| ObjectiveThe intestinal epithelial cells (IECs) not only are the major component of the intestinal mucosal barrier but also have many functions such as absorption and secretion, which contribute to its important role of maintaining the homeostasis of the body. Severe Gastrointestinal Syndrome occurred as human suffering the nuclear weapon explosion and nuclear casualty. It is incurable because of the lack of efficient treatments. IECs were sensitive to ionizing radiation and performed notable pathological process including cell proliferation inhibition, structure abnormality and deletion when exposed to enough high dose of radiation. Specially, the clongenic cell located in the crypt failed to survive and repopulate, which resulted in the collapse of the intestinal mucosal barrier and the failure of regeneration of the crypt and villi. These were regarded as the primary causes of the Gastrointestinal Syndrome. Because of the difficulty of purifying the crypt stem cell in vivo, IEC-6 cell line with the feature of undifferentiated crypt cells had been widely used in vitro for investigation of many kinds of side effects to the abdomen. In this study, we had identified and cloned a series of genes whose expression were enhanced in the IEC-6 cells after irradiated by 35Gyγray. The identification and functional study of these radiation-induced genes will be helpful to elucidate the molecular mechanisms of the damage and repair of intestinal epithelium exposed to high dose of radiation, provide new clues for the clinical treatment and offer the revelatory idea for the basic question such as proliferation, differentiation, apoptosis and transformation, etc. Methods Construction of a differentially expressed genes subtracted cDNA library of rat intestinal epithelium cell by suppression subtractive hybridization. 1) The adequate IEC-6 cells which in the logarithmic phase were divided into two groups .One of them were irradiated with a 60Co source at dose of 35Gy. After irradiation, the cells were cultivated for another day.2) Total RNA of the two group cells were isolated and the RNA's integrity and purity were examined.3)We took the irradiated IEC-6 cells as1. the tester and the normal one as the driver. The high quality smart double strand cDNAs were produced with the LD PCR based method. 4) Tester and driver cDNAs were separately digested to obtain shorter, blunt-ended molecules. 5) Two tester populations were created with different adaptors, but driver cDNA had no adaptors. 6)In the first hybridization, excess driver cDNA was added to each tester pool, heat denatured, and allowed to anneal. During the annealment, the single-stranded cDNA tester fraction was normalized (high- and low-abundant cDNAs became roughly equal) which helped prevent the loss of rare transcripts. In the second hybridization, the two separately subtracted tester reaction products were mixed together along with additional driver cDNA, and allowed to anneal. Thus, the tester fragments that were left unhybridized by the driver cDNA in the first hybridization could more easily find their complementary fragments and anneal. These newly formed hybrids had the two different adapters on their 5'ends. These two different adaptors allowed for exponential amplification during the PCR step. 7) The efficiency of subtraction was assessed by comparing the abundance of known cDNA before and after subtraction. 8)Took the normal IEC-6 cells as the tester and the irradiated one as the driver and repeat the step 1-8. 9) The production of the SSH was cloned to T/A vector and transformed the E.coli .2. Screened and identified the clones by reverse and forward northern blot. 1)Ninety six clones were picked out and were screened through reverse northern with the probe from the forward and reverse SSH production. The positive clones were sequenced and the similarity was searched against the DNA database in GenBank. 2)The expression of limited clones in different sample was identified by northern blot.Results1. High-quality RNA with perfe...
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