| Small cell lung cancer is very malignant tumor, its clinical characteristic is that the blood and lymph metastasis occur very early. Nowadays, there are not effective therapies in clinic. We hope we can gain a new breakthrough on the gene therapy. So it is a hotspot to screen out the target genes and extrinsic genes. The gene therapy of Small cell lung cancer mainly focuses on how to interdict the way of metastasis, so it is very urgent to screen out the correlative gene of metastasis. In 1992, liangpeng and Pardee setted up a new mRNA dif-feretial display technology, it was called DD - PCR. The virtues of DD - PCR are very obvious, it almost can check all of the gene that expressed by cells, and at the same time compare many types of cells, show the differences, check the up and down expressions of gene. In the past few years, there were many a-meliorations on thechnology, which focused on how to improve the efficiency of distinguishing the differetial genes and reduce the workload. With the consummation of mRNA - DD, it is broad applicated in the field of biomedicine, especially in the pathophysiological process to search the new genes. In this study, we make use of mRNA - DD to investigate the difference of gene expression in the original site of cellule lung cancer and metastasis lymph nodes, and to show a method which screen out the correlative metastasis gene of small cell lung cancer.ObjectTo scan out the correlative gene fragments of small cell lung cancer metastasis by differetial display technology, and show a way to study the mechanism of small cell lung cancer metastasis. At the same time, further to complete the differetial display technology of mRNA.Materials1. Materials 5 specimens of the original site of small cell lung cancer and metastasis of lymph node were obtained from the first affiliated hospital of china medical university, which were excised by thoracic department and comfirmed by pathology after operation.2. Reagents I used anchored primer - T7( dT12) Aps and stochastic primer - M13r - ARPs in my experience, the reagent of distilling and RNA produced by IBCOBRLG company. The peagent of PCR produced by TaKaRa company. Genomyx fluorescent differetial reagent produced by and main equipments ( Genonryx DNA sequencer electrophoretic system and fluorescent Imaging Scanner) produced by BECKMAN corp, USA.Method1. We distilled the cancer cells of original site of small cell lung cancer and lymph nodes, which about 8000,by Pix cell II LCM.2. We distilled the RNA of the original site of small cell lung cancer and lymph node with Trizol method, put the cap that seized the cells on 0.5mlEp-pendoff tube that had put 200 ul Trizol reagent in it, and then put it into the centrifugal tube for 30 minutes at room temperature, then put 40ul chloroform into it and surged tempestuously and lay at room temperature for 15 minutes, and then centrifuged as 4C 11000 circles for 15 minutesk, lay at -70C for 30 minutes, then centrifuged as 4C 11000 circles for 10 minutes and then diacardedthe super fluids and put 75% DEPC ethanol 200ul into it, and then put it into RNA enzyme fluid to hatch for 10 minutes at 55C and preserved at -70C.3. To synthesize the first chain of cDNA: using anchored primer to synthesize the first chain of cDNA in PCR instrument.4. PCR; took the first chain of cDNA as templet to proliferate PCR with anchored primer and stochastic primer.5. The reclaim and proliferation of different zone: the outcomes of DDRT -PCR were electrophoresised 3000V, 55C for 4 hours. The gel was put into 1.5 ml centrifugal tube and put 50ul TE for 37C water for 60 minutes as -20C. 4ul TE, T7 Promoter and Ml3 Reverse proliferated the fragment of DNA.6. To Purify and analyze of the list: to proliferate the outcomes of DDRT -PCR and purified the 12000g for 15min and gain the fragment of purified DNA and then to measure the list.Results1. In my experiment, I synthesized the first cDNA chain by 2 anchored primers and selected 4 primers to analyse... |
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