Studies On The Response Of Hep2 Cells Transfected With TNFR2 To Stimulation By TNF

Laryngeal cancer is a common malignant tumor on otolaryngology-headand neck surgery. It accounts for 35.4% in head and neck tumors, 7.6% inhuman malignant tumors. At present, the ways of curing laryngeal cancerinclude many multiple therapeutic methods, such as radiotherapy,chemotherapy, surgical operation, laser therapy and so on, but none of themleads to ideal effect. TNF-α is a pleiotropic cytokine that regulates various cellular responsesincluding growth, differentiation, immune regulation and apoptosis.The effectsof TNF-α are mediated by two distinct receptors, the p60 TNF receptor 1(TNFR1) and the p80 TNF receptor 2 (TNFR2). The clinic experiments ofusing TNF-α to cure malignant tumor have been carried out, but the side-effectis very serious.This study uses the Hep2 cell from patients with laryngeal squamouscancer as the research object, and Uses immunohistochemical method,TUNEL method, flow cytometry and gene transfection method to examine theexpression of TNFR1, TNFR2,TRAF2 and the apoptitic index in 33 laryngealsquamous carcinoma tissue. The research also assesse the effect of both Hep2cells and Hep2 cells transfected with TNFR2 to stimulation by TNF-α.The contents of this paper are as follows:1. Obtaining the clinical data of specimanSpecimens were obtained from 33 patients with laryngeal squamouscarcinoma at Bethune International Peaceful Hospital between 1990 and 2002.All patients did not undergo chemotherapy and radiotherapy. The average agewas 57.3 (37-80). 29 patients were male, and 4 patients were female. Therewere 7 patients with metastatic neck lymph node metastasis, and 31 patientswith smoking. The patients were divided into four stages according to TNM.There were 5 patients in stageⅠ , 10 patients in stage Ⅱ , 9 patients in stage Ⅲ ,and 9 patients in stage Ⅳ. The patients were divided into three gradesaccording to pathology. There were 5 patients in grade Ⅰ, 10 patients ingrade Ⅱ , and 9 patients in grade Ⅲ .2. Examining the expression of TNFR1,TNFR2 and TRAF2 inlaryngeal squamous carcinomaThe immunohistochemical method was used to test the expression ofTNFR1, TNFR2, TRAF2. The TUNEL method was used to test the apopticindex.The experiment results show: In 33 laryngeal squamous carcinomatissues, the positive rate of TNFR1 was 63.6%, the positive rate of TNFR2 was6.1%, the positive rate of TRAF2 was 69.7%. Neither TNFR1 nor TNFR2 wasrelated to the biological characters of laryngeal squamous carcinoma (P>0.05), and TRAF2 was related to the tumor pathological grading only (P<0.05).The expression of TNFR1 was strongly associated with the expression ofTRAF2 (P<0.01). The apoptotic index of the positive tissue of TNFR1 andTRAF2 (3.12±1.47 and 2.85±1.19)is significantly lower than that of thenegative tissues(4.26±1.34 and 5.10±0.83)(P<0.05 and P<0.01).3. Examining the expression of TNFR1 and TNFR2 in Hep2 cellBoth immunocytostaining and flow cytometry were used to evaluateexpression of TNFRs.The experiment results show: TNFR1 expression showed moderatestaining, and TNFR2 expression showed weak staining. Compared with thenegative control, the OD value of both TNFR1 and TNFR2 are significantlydifferent from one another, P<0.01. By flow cytometry, the quantity of TNFR1was 1.8 and the quantity of TNFR2 was 1.2 .4. Examining the effect of TNF-α on cell inhibition and cell cycle onHep2 cellsTo observe the effect of TNF-α on cell inhibition and cell cycle in Hep2cells by crystal violet staining,acridine orange staining and flow cytometry.The experiment results show: At the concentration of 102-,103-,104-,105-or 106pg/ml in 24 hours,the death rates of Hep-2 cells were 5.17±0.87%,16.4±0.59%, 23.0±0.97%, 26.3±1.10%, 31.9±0.96%;the apoptic rate were1.40±0.32%, 2.27±0.12%, 3.41±0.03%, 6.44±0.18%, 8.07±0.65% . In12-,24-,36-,48-or 72 hours, at the concentration of 104pg/ml, the death rates ofHep-2 cells were 14.3±1.00%, 23.0±0.97%, 28.0±0.75%, 35.7±2.10%,43.3±3.10%, the apoptosis rate were 1.61±0.20%, 3.41±0.33%, 7.71±0.47%,10.94±0.77%, 12.85±0.36%. With the increasing of concentration and time,both the death rate and the apoptic rate increased significantly. After treatmentwith TNF,the percentage of G2 cell cycle increased significantly from7.73±1.66% to 18.14±1.33% (P<0.01).5. Examining the Change of sensitivity to TNF on Hep2 cells withexogenous TNFR2 gene5.1 Measuring the the quantity of TNFR2 on Hep2 cells transientlytransfected with pcDNA3-TNFR2Cells were transfected using Lipofectamine 2000. TNFR2 expression wasassessed by cotransfection with GFP plasmid, immunocytostaining and flowcytometry.The experiment results show: GFP expression was the most strong in 48h,and did not increase between 48h and 72h.The OD values of TNFR1 were 153.8±3.2 and 152.0±5.0 in Hep2 cellstransfected with pcDNA3 and pcDNA3-TNFR2, the difference was notsignificant. The OD values of TNFR2 were 155.0±3.8 and 132.8±9.0 in Hep2cells transfected with pcDNA3 and pcDNA3-TNFR2, the difference wassignificant, P<0.01. By flow cytometry, the quantity of TNFR2 was twicetimes in Hep2 cells transfected with pcDNA3-TNFR2 as in Hep2 celltransfected with pcDNA3. TNFR2 expression was significantly strong.5.2 Measuring the effect of TNF on Hep2 cells with exogenousTNFR2 geneBoth the death rate and the apoptic rate were measured using crystalviolet staining and flow cytometry.The experiment results show: At the concentration of 102-,103-,104-,105-or 106pg/ml in 24 hours,in Hep2 cells transfected with pcDNA3-TNFR2, thedeath rates were 37.5±4.7%, 46.1±1.9%, 62.9±1.1%, 75.5±1.2%, 85.7±1.5%,the apoptic rates were 5.06±0.37%, 7.12±0.20%, 12.11±1.59%, 28.43±2.38%,63.54±5.50%, respectiveLy. In Hep2 cells transfected with pcDNA3, the deathrates were 5.8±1.5%, 14.5±4.9%, 21.0±4.9%, 29.5±2.1%, 35.3±2.9%, theapoptic rates were 1.53±0.53%, 2.37±0.22%, 4.02±0.23%, 6.78±0.27%,8.37±0.19%. At the same concentration, the inhibition rate of both cells wassignificantly different.In 12-,24-,36-,48-or 72 hours, at the concentration of 104pg/ml, the deathrates of Hep2 cells transfected with pcDNA3-TNFR2 were 52.1±2.7%,62.9±1.1%, 73.5±0.6%, 79.8±0.6%, 86.2±0.6%, the apoptic rates were6.86±0.34%, 12.11±1.59%, 23.09±2.63%, 37.29±2.13%, 57.12±4.44%. InHep2 cells transfected with pcDNA3, the death rates were 12.4±1.9%,21.0±4.9%, 26.5±2.4%, 32.8±2.6%, 39.8±3.1%, the apoptic rates were1.77±0.42%, 4.02±0.23%, 6.78±0.44%, 9.95±0.66%, 12.10±0.55%. At thesame time, the inhibition rate of both cells was significant difference.Taken together, the present research studied the response of Hep2 cellstransfected with TNFR2 to stimulation by TNF-α using the in vitroexperiments. The data will benefit the gene therapy of laryngeal squamouscarcinoma with TNF-α.