Effect Of Lipofectamine Induced CFLIP Antisense Oligodeoxynuclecotide On Human Lung Adenoma Cell SPCA-1

IntroductionLung cancer is on the first position of lethal cancer now. Scientist have used various kinds of treatments. But the therapeutic efficacy is not satisfactory. The cure rate is less than 15% . Human began to know the mechanism of the lung cancer in carcinogenesis, development and diversion by the rapid development of molecular biology. Antisense oligodeoxynuclecotide, ASODN is an artificial composition molecule lengh of mRNA or DNA's specific binding which block its expression. ASODN can block or inhibit key coding gene. The turner cell'prolifration can be specifically inhibited. We apply cFLIP_L/S mRNA mediated by lipidosome on anthropoadenoca cell line SPCA - 1 to observe if it can inhibit the growth of SPCA - 1 or encourage its apoptosis. This can be provided for the gene treament of lung cancer.Antisense drug is on the found of antisensenucleic. It's safe and effective. There are many antisense drugs in clinical test on the target of virus, oncogenes and cell active factors with the illuminating of oligodeoxynucleotides (ODN) and phosphorothioate oligodeoxynucleotides (PSODN).Our objectives of the experiments were to study the effect of Lipofectamine induced cFLIP antisense oligodeoxynuclecotide on the apoptosis in human lung adenoma cell SPCA - 1, and transplanted tumor of SPCA - 1 in Babl nude mouse, and to find out a new way for gene therapy of lung cancer.It is the exploitated phase for lung cancer target treatment study. Many an-tisense drugs are putting into clinical tests. This experiment certificate that anti-sense can inhibit tumour cell growth and induct it to be apoptosis after transfec-tion. This may be a reliable experiment base for the antisense treatment of lung cancer.Methods1. Cell culture high diversion anthropoadenoca cell line SPCA - 1 waw provided by Tumer Research Center of Harbin Medical University. Cells was cultured in routin RPMI1640 + 10% fetal bovine serum and 5%CO₂, 37C°constant temperature cabinet. After digesting and passage, the cells are seeding in 96 hole mask. Cell transfection beginning from the target of cFLIP gene' s translate initiation. There are three groups; Lipo - ASODN, Lipo - NSODN, Lipo-fectamine and negative control. The concentration are 0. 5, 1. 0, 2. 0μM/L in ASODN group and NASODN group. Lipid body group is at equal with its corresponding hole. The experment is in accordance with Lipofectamine TM2000 transfection 。 Then observe the cells by fluorescent inverted microscope 24 hours later. Select five high power field by trabant and count the cell in full as well as the green color fluorescence. Calculate the transformation efficiency according to the formula.2. Detection of growth inhibition rate by MTT: Every 104 logarithmic growth phase SPCA - 1 cells in 100μL were taken into 96 shadow mask These cells were cultured 2 hours after transfection. MTT(5g/L) was put in, then throw away the supernatant 4 hours later. Put 100μl DMSO in every hole. Determinate the absorbance( A₄₉₀) of 490nm wave length 10 minutes later. The experiment was repeated after transfection 6 hours, 12 hours and 24 hours later, then the cell inhibition rate was calculated.3. RT - PCR:collect cells after transfection and extract cell's RNA by Tr-izol. The first chain of cDNA was reverse transcription, and PCR products of cFLIP_L/S were amplified. After 2% agarose gel jel, images were made and PCR products were scanned by image analysis system.4. Western Blot. Throw away culture solution after SPCA - 1 cells transfect-ed 24 hours later. Determinate the protein concentricun by Bradford and scan the bands with Bio - Rad2000 image analysis software. Judge the power with grate extinction.5. TUNEL:Seed every 3. 0 × 10⁵ cells in 6 holes and add in 1. 0μm/L Lipo - ASODN and Lipo - NSODN when the 80% of cells fused. Fix them with 4%formalin for 10 minutes after cultured in 24 hours. Colorate by DAB. Count the full cells and the apoptosis cells in microscope.6. Flow cytometry: Fix the cells which were transfected 48 hours later with 70% alcohol in 4℃ , then incubation with RNA enzyme in 37℃ for 1h. Dyed by PI in 4℃. Detect the apoptosis by flow cytometry. Analyses the result with multicycle software.7. Cell morphology: the cells were fixed with glutaradehyde and osmic acid. after staining cells ultrastructures were observed and pictures were taken under H - 600A electron microscope.8. In animal experiment, Lipo - cFLICE - ASODN and Liposome control were administered to SPCA - 1 transpalnted tumor model. Tumor inhibitory rate was used to evaluate the tumor inhibitory effect of Lipo - cFLIP - ASODN, and pathologic changes was observed by light and electron microscope.Results1. Transfection mediated by lipid body. The transfection efficiency of lipo -ASODN and Lipo - NSODN is 50. 16% and 51. 34% in 2 hours;, 80. 25%and 78. 98% in 6 hours; 95.47% and 96. 80% 24 hours.2. After transfection, the cell growth inhibition ratio increase in different degree. Cellular inhibition rate went up to 71. 52% in Lipo - ASODN group higher than those in Lipo - NSODN group and liposome controls in MTT assay ( p<0.01).3. After transfection cFLIP for 24h gene expression in antisense oligodeoxy-nuclecotide group was less than that in control group in RT - PCR assay.4. cFLIP_L and cFLIP_S protein expression were also decreased in Western Blot. Protein of cFLIP_SLipo - ASODN 0. 5, 1.0, 2. 0μm/L are down regula-...