| Laryngeal cancer is the common malignant head and neck neoplasm, among these,more than 90% is glottic carcinoma. Surgery is the mainly treatment methods for laryngeal cancer.The patients can not be sound or their sound quality is damaged by rececting the vocal cord in whole or in part.This will affect the patient's quality of life seriously.Thermotherapy is considered as the fifth tumor treatment after surgery, radiotherapy, chemotherapy and biological therapy. In recent years, the gold nanoparticles of heat therapy received extensive attention. The principle of this treatment is:when under the irradiation of near infrared laser passing through the body without any damage, plasma resonance can occur on the surface of kernei of gold nanoparticles. These kernei can absorb the luminous energy and the luminous energy eventually converse to the thermal energy and these energy transfer to the environmental cell effectively to produce hot lethal effect. As cancer cells were poor heat-resistance,it can be killed by this way and then achieve therapeutic purposes. Research shows that gold nanoparticles can be easily modified and it can combind with anti-EGFR antibody and attach to the epicyte surface of squamous carcinomas and adenocarcinoma. So, the gold nanochains combine with anti-EGFR antibody and irradiate by near infrared ray, targeted treatment of laryngeal Hep-2 cells can be conducted.Objective:to evaluate the growth inhibition effect of gold nanochain and anti-EGFR/Au conjugate on treatment of Hep-2 human laryngeal cancer cell and the treatment effect of laryngeal Hep-2 cells on experimental model of the nude mice, and to explore the apoptosis approach to kill Hep-2 cell by thermotherapy.Methods:(1) In vitro test:To detect the cytotoxicity of gold nano-chain firstly by MTT method, and to observe the endocytosis and exocytosis of transmembrane when nanochain attach to Hep-2 through the electron microscope. Then to detect the growth inhibition effect of gold nanochain and anti-EGFR/Au conjugate under the irradiation of different power of the near infrared laser by the MTT method.To observe the change of cancer cellular morphology and by light-microscopy.To observe apoptosis by electron microscopy.To detect apoptosis rate by FCM.To detect the expression of apoptosis factor of Hep-2, bcl-2,bax, caspase and hsp70 by Westen blot.To explore possible apoptosis pathway by nanochain treatment.To compare the group difference and P<0.05,there is a significant difference(2) To establish laryngeal cancer nuke mice models. Gold nanoparticles chains and ant-EGFR/Au conjugate inject into the tumor body of different animal group respectively. To observe the growing and body weight change of nuke mice after irradiation of near infrared laser.To draw tumor growth curve, calculate the rate of tumour-inhibitory. To detect the expression of bcl-2,bax and hsp-70 in tumor tissue by immunohistochemical method after sacrifice of animal and further discuss Thermotherapy mechanism. To compare the group difference and P<0.05,there is a significant difference.Results:1.It can be found that gold nanochain can combine with anti-EGFR antibody through observing spectra. Toxicity experiments in vitro show that the gold nanoparticles combined by glucose synthesis method are no toxicity to Hep-2 and 50% concentration is the most suitable vitro concentration. Gold nanchain can enter into Hep-2 after 8 hours co-culture and can leave cell 48 hours later.So gold nanoparticles chain can enter and leave Hep-2 freely.2. Experiments in vitro show that there would be plenty of apoptosis of Hep-2 after six minutes illuminating of 8W/cm2 power laser both in gold nanochain heat therapy group and anti-EGFR/Au heat therapy group. Foaming epicyte can be observed under inverted microscope and cell adherece decreases. Apoptotic body can be observed on electron microscopy and swelling mitochondria. Damage is lighter in pure illumination group.Under the same condition, by FCM method,the apoptosis rate of pure illumination group, gold nanochain heat therapy group and anti-EGFR/Au heat therapy group is 9.5%,72.0%and 95.7%respectively. There is statistical difference between two group(P< 0.05). With the extension of irradiating time, there is a apoptosis increasing gradient change.3. Experiments in vitro show that the expression of Bcl-2 were significantly reduced and the expression of bax gene were increased significantly in gold nanochain heat therapy group and anti-EGFR/Au heat therapy group by westen blot testing.But there were no any obvious change in pure illumination group. Hsp70 protein expression were significantly higher in gold nano chain heat therapy testing group and anti-EGFR/Au heat treatment group in westen blot test and caspase-3 expression were significantly increased in ELIASA test.There were a significant difference comparing with control group and pure illumination group(P<0.05).4. Experiment in vivo of nuke mice show that the Tumor inhibition rate is 43% and 52% for gold nano chain illuminating group and anti-EGFR/Au heat therapy group respectively, but is 15% for pure illumination group only. Immunohistochemical result of oncogene show that the expression of bax, and Hsp70 were significantly higher but expression of bcl-2 were significantly lower both in the gold nanochain heat therapy group and anti-EGFR/Au heat therapy group. There were a significant difference comparing with control group and pure illumination group(P<0.05).Conclusion:1. Experiments in vitro show that the anti-EGFR/Au has a more lethal for Hep-2 cells than a gold nano chain of near infrared heat therapy.2. Experiment in vivo of nuke mice show that both the gold nanochain and anti-EGFR/Au near-infrared heat therapy could obviously inhibit tumor.3. The mechanism the gold nanoparticles near-infrared heat therapy kill Hep-2 cells may be conducted by mitochondria mediated by Bcl-2 and bax.
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