| Objective: The incidence of esophageal cancer in the middle and southareas of Hebei province is the highest in the world. Esophageal cancer is notso sensitive to radiation or chemotherapy. Surgical treatment is now the maintherapy. As for this reason, many kinds of biochemistry researches were madeto explore the etiology and pathogenesis of esophageal cancer so as to benefitpatients in early detection, early diagnosis and early treatment. At the sametime, in evaluating the prognosis of patients with esophageal cancer there shallbe a positive sense. It is especially helpful to find a new therapeutic method intreatment and improve the life quality and survive of patients with esophagealcancer. Metallothinein (MT) contributes to help cells to survive afterchemo-radiation therapy and its expression is significantly higher in patientswith esophageal cancer. Different MT subtypes present expression rulesdifferently. Many signs in experiments show that behind the high expressionsare genetic mutations. We selected patients with esophageal cancer those whohave had surgery therapy to obtain the tumor tissues and normal tissues ascontrol in this study. Polymerase chain reaction (PCR) was employed toamplify the three exons ragins of MT-2A gene to gain gene's sequencesstudied. Through a direct sequencing method, get amplification gene sequenceand study whether there was a mutation in MT protein coding areas whichcould contribute to a change in the gene encoding protein.Methods:1Get fresh esophageal cancer tissues and normal esophagealmucosal tissues. Select esophageal cancer tissues and neighbored tumor-freetissues from36patients who had operations in Thoracic Ward of the FourthAffiliated Hospital of Hebei Medical University from May2010to September2011. Among the patients,24were male while12female and age range from 37to71with a mean age63. Select fresh tumor tissues by operations andesophageal mucosal tissues called normal esophageal mucosa tissues whichwere more than5cm away from the cancer tissues as control. All theprocesses in gaining tissues were done in a low temperature environment, andafter the collection and registration, tissues were invested in liquid nitrogenlabel as storage. Tissues those postoperative pathology was esophagealsquamous cell carcinoma were taken as the ones used in the study.2Cut the collected tissues to get pieces sized about0.5mm x0.5mm x0.5mm. HE anastomosed slice of wax dyeing was used to get the esophagealsquamous cell carcinoma tissues and the normal esophageal mucosa tissuesconfirmed.3Extract DNA from tissues. After cutted to do HE dyed, the tissuesamples remaining were done by NaCl to extract DNA. The DNA extractedfrom tissues was solved in deionized water, and then use spectrophotometer toobtain the concentration and purity. Saved at-20℃.4Primer Designing. After application of MT-2A three exon areas DNAsequence from NCBI, the Sanggong as commission agent designs primers.Blast primers designed first to confirm specificity for the requirements andthen made them composed.5Polymerase chain reaction. Polymerase chain reaction with the primerswas employed to amplify destination genetic segments based on theexperimental best annealing temperatures gained by experiments in advance.Then an electrophoresis with agarose gel was done to confirm amplificationeffects and judge whether there was genetic mutation.6Test gene sequencing and analysis of mutations. After getting throughPCR and confirmed by agarose gel electrophoresis, the destination geneamplified was sent to SangGong biological engineering (Shanghai) Co., LTDto gain the sequence of the amplification products by direct sequencing test.Both the sense sequence and the reverse sequence were tested, and only thetwo sequences matched each other perfectly, was the result100percenttrustworthy. Finally obtain the specific sequences of amplification fragments which were the products of PCR. Then analyze the results compare with thehuman genome genetic sequence to check whether there was a mutation.Results:1All the tumor tissues collected from clinic were confirmed tobe esophageal squamous cell carcinoma which was matched with the result ofpostoperative pathology. And "normal" esophageal mucosa tissues were notcontaining tumor tissues.2The DNA extracted from tumor tissues and normal tissues wereamplified by polymerase chain reaction. The products all appeared destinationbands after electrophoresis strips, and the strips were single,clear and at thesame consistent position which meant there was no insert or missing geneticfragments, which is also a kind of mutation, between primers' design area.3After direct gene sequence test, no point mutation performance ofMT-2A DNA explicit branch area that could influence the protein codingchange was found in either the36tumor tissues or control tissues.4An A/G polymorphism site upstream MT-2A exon1was detected bothin the tumor tissues and the control tissues,31out of the36patients' sampleswere negative while the others positive. After comparing with the humangenome project gene pool, the existence of the polymorphism was confirmed.Comparing with the statistics from NCBI, no difference was found.Conclusion: MT-2A DNA sequence of esophageal cancer patients'tissues was highly conservative. That meant DNA mutations that could lead toMT-2A coding protein change was not found in esophageal squamous cellcarcinoma tissues. And we think that reason for the MT protein accumulationin esophageal squamous cell carcinoma tissues was the degradation of theprotein. And a further study is needed to reveal the mechanism.And moreresearches are need to confirm whether the polymorphism had an influence onthe transcription of MT-2A gene,the protein expression and patients' survive. |
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