is percent 57.07 in the treatment group.11,Immunity histochemistry show that MVD descend obviously in tumstatin treatment group. By electron microscope, we can observe that cell in the treatment group assume apoptosis.Conclusion1,19 peptide which is a direct Anti-tumor Peptide of Tumstatin can be expressed high performance and get ou.2,Vitro-experiment can prove that 19 peptide can induce laryngocarcinoma Hep2 cell apoptosis obviously.3,The mechanism that 19 peptide induce laryngocarcinoma Hep2 cell apoptosis may be through bioblast way.4,19 peptide can cure SC hairless mouse pattem efficiently.