Study On Aberrant Transcription And Expression Of FHIT Gene In Colorectal Carcinomas Induced By 1,2-dimethylhydrazine In Rats

Colorectal carcinoma(CRC) is one of the most common malignant tumors in human digestive tract. The mortality and incidence of CRC have been increasing in the past thirty years. It is proved that the carcinogenesis and development of CRC is a multi-stage process which may be initiated by many factors and involved with many genes. CRC is the result of cooperation of both genetic and environmental factors.FHIT gene, a proved tumor suppressor gene, was identified by Dr. Ohta in 1996. It is located at 3pl4.2 region of human chromosome, which contains the most active common fragile site in the human genome(the FRA3B locus). FHIT gene is the most commonly altered gene in human cancer today and is usually considered the target molecule of environmental carcinogens.To make clear the effective mechanism of FHIT gene in the carcinogenesis and development of CRC, especially the aberrant alteration of mRNA transcription and expression of FHIT gene in the CRC caused by chemical carcinogens, the CRC animal model of Wistar rats induced by l'2'-dimethylhydrazine(DMH) is introduced in this study. The alterative mode of FHIT gene during the carcinogenesis of CRC will be studied with immunohistochemical method, nested reverse-transcriptase polymerase chain reaction(nested RT-PCR) and DNA sequencing. All study consists of three parts as following:Part I: The construction of CRC animal model induced by DMH in Wistar rats. Objective: To construct CRC animal model induced by DMH in Wistar rats, and to observe the ratio of reproduction and apoptosis of crypt cells of colorectal mucosa in carcinogenesis of CRC. Methods: CRCs in Wistar rats were induced by percutaneous injection of DMH with regular one dose(20mg/kg body weight) a week for 20 weeks, and the incidence of CRC were observed by killing animals half at middle term(at the end of 10th week) and half at the end(at the end of 25th week). The reproduction and apoptosis state of crypt cells of colorectal mucosa were evaluated by BrdU-labelled cellular immunohistochemical technique and TUNEL method separately. Results: (1) Themiddle-term incidence of colorectal adenomas is 74.2%(23/31) and no CRC observed. (2) The end-term incidence of CRC is 87.1%(27/31). (3) The ratio between reproduction and apoptosis of crypt cells of colorectal mucosa in DMH group is significantly higher than that of control group. Conclusions: The CRC animal model induced by DMH in rats is successfully constructed and the 87.1%(27/31) incidence of CRC is achieved. The whole process of carcinogenesis of CRC, from normal mucosa to adenoma, and to carcinoma is observed step by step. The ratio between reproduction and apoptosis of crypt cells of colorectal mucosa has a good correlation with the incidence of CRC, which can evaluate the carcinogenesis of CRC efficiently.Part II: The experimental study of aberrant transcription of FHIT gene in CRCinduced by DMH in rats.Objective: To study the aberrant transcription of FHIT gene in CRC during the induction by DMH in rats. Methods: The FHIT cDNA of induced CRC, colorectal adenoma by DMH and colorectal mucosa in rats were detected by nested RT-PCR and direct DNA sequencing. Results: (1) The aberrant transcripts of FHIT gene were detected in 21.4%(3/14) colorectal adenoma and in 52.4%(22/42) CRC. (2) DNA sequencing found that the aberrant transcripts of FHIT gene were caused by deletion or insertion of nucleotide fragments. (3) The FHIT gene sequence of Wistar rats in China is different in two bases from that recorded in GenBank. Conclusions: (1) The aberrant transcription of FHIT gene is correlated with the initiation and development of CRC induced by DMH in rats. (2) It is first time to find the deletion mutation in aberrant transcription of FHIT gene in rats. (3) A new sequence of FHIT gene from China Wistar rat was discovered.Part HI: The experimental study of aberrant expression of Fhit protein in CRC induced by DMH in rats.Objective: To study the aberrant expression of Fhit protein in CRC during the induction by DMH in rats. Methods: The expressions...